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PCR & qPCR
qPCR Instrumentation
Q
Real-Time Quantitative PCR
SYBR Green Detection
PerfeCTa SYBR Green SuperMix
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PerfeCTa qPCR ToughMix
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PerfeCTa qPCR FastMix UNG
Multiplexed Pre-Amplification
PerfeCTa PreAmp SuperMix
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AccuStart II PCR SuperMix
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AccuStart II PCR ToughMix
AccuStart II GelTrack PCR SuperMix
AccuStart Long Range SuperMix
repliQa HiFi ToughMix
Cloning
repliQa HiFi ToughMix
Next Generation Sequencing (NGS)
sparQ HiFi PCR Master Mix
sparQ DNA Library Prep Kit
sparQ DNA Frag & Library Prep Kit
sparQ PureMag Beads
sparQ Universal Library Quant Kit
sparQ UDI Adapters (1-96)
Reverse Transcription
Conventional RT-PCR
qScript XLT 1-Step RT-PCR Kit
Quantitative RT-qPCR
qScript XLT 1-Step RT-qPCR ToughMix
qScript One-Step SYBR Green RT-qPCR
UltraPlex 1-Step ToughMix
qScript One-Step RT-qPCR Kit
Qscript lyo 1-step
qScript 1-Step Virus ToughMix
First-Strand cDNA Synthesis
qScript cDNA SuperMix
qScript XLT cDNA SuperMix
qScript cDNA Synthesis Kit
qScript Flex cDNA Kit
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Extracta DNA Prep
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AccuStart II PCR Genotyping Kit
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CofA (PSF)
PSF-95073-250 Lot# 66158253PSF-95166-500 Lot# 66170168PSF-95114-05K Lot 66158684PSF-172-5565 LOT# 66170162PSF-95048-100 Lot# 66167704PSF-172-5064-Lot#100820PSF- 95054-500 Lot #66170261PSF-95054-500 Lot# 66166267 PSF-95137-100 LOT#66168457PSF-95057-200, Lot# 66170461PSF-95191-024 Lot# 66169235PSF-172-5074-LOT#66179734PSF-95048-500 Lot# 66167705PSF-20015C-0.02-LOT#66181211PSF-172-5565 LOT# 66170162PSF-172-5588-Lot#66172319PSF PSF-95062-01K Lot # 66146449PSF-172-5064(02)-Lot#100820PSF-95191-096 Lot# 66169243PSF-95049-025 Lot# 66158690 PSF-20015C-LOT#66181210PSF-61071103-Lot#66177100PSF- 95091-250 Lot# 66158696PSF-95134-02K Lot# 66169592CoA-172-5568-Lot#029562PSF-95047-500 Lot# 66157817PSF 95139-250-LOT # 66160961PSF-172-5565-Lot#66170162PSF-95194-024 Lot# 66169250PSF-95061-250-LOT#66153582PSF-95131-Lot#66177652PSF-95049-100 Lot# 66159101 CoA-1077404-Lot#029894PSF- 95166-01K - Lot# 66156165PSF-95050-500 Lot# 66167990PSF-95063-01K Lot# 66166075PSF-172-5579-Lot#029816PSF-172-5587-Lot#66169344PSF-95194-096 Lot# 66169260PSF-95047-025 Lot# 66159095 PSF-95049-100 Lot# 66167779PSF-95054-100 LOT#66168779CoA-172-5527-Lot#029070PSF-95056-02K Lot# 66166083 PSF-95118-012 Lot# 66169815PSF- 95091-250 Lot 66158696PSF-172-5577-Lot#66175725PSF-172-5588-Lot#66169345PSF-CMK0022-96 Lot# 66168664PSF-95047-500 Lot# 66167776PSF-95050-100 Lot# 66163976 PSF-95131-02K LOT#66168584CoA-172-5561-Lot#029295PSF-172-5587 -LOT#66169344PSF-95059-050 Lot# 66165474 PSF-95091-250 Lot 66158696PSF-95119-012 Lot# 66169818 (01)PSF-95048-025 Lot# 66167707PSF-95052-100 Lot# 66164848 PSF-95131-100 LOT#66168656CoA-852-0035-Lot#029287(MMDLot#2834)PSF-172-5055-Lot#66172529PSF-95055-500 Lot 66168777PSF-172-5055-Lot#66172529PSF-172-5588-LOT#66169345PSF-95062-01K Lot# 66158157PSF-172-5054-LOT#66184311PSF-95166-01K, Lot# 66170167PSF-95112-05K. Lot 66158683PSF-CMK0022-16 Lot# 66168679PSF-95048-100 Lot# 66164355 PSF-95054-100 Lot# 66164452 PSF-95131-500 LOT#66168658CoA-CM0175C-1-Lot#026230
FAQs
  • PCR & qPCR
    qPCR Instrumentation
    Why are some Taq suited for fast cycling and others require the standard cycling profile?
    There are several commercially available Taq polymerases that differ in their enzymatic properties. Some display high processivity at temperatures above 60oC and are labelled as ‘fast cycling compatible’ polymerases because they can achieve PCR with a relatively short single annealing/extension step (<20 sec) without the need for a dedicated extension step at 68-72oC. These fast cycling compatible enzymes should be used with the ‘fast taq’ profile in the Q-qPCR software for details on how to set up a thermal profile. For Taq polymerases that are not compatible with fast cycling conditions, some consideration must be taken to select the appropriate thermal profile. For assays with an annealing temperature <60oC, a two-step cycling may not be achievable and a dedicated extension step and 68-72oC may be required. For assays with an annealing temperature >60oC, it may be possible to achieve a two-step cycling if the ‘standard taq’ profile is selected. This is because the standard taq setting will decrease the speed of transition between annealing and melt temperatures, which will increase the time that the reaction is held in the range of taq activity (a fast transition from annealing to melt temperature will quickly pass through this temperature range, while slowing this rate of increase will provide more time for taq to achieve amplicon extension).
    What is the ramp rate?
    We do not typically like to discuss ramp rates, except to point out that they are an arbitrary measurement that may be misleading. A ramp rate is the rate of temperature change over time, but there is no standardisation for how to take this measurement. For instance, you could measure a ramp rate for a metal component of a machine that in reality has no relationship with the heat over a block, and will certainly not reflect your sample reactions temperature. The lack of similarity in ways to define and/or measure ramp rates makes the calculated values not comparable (it is like comparing how much you like something. One person likes it heaps, another lots. Its meaningless to debate which person likes it more/less). The ramp rate for the Q is on our website, however it is much more indicative of machine performance to compare the time taken to complete a qPCR run
    How fast can the Q complete a run?
    While this will vary according to your particular assay (different annealing temperatures and hold times), the Q can typically perform a 35 cycle qPCR run complete with melt curve in under 25 min.
    Why is the efficiency presented as a percentage and not a number between 1→2?
    qPCR efficiency reflects the similarity of data to exponential growth, or a doubling of signal for each qPCR cycle. The efficiency can be expressed as the rate of change of the data (as a multiplication factor), with 1 representing no change in data per cycle and 2 representing a doubling of the data per cycle. Alternatively, this can be expressed as a percentage of amplification efficiency, with 0% representing no change in the data per cycle and 100% representing a doubling of data per cycle.
    What are the benefits of using the REST analysis rather than ∆∆Ct?
    The REST analysis has the key advantage that it incorporates reaction efficiency while calculating expression ratios (Pfaffl 2002). The inability for the 2-∆∆Ct method to incorporate reaction efficiency could lead to major biases in the calculated expression ratios (eg see Ruijter 2009). While the 2-∆∆Ct method has been modified in some cases to account for amplification efficiencies derived from serial dilution of a standard sample, the combination of linREGPCR and REST allows the reaction efficiency of each actual sample to be used, eliminating the bias of the standard curve method that is often referred to as being only assay specific, and not sample specific (the standard curve method assumes that the calculated efficiency is both a perfect derivation of that value, and that the value perfectly matches that of the actual samples in each instance. The linREGPCR method includes sample specific differences in efficiency and does not assume equivalence of a standard curve with observed values, increasing the accuracy of the calculated data). Finally, the REST analysis uses non-parametric randomisation to calculate the error, which is more suitable for qPCR data than more standard parametric tests (eg T-test). The REST randomisation retains the same power as parametric testing without being affected by data distribution, unlike parametric tests which rely on normal distribution of data which is often not true of qPCR data, and also requires equal variance among the datasets which non-parametric testing does not.
    Why did you choose linREGPCR for data analysis?
    LinREGPCR uses sophisticated algorithms to increase the accuracy and reproducibility of qPCR data analysis, and is in our opinion the all-round best performing qPCR cycling analysis method. For more information, see (Ramakers 2003, ruijter 2009). The algorithm places the threshold value within the best portion of collected data (the window of linearity), and can scan for the threshold value with the least variation among and between samples to increase the accuracy of the data analysis. Importantly, linREGPCR will calculate efficiency values for each qPCR curve in the data analysis (meaning you don’t need to assume equal efficiency between standards and unknowns, and any sample specific efficiency differences are accounted for). linREGPCR employs a superior baseline correction method that has been shown to reduce the error in efficiency values that can be caused by using other baseline correction methods. Finally, the linREGPCR cycling analysis functions synergistically with our relative quantification analysis to provide the most accurate and reproducible gene expression ratio calculation.
    If my computer has an issue during a run – how can I retrieve my data?
    If a computer loses communication with the Q, the data from that run is stored on the machine. If you are using the same computer to reconnect to the Q-qPCR, clicking on the serial number icon (top right hand corner of the software) will give an option to ‘recover the run’ file. Similarly, if you connect to the Q-qPCR with a separate computer, you can still recover the data by clicking on the serial number icon, but now the option provided will be to ‘reconstruct the run’.
    Which fluorophores can I use with the Q?
    The Q-qPCR’s optical detectors will work optimally with FAM (green), Cal Fluor Orange 560 (yellow detector), Cal Fluor Red 610 (orange channel) and Quasar 670 (red channel). This combination of fluorophores will produce less than 3% crosstalk between any/all channel/s used. Other fluorophores with similar spectral properties will also be suitable. For example, Texas Red/ROX have very similar spectral properties to Cal Fluor Red 610, and can be measure on the orange channel. A list of popular fluorophores that are suitable for a particular detector are available while setting up the assay chemistry, and a more comprehensive chart is provided in the Q-qPCR manual (page 143).
    Can I export the raw data?
    Yes. To export the raw data, expand the save as icon and select the save as excel workbook option. This will save all of the information in the run file, including the raw data.
    Do I really need water balance tubes?
    Yes, for optimal performance the Q-qPCR should have 48x reactions of equal volume (samples or H20). This is because the amount of material that you need to heat/cool (ie the thermal load) will affect the amount of energy required to heat it to a particular temperature. As an example, a kettle filled with 1000 mL water will take more energy to heat to boiling than that same kettle filled with 100 mL. The Q-qPCR will run to specification when using a fully stacked rotor, but changes to the thermal load (incompletely filling the rotor) may impact the thermal performance.
    Can the Q-qPCR perform HRM analysis?
    Yes. The Q-qPCR has excellent thermal accuracy (uniformity of ±0.05oC) and can run a melt curve at 0.025oC/sec.
    Can the samples be used for post-PCR analysis when the tubes have the oil overlay?
    Yes, you can break through the oil layer with a pipette tip to remove your PCR reaction without the oil.
    What level of ROX should I use?
    The Q-qPCR does not require ROX. Other qPCR machines may require ROX to track temperature and/or optical performance due to thermal drift and variation in the detector setup. The use of magnetic induction (which has no thermal drift and will continue to provide uniform thermal performance over time), combined with the lack of moving parts in the detectors means that the Q-qPCR machine is highly reproducible over time. This means that while other machines may require the ROX channel just to monitor machine performance, the Q-qPCR has the advantage of allowing all channels to be available for real samples.
    Can I use the Q-qPCR for fieldwork?
    The Q-qPCR is well suited for fieldwork. You can run the machine from any power source, using a power inverter as required. Because you don’t need to calibrate for optics or temperature, and because of its small size/weight, the Q is ideal to transport to field sites while retaining functionality – it stays plug-and-play when other machines would need calibration. Also see our Technical Note: Q in the Field - Alternative Power Sources
    Can I configure the Q-qPCR software to easily enter sample names from a 96-well plate?
    Yes, the left hand side of the 96-well plate view will list the samples in the A1::H6 format, and the icon beside that will list the samples in the A7::H12 format to match the right hand side of a plate (the A1::H6 and A7::H12 icons are located at the top right hand corner of the samples editor). Next to these icons are the ’samples by column’ and ‘samples by row’ icons. If the samples were originally oriented according to the columns of a 96-well plate then you can simply enter these in to the Q-PCR software as it is the default display. If the samples were originally oriented across the rows of the 96-well plate, the samples will be located in different positions in the rotor, but once you select the ‘samples by row’ icon the samples selector will re-orient the display so that you can easily enter the sample names
    Can I save my analysis settings to be automatically applied in subsequent runs?
    Yes. You can either click the ‘save analysis settings’ icon which is located at the top right hand corner of the analysis settings display (looks like an Erlenmeyer flask), or expand the assay in the navigation bar (left hand strip of the software) and click the + icon next to the analysis settings icon, fill out your information in the appropriate analysis setting and save (click on the assay name in the navigation pane and click the save icon).
    How can I find help about the Q-qPCR?
    The best initial resource is the Q-qPCR manual. You can access the manual by clicking on the ‘?’ icon located at the top left hand side of the software, and selecting the Q-qPCR manual option. Once open, you can scroll to the relevant section or use the ctrl+F search function to find what you need.
    Real-Time Quantitative PCR
    SYBR Green Detection
    We use Perfecta Sybr Green Fastmix. We are starting to use uMELT software to predict qPCR melt curves. Could you please provide me with this info so that I can accurately use uMELT? [Mono+] : Concentration of monovalent cations in solution (mM).
    The PerfeCta SYBR Green Fastmix has 50 mM salt and 2.2mM Free Mg at 1X. However, the co-solvents and the concentration of SYBR green I dye in the mastermix also affect amplicon Tm, so you may still not be able to accurately use uMELT. Our R&D has found uMELT mostly useful for predicting multiple melting domains in an amplicon.
    Can I use PerfeCTa® SYBR® Green FastMix® instead of SYBR Green SuperMix?
    You can use FastMix instead of SuperMix. We have observed similar results with both PCR mixes, however, slightly lower background was observed using SuperMix when using 10 ng of cDNA or more in the qPCR. The qScript microRNA Quantification system has been validated using SYBR Green SuperMix.
    What is the amount of cDNA used per TaqMan or SYBR qPCR Assay?
    Suggested input quantities of template are: cDNA corresponding to 1 pg to 100 ng of total RNA; 100 pg to 100 ng genomic DNA. For more information, please consult the PerfeCta qPCR Supermixes or PerfeCta SYBR Green Supermixes protocols.
    Is there a SYBR® Green Fast Master Mix currently available
    Yes. PerfeCTa SYBR Green Supermix is available. For further information, please consult the Kit protocol or product information.
    Probe-based Detection
    What are the advantages of PerfeCTa qPCR FastMix vs PerfeCTa qPCR Supermix?
    Amplification can be run in about X minutes, at least 3X faster than standard runs with the PerfeCTa qPCR Supermix, with comparable results. For more information on using these reagents, please consult the PerfeCTa qPCR FastMix and PerfeCTa qPCR Supermix Protocol.
    Storage of PerfeCTa qPCR FastMix
    We recommend a storage temperature of 2 – 8 ºC for the TaqMan® Fast Universal PCR Master Mix (P/N 4352042). The shelf life is 9 months from the manufacturing date.
    SNP assays with PerfeCTa qPCR FastMix?
    The TaqMan® SNP Genotyping assays have not been fully tested on the Applied Biosystems 7500 Fast System.
    What component in the PerfeCTa qPCR FastMix allows rapid amplification results?
    The hot-start DNA polymerase enzyme system in the PerfeCTa qPCR FastMix only requires a 30 seconds activation step, and supports significantly shorter hold times for the thermal cycling stages of PCR, thereby minimizing PCR run time. For more information on using this reagent, please consult the PerfeCTa qPCR FastMix (2X) protocol.
    How do assays performed with PerfeCTa qPCR FastMix compare with assays performed with PerfeCTa qPCR Supermix?
    In general, assays have been found to perform comparably. Please consult the PerfeCTa qPCR Supermix (2X) Protocol for more information on using this reagent.
    What are the applications for the PerfeCTa qPCR FastMix?
    Absolute Quantitation and Relative Quantitation assays can be run fast. Allelic Discrimination (SNP) assays and plus/minus assays using internal positive controls cannot be run fast.
    How stable is an experiment set up with the PerfeCTa qPCR Fastmix Kit at room temperature?
    For optimal results, it is recommended to directly run the reaction plate after setting up the reaction. If for any reason a reaction plate cannot be run within 2 hours of completing the reaction setup, then freeze/refrigerate the reaction plate until it can be loaded and run on a Real-Time PCR System.
  • Next Generation Sequencing (NGS)
    What is the recovery rate with sparQ PureMag Beads?
    The recovery rate depends on many factors, including the fragment size, sample volume, sample concentration, and elution volume. Up to 90% recovery rate can be expected under the optimal condition. The yield is also correlated with the ratio of beads to sample, and the lower ratio usually results in lower yield. When the ratio reaches to 1.8X, it generally produces maximal recovery.
    Is there a size limit for the DNA fragments that can be bound with sparQ PureMag Beads?
    sparQ PureMag Beads will bind DNA fragments 100 bp and above and exclude primers 50 bases and below. There will be some recovery for oligo or DNA between 50 and 100 bases.
    How do I dry my samples before elution?
    Air-dry the beads on the magnetic stand for 5 min or until the beads are dry and before they start to crack. Do not over-dry the beads as it can reduce the DNA recovery.
    Can I still use the sparQ PureMag Beads if it was accidentally frozen?
    sparQ PureMag Beads is manufactured and tested for the storage temperature indicated on the bottle and we cannot guarantee performance if it was stored outside of the recommended condition.
    Can I still use the sparQ PureMag Beads if I left it at room temperature for some time?
    sparQ PureMag Beads is manufactured and tested for the storage temperature indicated on the bottle and we cannot guarantee performance if it was stored outside of the recommended condition.
  • Reverse Transcription
    Quantitative RT-qPCR
    What is the amount of RNA used per TaqMan® or SYBR qPCR Assay?
    Suggested input quantities of template are: 1 pg to 1 μg total RNA; 10 fg to 100 ng poly A(+) RNA; 10 to 1x108 copies viral RNA. For more information, please consult the qScrip One-Step qRT-PCR Kits protocols.
    First-Strand cDNA Synthesis
    Is MMLV RT as stable in the qScript cDNA Supermix as in the qScript cDNA synthesis kit? Do you recommend storing these products at -80C?
    In the qScript cDNA synthesis kit, the RT/RNase inhibitor mix is provided as a concentrated enzyme separately from the 5X reaction mix. This enzyme is actually stable at -20C for a year. The cDNA SuperMix has a shelf life of 12 months at -20C. Neither product shows any diminished performance after storage in a REVCO at -70C to -80C; two years for the qScript cDNA Supermix and five years for the qScript cDNA synthesis kit. If you are looking to prolong shelf life, storage at -80C is best for the qScript cDNA Supermix. Storage at -20C is sufficient for the qScript cDNA Synthesis Kit.
    I am trying to amplify a very rare transcript. Can I scale up a cDNA synthesis reaction and use 2ug of mRNA?
    qScript cDNA SuperMix can easily handle up to 2 ug of total RNA in a typical 20-uL first-strand reaction without compromising cDNA synthesis efficiency. For the qScript cDNA Synthesis Kit we recommend doubling the reaction volume to 40-uL. Simply scale each component proportionally.
    Does the qScript™ cDNA Synthesis Kit include an RNase Inhibitor like the qScript™ cDNA SuperMix?
    Both kits contain an RNase inhibitor protein. qScript™ cDNA SuperMix is provided as a single tube reaction of 5X concentrated master mix. It provides all necessary components for first-strand synthesis including: buffer, dNTPs, MgCl2, primers, RNase inhibitor protein, qScript reverse transcriptase and stabilizers (except RNA template) . In the case of the qScript™ cDNA Synthesis Kit, Rnase inhibitor is premixed with the reverse transcriptase and is provided as a concentrated enzyme.
    Do the cDNA Supermix and the cDNA synthesis Kits have the same dynamic range in RNA quantity?
    Both qScript cDNA Supermix and qScript cDNA Synthesis Kits have a broad linear dynamic range. We recommend using them for first-strand cDNA synthesis using HeLa cell total RNA dilutions ranging from 1 µg to 1 pg. One tenth of each first-strand reaction should be used for qPCR amplification. Within this range of HeLa total RNA, qPCR results should be equivalent with both kits. qScript cDNA synthesis kit may have advantages over the qScript cDNA Supermix in certain situations such as working with small quantities of RNA. qScript cDNA Supermix outperforms the qScript cDNA Synthesis kit when starting with quantities of total RNA in excess of 100 ng. qScript cDNA SuperMix can easily handle up to 2 ug of total RNA in a typical 20-uL first-strand reaction without compromising cDNA synthesis efficiency. With the qScript cDNA Synthesis Kit we recommend doubling the reaction volume to 40-uL and simply scaling each component proportionally.
    What is the qScript™ Flex cDNA Kit? How is it different from the qScript™ cDNA Synthesis Kit?
    The qScript Flex cDNA Synthesis Kit is an easy-to-use and highly efficient kit for the synthesis of first-strand cDNA that enables your choice of cDNA priming method. Various RT-PCR applications may require different priming strategies for optimal performance and this kit provides optimized reagents for priming with oligo(dT)20, random primer, gene-specific primer (GSP), or any combination thereof. Therefore, qScript™ Flex cDNA Kit is ideal for developing the best protocol for your application. The qScript™ Flex cDNA Kit also allows the use of Oligo dT or gene-specific primers (GSP) for long RT-PCR of RNA targets up to 12 kb. The qScript cDNA Synthesis Kit consists of an optimized blend of random and oligo(dT) primers. It provides robust, consistent and unbiased first-strand synthesis over a broad range of RNA template concentrations. qScript™ cDNA Synthesis Kit is well suited for short RT-PCR apllications (i.e. qRT-PCR) and is not recommended for amplification of RNA's longer than 1kb. When using a mixture of the Oligo dT and Random Primer solutions, the qScript™ Flex cDNA Kit achieves first-strand synthesis over a broad range of RNA template concentrations and its performance is comparable to that of the qScript™ cDNA Synthesis Kit. With both kits, the resulting cDNA product is directly compatible with current real-time RT-PCR methods or end-point RT-PCR.
    What is the difference between qScript cDNA SuperMix and the qScript cDNA Synthesis kit? 
    qScript cDNA SuperMix is the first ""true"" SuperMix format commercially available for cDNA synthesis. A single tube reaction, of 5X concentrated master mix, provides all necessary components for first-strand synthesis (except RNA template) including: buffer, dNTPs, MgCl2, primers, RNase inhibitor protein, qScript reverse transcriptase and stabilizers. In the qScript cDNA Synthesis kit the qScript RT (mixture of RNase inhibitor protein and qScript reverse transcriptase) is provided as a concentrated enzyme. A separate tube contains a solution of 5X concentrated reaction buffer, dNTPs, MgCl2, and primers. Aside from the inclusion of the enzyme and the stabilizers in the qScript cDNA SuperMix, its formulation is also slightly different from the qScript cDNA Synthesis formulation.
    What is the Flex Kit? How is it different from the cDNA synthesis Kit? Can we use either an oligo dT or a random priming method?
    The Flex Kit supports the use of multiple priming strategies. When using a combination of the oligo dT primer solution and random primer solution, the linear dynamic range and qRT-PCR performance is comparable to that of the qScript cDNA Synthesis Kit. In addition to retaining high performance with limiting amounts of RNA, the Flex kit was also optimized to provide long cDNA synthesis with oligo dT primer. So its composition is different from that of the cDNA synthesis Kit. When using oligo dT primer alone, it is unquestionably one of the easiest to use and most sensitive cDNA kits on the market
    For first strand cDNA synthesis, is it better to use oligo(dT), random hexamers, gene specific primer (GSP) or combination of these primers?
    The choice of primer depends on your experimental goals. Oligo(dT) is recommended when using total RNA for cDNA synthesis. It is the key to full-length cDNA synthesis. Random hexamers give a series of short first-strand products spanning the entire mRNA. Use of random hexamers may be helpful if the PCR fragment is at the 5´ end of a large mRNA. To ensure full-length cDNA synthesis of large transcripts, oligo(dT) can be added along with random hexamers during first-strand synthesis. Gene specific primers (GSP) for cDNA synthesis may also be used and are required in a few applications such as 5´ RACE and qRT-PCR. For GC-rich templates, or templates rich in secondary structure, a GSP may not work as well as priming with oligo dT for first strand synthesis. If an RT-PCR is problematic, trying different options of oligo dT, random primers and/or GSP for priming first strand synthesis may find a solution. Oligo(dT)20 primer (Cat. No. 18418-020) is recommended for use with SuperScript III Reverse Transcriptase
    What is the smallest quantity of RNA detectable by the qScript™ cDNA Synthesis Kit?
    Lower limits of detection by the qScript™ cDNA Synthesis Kit are dependent on many factors, such as primer design, target size, and the abundance of message. However, this system was able to detect GAPDH mRNA from as little as 1.0 pg of total HeLa RNA when used in conjunction with AccuStart Taq DNA Polymerase.
    What is the RNA input for the qScript cDNA Synthesis Kit?
    The qScript cDNA Synthesis Kit provides for the quantitative conversion of 1µg to 10 pg total RNA to cDNA, with a reaction volume of 20 ul.
    What is the RNA input for qScript cDNA Supermix?
    The qScript cDNA Supermix provides for the quantitative conversion of 1µg to 10 pg total RNA to cDNA, with a reaction volume of 20 ul.
    In conventional cDNA-synthesis, an RNA-denaturation step is often included (typically 65 degrees C for 15 min.). Such a step is not mentioned in the protocol for the qSript cDNA SuperMix, is it not needed?
    We have not seen any need or benefit to including an RNA denaturation step with the qScript cDNA SuperMix. We have examined hundreds of different amplicons using real-time RT-PCR and have not seen any difference. However, there are exceptions to every rule – especially when it comes to RT-PCR. Disruption of RNA secondary structures, immediately prior to carrying out cDNA synthesis, is critical when using oligo-dT or gene-specific priming. Inclusion of the denaturation step is also important when using oligo-dT primer for generating long first strand products (i.e. amplification of full length genes). Our qScript Flex cDNA Synthesis kit allows the flexibility to choose the method of your choice (oligo-dT, random primer, gene-specific primer, or any combination thereof) for priming first-strand synthesis. We have been able to generate cDNAs that were over 15-kb long with this kit. How the denaturation step is carried out is very important. Often, incubation of RNA and primer in water alone at elevated temperatures can result in non-specific hydrolysis and degradation of the RNA. The qScript Flex kit includes a special additive in the primer solutions, as well as a separate tube of this enhancer solution for use with gene-specific primers, that protects the RNA from hydrolysis and facilitates efficient annealing of the primer(s). In the case of the cDNA SuperMix, it is possible that annealing of random primers to the RNA may facilitate disruption of secondary structure, and thus obviate the need for the denaturation step. Links to product information: qScript™ cDNA Synthesis, qScript™ cDNA, qScript™ Flex cDNA Links to product information: qScript™ cDNA Synthesis Kit:http://www.quantabio.com/pdf/manuals/95047%20(qScriptT%20cDNA%20Synthesis%20Kit%20PPS).pdf qScript™ cDNA SuperMix:http://www.quantabio.com/pdf/manuals/95048%20(qScript%20cDNA%20SuperMix%20PPS).pdf qScript™ Flex cDNA Kit:http://www.quantabio.com/pdf/manuals/95049%20(qScriptT%20Flex%20cDNA%20Synthesis%20Kit%20PPS).pdf
  • Sample Preparation
    What is the shelf life of the Extracta DBS product?
    The shelf life is 2 years stored at 2-8°C.
    What is the Extracta DBS kit and what are some of the advantages of using this kit?
    The Extracta DBS kit is a research use only reagent for rapid isolation of DNA from dried blood spots. It is supplied as a convenient “ready to use” solution that reduces sample preparation time and is compatible with automation. The numerous wash and/or proteolytic digestion steps of other protocols are not necessary with Extracta DBS and the eluted DNA solution does not interfere with downstream DNA amplification applications. Qiagen Beverly’s ISO13485 manufacturing process ensures consistent composition and performance across lots.
    Should all of the supernatant be removed after the first wash and centrifugation step?
    After the first wash and centrifugation step, remove and discard as much liquid and debris as possible. The DNA remains on the filter paper at this point.
    What Quantabio products have confirmed compatibility with DNA extracted by the Extracta DBS kit?
    The following Quantabio products have been demonstrated to perform well with the extracted DNA: Accustart Genotyping ToughMix (95115, 95116, 95117) PerfeCTa Multiplex qPCR ToughMix (95147, 95148, 95149) PerfeCTa qPCR ToughMix (95112, 95113, 95114, 95138, 95139, 95140)
    For what downstream applications can the extracted DNA be used?
    The extracted DNA is compatible with a variety of end-point PCR, real-time PCR, next generation sequencing, or Sanger sequencing procedures.
    Can the extracted DNA be stored?
    We recommend using the DNA in downstream applications immediately after extraction. If this is not possible, the extracted DNA can be refrigerated at 2-8°C for short term storage (1-2 days) or stored frozen at -15 to -25°C for prolonged storage. We have no data on the stability of extracted DNA after prolonged storage.
    What is the stability of the extracted DNA?
    We have no data on the stability of the extracted DNA. We recommend that the DNA be used for downstream applications as soon as possible following extraction.
    How should the extracted DNA be measured?
    Measurement of DNA by methods using DNA binding dyes or absorbance at 260 nm are not applicable to a crude lysis method such as the Extracta DBS method. The recommended way to accurately quantify DNA in an Extracta DBS lysate is to measure a specific DNA sequence (generally a single-copy gene in gDNA) by qPCR.
  • What is the amount of cDNA used per TaqMan or SYBR qPCR Assay?
    Suggested input quantities of template are: cDNA corresponding to 1 pg to 100 ng of total RNA; 100 pg to 100 ng genomic DNA. For more information, please consult the PerfeCta qPCR Supermixes or PerfeCta SYBR Green Supermixes protocols.
    Is there a SYBR® Green Fast Master Mix currently available
    Yes. PerfeCTa SYBR Green Supermix is available. For further information, please consult the Kit protocol or product information.
  • Miscellaneous Questions
    What is the purpose of the 25C incubation step when using Q-Script-RT during cDNA synthesis?
    The purpose of the 25C incubation step during cDNA synthesis is to allow annealing and extension of the random primers. Since the primers are short, a lower temperature is required. Omission of this step will result in inefficient cDNA synthesis.
    How are PerfeCTa® microRNA assays designed?
    PerfeCTa microRNA assays are designed with primer design software according to the following criteria: • Optimized primer Tm designed to match the Universal PCR Primer • Universal cycling conditions to ensure robust amplification for all assays in profiling experiments • No self-complementarity or primer dimer artifacts with the PerfeCTa Universal PCR Primer • Optimized PCR product size with melting temperature of 75-78°C
    Do the PerfeCTa microRNA assays distinguish between closely related family members?
    PerfeCTa microRNA Assays will distinguish closely related family members are distinguished to varying degrees depending on the specific assay. The current microRNA assays have been designed primarily as a general profiling reagent that work with maximum efficiency with common PCR cycling conditions. Greater assay specificity can be achieved by increasing the annealing temperature of the PCR cycling from 60°C to 63°C with some cost to assay sensitivity. In some cases assays have been designed (for example, the let-7 family) that will distinguish closely related family members.
    How are PerfeCTa microRNA assays validated?
    • Proper tissue or cell type specificity (where applicable) • No primer dimer or off-target amplification product • Good amplification efficiency tested with multiple input amounts of cDNA • Comparison of qPCR results to a no-poly(A) polymerase control must demonstrate significant differences in samples where the microRNA is present • A single melt peak observed at the expected amplicon melt temperature
    What should I do if I suspect that my RNA contains RNase activity?
    If it is suspected that an RNA prep contains RNase activity, add RNase inhibitor to the reverse transcription reaction at a final concentration of 0.4 U/µl. Q-Script RT is a mixture of MMLV RT reverse transcriptase and Rnase inhibitor and should provide some safeguard against this problem. Q-Script RT is used in all of Quanta's cDNA synthesis kits: qScript™ cDNA Synthesis Kit (Cat# 95047), qScript™ cDNA SuperMix (Cat# 95048), qScript™ Flex cDNA Kit (Cat# 95049). If the RNA is grossly contaminated, another RNA prep should be used.
    What are the sources of non-specific amplification products?
    The origin non-specific amplification could potentially arise from mRNA transcripts containing sequences similar to the microRNA assay sequence close to their 3’-ends. Specificity of the microRNA assay comes from a single microRNA-specific assay primer, thus, there is a slightly higher probability of non-specific amplification for microRNA detection than a typical two-step RT-qPCR assay for mRNA which employs two gene-specific primers. In general, amplification products are not produced from single primer reactions (microRNA-specific primer alone or UAP alone). Positive signals are dependent on inclusion of both the UAP and the microRNA-specific primer in the qPCR. The probability non-specific amplification products increases with increasing cDNA template in the qPCR reaction and when the microRNA of interest is rare or absent from the RNA sample. When observed, non-specific amplification can be reduced by increasing the temperature of the reverse transcriptase reaction to 45°C without compromising assay sensitivity. In addition, increasing the annealing temperature of the PCR cycling condition (up to 63°C) will reduce non-specific amplification at some cost to assay sensitivity. The most useful control to measure non-specific amplification is the no-poly(A) polymerase (no-PAP) control. Assay results should be considered negative if the difference in CTs from the plus-PAP and no-PAP reactions is less than 2 CTs.
    What is the optimal amount of RNA input?
    The kit can is quantitative with 1 ug to 10 pg of total RNA in a 20 uL cDNA reaction. More than 1 ug can be used in larger reaction volumes scaled appropriately.
    Does the RNA need to be DNAse treated?
    DNAse treatment is not necessary and not recommended for microRNA detection. qPCR reactions of no-RT controls or using genomic DNA as template do not produce amplification products. DNAse is often difficult to inactivate and residual DNAse activity will greatly decrease sensitivity of any RT-qPCR assay.
    What assays should I use for normalization of the data?
    Use any assay or set of assays that are stable (do not change) with respect to the treatment or conditions of your experiment in you biological model system (for example control vs. treated or normal vs. disease). You can learn much more about the subject at: http://normalisation.gene-quantification.info/. Click on microRNA on the panel on the left and click on microRNA normalization link at the top of the microRNA page.
    Why are the PAP and RT reactions not combined into a single reaction?
    A combined poly(A)-tailing and reverse transcription reactions means compromising optimal assay conditions for both steps and reduces the flexibility of the system to accommodate different end user applications. For example, the PAP reaction can be adjusted and scaled to accommodate a wide range of RNA inputs. When using less than 100 ng of total RNA the PAP reaction can be shortened to 20 minutes and/or less PAP enzyme can be used in the reaction. When using more than 1 ug of total RNA the PAP reaction can be scaled up and stored for later use into multiple RT reactions. The specificity of some microRNA assays can be increased by increasing the temperature of the cDNA synthesis reaction from 42 ˚C to 45 ˚C or higher which is good for RT but not so good for PAP. Combining the two reactions does not permit a no-poly(A) polymerase control which is a critical measurement of microRNA assay background signal and allows for detection of false-positive signals. In addition, the poly(A) polymerase would have the potential to tail the oligo dT adapter primer interfering with specificity of the cDNA synthesis reaction.
    What are the -3p and -5p designations on each microRNA?
    Previously the mature microRNAs were referred to as major miRs and minor miRs (according to their relative abundance in specific tissues) with the minor miRs being designated with an asterisk. The nomenclature at miRBase has now changed such that for each precursor microRNA there are potentially two mature microRNAs designated with a -5p and -3p which refer to the position (5-prime arm or 3-prime arm) that the mature microRNAs occupy within the precursor stem-loop structure. On the PerfeCta microRNA Assay website click on the links for both the -5p and -3p microRNAs. In red text there is a reference to the former miRBase IDs. There is also a link to the miRBase Entry (blue button) where you can cross-check all of the information.
    What should I do if I see a split cluster on either axis?
    This usually occurs if an alternate SNP site is present in the template, in the region complementary to the SNP-IT primer. This potential single base mismatch in some of the samples, at the alternate SNP site, may cause inefficient SNP-IT primer/template hybridization. Even though the extension step occurs, the signal is weaker in these samples and hence will show up as a distinct cluster in the scatter plot. This can be overcome by redesigning the SNP-IT for the opposite strand and on the other side of the SNP, so that the alternate SNP site is avoided altogether at the SNP-IT annealing step.
    Should I be concerned if I observe three clusters in the scatter plot but one of them seems to be shifting to the other or are very close to each other?
    The observation is usually due to template dependent noise. This happens when the SNP-IT primer anneals to more than one site on the PCR template, other than its intended location (immediately adjacent to the SNP of interest). This phenomenon leads to multiple extensions occurring at different sites and often produces a significant level of background in the SNP-IT assay. This is commonly observed as a shift towards heterozygotes in one or more of the genotype clusters. Since this is template specific, it is usually best to choose an alternate design for the assay primers.
    What should I do if my homozygous samples controls look like heterozygotes in the assay?
    The observation is usually due to template dependent noise. This happens when the SNP-IT primer anneals to more than one site on the PCR template, other than its intended location (immediately adjacent to the SNP of interest). This phenomenon leads to multiple extensions occurring at different sites and often produces a significant level of background in the SNP-IT assay. This is commonly observed as a shift towards heterozygotes in one or more of the genotype clusters. Since this is template specific, it is usually best to choose an alternate design for the assay primers.
    How much of the first strand reaction should I add to the PCR?
    The volume will depend on the starting amount of RNA used for first-strand synthesis, and the abundance of the target gene. We recommend starting with 10% of the first-strand reaction. More than 10% may inhibit the PCR.
    How do I eliminate non-specific bands in PCR?
    Here are some suggestions for optimizing your PCR under such conditions: - Make sure primers don't have complementary sequences at the 3' ends - Optimize the annealing step by increasing the temperature in 2-5C increments, and minimizing the annealing time. You can try higher annealing temperatures in the first few cycles, and lower annealing temperatures in the subsequent cycles. - Try hot-start protocols - Optimize the magnesium concentration for each template and primer combination - To minimize chances of amplifying contaminating DNA, use aerosol-resistant tips and UDG
    What is the control date? (expiration Date)
    The control date is not the expiration date, but rather the date through which we guarantee performance of the product. If stored under the recommended conditions, the product will maintain performance through the date printed on the label.
    One-step versus Two-step RT-PCR
    One-Step RT-PCR allows easier processing of large numbers of samples, and helps minimize carry-over contamination since tubes are not opened between cDNA synthesis and amplification. By amplifying the entire cDNA sample, one-step RT-PCR can provide greater sensitivity-down to 0.01 pg total RNA. You can only use gene specific primers with these kits. Two-Step RT-PCR is useful for detecting multiple messages from a single RNA sample. You’ll get greater flexibility when choosing polymerase and primers than with one-step RT-PCR systems. When performing two-step RT-PCR you have the option of using either oligo(dT), random hexamers, or gene-specific primers, and then performing PCR in combination with either AccuStart Taq DNA Polymerase, or your choice of other PCR enzymes.
    What controls should be run, no-PAP, no-RT or both and why?
    When the Ct values are high (approaching 30) a no-PAP control can be used to add confidence in the results. If there is a significant difference between the plus-PAP and no-PAP reactions the results can be considered real. The no-RT control should always be negative. The kit comes with 20% extra 5x PAP reaction buffer and cDNA mix to accommodate the use of controls.
    Is the use of UNG necessary for performing reverse transcription reactions?
    No. It is not recommended to use UNG when performing reverse transcription. When using a dNTP mix with dUTP in a RT reaction, uracil will be incorporated into the cDNA generated from your RNA template. UNG (uracil N-glycosylase) is capable of cleaving single- or double stranded DNA containing dUTP sequences. Therefore, use of UNG during a reverse transcription step will cleave the dU containing cDNA and result in significantly lower amplification or absence of amplification.
    I am using Uracil N-glycosylase (UNG) and dUTP in my PCR reactions and would like to use the PCR product in a post-PCR application. Does the dUTP affect my ability to hybridize, sequence, clone, or digest the PCR product?
    Uracyl residues are roughly equivalent to dT-containing PCR products as hybridization targets, if long fragments (>200bases) are used. With very short fragments (<30bases), hybridization of dU containing templates will require lower temperatures depending on the dU content of DNA. Uracil residues serve in an equivalent manner as dT-containing PCR products as templates for dideoxy-terminated sequencing reactions. Uracyl residues are equivalent to dT-containing PCR products if transferred into UNG-minus bacterial hosts as targets for direct cloning. The recognition of dU-containing DNA by restriction endonucleases has been studied. Depending on the specific endonuclease, there may be no effect of the substitution of dU for dT on enzymatic activity (e.g., EcoR1 and BamH1), or the dU-containing DNA is cleaved more slowly than dT-containing DNA (e.g., Hpa1, HindII, and HindIII). For other endonucleases the effect of substituting dU for dT on enzyme activity will need to be examined empirically on an individual enzyme basis.
    What is UNG (Uracil N-glycosylase)?
    UNG (Uracil N-glycosylase) is an enzyme used in a powerful method for the elimination of carryover PCR product. This method modifies the PCR products such that the products from previous PCR amplifications will be digested by UNG prior to initiation of amplification. During amplification dUTP is substituted for dTTP resulting in dUTP containing products. UNG is active on single and double stranded dUTP containing DNA. A short pre-PCR incubation step in subsequent reactions will allow the UNG to digest any dUTP containing DNA. Since UNG is active on single and double stranded dUTP containing DNA, the procedure should work on dU-containing PCR products from standard or asymmetric PCR amplifications. Uracil ribonucleotide residues in RNA, novel DNA containing dTTP or cDNA containing dTTP are not suitable substrates for UNG. This method is best put in place prior to the appearance of contamination problem, because it is effective only against contamination with dUTP labeled PCR products.
    Purpose of the 25C incubation step when using Q-Script RT and Rnase Inhibitor mix
    The purpose of the 25 C incubation step when using random hexamers is to allow annealing and extention of random primers. Since the primers are short, a lower temperature is required. Omission of this step will result in inefficient cDNA synthesis.
    Inactivation of reverse transcriptases: protocol.
    The enzymes can be inactivated by adding a chelating agent such as EDTA. They should be heated to 85°C for 5 minutes for complete inactivation.
    M-MLV RT: Storage & Stability
    MMLV RT in the Q-Script RT and Rnase Inhibitor mix is stable up to 2 years when stored at -20°C in a non-frost-free freezer. Enzymes may remain at 4°C for up to 48 hours without loss of activity
    What is the highest temperature that a reverse trascriptase can be used?
    The optimal temperature for MMLV is 42 C. Therefore, for optimal results, we recommend carrying out cDNA synthesis reactions at 42°C. Only in rare cases, such as One-Step qRT-PCR, where shorter and more specific RNA regions are transcribed, may it be effective to raise the temperature to 48° although a slight reduction in RT activity and half-life may occur at these temperatures. Discuss 1 step Kit temp. 45-50 and 2step kit protocols
    Mw and Size of Taq Polymerase
    Taq DNA Polymerase is an 832 amino acid, single subunit enzyme with a MW of 94,000.
    Optimal pH for Taq polymerase
    Taq polymerase is active from pH 7.5-9.5. The unit assay is performed at pH 9.3 in TAPs buffer.
    Extension rate of Taq
    Taq has been reported to have an extension rate of 35-100 nt/sec at 75°C. For further information, see Wittwer (1991) BioTechniques 10(1), 76. It should be noted that the extention rate will vary depending on the conditions in which the enzyme is being used.
    Is a probe assay more sensitive than a SYBR® Green I assay?
    A probe assay and a SYBR® Green I assay can be equally sensitive. In cases of difficult to optimize PCRs the SYBR® Green I assay might be less beneficial as it shows the total fluorescent signal of primer dimers, aspecific product and wanted product.
    What is the advantage of working with a probe system?
    A probe system is always specific (except Amplifluor™ probes) and therefore does only detect the gene of interest. If you have a difficult to optimize PCR it will not show you any primer dimers or aspecific products. With a probe system it is also possible to distinguish between similar sequences with small differences like SNPs or mutations. In general, probe assays need less optimisation than SYBR® Green I assays.
    What is the advantage of working with SYBR® Green I?
    SYBR® Green I is an inexpensive, universal dye which binds to all dsDNA. It can be easily used in combination with a simple primer pair to detect PCR products in Real-Time. This dye is mainly very attractive for researchers analysis lots of different genes. However it is important to do a good primer design to avoid primer dimers, which will also be detected by SYBR® Green I.
    What is the difference in sensitivity between TaqMan® chemistry vs. SYBR® Green reagent chemistry?
    Sensitivity is equivalent when using TaqMan® chemistry and SYBR® Green reagent chemistry. Since a fluorescent signal is generated by a sequence specific TaqMan® probe, users might think a TaqMan® assay is more sensitive than a SYBR® Green reagent assay. This is not always true. A poorly designed TaqMan® assay could theoretically be less specific than a well-designed SYBR® Green reagent assay. The potential for detection of primer dimers and non-specific products using SYBR® Green reagent chemistry may result in loss of sensitivity when attempting to quantitate lower copy numbers.
    What is the concentration of my primers and TaqMan probe to be used with Taqman assays?
    Optimal results may require titration of primer concentration between 100 and 900 nM. A final concentration of 300 nM each primer and 100 to 250 nM probe is effective for most applications. However, increasing the concentration of the primer that initiates synthesis of the target strand that is complementary to the probe can improve fluorescent signal for some primer/probe systems.
    How should I store my cDNA?
    The cDNA can be diluted in low EDTA TE (10 mM Tris-HCl pH 8, 0.1 mM EDTA) or water and stored at 4 C or at -20 C.
    How much cDNA should be used in each qPCR reaction?
    You can use from 10 ng down to 0.1 pg of cDNA in each qPCR reaction. The kit provides maximum flexibility with regards to the amount of starting RNA and the amount of cDNA used in the qPCR. The microRNA cDNA can be diluted appropriately to accommodate the amount of total RNA used in the cDNA synthesis reaction and the relative abundance of the microRNAs of interest. We recommend starting with 1 ng of cDNA in each qPCR. If an individual microRNA is relatively abundant then 0.1 ng may work fine. If the microRNA is rare or absent you can add up to 10 ng of cDNA to the qPCR reaction. In this case we recommend comparing your results to a no-poly(A) polymerase reaction. A significant difference between reactions with and without poly(A) polymerase will help determine if the sample is positive or negative for the microRNA of interest. Three examples are provided below: For abundant microRNAs where total RNA sample is limiting: • 20 ng of total RNA in a 20 uL cDNA synthesis reaction = 1 ng /uL • Dilute with TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) to 0.02 ng/uL (1/50 dilution) • Add 5 uL (0.1 ng) to a 20 uL qPCR • 200 total qPCRs For rare microRNAs: • 200 ng of total RNA in a 20 uL cDNA synthesis reaction = 10 ng/uL • Dilute with TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) to 2 ng/uL (1/2.5 dilution) • Add 5 uL (10 ng) to a 20 uL qPCR • 20 total qPCRs For profiling experiments: • 1000 ng total RNA in 20 uL cDNA synthesis reaction = 50 ng/uL • Dilute with TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) to 0.2 ng/uL (1/250 dilution) • Add 5 uL (1 ng) to a 20 uL qPCR • 1000 total qPCRs
    What is the function of the antibody in AccuStart Taq Polymerase? How does this contribute to hot start PCR?
    AccuStart Taq DNA Polymerase is recombinant Taq DNA polymerase complexed with proprietary antibodies that inhibit polymerase activity. Due to specific binding of the antibodies, the activity of Taq DNA polymerase is blocked at ambient temperatures. Therefore, AccuStart Taq Polymerase remains inactive during reaction assembly and initial temperature ramp up. Antibodies are inactivated at the initial PCR denaturation step and release fully active Taq DNA Polymerase. This process provides an automatic hot start and improves PCR specificity, sensitivity and yield significantly. It also allows room temperature reaction assembly for high though put applications. By increasing the effectiveness of Taq DNA polymerase through the use of this product, it is possible to reduce the optimization and handling of reaction components and improve PCR results.
    Stability of Taq polymerase
    The AccuStart Taq Polymerase and antibody should be stable a minimum of 18 months if stored properly at -20C in a non frost free freezer.
    Components of Taq buffer
    The Accustart Taq PCR Buffer is supplied as a 10X concentrate and should be diluted 1:10 (1 part buffer + 9 parts other components = 10 parts final reaction volume). Buffer Composition (10X): 200 mM Tris-HCl (pH 8.4), 500 mM KCl. The Taq PCR buffer does not contain Magnesium Chloride. This is provided in a separate tube as 50 mM Magnesium Chloride
    Units of AccuStart Taq DNA polymerase to be used in PCR
    1-1.5 units per 50 ul reaction are sufficient for most applications, but in some instances it may be necessary to use more enzyme.
    DMSO inhibition of Taq DNA polymerase
    Taq DNA polymerase is inhibited 47% at a DMSO concentration of 10%. 10% DMSO is used in PCR or cycle sequencing reactions of GC rich DNA in the presence of 2X the amount of Taq DNA polymerase (see Sun, (1993) BioTechniques)
    Does Taq DNA polymerase have RT activity
    Taq DNA Polymerase has some reverse transcriptase (RT) activity at 68-78°C. However, this activity is negligible under ordinary PCR conditions. We don’t recommend using Taq Polymerase as a reverse transcriptase.
    Benefits of AccuStart Taq DNA Polymerase compared to regular Taq
    AccuStart Taq DNA Polymerase is a recombinant Taq DNA polymerase preparation which contains monoclonal antibodies that bind to the polymerase and keep it inactive before PCR thermal cycling. It allows for automatic hot start PCR, without extra work by the scientist:  - Room temperature reaction assembly.  - Broader optimal Magnesium concentration.  - Less primer optimization. Upon heat activation (1 minute at 94ºC), the antibodies denature irreversibly, releasing fully active Taq DNA polymerase. Non-specific extension of primers at low temperatures is a common cause of artifacts and poor sensitivity in PCR. The AccuStart automatic hot-start enables specific and efficient primer extension in the PCR process with the added convenience of room temperature reaction assembly. Activated AccuStart Taq DNA polymerase possesses 5’→3’ DNA polymerase activity and a double-strand specific 5’→3’ exonuclease. The polymerase does not have 3’-exonuclease activity and is free of any contaminating endo or exonuclease activities. One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble material in 30 minutes at 74°C. AccuStart Taq DNA polymerase is stable for 2 years when stored in a constant temperature freezer at 20ºC.
    Does AccuStart Taq DNA Polymerase remain in an active state once it is activated?
    Yes, once activated, AccuStart Taq DNA Polymerase remains active. Lowering the temperature will not inactivate AccuStart Taq DNA Polymerase.
    Unit definitions for AccuStart Taq DNA Polymerase
    One unit (U) of enzyme is defined as the amount that will incorporate 10nmoles of dNTPs into acid insoluble material per 30 minutes in a 10-minute incubation at 74 oC under the analysis conditions provided in the product insert. AccuStart Taq DNA Polymerase is premixed with anti Taq Antibodies. It can be activated by heating at 95C for 1-3 minutes.
    Does AccuStart Taq DNA Polymerase have proofreading activity?
    No, AccuStart Taq DNA Polymerase does not have proofreading activity. Fidelity of this Polymerase in PCR amplifications may be improved, by: 1. Decreasing the final concentration of each nucleotide to 40-50 uM. 2. Using the lowest MgCl2 concentration possible. 3. Using less enzyme. 4. Decreasing extension times. 5. Using the highest annealing temperature possible. 6. Using as few cycles as possible.
    What is the activation time for AccuStart™ Taq DNA Polymerase and AccuFast™ Taq DNA Polymerase ?
    "The recommended activation time depends on which Supermix is being used: Activation Conditions for AccuStart™ Taq DNA Polymerase in: PerfeCTa qPCR Supermixes 1-2 min, 95C PerfeCTa SYBR Green Supermixes 1-2 min, 95C Activation Conditions for AccuFast™ Taq DNA Polymerase in: PerfeCTa qPCR FastMixes 20 sec, 95C PerfeCTa SYBR Green FastMixes 20 sec, 95C
Publications
  • PCR & qPCR
    qPCR Instrumentation
    Activation of LXR Receptors and Inhibition of TRAP1 Causes Synthetic Lethality in Solid Tumors
    Trang Thi Thu Nguyen, Cancers - 2019
    Abstract
    Cholesterol is a pivotal factor for cancer cells to entertain their relentless growth. In this case, we provide a novel strategy to inhibit tumor growth by simultaneous activation of liver-X-receptors and interference with Tumor Necrosis Factor Receptor-associated Protein 1 (TRAP1). Informed by a transcriptomic and subsequent gene set enrichment analysis, we demonstrate that inhibition of TRAP1 results in suppression of the cholesterol synthesis pathway in stem-like and established glioblastoma (GBM) cells by destabilizing the transcription factor SREBP2. Notably, TRAP1 inhibition induced cell death, which was rescued by cholesterol and mevalonate. Activation of liver X receptor (LXR) by a clinically validated LXR agonist, LXR623, along with the TRAP1 inhibitor, gamitrinib (GTPP), results in synergistic reduction of tumor growth and cell death induction in a broad range of solid tumors, which is rescued by exogenous cholesterol. The LXR agonist and TRAP1 inhibitor mediated cell death is regulated at the level of Bcl-2 family proteins with an elevation of pro-apoptotic Noxa. Silencing of Noxa and its effector BAK attenuates cell death mediated by the combination treatment of LXR agonists and TRAP1 inhibition. Combined inhibition of TRAP1 and LXR agonists elicits a synergistic activation of the integrated stress response with an increase in activating transcription factor 4 (ATF4) driven by protein kinase RNA-like endoplasmic reticulum kinase (PERK). Silencing of ATF4 attenuates the increase of Noxa by using the combination treatment. Lastly, we demonstrate in patient-derived xenografts that the combination treatment of LXR623 and gamitrinib reduces tumor growth more potent than each compound. Taken together, these results suggest that TRAP1 inhibition and simultaneous activation of LXR might be a potent novel treatment strategy for solid malignancies.
    Real-Time Quantitative PCR
    SYBR Green Detection
    Effect of Periodized Resistance Training on Skeletal Muscle During Androgen Deprivation Therapy for Prostate Cancer: A Pilot Randomized Trial
    Jacqueline K. Dawson, PhD, Integrative Cancer Therapies - 2021
    Abstract
    Purpose: Prostate cancer survivors (PCS) receive androgen deprivation therapy (ADT) as treatment for recurrent cancer, yet ADT is associated with loss of skeletal muscle and physical function. Resistance training can counter both muscle and physical function loss; however, an understanding of the molecular responses of skeletal muscle to resistance training during ADT is still undefined. This sub-analysis of the original randomized, controlled pilot trial investigated effects of 12 weeks of periodized resistance training on mRNA expression of the anabolic genes IGF-1, myogenin, PGC-1α4 and the catabolic genes myostatin and MuRF-1 in skeletal muscle of PCS on ADT. Secondary aims investigated if changes in lean mass and physical function correlated with changes in mRNA expression. Methods: PCS on ADT (n=17) were randomized to 12 weeks of supervised resistance training (EXE, n=9) or home-based stretching (STRETCH, n=8) 3 days per week. Outcomes were assessed at baseline and post-intervention. Muscle biopsies were analyzed by RT-PCR for mRNA expression. Body composition was assessed through dual-energy X-ray absorptiometry, and physical function through muscular strength, timed up and go, stair climb, and 400m walk. Results: MuRF-1 mRNA expression was significantly greater in EXE compared to STRETCH post-intervention (P=.005). Change in MuRF-1 mRNA expression significantly correlated with improvements in strength and physical function (P < .05), while change in IGF-1 expression correlated with change in lean mass (P =.015). Conclusion: Twelve weeks of resistance training increased mRNA expression of MuRF-1 in skeletal muscle of PCS on ADT. Elevations in resting mRNA expression of IGF-1, myogenin and PGC-1α4, and reduction in mRNA expression of myostatin that are typically expected following resistance training were not observed.
    Influenza A virus infection in turkeys induces respiratory and enteric bacterial dysbiosis correlating with cytokine gene expression
    Ngunjiri JM, Microbiology - 2021
    Abstract
    Turkey respiratory and gut microbiota play important roles in promoting health and production performance. Loss of microbiota homeostasis due to pathogen infection can worsen the disease or predispose the bird to infection by other pathogens. While turkeys are highly susceptible to influenza viruses of different origins, the impact of influenza virus infection on turkey gut and respiratory microbiota has not been demonstrated. In this study, we investigated the relationships between low pathogenicity avian influenza (LPAI) virus replication, cytokine gene expression, and respiratory and gut microbiota disruption in specific-pathogen-free turkeys. Differential replication of two LPAI H5N2 viruses paralleled the levels of clinical signs and cytokine gene expression. During active virus shedding, there was significant increase of ileal and nasal bacterial contents, which inversely corresponded with bacterial species diversity. Spearman’s correlation tests between bacterial abundance and local viral titers revealed that LPAI virus-induced dysbiosis was strongest in the nasal cavity followed by trachea, and weakest in the gut. Significant correlations were also observed between cytokine gene expression levels and relative abundances of several bacteria in tracheas of infected turkeys. For example, interferon γ/λ and interleukin-6 gene expression levels were correlated positively with Staphylococcus and Pseudomonas abundances, and negatively with Lactobacillus abundance. Overall, our data suggest a potential relationship where bacterial community diversity and enrichment or depletion of several bacterial genera in the gut and respiratory tract are dependent on the level of LPAI virus replication. Further work is needed to establish whether respiratory and enteric dysbiosis in LPAI virus-infected turkeys is a result of host immunological responses or other causes such as changes in nutritional uptake.
    ENTRY AND EARLY INFECTION OF NON-SEGMENTED NEGATIVE SENSE RNA VIRUSES
    Jean Mawuena Branttie, - 2021
    Abstract
    Paramyxoviruses, pneumoviruses, and other non-segmented negative sense (NNS) RNA viruses have historically been of public health concern. Although their genomes are typically small (up to 19kbs) they are able to inflict large-scale detrimental pathologies on host cells. Human metapneumovirus (HMPV) is a widespread pathogen and is a NNS RNA virus. HMPV results respiratory tract infections and is particularly dangerous for preterm infants, the elderly, and immunocompromised individuals. Other viruses within the NNS RNA virus order include the deadly Ebola, Hendra, and Nipah viruses (EBOV, HeV, and NiV), as well as the re-emerging measles virus (MeV). Despite their public impact, there are currently very limited available FDA-approved therapeutics and antivirals against NNS RNA viruses. During the infectious cycle, viral surface glycoproteins play critical roles in establishing infection. For most NNS RNA viruses, the attachment protein is important for the tethering of a viral membrane to host cells, while the fusion protein is responsible for the membrane merger of the virus and host. The fusion protein of paramyxo-and pneumovirus proteins are class I proteins that are folded into trimers, must be proteolytically cleaved to be functional, and are held in a metastable prefusion conformation until the signal for fusion occurs. Upon being signaled, the fusion protein undergoes dramatic essentially irreversible conformational changes for membrane mixing. Because of its important role in starting infection, F has garnered interest as a potentially powerful target against infection. For paramyxoviruses, the ectodomain regions of F have been wellstudied; however, the hydrophobic nature of the transmembrane domain (TMD) of the protein has resulted in difficulties in crystallization. To address this, several biochemical assays have been utilized to address the function of the TMDs of paramyxo-and pneumovirus fusion proteins. Although initially thought to be solely a membrane anchor, the transmembrane domains of several viruses have been shown to be important for the functionality of fusion proteins. For some paramyxoviruses, replacement of the proteinaceous TMD resulted in the premature triggering. Further studies showed that the TMDs of paramyxoviruses and several other viral F proteins exist in isolation as trimers, and these trimeric associations in turn drive trimeric associations of the full protein. Studies of the HeV F TMD in isolation identified a leucine/isoleucine (L/I) zipper as an important motif for TMD-TMD trimerization. Mutations to this L/I zipper motif in the context of the full protein resulted in reduced surface expression, and a loss of functionality. The L/I zipper was found to be present in 140 paramyxo- and pneumovirus fusion protein TMDs. This work examines whether the importance of the wh iimporether L/Izipper in the context of another paramyxvovirus. Weused the model system, PIV5 F to dissect the role of the TMD L/I zipper fusogenic activity. We found that the (L/I) zipper plays important roles in in expression and functionality of the PIV5 F protein, but not surface expression of the protein. Following membrane merging, a series of events occur that facilitate the release of viral contents into the host cell. The NNS RNA carried by the virus into the cell is used as a template for viral replication and transcription; two important steps in generation of viral progeny. In the life cycle of NNS viruses, viral proteins assume multi-functional roles to optimize their replication and spread. One of the key players during the course of infection is the matrix protein (M). The matrix protein has been identified as a master regulator of viral infection with most studies focusing on its roles in late-stage infection, during assembly and budding of viral progeny. The matrix proteins of many enveloped viruses have been shown to associate in high order oligomers to form a gridlike array underneath the plasma membrane, where they can induce membrane curvature to allow for the budding of viral particles. Not surprisingly, the absence of M in some NNS RNA viruses results in a significant viral titer decrease. Interestingly, some recent studies show that the matrix protein has other critical roles in viral infection such as immune modulation and host cell translation antagonism. One of these newly uncovered roles for viral matrix proteins involves the regulation of viral RNA synthesis. Studies with EBOV and MeV demonstrate that the matrix protein is involved in early infection events, as inhibits viral replication. To study the roles of the HMPV M protein in early infection, we performed a spatiotemporal analysis of M in HMPV-infected cells. We noted the presence of HMPV M within the nucleus during early infection. Our knockdown studies of HMPV M indicate that HMPV M is a positive regulator of viral replication and transcription, as in its absence, the rates of mRNA and viral genomic RNA synthesis are dramatically reduced. Additionally, within the NNS RNA virus order, HMPV M is the only matrix protein found to bind calcium. We created alanine mutants to the calcium coordinating residues of HMPV M and found that these residues were important in properly folding the protein. Together, these findings contribute to our understanding of the mechanisms of NNS RNA viral infection
    Polycomb-group recruitment to a Drosophila target gene is the default state that is inhibited by a transcriptional activator
    Elnaz Ghotbi, Science Advances - 2021
    Abstract
    Polycomb-group (PcG) proteins are epigenetic regulators that maintain the transcriptional repression of target genes following their initial repression by transcription factors. PcG target genes are repressed in some cells, but active in others. Therefore, a mechanism must exist by which PcG proteins distinguish between the repressed and active states and only assemble repressive chromatin environments at target genes that are repressed. Here, we present experimental evidence that the repressed state of a Drosophila PcG target gene, giant (gt), is not identified by the presence of a repressor. Rather, de novo establishment of PcG-mediated silencing at gt is the default state that is prevented by the presence of an activator or coactivator, which may inhibit the catalytic activity of Polycomb-repressive complex 2 (PRC2).
    SIRT4 is an early regulatorof branched-chain amino acid catabolismthat promotes adipogenesis
    Elma Zaganjor, Cell Reports - 2021
    Abstract
    Upon nutrient stimulation, pre-adipocytes undergo differentiation to transform into mature adipocytescapable of storing nutrients as fat. We profiled cellular metabolite consumption to identify early metabolicdrivers of adipocyte differentiation. We find that adipocyte differentiation raises the uptake and consumptionof numerous amino acids. In particular, branched-chain amino acid (BCAA) catabolism precedes and pro-motes peroxisome proliferator-activated receptor gamma (PPARg), a key regulator of adipogenesis. In earlyadipogenesis, the mitochondrial sirtuin SIRT4 elevates BCAA catabolism through the activation of methylcro-tonyl-coenzyme A (CoA) carboxylase (MCCC). MCCC supports leucine oxidation by catalyzing the carboxyl-ation of 3-methylcrotonyl-CoA to 3-methylglutaconyl-CoA. Sirtuin 4 (SIRT4) expression is decreased in adi-pose tissue of numerous diabetic mouse models, and its expression is most correlated with BCAA enzymes,suggesting a potential role for SIRT4 in adipose pathology through the alteration of BCAA metabolism. Insummary, this work provides a temporal analysis of adipocyte differentiation and uncovers early metabolicevents that stimulate transcriptional reprogramming.
    Novel Role of Tieg1 in Muscle Metabolism and Mitochondrial Oxidative Capacities
    Malek Kammoun, HAL - 2021
    Abstract
    Aim: Tieg1 is involved in multiple signaling pathways, human diseases, and is highly expressed in muscle where its functions are poorly understood. Methods: We have utilized Tieg1 KO mice to identify novel and important roles for this transcription factor in regulating muscle ultrastructure, metabolism and mitochondrial functions in the soleus and EDL muscles. RNA sequencing, immunoblotting, TEM, MRI, NMR, histochemical and mitochondrial function assays were performed. Results: Loss of Tieg1 expression resulted in altered sarcomere organization and a significant decrease in mitochondrial number. Histochemical analyses demonstrated an absence of SDH staining and a decrease in COX enzyme activity in KO soleus with similar, but diminished, effects in the EDL. Decreased complex I, COX and citrate synthase activities were detected in the soleus muscle of KO mice indicating altered mitochondrial function. Complex I activity was also diminished in KO EDL. Significant decreases in citrate synthase and respiratory chain complex activities were identified in KO soleus. 1H-NMR spectra revealed no significant metabolic difference between WT and KO muscles. However, 31P spectra revealed a significant decrease in phosphocreatine and ATP. Altered expression of 279 genes, many of which play roles in mitochondrial and muscle function, were identified in KO soleus muscle. Ultimately, all of these changes resulted in an exercise intolerance phenotype in Tieg1 KO mice. Conclusion: Our findings have implicated novel roles for Tieg1 in muscle including regulation of gene expression, metabolic activity and organization of tissue ultrastructure. This muscle phenotype resembles diseases associated with exercise intolerance and myopathies of unknown consequence.
    Cardiomyocytes Derived from Induced Pluripotent Stem Cells as a Disease Model for Propionic Acidemia
    Esmeralda Alonso-Barroso, International Journal of Molecular Sciences - 2021
    Abstract
    Propionic acidemia (PA), one of the most frequent life-threatening organic acidemias, is caused by mutations in either the PCCA or PCCB genes encoding both subunits of the mitochondrial propionyl-CoA carboxylase (PCC) enzyme. Cardiac alterations (hypertrophy, dilated cardiomyopathy, long QT) are one of the major causes of mortality in patients surviving the neonatal period. To overcome limitations of current cellular models of PA, we generated induced pluripotent stem cells (iPSCs) from a PA patient with defects in the PCCA gene, and successfully differentiated them into cardiomyocytes. PCCA iPSC-derived cardiomyocytes exhibited reduced oxygen consumption, an accumulation of residual bodies and lipid droplets, and increased ribosomal biogenesis. Furthermore, we found increased protein levels of HERP, GRP78, GRP75, SIG-1R and MFN2, suggesting endoplasmic reticulum stress and calcium perturbations in these cells. We also analyzed a series of heart-enriched miRNAs previously found deregulated in the heart tissue of a PA murine model and confirmed their altered expression. Our novel results show that PA iPSC-cardiomyocytes represent a promising model for investigating the pathological mechanisms underlying PA cardiomyopathies, also serving as an ex vivo platform for therapeutic evaluation.
    An ionising radiation-induced specic transcriptional signature of inammationassociated genes in human whole blood: a pilot study
    Lourdes Cruz-Garcia, Research Square - 2021
    Abstract
    This communication reports the identication of a new panel of transcriptional changes in inammationassociated genes observed in response to ionising radiation received by radiotherapy patients. Peripheral blood samples were taken with ethical approval and informed consent from a total of 20 patients undergoing external beam radiotherapy for breast, lung, gastrointestinal or genitourinary tumours. Nanostring nCounter analysis of transcriptional changes was carried out in samples prior and 24 hours post-delivery of the 1st radiotherapy fraction, just prior to the 5th or 6th fraction, and just before the last fraction. Statistical analysis with BRB Array Tools, GLM MANOVA and nSolver, revealed a radiation responsive panel of genes which varied by patient group (type of cancer) and with time since exposure (as an analogue for dose received), which may be useful as a biomarker of radiation response. Further validation in a wider group of patients is ongoing, together with work towards a full understanding of patient specic responses in support of personalised approaches to radiation medicine.
    SIRT7 deficiency suppresses inflammation, induces EndoMT, and increases vascular permeability in primary pulmonary endothelial cells
    Anne E. Wyman, Scientific Reports - 2020
    Abstract
    Acute lung injury (ALI), a common condition in critically ill patients, has limited treatments and high mortality. Aging is a risk factor for ALI. Sirtuins (SIRTs), central regulators of the aging process, decrease during normal aging and in aging-related diseases. We recently showed decreased SIRT7 expression in lung tissues and fibroblasts from patients with pulmonary fibrosis compared to controls. To gain insight into aging-related mechanisms in ALI, we investigated the effects of SIRT7 depletion on lipopolysaccharide (LPS)-induced inflammatory responses and endothelial barrier permeability in human primary pulmonary endothelial cells. Silencing SIRT7 in pulmonary artery or microvascular endothelial cells attenuated LPS-induced increases in ICAM1, VCAM1, IL8, and IL6 and induced endomesenchymal transition (EndoMT) with decreases in VE-Cadherin and PECAM1 and increases in collagen, alpha-smooth muscle actin, TGFβ receptor 1, and the transcription factor Snail. Loss of endothelial adhesion molecules was accompanied by increased F-actin stress fibers and increased endothelial barrier permeability. Together, these results show that an aging phenotype induced by SIRT7 deficiency promotes EndoMT with impaired inflammatory responses and dysfunction of the lung vascular barrier.
    CTCF loss mediates unique DNA hypermethylation landscapes in human cancers
    Nathan A. Damaschke, Clinical Epigenetics - 2020
    Abstract
    Background The chromatin insulator CCCTC-binding factor (CTCF) displays tissue-specific DNA binding sites that regulate transcription and chromatin organization. Despite evidence linking CTCF to the protection of epigenetic states through barrier insulation, the impact of CTCF loss on genome-wide DNA methylation sites in human cancer remains undefined. Results Here, we demonstrate that prostate and breast cancers within The Cancer Genome Atlas (TCGA) exhibit frequent copy number loss of CTCF and that this loss is associated with increased DNA methylation events that occur preferentially at CTCF binding sites. CTCF sites differ among tumor types and result in tissue-specific methylation patterns with little overlap between breast and prostate cancers. DNA methylation and transcriptome profiling in vitro establish that forced downregulation of CTCF leads to spatially distinct DNA hypermethylation surrounding CTCF binding sites, loss of CTCF binding, and decreased gene expression that is also seen in human tumors. DNA methylation inhibition reverses loss of expression at these CTCF-regulated genes. Conclusion These findings establish CTCF loss as a major mediator in directing localized DNA hypermethylation events in a tissue-specific fashion and further support its role as a driver of the cancer phenotype.
    Mutations that increase expression of the EmrAB-TolC efflux pump confer increased resistance to nitroxoline in Escherichia coli
    Fabiola Puértolas-Balint, Journal of Antimicrobial Chemotherapy - 2020
    Abstract
    Objectives To determine the mechanism of resistance to the antibiotic nitroxoline in Escherichia coli. Methods Spontaneous nitroxoline-resistant mutants were selected at different concentrations of nitroxoline. WGS and strain reconstruction were used to define the genetic basis for the resistance. The mechanistic basis of resistance was determined by quantitative PCR (qPCR) and by overexpression of target genes. Fitness costs of the resistance mutations and cross-resistance to other antibiotics were also determined. Results Mutations in the transcriptional repressor emrR conferred low-level resistance to nitroxoline [nitroxoline MIC (MICNOX)=16 mg/L] by increasing the expression of the emrA and emrB genes of the EmrAB-TolC efflux pump. These resistant mutants showed no fitness reduction and displayed cross-resistance to nalidixic acid. Second-step mutants with higher-level resistance (MICNOX=32–64 mg/L) had mutations in the emrR gene, together with either a 50 kb amplification, a mutation in the gene marA, or an IS upstream of the lon gene. The latter mutations resulted in higher-level nitroxoline resistance due to increased expression of the tolC gene, which was confirmed by overexpressing tolC from an inducible plasmid in a low-level resistance mutant. Furthermore, the emrR mutations conferred a small increase in resistance to nitrofurantoin only when combined with an nfsAB double-knockout mutation. However, nitrofurantoin-resistant nfsAB mutants showed no cross-resistance to nitroxoline. Conclusions Mutations in different genes causing increased expression of the EmrAB-TolC pump lead to an increased resistance to nitroxoline. The structurally similar antibiotics nitroxoline and nitrofurantoin appear to have different modes of action and resistance mechanisms.
    Microbial community similarity and dissimilarity inside and across full-scale activated sludge processes for simultaneous nitrification and denitrification
    Jianfeng Wen, Water Science and Technology - 2020
    Abstract
    Simultaneous nitrification and denitrification under low dissolved oxygen conditions is an energy-saving modification of the activated sludge process to achieve efficient nitrogen removal. Geographically distinct full-scale treatment plants are excellent platforms to address the links of microbial community with operating parameters. Mixed liquor samples were collected from a sequencing batch reactor plant, oxidation ditch plant, and step-feed activated sludge plant. Next-Generation Sequencing of the samples showed that the microbial communities were similar at the phylum level among the plants, being dominated by Proteobacteria. Microbial composition of functional groups was similar between the react fill and react phases of the sequencing batch reactors, among four sequencing batch reactors, and among four oxidation ditches. Nitrospira was the only identified genus of autotropic nitrifying bacteria with a relative abundance of 2.2–2.5% in the oxidation ditches and 0.4–0.7% at the other plants. Heterotrophic nitrifying–aerobic denitrifying bacteria were dominated by Dechloromonas with a relative abundance of 0.4–1.0%. Microbial community composition and nitrogen removal mechanisms were related to overall level and local zonation of dissolved oxygen, mixed liquor suspended solids concentration, nitrogen and organic loadings, and solids retention time. Low dissolved oxygen and low organic and nitrogen loadings favored growth of Nitrospira.
    Sphingomyelin-Based Nanosystems (SNs) for the Development of Anticancer miRNA Therapeutics
    Surasa Nagachinta, Pharmaceutics - 2020
    Abstract
    Gene replacement therapy with oncosuppressor microRNAs (miRNAs) is a promising alternative to interfere with cancer progression. However, miRNAs are highly inefficient in a biological environment, hampering a successful translation to clinics. Nanotechnology can tackle this drawback by providing delivery systems able to efficiently deliver them to cancer cells. Thus, the objective of this work was to develop biocompatible nanosystems based on sphingomyelin (SM) for the intracellular delivery of miRNAs to colorectal cancer cells. We pursued two different approaches to select the most appropriate composition for miRNA delivery. On the one hand, we prepared sphingomyelin-based nanosystems (SNs) that incorporate the cationic lipid stearylamine (ST) to support the association of miRNA by the establishment of electrostatic interactions (SNs–ST). On the other hand, the cationic surfactant (DOTAP) was used to preform lipidic complexes with miRNA (Lpx), which were further encapsulated into SNs (SNs-Lpx). Restitution of miRNA145 levels after transfection with SNs-Lpx was related to the strongest anticancer effect in terms of tumor proliferation, colony forming, and migration capacity assays. Altogether, our results suggest that SNs have the potential for miRNA delivery to develop innovative anticancer therapies.
    Inhibition of PIM2 in liver cancer decreases tumor cell proliferation in vitro and in vivo primarily through the modulation of cell cycle progression
    Pia Kronschnabl, International Journal of Oncology - 2020
    Abstract
    Liver cancer is the fourth leading cause of cancer‑related mortality worldwide with limited therapeutic options. Thus, novel treatment strategies are urgently required. While the oncogenic kinase, proviral integration site for Moloney murine leukemia virus 2 (PIM2), has been shown to be overexpressed in liver cancer, little is known about the role of PIM2 in this tumor entity. In this study, we explored the functional relevance and therapeutic potential of PIM2 in liver cancer. Using PIM2‑specific siRNAs, we examined the effects of PIM2 knockdown on proliferation (WST‑1 assays and spheroid assays), 3D‑colony formation and colony spread, apoptosis (flow cytometry and caspase 3/caspase 7 activity), as well as cell cycle progression (flow cytometry, RT‑qPCR and western blot analysis) in the two liver cancer cell lines, HepG2 and Huh‑7. In subcutaneous liver cancer xenografts, we assessed the effects of PIM2 knockdown on tumor growth via the systemic delivery of polyethylenimine (PEI)‑complexed siRNA. The knockdown of PIM2 resulted in potent anti‑proliferative effects in cells grown on plastic dishes, as well as in spheroids. This was due to G0/G1 cell cycle blockade and the subsequent downregulation of genes related to the S phase as well as the G2/M phase of the cell cycle, whereas the apoptotic rates remained unaltered. Furthermore, colony formation and colony spread were markedly inhibited by PIM2 knockdown. Notably, we found that HepG2 cells were more sensitive to PIM2 knockdown than the Huh‑7 cells. In vivo, the therapeutic nanoparticle‑mediated delivery of PIM2 siRNA led to profound anti‑tumor effects in a liver cancer xenograft mouse model. On the whole, the findings of this study underscore the oncogenic role of PIM2 and emphasize the potential of targeted therapies based on the specific inhibition of PIM2 in liver cancer.
    Assessment of the ptxD gene as a growth and selective marker in Trichoderma atroviride using Pccg6, a novel constitutive promoter
    Nohemí Carreras-Villaseñor, Springer Link - 2020
    Abstract
    Background Trichoderma species are among the most effective cell factories to produce recombinant proteins, whose productivity relies on the molecular toolkit and promoters available for the expression of the target protein. Although inducible promoter systems have been developed for producing recombinant proteins in Trichoderma, constitutive promoters are often a desirable alternative. Constitutive promoters are simple to use, do not require external stimuli or chemical inducers to be activated, and lead to purer enzyme preparations. Moreover, most of the promoters for homologous and heterologous expression reported in Trichoderma have been commonly evaluated by directly assessing production of industrial enzymes, requiring optimization of laborious protocols. Results Here we report the identification of Pccg6, a novel Trichoderma atroviride constitutive promoter, that has similar transcriptional strength as that of the commonly used pki1 promoter. Pccg6 displayed conserved arrangements of transcription factor binding sites between promoter sequences of Trichoderma ccg6 orthologues genes, potentially involved in their regulatory properties. The predicted ccg6-encoded protein potentially belongs to the SPE1/SPI1 protein family and shares high identity with CCG6 orthologue sequences from other fungal species including Trichoderma reesei, Trichoderma virens, Trichoderma asperellum, and to a lesser extent to that of Neurospora crassa. We also report the use of the Pccg6 promoter to drive the expression of PTXD, a phosphite oxidoreductase of bacterial origin, which allowed T. atroviride to utilize phosphite as a sole source of phosphorus. We propose ptxD as a growth reporter gene that allows real-time comparison of the functionality of different promoters by monitoring growth of Trichoderma transgenic lines and enzymatic activity of PTXD. Finally, we show that constitutive expression of ptxD provided T. atroviride a competitive advantage to outgrow bacterial contaminants when supplied with phosphite as a sole source of phosphorus. Conclusions A new constitutive promoter, ccg6, for expression of homologous and heterologous proteins has been identified and tested in T. atroviride to express PTXD, which resulted in an effective and visible phenotype to evaluate transcriptional activity of sequence promoters. Use of PTXD as a growth marker holds great potential for assessing activity of other promoters and for biotechnological applications as a contamination control system.
    The Cutaneous Inflammatory Response to Thermal Burn Injury in a Murine Model
    Zabeen Lateef, International Journal of Molecular Sciences - 2019
    Abstract
    Many burn interventions aim to target the inflammatory response as a means of enhancing healing or limiting hypertrophic scarring. Murine models of human burns have been developed, but the inflammatory response to injury in these models has not been well defined. The aim of this study was to profile inflammatory cell populations and gene expression relative to healing and scarring in a murine model of thermal burns. Cutaneous injuries were created on the dorsal region of C57Bl/6 mice using a heated metal rod. Animals were euthanized at selected time points over ten weeks, with the lesions evaluated using macroscopic measurements, histology, immunofluorescent histochemistry and quantitative PCR. The burn method generated a reproducible, partial-thickness injury that healed within two weeks through both contraction and re-epithelialization, in a manner similar to human burns. The injury caused an immediate increase in pro-inflammatory cytokine and chemokine expression, coinciding with an influx of neutrophils, and the disappearance of Langerhans cells and mast cells. This preceded an influx of dendritic cells and macrophages, a quarter of which displayed an inflammatory (M1) phenotype, with both populations peaking at closure. As with human burns, the residual scar increased in size, epidermal and dermal thickness, and mast cell numbers over 10 weeks, but abnormal collagen I-collagen III ratios, fibre organization and macrophage populations resolved 3–4 weeks after closure. Characterisation of the inflammatory response in this promising murine burn model will assist future studies of burn complications and aid in the preclinical testing of new anti-inflammatory and anti-scarring therapies.
    Fructose-1,6-bisphosphate prevents pulmonary fibrosis by regulating extracellular matrix deposition and inducing phenotype reversal of lung myofibroblasts
    Henrique Bregolin Dias, PLOS ONE - 2019
    Abstract
    Pulmonary fibrosis (PF) is the result of chronic injury where fibroblasts become activated and secrete large amounts of extracellular matrix (ECM), leading to impaired fibroblasts degradation followed by stiffness and loss of lung function. Fructose-1,6-bisphosphate (FBP), an intermediate of glycolytic pathway, decreases PF development, but the underlying mechanism is unknown. To address this issue, PF was induced in vivo using a mouse model, and pulmonary fibroblasts were isolated from healthy and fibrotic animals. In PF model mice, lung function was improved by FBP as revealed by reduced collagen deposition and downregulation of ECM gene expression such as collagens and fibronectin. Fibrotic lung fibroblasts (FLF) treated with FBP for 3 days in vitro showed decreased proliferation, contraction, and migration, which are characteristic of myofibroblast to fibroblast phenotype reversal. ECM-related genes and proteins such as collagens, fibronectin and α-smooth muscle actin, were also downregulated in FBP-treated FLF. Moreover, matrix metalloproteinase (MMP) 1, responsible for ECM degradation, was produced only in fibroblasts obtained from healthy lungs (HLF) and FBP did not alter its expression. On the other hand, tissue inhibitor of metalloproteinase (TIMP)-1, a MMP1 inhibitor, and MMP2, related to fibroblast tissue-invasion, were predominantly produced by FLF and FBP was able to downregulate its expression. These results demonstrate that FBP may prevent bleomycin-induced PF development through reduced expression of collagen and other ECM components mediated by a reduced TIMP-1 and MMP2 expression.
    Glucocorticoid receptor modulation decreases ER-positive breast cancer cell proliferation and suppresses wild-type and mutant ER chromatin association
    Eva Tonsing-Carter, Breast Cancer Research - 2019
    Abstract
    Background: Non-ER nuclear receptor activity can alter estrogen receptor (ER) chromatin association and resultant ER-mediated transcription. Consistent with GR modulation of ER activity, high tumor glucocorticoid receptor (GR) expression correlates with improved relapse-free survival in ER+ breast cancer (BC) patients. Methods: In vitro cell proliferation assays were used to assess ER-mediated BC cell proliferation following GR modulation. ER chromatin association following ER/GR co-liganding was measured using global ChIP sequencing and directed ChIP analysis of proliferative gene enhancers. Results: We found that GR liganding with either a pure agonist or a selective GR modulator (SGRM) slowed estradiol (E2)-mediated proliferation in ER+ BC models. SGRMs that antagonized transcription of GR-unique genes both promoted GR chromatin association and inhibited ER chromatin localization at common DNA enhancer sites. Gene expression analysis revealed that ER and GR co-activation decreased proliferative gene activation (compared to ER activation alone), specifically reducing CCND1, CDK2, and CDK6 gene expression. We also found that ligand-dependent GR occupancy of common ER-bound enhancer regions suppressed both wild-type and mutant ER chromatin association and decreased corresponding gene expression. In vivo, treatment with structurally diverse SGRMs also reduced MCF-7 Y537S ER-expressing BC xenograft growth. Conclusion: These studies demonstrate that liganded GR can suppress ER chromatin occupancy at shared ER-regulated enhancers, including CCND1 (Cyclin D1), regardless of whether the ligand is a classic GR agonist or antagonist. Resulting GR-mediated suppression of ER+ BC proliferative gene expression and cell division suggests that SGRMs could decrease ER-driven gene expression.
    THROUGH THE LOOKING GLASS: Real-Time Imaging in Brachypodium Roots and Osmotic Stress Analysis
    Zaemma Khan, Plants - 2019
    Abstract
    To elucidate dynamic developmental processes in plants, live tissues and organs must be visualised frequently and for extended periods. The development of roots is studied at a cellular resolution not only to comprehend the basic processes fundamental to maintenance and pattern formation but also study stress tolerance adaptation in plants. Despite technological advancements, maintaining continuous access to samples and simultaneously preserving their morphological structures and physiological conditions without causing damage presents hindrances in the measurement, visualisation and analyses of growing organs including plant roots. We propose a preliminary system which integrates the optical real-time visualisation through light microscopy with a liquid culture which enables us to image at the tissue and cellular level horizontally growing Brachypodium roots every few minutes and up to 24 h. We describe a simple setup which can be used to track the growth of the root as it grows including the root tip growth and osmotic stress dynamics. We demonstrate the system’s capability to scale down the PEG-mediated osmotic stress analysis and collected data on gene expression under osmotic stress.
    The TLR4 adaptor TRAM controls the phagocytosis of Gram-negative bacteria by interacting with the Rab11-family interacting protein 2
    Astrid Skjesol, Plos Pathogens - 2019
    Abstract
    Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria.
    α-Lipoic Acid Reduces Iron-induced Toxicity and Oxidative Stress in a Model of Iron Overload
    Giuseppina Camiolo, International Journal of Molecular Sciences - 2019
    Abstract
    Iron toxicity is associated with organ injury and has been reported in various clinical conditions, such as hemochromatosis, thalassemia major, and myelodysplastic syndromes. Therefore, iron chelation therapy represents a pivotal therapy for these patients during their lifetime. The aim of the present study was to assess the iron chelating properties of α-lipoic acid (ALA) and how such an effect impacts on iron overload mediated toxicity. Human mesenchymal stem cells (HS-5) and animals (zebrafish, n = 10 for each group) were treated for 24 h with ferric ammonium citrate (FAC, 120 µg/mL) in the presence or absence of ALA (20 µg/mL). Oxidative stress was evaluated by reduced glutathione content, reactive oxygen species formation, mitochondrial dysfunction, and gene expression of heme oxygenase-1b and mitochondrial superoxide dismutase; organ injury, iron accumulation, and autophagy were measured by microscopical, cytofluorimetric analyses, and inductively coupled plasma-optical mission Spectrometer (ICP-OES). Our results showed that FAC results in a significant increase of tissue iron accumulation, oxidative stress, and autophagy and such detrimental effects were reversed by ALA treatment. In conclusion, ALA possesses excellent iron chelating properties that may be exploited in a clinical setting for organ preservation, as well as exhibiting a good safety profile and low cost for the national health system.
    Influence of the concentration of dietary digestible calcium on growth performance, bone mineralization, plasma calcium, and abundance of genes involved in intestinal absorption of calcium in pigs from 11 to 22 kg fed diets with different concentrations o
    L. Vanessa Lagos, Journal of Animal Science and Biotechnology - 2019
    Abstract
    A 21-day experiment was conducted to test the hypothesis that Ca requirements to maximize growth performance expressed as the standardized total tract digestible (STTD) Ca to STTD P ratio is less than 1.40:1. The second hypothesis was that increasing dietary Ca increases plasma Ca concentration and downregulates abundance of genes related to Ca absorption (TRPV6, S100G, and ATP2B1) in the duodenum, and tight junction proteins (OCLN, CLDN1, and ZO1) in the duodenum and ileum. Methods Twenty corn-soybean meal diets were formulated using a 4 × 5 factorial design with diets containing 0.16%, 0.33%, 0.42%, or 0.50% STTD P, and 0.14%, 0.29%, 0.44%, 0.59%, or 0.74% STTD Ca. Six hundred and forty pigs (initial weight: 11.1 ± 1.4 kg) were allotted to 20 diets and 5 blocks in a randomized complete block design. On day 21, weights of pigs and feed left in feeders were recorded and blood, duodenal tissue, ileal mucosa, and the right femur were collected from 1 pig per pen. Abundance of mRNA was determined in duodenal and ileal tissue via quantitative RT-PCR. Data were analyzed using a response surface model. Results The predicted maximum ADG (614 g), G:F (0.65), and bone ash (11.68 g) was obtained at STTD Ca:STTD P ratios of 1.39:1, 1.25:1, and 1.66:1, respectively, when STTD P was provided at the requirement (0.33%). If dietary STTD P was below the requirement, increasing dietary Ca resulted in reduced (P < 0.05) ADG and G:F. However, if dietary STTD P was above the requirement, negative effects (P < 0.05) on ADG and G:F of increasing STTD Ca were observed only if dietary STTD Ca exceeded 0.6%. Plasma Ca concentration was positively affected by STTD Ca over the range studied (quadratic, P < 0.01) and negatively affected by increasing STTD P (linear, P < 0.01). There was a linear negative effect (P < 0.05) of STTD Ca on the abundance of S100G, TRPV6, OCLN, and ZO1 in duodenum, and CLDN and ZO1 in ileum. Conclusions The STTD Ca:STTD P ratio needed to maximize growth performance of 11- to 25-kg pigs is less than 1.40:1, if P is at the estimated requirement. Increasing dietary Ca reduces transcellular absorption of Ca and increases paracellular absorption of Ca.
    Brf1 loss and not overexpression disrupts tissues homeostasis in the intestine, liver and pancreas
    Dritan Liko, Cell Death and Differentiation - 2019
    Abstract
    RNA polymerase III (Pol-III) transcribes tRNAs and other small RNAs essential for protein synthesis and cell growth. Pol-III is deregulated during carcinogenesis; however, its role in vivo has not been studied. To address this issue, we manipulated levels of Brf1, a Pol-III transcription factor that is essential for recruitment of Pol-III holoenzyme at tRNA genes in vivo. Knockout of Brf1 led to embryonic lethality at blastocyst stage. In contrast, heterozygous Brf1 mice were viable, fertile and of a normal size. Conditional deletion of Brf1 in gastrointestinal epithelial tissues, intestine, liver and pancreas, was incompatible with organ homeostasis. Deletion of Brf1 in adult intestine and liver induced apoptosis. However, Brf1 heterozygosity neither had gross effects in these epithelia nor did it modify tumorigenesis in the intestine or pancreas. Overexpression of BRF1 rescued the phenotypes of Brf1 deletion in intestine and liver but was unable to initiate tumorigenesis. Thus, Brf1 and Pol-III activity are absolutely essential for normal homeostasis during development and in adult epithelia. However, Brf1 overexpression or heterozygosity are unable to modify tumorigenesis, suggesting a permissive, but not driving role for Brf1 in the development of epithelial cancers of the pancreas and gut.
    Single-Cell Heterogeneity Analysis and CRISPR Screen Identify Key β-Cell-Specific Disease Genes
    Zhou Fang, Cell Reports - 2019
    Abstract
    Identification of human disease signature genes typically requires samples from many donors to achieve statistical significance. Here, we show that single-cell heterogeneity analysis may overcome this hurdle by significantly improving the test sensitivity. We analyzed the transcriptome of 39,905 single islets cells from 9 donors and observed distinct β cell heterogeneity trajectories associated with obesity or type 2 diabetes (T2D). We therefore developed RePACT, a sensitive single-cell analysis algorithm to identify both common and specific signature genes for obesity and T2D. We mapped both β-cell-specific genes and disease signature genes to the insulin regulatory network identified from a genome-wide CRISPR screen. Our integrative analysis discovered the previously unrecognized roles of the cohesin loading complex and the NuA4/Tip60 histone acetyltransferase complex in regulating insulin transcription and release. Our study demonstrated the power of combining single-cell heterogeneity analysis and functional genomics to dissect the etiology of complex diseases.
    No evidence that gut microbiota impose a net cost on their butterfly host
    Alison Ravenscraft, Molecular Ecology - 2019
    Abstract
    Gut microbes are believed to play a critical role in most animal life, yet fitness effects and cost–benefit trade‐offs incurred by the host are poorly understood. Unlike most hosts studied to date, butterflies largely acquire their nutrients from larval feeding, leaving relatively little opportunity for nutritive contributions by the adult's microbiota. This provides an opportunity to measure whether hosting gut microbiota comes at a net nutritional price. Because host and bacteria may compete for sugars, we hypothesized that gut flora would be nutritionally neutral to adult butterflies with plentiful food, but detrimental to semistarved hosts, especially when at high density. We held field‐caught adult Speyeria mormonia under abundant or restricted food conditions. Because antibiotic treatments did not generate consistent variation in their gut microbiota, we used interindividual variability in bacterial loads and operational taxonomic unit abundances to examine correlations between host fitness and the abdominal microbiota present upon natural death. We detected strikingly few relationships between microbial flora and host fitness. Neither total bacterial load nor the abundances of dominant bacterial taxa were related to butterfly fecundity, egg mass or egg chemical content. Increased abundance of a Commensalibacter species did correlate with longer host life span, while increased abundance of a Rhodococcus species correlated with shorter life span. Contrary to our expectations, these relationships were unchanged by food availability to the host and were unrelated to reproductive output. Our results suggest the butterfly microbiota comprises parasitic, commensal and beneficial taxa that together do not impose a net reproductive cost, even under caloric stress.
    Altered Gene Response to Aflatoxin B1 in the Spleens of Susceptible and Resistant Turkeys
    Kent M. Reed, Toxins - 2019
    Abstract
    Susceptibility and/or resistance to aflatoxin B1 (AFB1) is a threshold trait governed principally by glutathione S transferase (GST)-mediated detoxification. In poultry, domesticated turkeys are highly sensitive to AFB1, most likely due to dysfunction in hepatic GSTs. In contrast, wild turkeys are comparatively resistant to aflatoxicosis due to the presence of functional hepatic GSTAs and other possible physiological and immunological interactions. The underlying genetic basis for the disparate GST function in turkeys is unknown as are the broader molecular interactions that control the systemic response. This study quantifies the effects of dietary AFB1 on gene expression in the turkey spleen, specifically contrasting genetically distinct domesticated (DT, susceptible) and Eastern wild (EW, resistant) birds. Male turkey poults were subjected to a short-term AFB1 treatment protocol with feed supplemented with 320 ppb AFB1 beginning on day 15 of age and continuing for 14 days. Spleen tissues were harvested and subjected to deep RNA sequencing and transcriptome analysis. Analysis of differential gene expression found the effects of AFB1 treatment on the spleen transcriptomes considerably more prominent in the DT birds compared to EW. However, expression of the differentially expressed genes (DEGs) was directionally biased, with the majority showing higher expression in EW (i.e., down-regulation in DT). Significantly altered pathways included FXR/RXR and LXR/RXR activation, coagulation system, prothrombin activation, acute phase response, and atherosclerosis signaling. Differential extra-hepatic expression of acute phase protein genes was confirmed by quantitative real time PCR (qRT-PCR) in the original experiment and additional turkey lines. Results demonstrate that wild turkeys possess a capacity to more effectively respond to AFB1 exposure
    Rhodnius prolixus: Identification of missing components of the IMD immune signaling pathway and functional characterization of its role in eliminating bacteria
    Nicolas Salcedo-Porras, PLOS ONE - 2019
    Abstract
    The innate immune system in insects is regulated by specific signalling pathways. Most immune related pathways were identified and characterized in holometabolous insects such as Drosophila melanogaster, and it was assumed they would be highly conserved in all insects. The hemimetabolous insect, Rhodnius prolixus, has served as a model to study basic insect physiology, but also is a major vector of the human parasite, Trypanosoma cruzi, that causes 10,000 deaths annually. The publication of the R. prolixus genome revealed that one of the main immune pathways, the Immune-deficiency pathway (IMD), was incomplete and probably non-functional, an observation shared with other hemimetabolous insects including the pea aphid (Acyrthosiphon pisum) and the bedbug (Cimex lectularius). It was proposed that the IMD pathway is inactive in R. prolixus as an adaptation to prevent eliminating beneficial symbiont gut bacteria. We used bioinformatic analyses based on reciprocal BLAST and HMM-profile searches to find orthologs for most of the “missing” elements of the IMD pathway and provide data that these are regulated in response to infection with Gram-negative bacteria. We used RNAi strategies to demonstrate the role of the IMD pathway in regulating the expression of specific antimicrobial peptides (AMPs) in the fat body of R. prolixus. The data indicate that the IMD pathway is present and active in R. prolixus, which opens up new avenues of research on R. prolixus-T. cruzi interactions.
    TCO, a Putative Transcriptional Regulator in Arabidopsis, Is a Target of the Protein Kinase CK2
    Laina M. Weinman, International Journal of Molecular Sciences - 2019
    Abstract
    As multicellular organisms grow, spatial and temporal patterns of gene expression are strictly regulated to ensure that developmental programs are invoked at appropriate stages. In this work, we describe a putative transcriptional regulator in Arabidopsis, TACO LEAF (TCO), whose overexpression results in the ectopic activation of reproductive genes during vegetative growth. Isolated as an activation-tagged allele, tco-1D displays gene misexpression and phenotypic abnormalities, such as curled leaves and early flowering, characteristic of chromatin regulatory mutants. A role for TCO in this mode of transcriptional regulation is further supported by the subnuclear accumulation patterns of TCO protein and genetic interactions between tco-1D and chromatin modifier mutants. The endogenous expression pattern of TCO and gene misregulation in tco loss-of-function mutants indicate that this factor is involved in seed development. We also demonstrate that specific serine residues of TCO protein are targeted by the ubiquitous kinase CK2. Collectively, these results identify TCO as a novel regulator of gene expression whose activity is likely influenced by phosphorylation, as is the case with many chromatin regulators.
    Octopus maya white body show sex-specific transcriptomic profiles during the reproductive phase, with high differentiation in signaling pathways
    Oscar E. Juarez, PLOS ONE - 2019
    Abstract
    White bodies (WB), multilobulated soft tissue that wraps the optic tracts and optic lobes, have been considered the hematopoietic organ of the cephalopods. Its glandular appearance and its lobular morphology suggest that different parts of the WB may perform different functions, but a detailed functional analysis of the octopus WB is lacking. The aim of this study is to describe the transcriptomic profile of WB to better understand its functions, with emphasis on the difference between sexes during reproductive events. Then, validation via qPCR was performed using different tissues to find out tissue-specific transcripts. High differentiation in signaling pathways was observed in the comparison of female and male transcriptomic profiles. For instance, the expression of genes involved in the androgen receptorsignaling pathway were detected only in males, whereas estrogen receptor showed higher expression in females. Highly expressed genes in males enriched oxidation-reduction and apoptotic processes, which are related to the immune response. On the other hand, expression of genes involved in replicative senescence and the response to cortisol were only detected in females. Moreover, the transcripts with higher expression in females enriched a wide variety of signaling pathways mediated by molecules like neuropeptides, integrins, MAPKs and receptors like TNF and Toll-like. In addition, these putative neuropeptide transcripts, showed higher expression in females’ WB and were not detected in other analyzed tissues. These results suggest that the differentiation in signaling pathways in white bodies of O. maya influences the physiological dimorphism between females and males during the reproductive phase.
    Specific sequences of infectious challenge lead to secondary hemophagocytic lymphohistiocytosis-like disease in mice
    Andrew Wang, PNAS - 2019
    Abstract
    Secondary hemophagocytic lymphohistiocytosis (sHLH) is a highly mortal complication associated with sepsis. In adults, it is often seen in the setting of infections, especially viral infections, but the mechanisms that underlie pathogenesis are unknown. sHLH is characterized by a hyperinflammatory state and the presence hemophagocytosis. We found that sequential challenging of mice with a nonlethal dose of viral toll-like receptor (TLR) agonist followed by a nonlethal dose of TLR4 agonist, but not other permutations, produced a highly lethal state that recapitulates many aspects of human HLH. We found that this hyperinflammatory response could be recapitulated in vitro in bone marrow-derived macrophages. RNA sequencing analyses revealed dramatic up-regulation of the red-pulp macrophage lineage-defining transcription factor SpiC and its associated transcriptional program, which was also present in bone marrow macrophages sorted from patients with sHLH. Transcriptional profiling also revealed a unique metabolic transcriptional profile in these macrophages, and immunometabolic phenotyping revealed impaired mitochondrial function and oxidative metabolism and a reliance on glycolytic metabolism. Subsequently, we show that therapeutic administration of the glycolysis inhibitor 2-deoxyglucose was sufficient to rescue animals from HLH. Together, these data identify a potential mechanism for the pathogenesis of sHLH and a potentially useful therapeutic strategy for its treatment.
    Impact of malaria and hepatitis B co-infection on clinical and cytokine profiles among pregnant women
    Nsoh Godwin Anabire, PLOS ONE - 2019
    Abstract
    Background The overlap of malaria and chronic hepatitis B (CHB) is common in endemic regions, however, it is not known if this co-infection could adversely influence clinical and immunological responses. This study investigated these interactions in pregnant women reporting to antenatal clinics in Ghana. Methods Clinical parameters (hemoglobin, liver function biomarker, peripheral malaria parasitemia, and hepatitis B viremia) and cytokine profiles were assayed and compared across four categories of pregnant women: un-infected, mono-infected with Plasmodium falciparum (Malaria group), mono-infected with chronic hepatitis B virus (CHB group) and co-infected (Malaria+CHB group). Results Women with Malaria+CHB maintained appreciably normal hemoglobin levels (mean±SEM = 10.3±0.3 g/dL). That notwithstanding, Liver function test showed significantly elevated levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin [P<0.001 for all comparisons]. Similarly, the Malaria+CHB group had significantly elevated pro-inflammatory cytokines, including tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6 [P<0.05 for all comparisons]. In women with Malaria+CHB, correlation analysis showed significant negative association of the pro-inflammatory cytokines responses with malaria parasitemia [IL-1β (P<0.001; r = -0.645), IL-6 (P = 0.046; r = -0.394) and IL-12 (P = 0.011; r = -0.49)]. On the other hand, the pro-inflammatory cytokine levels positively correlated with HBV viremia [TNF-α (P = 0.004; r = 0.549), IL-1β (P<0.001; r = 0.920), IL-6 (P<0.001; r = 0.777), IFN-γ (P = 0.002; r = 0.579), IL-2 (P = 0.008; r = 0.512) and IL-12 (P<0.001; r = 0.655)]. Also, for women in the Malaria+CHB group, parasitemia was observed to diminish HBV viremia [P = 0.003, r = -0.489]. Conclusion Put together the findings suggests that Malaria+CHB could exacerbate inflammatory cytokine responses and increase susceptibility to liver injury among pregnant women in endemic settings.
    Selection for novel metabolic capabilities in Salmonella enterica
    Omar Warsi, Evolution International Journal of Evolution - 2019
    Abstract
    Bacteria are known to display extensive metabolic diversity and many studies have shown that they can use an extensive repertoire of small molecules as carbon‐ and energy sources. However, it is less clear to what extent a bacterium can expand its existing metabolic capabilities by acquiring mutations that, for example, rewire its metabolic pathways. To investigate this capability and potential for evolution of novel phenotypes, we sampled large populations of mutagenized Salmonella enterica to select very rare mutants that can grow on minimal media containing 124 low molecular weight compounds as sole carbon sources. We found mutants growing on 18 of these novel carbon sources, and identified the causal mutations that allowed growth for four of them. Mutations that relieve physiological constraints or increase expression of existing pathways were found to be important contributors to the novel phenotypes. For the remaining 14 novel phenotypes, whole genome sequencing of independent mutants and genetic analysis suggested that these novel metabolic phenotypes result from a combination of multiple mutations. This work, by virtue of identifying the genetic and mechanistic basis for new metabolic capabilities, sheds light on the properties of adaptive landscapes underlying the evolution of novel phenotypes.
    Plant growth-promoting rhizobacteria induce changes in Arabidopsis thaliana gene expression of nitrate and ammonium uptake genes
    Pamela Calvo, Journal of Plant Interactions - 2019
    Abstract
    Plant growth-promoting rhizobacteria (PGPR) enhance plant growth under the influence of multigenic processes, including nitrate (NO−3) and ammonium (NH+4) uptake genes, which could potentially explain the improvement in plant nutrition and plant growth promotion. Studies on the effects of PGPR inoculation on regulation of NO−3 and NH+4 plant uptake genes and nutrient accumulation using soil or soil-like substrates are limited. Here, we tested the hypothesis that the application of PGPR Bacillus mixtures increases overall plant growth, nutrient uptake and the transcript levels of nitrate and ammonium uptake genes in Arabidopsis thaliana. All three PGPR mixtures tested in this study significantly increased plant shoot fresh weight, root fresh, chlorophyll content, nutrient uptake and plant diameter. The transcript levels of five nitrate and four ammonium uptake genes were significantly higher in PGPR-treated plants compared to untreated plants. These results demonstrate that plant growth promotion and enhanced nutrient uptake by select PGPR mixtures.
    The effect of maxillary sinus antrostomy size on the sinus microbiome
    Alexander S. Kim BSE, Allergy and Rhinology - 2019
    Abstract
    Background The optimal maxillary antrostomy size to surgically treat sinusitis is not well known. In this study, we examined clinical metrics of disease severity and symptom scores, measured secreted inflammatory markers, and characterized the sinus microbiome to determine if there were significant differences in outcome between different maxillary ostial sizes. Methods Prospective randomized, single‐blinded clinical trial enrolling 12 individuals diagnosed with recurrent acute or chronic rhinosinusitis. Each patient was blinded and randomized to receive minimal maxillary ostial dilation via balloon sinuplasty on 1 side vs a mega‐antrostomy on the contralateral side. Data collected included symptom scores (20‐item Sino‐Nasal Outcome Test [SNOT‐20]), endoscopy, and radiologic Lund‐Mackay scores. During surgery and at their postoperative visit swabs were obtained from each maxillary sinus, and 16S DNA and inflammatory cytokine levels analyzed. The use of each patient as their own control allowed us to minimize confounding variables. Results There was statistically significant improvement in SNOT‐20 symptom scores postoperatively in all patients. There were no significant differences between maxillary ostial size in postoperative endoscopy scores, cytokine profile, or bacterial burden. There were statistically significant differences in relative postoperative abundance of Staphylococcus, Lactococcus, and Cyanobacteria between the mega‐antrostomy and mini‐antrostomy. Conclusions The method used in surgical maxillary antrostomies had no effect on endoscopy scores or cytokine profiles. Microbiome analysis determined significant differences between the different antrostomy sizes in postoperative Staphylococcus, Lactococcus, and Cyanobacteria abundance. The clinical significance of these changes in the sinus microbiome are not known but may be a result of increased access to postoperative sinonasal irrigations.
    An Fc-Optimized CD133 Antibody for Induction of Natural Killer Cell Reactivity Against Colorectal Cancer
    Bastian J. Schmied, Cancers - 2019
    Abstract
    The introduction of monoclonal antibodies (mAbs) has largely improved treatment options for cancer patients. The ability of antitumor mAbs to elicit antibody-dependent cellular cytotoxicity (ADCC) contributes to a large extent to their therapeutic efficacy. Many efforts accordingly aim to improve this important function by engineering mAbs with Fc parts that display enhanced affinity to the Fc receptor CD16 expressed, e.g., on natural killer (NK) cells. Here we characterized the CD133 mAb 293C3-SDIE that contains an engineered Fc part modified by the amino acid exchanges S239D/I332E—that reportedly increase the affinity to CD16—with regard to its ability to induce NK reactivity against colorectal cancer (CRC). 293C3-SDIE was found to be a stable protein with favorable binding characteristics achieving saturating binding to CRC cells at concentrations of approximately 1 µg/mL. While not directly affecting CRC cell growth and viability, 293C3-SDIE potently induced NK cell activation, degranulation, secretion of Interferon-γ, as well as ADCC resulting in potent lysis of CRC cell lines. Based on the preclinical characterization presented in this study and the available data indicating that CD133 is broadly expressed in CRC and represents a negative prognostic marker, we conclude that 293C3-SDIE constitutes a promising therapeutic agent for the treatment of CRC and thus warrants clinical evaluation.
    Bacterial diet and weak cadmium stress affect the survivability of Caenorhabditis elegans and its resistance to severe stress
    Ramona Dolling, Heliyon - 2019
    Abstract
    Stress may have negative or positive effects in dependence of its intensity (hormesis). We studied this phenomenon in Caenorhabditis elegans by applying weak or severe abiotic (cadmium, CdCl2) and/or biotic stress (different bacterial diets) during cultivation/breeding of the worms and determining their developmental speed or survival and performing transcriptome profiling and RT-qPCR analyses to explore the genetic basis of the detected phenotypic differences. To specify weak or severe stress, developmental speed was measured at different cadmium concentrations, and survival assays were carried out on different bacterial species as feed for the worms. These studies showed that 0.1 μmol/L or 10 mmol/L of CdCl2 were weak or severe abiotic stressors, and that E. coli HT115 or Chitinophaga arvensicola feeding can be considered as weak or severe biotic stress. Extensive phenotypic studies on wild type (WT) and different signaling mutants (e.g., kgb-1Δ and pmk-1Δ) and genetic studies on WT revealed, inter alia, the following results. WT worms bred on E. coli OP50, which is a known cause of high lipid levels in the worms, showed high resistance to severe abiotic stress and elevated gene expression for protein biosynthesis. WT worms bred under weak biotic stress (E. coli HT115 feeding which causes lower lipid levels) showed an elevated resistance to severe biotic stress, elevated gene expression for the innate immune response and signaling but reduced gene expression for protein biosynthesis. WT worms bred under weak biotic and abiotic stress (E. coli HT115 feeding plus 0.1 μmol/L of CdCl2) showed high resistance to severe biotic stress, elevated expression of DAF-16 target genes (e.g., genes for small heat shock proteins) but further reduced gene expression for protein biosynthesis. WT worms bred under weak biotic but higher abiotic stress (E. coli HT115 feeding plus 10 μmol/L of CdCl2) showed re-intensified gene expression for the innate immune response, signaling, and protein biosynthesis, which, however, did not caused a higher resistance to severe biotic stress. E. coli OP50 feeding as well as weak abiotic and biotic stress during incubations also improved the age-specific survival probability of adult WT worms. Thus, this study showed that a bacterial diet resulting in higher levels of energy resources in the worms (E. coli OP50 feeding) or weak abiotic and biotic stress promote the resistance to severe abiotic or biotic stress and the age-specific survival probability of WT.
    Epigenetic Changes at the Birc5 Promoter Induced by YM155 in Synovial Sarcoma
    Aleksander Mika, Journal of Clinical Medicine - 2019
    Abstract
    YM155 is an anti-cancer therapy that has advanced into 11 different human clinical trials to treat various cancers. This apoptosis-inducing therapy indirectly affects the protein levels of survivin (gene: Birc5), but the molecular underpinnings of the mechanism remain largely unknown. Synovial sarcoma is a rare soft-tissue malignancy with high protein expression of survivin. We investigated whether YM155 would be a viable therapeutic option to treat synovial sarcoma. YM155 therapy was applied to human synovial sarcoma cell lines and to a genetically engineered mouse model of synovial sarcoma. We discovered that YM155 exhibited nanomolar potency against human synovial sarcoma cell lines and the treated mice with synovial sarcoma demonstrated a 50% reduction in tumor volume compared to control treated mice. We further investigated the mechanism of action of YM155 by looking at the change of lysine modifications of the histone tails that were within 250 base pairs of the Birc5 promoter. Using chromatin immunoprecipitation (ChIP)-qPCR, we discovered that the histone epigenetic marks of H3K27 for the Birc5 promoter changed upon YM155 treatment. H3K27me3 and H3K27ac increased, but the net result was decreased Birc5/survivin expression. Furthermore, the combination of molecular events resulted in caspase 3/7/8 upregulation and death of the sarcoma cells.
    Chemokine Receptor Redundancy and Specificity Are Context Dependent
    Douglas P. Dyer, Immunity - 2019
    Abstract
    Currently, we lack an understanding of the individual and combinatorial roles for chemokine receptors in the inflammatory process. We report studies on mice with a compound deletion of Ccr1, Ccr2, Ccr3, and Ccr5, which together control monocytic and eosinophilic recruitment to resting and inflamed sites. Analysis of resting tissues from these mice, and mice deficient in each individual receptor, provides clear evidence for redundant use of these receptors in establishing tissue-resident monocytic cell populations. In contrast, analysis of cellular recruitment to inflamed sites provides evidence of specificity of receptor use for distinct leukocyte subtypes and no indication of comprehensive redundancy. We find no evidence of involvement of any of these receptors in the recruitment of neutrophils or lymphocytes to resting or acutely inflamed tissues. Our data shed important light on combinatorial inflammatory chemokine receptor function and highlight Ccr2 as the primary driver of myelomonocytic cell recruitment in acutely inflamed contexts.
    Localization of the 1,25-dihydroxyvitamin d-mediated response in the intestines of mice
    Carmen J. Reynolds, The Journal of Steroid Biochemistry and Molecular Biology - 2019
    Abstract
    1,25-Dihydroxyvitamin D3 (1,25(OH)2D) elicits a transcriptional response in the intestines. Assessments of this response are often derived from crude tissue homogenates and eliminate the ability to discriminate among different cell types. Here, we used an RNA in situ hybridization assay, RNAScope (Advanced Cell Diagnostics, Newark, CA), to identify the cells in the intestine that respond to 1,25(OH)2D with expression of cytochrome P450 family 24 subfamily A member 1 (Cyp24a1) mRNA. Mice were gavaged with a single bolus dose of 1,25(OH)2D to target the duodenum or a glucuronic acid conjugate of 1,25(OH)2D, β-G-1,25(OH)2D, to target the colon. QRT-PCR analysis of Cyp24a1 mRNA verified that the 1,25(OH)2D-induced responses were present. RNAScope revealed that the mRNA response present after six hours is limited to mature enterocytes exposed to the intestinal lumen in both the duodenum and colon. No detectable expression was observed in goblet cells, lamina propria, muscularis mucosa muscle, submucosa and submucosal lymphoid follicles, or tunica muscularis. Our findings have identified epithelial enterocytes to be the intestinal targets for 1,25(OH)2D in both the duodenum and colon.
    Progesterone decreases gut permeability through upregulating occludin expression in primary human gut tissues and Caco-2 cells
    Zejun Zhou, Scientific Reports - 2019
    Abstract
    Progesterone plays a protective role in preventing inflammation and preterm delivery during pregnancy. However, the mechanism involved is unknown. Microbial product translocation from a permeable mucosa is demonstrated as a driver of inflammation. To study the mechanism of the protective role of progesterone during pregnancy, we investigated the effect of physiologic concentrations of progesterone on tight junction protein occludin expression and human gut permeability in vitro and systemic microbial translocation in pregnant women in vivo. Plasma bacterial lipopolysaccharide (LPS), a representative marker of in vivo systemic microbial translocation was measured. We found that plasma LPS levels were significantly decreased during 24 to 28 weeks of gestation compared to 8 to 12 weeks of gestation. Moreover, plasma LPS levels were negatively correlated with plasma progesterone levels but positively correlated with plasma tumor necrosis factor-alpha (TNF-α) levels at 8 to 12 weeks of gestation but not at 24 to 28 weeks of gestation. Progesterone treatment increased intestinal trans-epithelial electrical resistance (TEER) in primary human colon tissues and Caco-2 cells in vitro through upregulating tight junction protein occludin expression. Furthermore, progesterone exhibited an inhibitory effect on nuclear factor kappa B (NF-κB) activation following LPS stimulation in Caco-2 cells. These results reveal a novel mechanism that progesterone may play an important role in decreasing mucosal permeability, systemic microbial translocation, and inflammation during pregnancy.
    Chronic liver injury drives non‐traditional intrahepatic fibrin(ogen) crosslinking via tissue transglutaminase
    L. G. Poole, Journal of Thrombosis and Haemostasis - 2019
    Abstract
    Background Intravascular fibrin clots and extravascular fibrin deposits are often implicated in the progression of liver fibrosis. However, evidence supporting a pathological role of fibrin in hepatic fibrosis is indirect and based largely on studies using anticoagulant drugs that inhibit activation of the coagulation protease thrombin, which has other downstream targets that promote fibrosis. Therefore, the goal of this study was to determine the precise role of fibrin deposits in experimental hepatic fibrosis. Methods Liver fibrosis was induced in mice expressing mutant fibrinogen insensitive to thrombin‐mediated proteolysis (i.e. locked in the monomeric form), termed FibAEK mice, and factor XIII A2 subunit‐deficient (FXIII−/−) mice. Female wild‐type mice, FXIII−/− mice and homozygous FibAEK mice were challenged with carbon tetrachloride (CCl4) twice weekly for 4 weeks or 6 weeks (1 mL kg−1, intraperitoneal). Results Hepatic injury and fibrosis induced by CCl4 challenge were unaffected by FXIII deficiency or inhibition of thrombin‐catalyzed fibrin polymer formation (in FibAEK mice). Surprisingly, hepatic deposition of crosslinked fibrin(ogen) was not reduced in CCl4‐challenged FXIII−/− mice or FibAEK mice as compared with wild‐type mice. Rather, deposition of crosslinked hepatic fibrin(ogen) following CCl4 challenge was dramatically reduced in tissue transglutaminase‐2 (TGM2)‐deficient (TGM2−/−) mice. However, the reduction in crosslinked fibrin(ogen) in TGM2−/− mice did not affect CCl4‐induced liver fibrosis. Conclusions These results indicate that neither traditional fibrin clots, formed by the thrombin–activated FXIII pathway nor atypical TGM2‐crosslinked fibrin(ogen) contribute to experimental CCl4‐induced liver fibrosis. Collectively, the results indicate that liver fibrosis occurs independently of intrahepatic fibrin(ogen) deposition.
    Characterization of myofibroblasts isolated from the intestine of patients with inflammatory bowel disease [version 1; peer review: 1 approved, 1 approved with reservations]
    Serge Dionne, F1000 Research Open for Science - 2019
    Abstract
    Background: Intestinal fibrosis represents a serious complication of inflammatory bowel diseases (IBD), often necessitating surgical resections. Myofibroblasts are primarily responsible for interstitial matrix accumulation in fibrotic diseases. However intestinal myofibroblasts (IMF) remain inadequately characterized. The aim was to examine fibroblast markers and fibrosis-associated gene expression in IMF isolated from resected intestine from IBD and control patients. As well as determining the effect of the fibrogenic cytokine TGFβ. Methods: Intestinal resections were obtained (n =35) from consenting patients undergoing elective surgery (2014-16). Primary cultures of IMF were isolated using DTT and EDTA and cultured. Viability and phenotypic characterization of IMF was carried out by flow cytometry and fluorescence microscopy. IMF (passages 3-8) were treated for 24 hours. Cytokines were quantified in IMF by real time PCR and in supernatants using the human pro-inflammatory cytokine panel Results: All markers and most fibrosis mediators studied were preferentially expressed by IMF compared to mucosal tissue. Metalloproteinases (MMP) 2 and 3, as well as their inhibitor TIMP1, are highly expressed by IMF. They also highly expressed inflammatory mediators, including IL-6, IL-8, CCL2 and PTGS2. Whereas mucosal expression of pro-inflammatory cytokines such as TNFα and IL-17 is increased in IBD, that of fibrosis mediators was not different. Fibrosis-related gene expression in IMF from IBD patients and controls was similar, but IMF from IBD expressed higher levels of several inflammatory genes. IMF from CD and UC had mostly similar expression profiles. TGFβ induced expression of fibrogenic genes αSMA, COL1A1, CTGF, FN1 and LOX. TGFβ-stimulated IMF released increased levels of IL-6, whereas IL-6, IL-8, as well as small amounts of IFN-γ and IL12p70 were produced following stimulation with IL-1β+IL-23. Conclusions: This study extends knowledge about the pathogenesis of fibrosis in IBD. Further research in the identification of mechanisms involved in IMF activation and fibrogenesis are required.
    Hairless regulates p53 target genes to exert tumor suppressive functions in glioblastoma
    Lemlem Brook, Journal of Cellular Biochemistry - 2019
    Abstract
    Glioblastoma (GBM) is the most common malignant brain tumor and is associated with a poor prognosis, with most patients living less than a year after diagnosis. Given that GBM nearly always recurs after conventional treatments, there is an urgent need to identify novel molecular targets. Hairless (HR) is a nuclear factor enriched in the skin and has been previously implicated in hair cycling. HR is also highly expressed in the brain, but its significance is unknown. We found that human hairless gene (HR) expression is significantly decreased in all GBM subtypes compared with normal brain tissue and is predictive of prognosis, which suggests that loss of HR expression can contribute to GBM pathogenesis. HR was recently discovered to bind to and regulate p53 responsive elements, and thus we hypothesized that HR may have a tumor suppressive function in GBM by modulating p53 target gene expression. We found that HR indeed regulates p53 target genes, including those implicated in cell cycle progression and apoptosis in the GBM‐derived U87 cell line, and restoring HR expression triggered G2/M arrest and apoptosis. An analysis of sequenced genomes from patients with GBM revealed 10 HR somatic mutations in patients with glioma, two of which are located in the histone demethylase domain of HR. These two mutations, P996S and K1004N, were reconstructed and found to have impaired p53 transactivating properties. Collectively, the results of our study suggest that HR has tumor suppressive functions in GBM, which may be clinically relevant and a potential avenue for therapeutic intervention.
    Neuroendocrine Whiplash: Slamming the Breaks on Anabolic-Androgenic Steroids Following Repetitive Mild Traumatic Brain Injury in Rats May Worsen Outcomes
    Jason Tabor, Frontiers in Neurology - 2019
    Abstract
    Sport-related concussion is an increasingly common injury among adolescents, with repetitive mild traumatic brain injuries (RmTBI) being a significant risk factor for long-term neurobiological and psychological consequences. It is not uncommon for younger professional athletes to consume anabolic-androgenic steroids (AAS) in an attempt to enhance their performance, subjecting their hormonally sensitive brains to potential impairment during neurodevelopment. Furthermore, RmTBI produces acute neuroendocrine dysfunction, specifically in the anterior pituitary, disrupting the hypothalamic-pituitary adrenal axis, lowering cortisol secretion that is needed to appropriately respond to injury. Some AAS users exhibit worse symptoms post-RmTBI if they quit their steroid regime. We sought to examine the pathophysiological outcomes associated with the abrupt cessation of the commonly abused AAS, Metandienone (Met) on RmTBI outcomes in rats. Prior to injury, adolescent male rats received either Met or placebo, and exercise. Rats were then administered RmTBIs or sham injuries, followed by steroid and exercise cessation (SEC) or continued treatment. A behavioral battery was conducted to measure outcomes consistent with clinical representations of post-concussion syndrome and chronic AAS exposure, followed by analysis of serum hormone levels, and qRT-PCR for mRNA expression and telomere length. RmTBI increased loss of consciousness and anxiety-like behavior, while also impairing balance and short-term working memory. SEC induced hyperactivity while Met treatment alone increased depressive-like behavior. There were cumulative effects whereby RmTBI and SEC exacerbated anxiety and short-term memory outcomes. mRNA expression in the prefrontal cortex, amygdala, hippocampus, and pituitary were modified in response to Met and SEC. Analysis of telomere length revealed the negative impact of SEC while Met and SEC produced changes in serum levels of testosterone and corticosterone. We identified robust changes in mRNA to serotonergic circuitry, neuroinflammation, and an enhanced stress response. Interestingly, Met treatment promoted glucocorticoid secretion after injury, suggesting that maintained AAS may be more beneficial than abstaining after mTBI.
    MiRNA-27a sensitizes breast cancer cells to treatment with Selective Estrogen Receptor Modulators
    Bojan Ljepoja, The Breast - 2019
    Abstract
    Background MicroRNA-27a (miR-27a) is a small non-coding RNA, shown to play a role in multiple cancers, including the regulation of ERα expression in breast cancer. Most ERα positive tumors are treated with Selective Estrogen Receptor Modulators (SERMs) and thus the role of miR-27a expression in response to SERM treatment is of interest. Methods Tamoxifen resistant cells were generated by molecular evolution with six cycles of tamoxifen treatment. MCF7 and T47D luminal A breast cancer cell lines were either treated with miR-27a mimics, or ER-signaling was modulated ectopically. The changes were analyzed with RT-qPCR, western blotting and transcriptional activity ERE-reporter assays. Moreover, the response to SERM treatments (tamoxifen, endoxifen and toremifen) was investigated by cell viability and apoptosis measurements. An in silico analysis of survival data from the METABRIC study was performed in order to assess the prognostic value of miR-27a for response to SERM treatment. Results Tamoxifen-resistant cells showed decreased expression of ERα and miR-27a. The overexpression of miR-27a increased the levels of ERα, while modulation of ERα decreased miR-27a expression. High miR-27a expression increased the sensitivity of MCF7 and T47D cells to SERM treatments and re-sensitized the cells to tamoxifen. Patient survival of luminal A breast cancer patients that underwent endocrine therapies was better in groups with high miR-27a expression. Conclusion MiR-27a sensitizes luminal A breast cancer cells to SERM treatments based on a positive feedback loop with ERα. An increased overall-survival of ER-positive breast cancer patients that underwent endocrine treatments and displayed high miR-27a levels was found.
    Fine-scale spatial and temporal dynamics of kdr haplotypes in Aedes aegypti from Mexico
    Marissa K. Grossman, BMC - 2019
    Abstract
    Background As resistance to insecticides increases in disease vectors, it has become exceedingly important to monitor populations for susceptibility. Most studies of field populations of Aedes aegypti have largely characterized resistance patterns at the spatial scale of the city or country, which may not be completely informative given that insecticide application occurs at the scale of the house or city block. Phenotypic resistance to pyrethroids dominates in Ae. aegypti, and it has been partially explained by mutations in the voltage-gated sodium channel gene. Here, we assess community-level patterns of four knockdown resistance (kdr) haplotypes (C1534/I1016, F1534/I1016, C1534/V1016 and F1534/V1016) in Ae. aegypti in 24 randomly chosen city blocks from a city in Yucatán State, Mexico, during both the dry and wet season and over two years. Results Three of the four haplotypes, C1534/I1016, C1534/V1016 and F1534/V1016 were heterogeneous between city blocks at all four sampling time points, and the double mutant haplotype, C1534/I1016, showed a significant increase following the wet season. The F1534/I1016 haplotype was rarely detected, similar to other studies. However, when haplotype frequencies were aggregated to a coarser spatial scale, the differences in space and time were obscured. Conclusions Our results provide empirical evidence that the selection of kdr alleles is occurring at fine spatial scales, indicating that future studies should include this scale to better understand evolutionary processes of resistance in natural populations.
    Loss-of-function mutations in caspase recruitment domain-containing protein 14 (CARD14) are associated with a severe variant of atopic dermatitis
    Alon Peled BMedSci, Science Direct - 2019
    Abstract
    Background Atopic dermatitis (AD) is a highly prevalent chronic inflammatory skin disease that is known to be, at least in part, genetically determined. Mutations in caspase recruitment domain-containing protein 14 (CARD14) have been shown to result in various forms of psoriasis and related disorders. Objective We aimed to identify rare DNA variants conferring a significant risk for AD through genetic and functional studies in a cohort of patients affected with severe AD. Methods Whole-exome and direct gene sequencing, immunohistochemistry, real-time PCR, ELISA, and functional assays in human keratinocytes were used. Results In a cohort of patients referred with severe AD, DNA sequencing revealed in 4 patients 2 rare heterozygous missense mutations in the gene encoding CARD14, a major regulator of nuclear factor κB (NF-κB). A dual luciferase reporter assay demonstrated that both mutations exert a dominant loss-of-function effect and result in decreased NF-κB signaling. Accordingly, immunohistochemistry staining showed decreased expression of CARD14 in patients' skin, as well as decreased levels of activated p65, a surrogate marker for NF-κB activity. CARD14-deficient or mutant-expressing keratinocytes displayed abnormal secretion of key mediators of innate immunity. Conclusions Although dominant gain-of-function mutations in CARD14 are associated with psoriasis and related diseases, loss-of-function mutations in the same gene are associated with a severe variant of AD.
    Presence of Circulating miR-145, miR-155, and miR-382 in Exosomes Isolated from Serum of Breast Cancer Patients and Healthy Donors
    Vianey Gonzalez-Villasana, Hindawi - 2019
    Abstract
    miR-145, miR-155, and miR-382 have been proposed as noninvasive biomarkers to distinguish breast cancer patients from healthy individuals. However, it is unknown if these three miRNAs are secreted by exosomes. Thus, we hypothesized that miR-145, miR-155, and miR-382 in breast cancer patients are present in exosomes. We isolated exosomes from serum of breast cancer patients and healthy donors, then we characterized them according to their shape, size, and exosome markers by scanning electron microscopy, atomic force microscopy, nanoparticle tracking analysis (NTA), and Western blot and determined the exosome concentration in all samples by NTA. Later, exosomal small RNA extraction was done to determine the expression levels of miR-145, miR-155, and miR-382 by qRT-PCR. We observed a round shape of exosomes with a mean size of 119.84 nm in breast cancer patients and 115.4 nm in healthy donors. All exosomes present the proteins CD63, Alix, Tsg, CD9, and CD81 commonly used as markers. Moreover, we found a significantly high concentration of exosomes in breast cancer patients with stages I, III, and IV compared to healthy donors. We detected miR-145, miR-155, and miR-382 in the exosomes isolated from serum of breast cancer patients and healthy donors. Our results show that the exosomes isolated from the serum of breast cancer patients and healthy donors contains miR-145, miR-155, and miR-382 but not in a selective manner in breast cancer patients. Moreover, our data support the association between exosome concentration and the presence of breast cancer, opening the possibility to study how miRNAs packaged into exosomes play a role in BC progression.
    Quantitative PCR Measurement of miR-371a-3p and miR-372-p Is Influenced by Hemolysis
    Mette Pernille Myklebust, Frontiers in Genetics - 2019
    Abstract
    Cell-free microRNAs have been reported as biomarkers for several diseases. For testicular germ cell tumors (GCT), circulating microRNAs 371a-3p and 372-3p in serum and plasma have been proposed as biomarkers for diagnostic and disease monitoring purposes. The most widely used method for quantification of specific microRNAs in serum and plasma is reverse transcriptase real-time quantitative PCR (RT-qPCR) by the comparative Ct-method. In this method one or several reference genes or reference microRNAs are needed in order to normalize and calculate the relative microRNA levels across samples. One of the pitfalls in analysis of microRNAs from serum and plasma is the release of microRNAs from blood cells during hemolysis. This is an important issue because varying degrees of hemolysis are not uncommon in routine blood sampling. Thus, hemolysis must be taken into consideration when working with circulating microRNAs from blood. miR-93-5p, miR-30b-5p, and miR-20a-5p have been reported as reference microRNA in analysis of the miR-371a-373 cluster. We here show how these three microRNAs are influenced by hemolysis. We also propose a new reference microRNA, miR-191-5p, which is relatively stable in serum samples with mild hemolysis. In addition, we show how hemolysis can have effect on the reported microRNA levels in patient samples when these reference microRNAs are used in samples with varying levels of hemolysis.
    Superresolution microscopy reveals linkages between ribosomal DNA on heterologous chromosomes
    Tamara A. Potapova, Journal of Cell Biology - 2019
    Abstract
    The spatial organization of the genome is enigmatic. Direct evidence of physical contacts between chromosomes and their visualization at nanoscale resolution has been limited. We used superresolution microscopy to demonstrate that ribosomal DNA (rDNA) can form linkages between chromosomes. We observed rDNA linkages in many different human cell types and demonstrated their resolution in anaphase. rDNA linkages are coated by the transcription factor UBF and their formation depends on UBF, indicating that they regularly occur between transcriptionally active loci. Overexpression of c-Myc increases rDNA transcription and the frequency of rDNA linkages, further suggesting that their formation depends on active transcription. Linkages persist in the absence of cohesion, but inhibition of topoisomerase II prevents their resolution in anaphase. We propose that linkages are topological intertwines occurring between transcriptionally active rDNA loci spatially colocated in the same nucleolar compartment. Our findings suggest that active DNA loci engage in physical interchromosomal connections that are an integral and pervasive feature of genome organization
    Serum FHR1 binding to necrotic-type cells activates monocytic inflammasome and marks necrotic sites in vasculopathies
    Sarah Irmscher, Nature Communications - 2019
    Abstract
    Persistent inflammation is a hallmark of many human diseases, including anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) and atherosclerosis. Here, we describe a dominant trigger of inflammation: human serum factor H-related protein FHR1. In vitro, this protein selectively binds to necrotic cells via its N-terminus; in addition, it binds near necrotic glomerular sites of AAV patients and necrotic areas in atherosclerotic plaques. FHR1, but not factor H, FHR2 or FHR3 strongly induces inflammasome NLRP3 in blood-derived human monocytes, which subsequently secrete IL-1β, TNFα, IL-18 and IL-6. FHR1 triggers the phospholipase C-pathway via the G-protein coupled receptor EMR2 independent of complement. Moreover, FHR1 concentrations of AAV patients negatively correlate with glomerular filtration rates and associate with the levels of inflammation and progressive disease. These data highlight an unexpected role for FHR1 during sterile inflammation, may explain why FHR1-deficiency protects against certain diseases, and identifies potential targets for treatment of auto-inflammatory diseases.
    RAL GTPases Drive Intestinal Stem Cell Function and Regeneration through Internalization of WNT Signalosomes
    Joel Johansson, Cell Stem Cell - 2019
    Abstract
    Ral GTPases are RAS effector molecules and by implication a potential therapeutic target for RAS mutant cancer. However, very little is known about their roles in stem cells and tissue homeostasis. Using Drosophila, we identified expression of RalA in intestinal stem cells (ISCs) and progenitor cells of the fly midgut. RalA was required within ISCs for efficient regeneration downstream of Wnt signaling. Within the murine intestine, genetic deletion of either mammalian ortholog, Rala or Ralb, reduced ISC function and Lgr5 positivity, drove hypersensitivity to Wnt inhibition, and impaired tissue regeneration following damage. Ablation of both genes resulted in rapid crypt death. Mechanistically, RALA and RALB were required for efficient internalization of the Wnt receptor Frizzled-7. Together, we identify a conserved role for RAL GTPases in the promotion of optimal Wnt signaling, which defines ISC number and regenerative potential.
    A Neuronal Relay Mediates a Nutrient Responsive Gut/Fat Body Axis Regulating Energy Homeostasis in Adult Drosophila
    Alessandro Scopelliti, Cell Metabolism - 2019
    Abstract
    The control of systemic metabolic homeostasis involves complex inter-tissue programs that coordinate energy production, storage, and consumption, to maintain organismal fitness upon environmental challenges. The mechanisms driving such programs are largely unknown. Here, we show that enteroendocrine cells in the adult Drosophila intestine respond to nutrients by secreting the hormone Bursicon α, which signals via its neuronal receptor DLgr2. Bursicon α/DLgr2 regulate energy metabolism through a neuronal relay leading to the restriction of glucagon-like, adipokinetic hormone (AKH) production by the corpora cardiaca and subsequent modulation of AKH receptor signaling within the adipose tissue. Impaired Bursicon α/DLgr2 signaling leads to exacerbated glucose oxidation and depletion of energy stores with consequent reduced organismal resistance to nutrient restrictive conditions. Altogether, our work reveals an intestinal/neuronal/adipose tissue inter-organ communication network that is essential to restrict the use of energy and that may provide insights into the physiopathology of endocrine-regulated metabolic homeostasis.
    A Role for FACT in RNA Polymerase II Promoter-Proximal Pausing
    Theophilus T. Tettey, Cell Reports - 2019
    Abstract
    FACT (facilitates chromatin transcription) is an evolutionarily conserved histone chaperone that was initially identified as an activity capable of promoting RNA polymerase II (Pol II) transcription through nucleosomes in vitro. In this report, we describe a global analysis of FACT function in Pol II transcription in Drosophila. We present evidence that loss of FACT has a dramatic impact on Pol II elongation-coupled processes including histone H3 lysine 4 (H3K4) and H3K36 methylation, consistent with a role for FACT in coordinating histone modification and chromatin architecture during Pol II transcription. Importantly, we identify a role for FACT in the maintenance of promoter-proximal Pol II pausing, a key step in transcription activation in higher eukaryotes. These findings bring to light a broader role for FACT in the regulation of Pol II transcription.
    Suppression of UV-B stress induced flavonoids by biotic stress: Is there reciprocal crosstalk?
    Dirk Schenke, Plant Physiology and Biochemistry - 2019
    Abstract
    Plants respond to abiotic UV-B stress with enhanced expression of genes for flavonoid production, especially the key-enzyme chalcone synthase (CHS). Some flavonoids are antioxidative, antimicrobial and/or UV-B protective secondary metabolites. However, when plants are challenged with concomitant biotic stress (simulated e.g. by the bacterial peptide flg22, which induces MAMP triggered immunity, MTI), the production of flavonoids is strongly suppressed in both Arabidopsis thaliana cell cultures and plants. On the other hand, flg22 induces the production of defense related compounds, such as the phytoalexin scopoletin, as well as lignin, a structural barrier thought to restrict pathogen spread within the host tissue. Since all these metabolites require the precursor phenylalanine for their production, suppression of the flavonoid production appears to allow the plant to focus its secondary metabolism on the production of pathogen defense related compounds during MTI. Interestingly, several flavonoids have been reported to display anti-microbial activities. For example, the plant flavonoid phloretin targets the Pseudomonas syringae virulence factors flagella and type 3 secretion system. That is, suppression of flavonoid synthesis during MTI might have also negative side-effects on the pathogen defense. To clarify this issue, we deployed an Arabidopsis flavonoid mutant and obtained genetic evidence that flavonoids indeed contribute to ward off the virulent bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Finally, we show that UV-B attenuates expression of the flg22 receptor FLS2, indicating that there is negative and reciprocal interaction between this abiotic stress and the plant-pathogen defense responses.
    Diet and diet‐associated bacteria shape early microbiome development in Yellowtail Kingfish (Seriola lalandi)
    Jackson Wilkes Walburn, Microbial Biotechnology - 2019
    Abstract
    The supply of quality juveniles via land‐based larviculture represents a major bottleneck to the growing finfish aquaculture industry. As the microbiome plays a key role in animal health, this study aimed to assess the microbial community associated with early larval development of commercially raised Yellowtail Kingfish (Seriola lalandi). We used qPCR and 16S rRNA gene amplicon sequencing to monitor changes in the microbiome associated with the development of S. lalandi from larvae to juveniles. We observed an increase in the bacterial load during larval development, which consisted of a small but abundant core microbiota including taxa belonging to the families Rhodobacteraceae, Lactobacillaceae and Vibrionaceae. The greatest change in the microbiome occurred as larvae moved from a diet of live feeds to formulated pellets, characterized by a transition from Proteobacteria to Firmicutes as the dominant phylum. A prediction of bacterial gene functions found lipid metabolism and secondary metabolite production were abundant in the early larval stages, with carbohydrate and thiamine metabolism functions increasing in abundance as the larvae age and are fed formulated diets. Together, these results suggest that diet is a major contributor to the early microbiome development of commercially raised S. lalandi.
    Therapeutic Targeting of Stat3 Using Lipopolyplex Nanoparticle-Formulated siRNA in a Syngeneic Orthotopic Mouse Glioma Model
    Benedikt Linder, Cancers - 2019
    Abstract
    Glioblastoma (GBM), WHO grade IV, is the most aggressive primary brain tumor in adults. The median survival time using standard therapy is only 12–15 months with a 5-year survival rate of around 5%. Thus, new and effective treatment modalities are of significant importance. Signal transducer and activator of transcription 3 (Stat3) is a key signaling protein driving major hallmarks of cancer and represents a promising target for the development of targeted glioblastoma therapies. Here we present data showing that the therapeutic application of siRNAs, formulated in nanoscale lipopolyplexes (LPP) based on polyethylenimine (PEI) and the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), represents a promising new approach to target Stat3 in glioma. We demonstrate that the LPP-mediated delivery of siRNA mediates efficient knockdown of Stat3, suppresses Stat3 activity and limits cell growth in murine (Tu2449) and human (U87, Mz18) glioma cells in vitro. In a therapeutic setting, intracranial application of the siRNA-containing LPP leads to knockdown of STAT3 target gene expression, decreased tumor growth and significantly prolonged survival in Tu2449 glioma-bearing mice compared to negative control-treated animals. This is a proof-of-concept study introducing PEI-based lipopolyplexes as an efficient strategy for therapeutically targeting oncoproteins with otherwise limited druggability
    On resolving ambiguities in microbial community analysis of partial nitritation anammox reactors
    Laura Orschler, Scientific Reports - 2019
    Abstract
    PCR-based methods have caused a surge for integration of eco-physiological approaches into research on partial nitritation anammox (PNA). However, a lack of rigorous standards for molecular analyses resulted in widespread data misinterpretation and consequently lack of consensus. Data consistency and accuracy strongly depend on the primer selection and data interpretation. An in-silico evaluation of 16S rRNA gene eubacterial primers used in PNA studies from the last ten years unraveled the difficulty of comparing ecological data from different studies due to a variation in the coverage of these primers. Our 16S amplicon sequencing approach, which includes parallel sequencing of six 16S rRNA hypervariable regions, showed that there is no perfect hypervariable region for PNA microbial communities. Using qPCR analysis, we emphasize the significance of primer choice for quantification and caution with data interpretation. We also provide a framework for PCR based analyses that will improve and assist to objectively interpret and compare such results.
    Infection with Toxoplasma gondii (Eucoccidiorida: Sarcocystidae) in bats of Campeche and Yucatán, Mexico
    Marco Torres-Castro, International Journal of Tropical Biology and Conservation - 2019
    Abstract
    Toxoplasma gondii is a protozoan parasite recognized as the causative agent of toxoplasmosis, a zoonotic disease that affects humans and domestic or wild animals. In Mexico, it represents a public and animal health problem, especially in regions with tropical and subtropical climates. Bats have been reported as accidental hosts in the transmission cycle; however, there is no preceding information in Mexico. Therefore, the aim of the present study is to report the T. gondii infection in bats captured in sites of Campeche and Yucatan states, Mexico. Bats were captured in two sites in Yucatan (X’matkuil and Panaba) and one in Campeche (Hampolol), located in the Yucatan Peninsula. Kidneys, spleen, and liver were collected and used in the total DNA extraction. Toxoplasma gondii infection was detected through the amplification of a B1 gene fragment, using nested PCR. The positive PCR products were purified and sent to sequencing for a posterior sequence identity analysis. Additionally, a phylogenetic tree was made. A total of 69 bats belonging to eight different species were processed: 41 (59.4 %, 41/69) Artibeus jamaicensis; six (8.7 %, 6/69) Pteronotus parnellii; six (8.7 %, 6/69) Noctilio leporinus; six (8.7 %, 6/69) Chiroderma villosum; four (5.8 %, 4/69) Glossophaga soricina; two (2.9 %, 2/69) Carollia sowelli; two (2.89 %, 2/69) Artibeus lituratus; and two (2.9 %, 2/69) Rhogeessa aeneus. The nested PCR identified eight (11.6 %, 8/69) infected bats: six (75 %, 6/8) A. jamaicensis, captured in X'matkuil and Panaba, one (12.5 %, 1/8) G. soricina, and one (12.5 %, 1/8) C. villosum, both captured in Panaba. The alignment analysis yielded 99-100 % for cover and 97-99 % for identity to T. gondii sequences. Our results contribute to the understanding of the T. gondii transmission cycle in the region; however, future research is needed to determine circulating genotypes, as previous studies have demonstrated that these animals might be infected with identified genotypes in other domestic or wild animals and even in humans.
    Overlapping Activities of Two Neuronal Splicing Factors Switch the GABA Effect from Excitatory to Inhibitory by Regulating REST
    Yoko Nakano, Cell Reports - 2019
    Abstract
    A truncating mutation in the mouse Srrm4 gene, which encodes a neuronal splicing factor, causes alternative splicing defects selectively in the ear. The mechanism by which splicing is preserved in the brain of these mice is not known. Here, we show that SRRM3 limits the Srrm4 mutation-associated defects to the ear and that, in cortical neurons, overlapping SRRM3-SRRM4 activity regulates the development of interneuronal inhibition. In vitro, SRRM3 and SRRM4 regulate the same splicing events, but a mutation in mouse Srrm3 causes tremors and mild defects in neuronal alternative splicing, demonstrating unique SRRM3 roles in vivo. Mice harboring mutations in both Srrm3 and Srrm4 die neonatally and exhibit severe splicing defects. In these mice, splicing alterations prevent inactivation of the gene repressor REST, which maintains immature excitatory GABAergic neurotransmission by repressing K-Cl cotransporter 2. Thus, our data reveal that SRRM3 and SRRM4 act redundantly to regulate GABAergic neurotransmission by inactivating REST.
    Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts
    Zoe E. Gillespie, Scientific Reports - 2019
    Abstract
    Metformin is a widely-used treatment for type 2 diabetes and is reported to extend health and lifespan as a caloric restriction (CR) mimetic. Although the benefits of metformin are well documented, the impact of this compound on the function and organization of the genome in normal tissues is unclear. To explore this impact, primary human fibroblasts were treated in culture with metformin resulting in a significant decrease in cell proliferation without evidence of cell death. Furthermore, metformin induced repositioning of chromosomes 10 and 18 within the nuclear volume indicating altered genome organization. Transcriptome analyses from RNA sequencing datasets revealed that alteration in growth profiles and chromosome positioning occurred concomitantly with changes in gene expression profiles. We further identified that different concentrations of metformin induced different transcript profiles; however, significant enrichment in the activator protein 1 (AP-1) transcription factor network was common between the different treatments. Comparative analyses revealed that metformin induced divergent changes in the transcriptome than that of rapamycin, another proposed mimetic of CR. Promoter analysis and chromatin immunoprecipitation assays of genes that changed expression in response to metformin revealed enrichment of the transcriptional regulator forkhead box O3a (FOXO3a) in normal human fibroblasts, but not of the predicted serum response factor (SRF). Therefore, we have demonstrated that metformin has significant impacts on genome organization and function in normal human fibroblasts, different from those of rapamycin, with FOXO3a likely playing a role in this response.
    The chemokine receptor CXCR2 contributes to murine adipocyte development
    Douglas P. Dyer, Journal of Leukocyte Biology - 2019
    Abstract
    Chemokines are members of a large family of chemotactic cytokines that signal through their receptors to mediate leukocyte recruitment during inflammation and homeostasis. The chemokine receptor CXCR2 has largely been associated with neutrophil recruitment. However, there is emerging evidence of roles for chemokines and their receptors in processes other than leukocyte migration. We have previously demonstrated that CXCR2 knockout (KO) mice have thinner skin compared to wild‐type mice. Herein we demonstrate that this is due to a thinner subcutaneous adipose layer, as a result of fewer and smaller individual adipocytes. We observe a similar phenotype in other fat depots and present data that suggests this may be due to reduced expression of adipogenesis related genes associated with adipocyte specific CXCR2 signaling. Interestingly, this phenotype is evident in female, but not male, CXCR2 KO mice. These findings expand our understanding of nonleukocyte related chemokine receptor functions and help to explain some previously observed adipose‐related phenotypes in CXCR2 KO mice.
    Cytoprotective effects of Avenathramide C against oxidative and inflammatory stress in normal human dermal fibroblasts
    Chenxuan Wang, Scientific Reports - 2019
    Abstract
    Natural polyphenols are promising anti-aging compounds not only for their antioxidant activity, but also their ability to activate specific cellular pathways mediating the aging process. Avenanthramide C (Avn C), found exclusively in oats, is a natural antioxidant associated with free radical scavenging; however, it is how this compound elicits other protective effects. We investigated the intracellular antioxidant activity of Avn C and other cytoprotective potential in normal human skin fibroblasts exposed to extracellular stress. Avn C reduced H2O2-induced oxidative stress by reducing intracellular free radical levels and antioxidant gene transcripts. Avn C also resulted in decreased levels of gene transcripts encoding pro-inflammatory cytokines in response to H2O2 or tumor necrosis factor-α (TNF-α). This reduction in cytokine gene transcription occurred concomitantly with reduced phosphorylated nuclear factor-κB (NF-κB) p65, and decreased NF-κB DNA binding. Avn C further induced heme oxygense-1 (HO-1) expression through increased Nrf2 DNA binding activity, demonstrating a second mechanism by which Avn C attenuates cellular stress. Collectively, our findings indicate that Avn C protects normal human skin fibroblasts against oxidative stress and inflammatory response through NF-κB inhibition and Nrf2/HO-1 activation
    Effects of 50 Hz magnetic fields on circadian rhythm control in mice
    Louise Lundberg, Bio Electro Magnetics - 2019
    Abstract
    Artificial light and power frequency magnetic fields are ubiquitous in the built environment. Light is a potent zeitgeber but it is unclear whether power frequency magnetic fields can influence circadian rhythm control. To study this possibility, 8–12‐week‐old male C57BL/6J mice were exposed for 30 min starting at zeitgeber time 14 (ZT14, 2 h into the dark period of the day) to 50 Hz magnetic fields at 580 μT using a pair of Helmholtz coils and/or a blue LED light at 700 lux or neither. Our experiments revealed an acute adrenal response to blue light, in terms of increased adrenal per1 gene expression, increased serum corticosterone levels, increased time spent sleeping, and decreased locomotor activity (in all cases, P < 0.0001) compared to an unexposed control group. There appeared to be no modulating effect of the magnetic fields on the response to light, and there was also no effect of the magnetic fields alone (in both cases, P > 0.05) except for a decrease in locomotor activity (P < 0.03). Gene expression of the cryptochromes cry1 and cry2 in the adrenals, liver, and hippocampus was also not affected by exposures (in all cases, P > 0.05). In conclusion, these results suggest that 50 Hz magnetic fields do not significantly affect the acute light response to a degree that can be detected in the adrenal response. Bioelectromagnetics. 2019;9999:XX–XX. © 2019 Bioelectromagnetics Society.
    Three Types of Functional Regulatory T Cells Control T Cell Responses at the Human Maternal-Fetal Interface
    Maria Salvany-Celades, Cell Reports - 2019
    Abstract
    During pregnancy, maternal regulatory T cells (Tregs) are important in establishing immune tolerance to invading fetal extravillous trophoblasts (EVTs). CD25HIFOXP3+ Tregs are found at high levels in decidual tissues and have been shown to suppress fetus-specific and nonspecific responses. However, limited data are available on additional decidual Treg types and the mechanisms by which they are induced. This study investigated three distinct decidual CD4+ Treg types in healthy pregnancies with a regulatory phenotype and the ability to suppress T cell responses: CD25HIFOXP3+, PD1HIIL-10+, and TIGIT+FOXP3dim. Moreover, co-culture of HLA-G+ EVTs or decidual macrophages with blood CD4+ T cells directly increased the proportions of CD25HIFOXP3+ Tregs compared to T cells cultured alone. EVTs also increased PD1HI Tregs that could be inhibited by HLA-C and CD3 antibodies, suggesting an antigen-specific induction. The presence of distinct Treg types may allow for the modulation of a variety of inflammatory responses in the placenta.
    Surmounting Cytarabine-resistance in acute myeloblastic leukemia cells and specimens with a synergistic combination of hydroxyurea and azidothymidine
    May Levin, Cell Death and Disease - 2019
    Abstract
    Acute myeloid leukemia (AML) patients display dismal prognosis due to high prevalence of refractory and relapsed disease resulting from chemoresistance. Treatment protocols, primarily based on the anchor drug Cytarabine, remained chiefly unchanged in the past 50 years with no standardized salvage regimens. Herein we aimed at exploring potential pre-clinical treatment strategies to surmount Cytarabine resistance in human AML cells. We established Cytarabine-resistant sublines derived from human leukemia K562 and Kasumi cells, and characterized the expression of Cytarabine-related genes using real-time PCR and Western blot analyses to uncover the mechanisms underlying their Cytarabine resistance. This was followed by growth inhibition assays and isobologram analyses testing the sublines’ sensitivity to the clinically approved drugs hydroxyurea (HU) and azidothymidine (AZT), compared to their parental cells. All Cytarabine-resistant sublines lost deoxycytidine kinase (dCK) expression, rendering them refractory to Cytarabine. Loss of dCK function involved dCK gene deletions and/or a novel frameshift mutation leading to dCK transcript degradation via nonsense-mediated decay. Cytarabine-resistant sublines displayed hypersensitivity to HU and AZT compared to parental cells; HU and AZT combinations exhibited a marked synergistic growth inhibition effect on leukemic cells, which was intensified upon acquisition of Cytarabine-resistance. In contrast, HU and AZT combination showed an antagonistic effect in non-malignant cells. Finally, HU and AZT synergism was demonstrated on peripheral blood specimens from AML patients. These findings identify a promising HU and AZT combination for the possible future treatment of relapsed and refractory AML, while sparing normal tissues from untoward toxicity.
    Transcriptome Response of Female Culicoides sonorensis Biting Midges (Diptera: Ceratopogonidae) to Early Infection with Epizootic Hemorrhagic Disease Virus (EHDV-2)
    Dana Nayduch, Viruses - 2019
    Abstract
    Female Culicoides sonorensis biting midges are vectors of epizootic hemorrhagic disease virus (EHDV), which causes morbidity and mortality in wild and domesticated ruminants. The aims in this study were to identify key changes in female midge transcriptome profiles occurring during early infection with EHDV-2. Midges were fed either negative control bloodmeals or bloodmeals containing EHDV-2 and transcriptomes were acquired at 36 h through deep sequencing. Reads were de novo assembled into a transcriptome comprised of 18,754 unigenes. Overall, there were 2401 differentially expressed unigenes and ~60% were downregulated in response to the virus (953 up; 1448 down). Downstream Gene Ontology enrichment, KEGG pathway mapping, and manual analyses were used to identify the effect of virus ingestion at both the gene and pathway levels. Downregulated unigenes were predominantly assigned to pathways related to cell/tissue structure and integrity (actin cytoskeleton, adherens junction, focal adhesion, hippo signaling), calcium signaling, eye morphogenesis and axon guidance. Unigenes attributed to sensory functions (especially vision), behavior, learning and memory were largely downregulated. Upregulated unigenes included those coding for innate immune processes, olfaction and photoreceptor pigments. Our results suggest that midges respond to virus infection as soon as 36 h post-ingestion, and that EHDV-2 may have a significant phenotypic effect on sensory and neural tissues.
    CYLD Regulates Centriolar Satellites Proteostasis by Counteracting the E3 Ligase MIB1
    Tiphaine Douanne, Cell Reports - 2019
    Abstract
    The tumor suppressor CYLD is a deubiquitinatingenzyme that removes non-degradative ubiquitin link-ages bound to a variety of signal transduction adap-tors. CYLD participates in the formation of primarycilia, a microtubule-based structure that protrudesfrom the cell body to act as a ‘‘sensing antenna.’’Yet, how exactly CYLD regulates ciliogenesis is notfully understood. Here, we conducted an unbiasedproteomic screen of CYLD binding partners andidentified components of the centriolar satellites.These small granular structures, tethered to the scaf-fold protein pericentriolar matrix protein 1 (PCM1),gravitate toward the centrosome and orchestrateciliogenesis. CYLD knockdown promotes PCM1degradation and the subsequent dismantling of thecentriolar satellites. We found that CYLD marshalsthe centriolar satellites by deubiquitinating andpreventing the E3 ligase Mindbomb 1 (MIB1) frommarking PCM1 for proteasomal degradation. Theseresults link CYLD to the regulation of centriolar satel-lites proteostasis and provide insight into howreversible ubiquitination finely tunes ciliogenesis.
    Disruption of Intestinal Homeostasis and Intestinal Microbiota During Experimental Autoimmune Uveitis
    Cathleen Janowitz, Investigative Ophthalmology & Visual Science - 2019
    Abstract
    Purpose: We determine the changes in intestinal microbiota and/or disruptions in intestinal homeostasis during uveitis. Methods: Experimental autoimmune uveitis (EAU) was induced in B10.RIII mice with coadministration of interphotoreceptor retinoid-binding protein peptide (IRBP) and killed mycobacterial antigen (MTB) as an adjuvant. Using 16S rRNA gene sequencing, we looked at intestinal microbial differences during the course of uveitis, as well as intestinal morphologic changes, changes in intestinal permeability by FITC-dextran leakage, antimicrobial peptide expression in the gastrointstinal tract, and T lymphocyte prevalence before and at peak intraocular inflammation. Results: We demonstrate that increased intestinal permeability and antimicrobial peptide expression in the intestinal tract coincide in timing with increased effector T cells in the mesenteric lymph nodes, during the early stages of uveitis, before peak inflammation. Morphologic changes in the intestine were most prominent during this phase, but also occurred with adjuvant MTB alone, whereas increased intestinal permeability was found only in IRBP-immunized mice that develop uveitis. We also demonstrate that the intestinal microbiota were altered during the course of uveitis, and that some of these changes are specific to uveitic animals, whereas others are influenced by adjuvant MTB alone. Intestinal permeability peaked at 2 weeks, coincident with an increase in intestinal bacterial strain differences, peak lipocalin production, and peak uveitis. Conclusions: An intestinal dysbiosis accompanies a disruption in intestinal homeostasis in autoimmune uveitis, although adjuvant MTB alone promotes intestinal disruption as well. This may indicate a novel axis for future therapeutic targeting experimentally or clinically.
    Tart Cherry Prevents Bone Loss through Inhibition of RANKL in TNF-Overexpressing Mice
    Nicholas Moon, Nutrients - 2019
    Abstract
    Current drugs for the treatment of rheumatoid arthritis-associated bone loss come with concerns about their continued use. Thus, it is necessary to identify natural products with similar effects, but with fewer or no side effects. We determined whether tart cherry (TC) could be used as a supplement to prevent inflammation-mediated bone loss in tumor necrosis factor (TNF)-overexpressing transgenic (TG) mice. TG mice were assigned to a 0%, 5%, or 10% TC diet, with a group receiving infliximab as a positive control. Age-matched wild-type (WT) littermates fed a 0% TC diet were used as a normal control. Mice were monitored by measurement of body weight. Bone health was evaluated via serum biomarkers, microcomputed tomography (µCT), molecular assessments, and mechanical testing. TC prevented TNF-mediated weight loss, while it did not suppress elevated levels of interleukin (IL)-1β and IL-6. TC also protected bone structure from inflammation-induced bone loss with a reduced ratio of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) to a degree comparable to infliximab. Furthermore, unlike with infliximab, TC exhibited a moderate improvement in TNF-mediated decline in bone stiffness. Thus, TC could be used as a prophylactic regimen against future fragility fractures in the context of highly chronic inflammation.
    Insulin-like Growth Factor (IGF)-1 treatment stabilizes the microvascularcytoskeleton under ischemic conditions
    Shameena Bake, Experimental Neurology - 2019
    Abstract
    Our previous studies showed that Insulin-like Growth Factor (IGF)-1 reduced blood brain barrier permeabilityand decreased infarct volume caused by middle cerebral artery occlusion (MCAo) in middle aged female rats.Similarly, cultures of primary brain microvessel endothelial cells from middle-aged female rats and exposed tostroke-like conditions (oxygen glucose deprivation; OGD) confirmed that IGF-1 reduced dye transfer across thiscell monolayer. Surprisingly, IGF-1 did not attenuate endothelial cell death caused by OGD. To reconcile thesefindings, the present study tested the hypothesis that, at the earliest phase of ischemia, IGF-1 promotes barrierfunction by increasing anchorage and stabilizing cell geometry of surviving endothelial cells. Cultures of humanbrain microvessel endothelial cells were subject to oxygen-glucose deprivation (OGD) in the presence of IGF-1,IGF-1 + JB-1 (IGFR inhibitor) or vehicle. OGD disrupted the cell monolayer and reduced cell-cell interactions,which was preserved in IGF-1-treated cultures and reversed by concurrent treatment with JB-1. IGF-1-mediatedpreservation of the endothelial monolayer was reversed with LY294002 treatment, but not by Rapamycin, in-dicating that IGF-1 s actions on cell-cell contacts are likely mediated via the PI3K pathway. In vivo, microvesselmorphology was evaluated in middle-aged female rats that were subjected to ischemia by MCAo, and treated ICVwith IGFeI, IGF-1 + JB-1, or artificial CSF (aCSF; vehicle) after reperfusion. Compared to vehicle controls, IGF-1treated animals displayed larger microvessel diameters in the peri-infarct area and increased staining density forvinculin, an anchorage protein. Both these measures were reversed by concurrent IGF-1 + JB-1 treatment.Moreover these effects were restricted to 24 h after ischemia-reperfusion and no treatment effects were seen at5d post stroke. Collectively, these data suggest that in the earliest hours during ischemia, IGF-1 promotes re-ceptor-mediated anchorage of endothelial cells, and its actions may be accurately characterized as vasculo-protective.
    High diagnostic yield and novel variants in very late-onset spasticity
    Momen Almomen, Journal of Neurogenetics - 2019
    Abstract
    Hereditary spastic paraplegias (HSPs) are a diverse group of genetic conditions with variable severity and onset age. From a neurogenetic clinic, we identified 14 patients with very late-onset HSP, with symptoms starting after the age of 35. In this cohort, sequencing of known genetic causes was performed using clinically available HSP sequencing panels. We identified 4 patients with mutations in SPG7 and 3 patients with SPAST mutations, representing 50% of the cohort and indicating a very high diagnostic yield. In the SPG7 group, we identified novel variants in two patients. We have also identified two novel mutations in the SPAST group. We present sequencing data from cDNA and RT-qPCR to support the pathogenicity of these variants, and provide observations regarding the poor genotypephenotype correlation in these conditions that should be the subject of future study
    Three-Dimensional Printed Polylactic Acid Scaffolds Promote Bonelike Matrix Deposition in Vitro
    Rayan Fairag, ACS Applied Materials & Interfaces - 2019
    Abstract
    Large bone defects represent a significant challenge for clinicians and surgeons. Tissue engineering for bone regeneration represents an innovative solution for this dilemma and may yield attractive alternate bone substitutes. Three-dimensional (3D) printing with inexpensive desktop printers shows promise in generating high-resolution structures mimicking native tissues using biocompatible, biodegradable, and cost-effective thermoplastics, which are already FDA-approved for food use, drug delivery, and many medical devices. Microporous 3D-printed polylactic acid scaffolds, with different pore sizes (500, 750, and 1000 μm), were designed and manufactured using an inexpensive desktop 3D printer, and the mechanical properties were assessed. The scaffolds were compared for cell growth, activity, and bone-like tissue formation using primary human osteoblasts. Osteoblasts showed high proliferation, metabolic activity, and osteogenic matrix protein production, in which 750 μm pore-size scaffolds showed superiority. Further experimentation using human mesenchymal stem cells on 750 μm pore scaffolds showed their ability in supporting osteogenic differentiation. These findings suggest that even in the absence of any surface modifications, low-cost 750 μm pore-size 3D-printed scaffolds may be suitable as a bone substitute for repair of large bone defects.
    Biallelic CCM3 mutations cause a clonogenic survival advantage and endothelial cell stiffening
    Konrad Schwefel, Journal of Cellular and Molecular Medicine - 2019
    Abstract
    CCM3, originally described as PDCD10, regulates blood‐brain barrier integrity and vascular maturation in vivo. CCM3 loss‐of‐function variants predispose to cerebral cavernous malformations (CCM). Using CRISPR/Cas9 genome editing, we here present a model which mimics complete CCM3 inactivation in cavernous endothelial cells (ECs) of heterozygous mutation carriers. Notably, we established a viral‐ and plasmid‐free crRNA:tracrRNA:Cas9 ribonucleoprotein approach to introduce homozygous or compound heterozygous loss‐of‐function CCM3 variants into human ECs and studied the molecular and functional effects of long‐term CCM3 inactivation. Induction of apoptosis, sprouting, migration, network and spheroid formation were significantly impaired upon prolonged CCM3 deficiency. Real‐time deformability cytometry demonstrated that loss of CCM3 induces profound changes in cell morphology and mechanics: CCM3‐deficient ECs have an increased cell area and elastic modulus. Small RNA profiling disclosed that CCM3 modulates the expression of miRNAs that are associated with endothelial ageing. In conclusion, the use of CRISPR/Cas9 genome editing provides new insight into the consequences of long‐term CCM3 inactivation in human ECs and supports the hypothesis that clonal expansion of CCM3‐deficient dysfunctional ECs contributes to CCM formation.
    S-adenosyl methionine prevents ASD like behaviors triggered by earlypostnatal valproic acid exposure in very young mice
    asher Ornoy, Neurotoxicology and Terattology - 2019
    Abstract
    Introduction:A common animal model of ASD is the one induced by valproic acid (VPA), inducing epigeneticchanges and oxidative stress. We studied the possible preventive effect of the methyl donor for epigenetic en-zymatic reactions, S-adenosine methionine (SAM), on ASD like behavioral changes and on redox potential in thebrain and liver in this model.Methods:ICR albino mice were injected on postnatal day 4 with one dose of 300 mg/kg of VPA, with normalsaline (controls) or with VPA and SAM that was given orally for 3 days at the dose of 30 mg/kg body weight.From day 50, we carried out neurobehavioral tests and assessment of the antioxidant status of the prefrontalcerebral cortex, liver assessing SOD and CAT activity, lipid peroxidation and the expression of antioxidant genes.Results:Mice injected with VPA exhibited neurobehavioral deficits typical of ASD that were more prominent inmales. Changes in the activity of SOD and CAT increased lipid peroxidation and changes in the expression ofantioxidant genes were observed in the prefrontal cortex of VPA treated mice, more prominent in females, whileASD like behavior was more prominent in males. There were no changes in the redox potential of the liver. Theco-administration of VPA and SAM alleviated most ASD like neurobehavioral symptoms and normalized theredox potential in the prefrontal cortex.Conclusions:Early postnatal VPA administration induces ASD like behavior that is more severe in males, whilethe redox status changes are more severe in females; SAM corrects both. VPA-induced ASD seems to result fromepigenetic changes, while the redox status changes may be secondary.
    Human-based fibrillar nanocomposite hydrogels as bioinstructive matrices to tune stem cell behavior
    Bárbara B. Mendes, Nanoscale - 2018
    Abstract
    The extracellular matrix (ECM)-biomimetic fibrillar structure of platelet lysate (PL) gels along with their enriched milieu of biomolecules has drawn significant interest in regenerative medicine applications. However, PL-based gels have poor structural stability, which severely limits their performance as a bioinstructive biomaterial. Here, rod-shaped cellulose nanocrystals (CNC) are used as a novel approach to modulate the physical and biochemical microenvironment of PL gels enabling their effective use as injectable human-based cell scaffolds with a level of biomimicry that is difficult to recreate with synthetic biomaterials. The incorporation of CNC (0 to 0.61 wt%) into the PL fibrillar network during the coagulation cascade leads to decreased fiber branching, increased interfiber porosity (from 66 to 83%) and modulates fiber (from 1.4 ± 0.7 to 27 ± 12 kPa) and bulk hydrogel (from 18 ± 4 to 1256 ± 82 Pa) mechanical properties. As a result of these physicochemical alterations, nanocomposite PL hydrogels resist the typical extensive clot retraction (from 76 ± 1 to 24 ± 3 at day 7) and show favored retention of PL bioactive molecules. The feedback of these cues on the fate of human adipose-derived stem cells is evaluated, showing how it can be explored to modulate the commitment of encapsulated stem cells toward different genetic phenotypes without the need for additional external biological stimuli. These fibrillar nanocomposite hydrogels allow therefore the exploration of the outstanding biological properties of human-based PL as an efficient engineered ECM which can be tailored to trigger specific regenerative pathways in minimal invasive strategies.
    Maternal malnourishment induced upregulation of fetuin-B blunts nephrogenesis in the low birth weight neonate
    May M. Rabadi, Developmental Biology - 2018
    Abstract
    Maternal undernutrition during pregnancy (MUN) often leads to low birth weight (LBW) neonates that have a reduced total nephron endowment, leaving these neonates susceptible to kidney disease throughout their lives. For reasons unknown, these LBW neonates have impaired kidney development due to a severe reduction in renal SIX2+ stem cells during nephrogenesis. Using a mouse model of MUN, we investigated SIX2+ stem cell reduction in the LBW neonate. Significant upregulation of the protein fetuin-B (measured by PCR and immunoblotting) in the MUN mother's placenta, organs and circulation yielded a 3-fold increase of this protein in the embryonic kidney. Recombinant fetuin-B, administered to healthy pregnant mothers at the concentration equivalent to that in the MUN mother, crossed the placenta and reduced both SIX2+ stem cells by 50% and nephron formation by 66% in embryonic kidneys (measured by immunofluorescence and the physical dissector/fractionator stereological method). Administration of fetuin-B to kidney explants yielded similar reductions in renal SIX2+ stem cells and nephron formation. Fetuin-B treatment of isolated embryonic renal SIX2+ stem cell primary cultures 1) increased NF-kB activity and apoptosis, 2) reduced cell proliferation due to upregulated p21 nuclear activity and subsequent cell cycle arrest, and 3) enhanced generation of reactive oxygen species (measured by fluorescence microscopy). In conclusion, MUN increases fetuin-B in the developing embryonic kidney. The increase in fetuin-B blunts nephrogenesis by reducing SIX2+ stem cells by promoting their apoptosis (via NF-kB upregulation), blunting their proliferative renewal (via p21 upregulation) and enhancing oxidative stress.
    Polyunsaturated Fatty Acids Induce ROS Synthesis in Microvascular Endothelial Cells
    Simon Trommer, Oxygen Transport to Tissue XL - 2018
    Abstract
    In sepsis, endothelial dysfunction is a crucial driver known to limit the survival rate of affected patients. For this, ROS-mediated signaling plays an important role in endothelial communication and functionality. In the management of sepsis, polyunsaturated fatty acids (PUFA) have received increasing attention regarding their anti-inflammatory potential neglecting the oxidative properties of these substances. Therefore, in the present study we examined the capacity of PUFA to interfere with the expression of major ROS-producing enzymes, as well as endothelial ROS production itself. The human microvascular endothelial cells TIME (ATCC number: CRL-4025) were used. Cells were cultured in medium enriched with LNA (C18:3n3), EPA (C20:5n3), DHA (C22:6n3), LA (C18:2n6), or AA (C20:4n6) in concentrations of 15 μM totaling 144 h. Stimulation of cells was performed in the last 24 h of fatty acid supplementation by addition of the cytokines TNF-α + IL-1β + IFN-γ (5 ng/ml each). Gene expression of eNOS, COX-2, and NOX-4 was evaluated by qPCR. ROS synthesis was analyzed by means of a flow cytometry-based rhodamine 123 assay. Cytokine stimulation was found to differentially affect gene expression of major ROS synthesizing enzymes: eNOS was decreased whereas COX-2 and NOX-4 were increased. As a consequence, cytokine stimulation had no effect on rhodamine accumulation in endothelial cells. PUFA supplementation alone did not affect the gene expression of eNOS, COX-2, and NOX-4. Nevertheless, an increasing action of PUFA on the stimulation-induced reduction in eNOS expression was found. More importantly, the number of rhodamine positive endothelial cells almost doubled following enrichment with the PUFA EPA, DHA or AA. This effect was independent of the stimulation status of the cells but seemed to be related to the number of double bonds of a supplemented fatty acid. Our data warrant further studies to ensure that increased endothelial cell oxidative stress is not boosted by PUFA in septic patients.
    Detection of coliphages and human adenoviruses in a subtropical estuarine lake
    Emily M. Cooksey, Science of The Total Environment - 2018
    Abstract
    Fecal indicator bacteria (FIB) have been used to assess fecal contamination in recreational water. However, enteric viruses have been shown to be more persistent in the environment and resistant to wastewater treatment than bacteria. Recently, U.S Environmental Protection Agency has proposed the use of coliphages as viral indicators to better protect against viral waterborne outbreaks. This study aimed to detect and determine correlation between coliphages (F-specific and somatic), fecal indicator bacteria (enterococci and fecal coliforms), and human enteric viruses (human adenovirus) in a subtropical brackish estuarine lake. Water samples were collected from 9 estuarine recreation sites on Lake Pontchartrain in southeast Louisiana. Water samples (n = 222, collected weekly) were analyzed for coliphages and fecal indicator bacteria using culture-based methods and large volume water samples (n = 54, collected monthly) were analyzed for human adenovirus using quantitative PCR. Somatic coliphage and F-specific coliphage were found in 93.7 and 65.2% of samples with geometric mean concentrations of 30 and 3 plaque forming units (PFU) per 100 mL, respectively. Enterococci, fecal coliforms, and adenovirus were found in all samples with geometric mean concentrations of 27 most probable number (MPN), 77 MPN, and 3.0 × 104 gene copies per 100 mL, respectively. Watersheds in suburban areas exhibited significantly higher concentrations of coliphages and fecal indicator bacteria, indicating potential fecal contamination from septic systems. There was no significant correlation (p > 0.05) observed between the presence of adenoviruses and fecal indicator bacteria and coliphages. The presence of human adenovirus in Lake Pontchartrain poses a significant public health problem for both recreational use and seafood harvesting as it increases exposure risks. This study demonstrated the lack of relationship between fecal indicators and human viral pathogen in Lake Pontchartrain supporting an alternative microbial surveillance system such as direct pathogen detection.
    Restoring Endothelial Function by Targeting Desert Hedgehog Downstream of Klf2 Improves Critical Limb Ischemia in Adults
    Caroline Caradu, Circulation Research - 2018
    Abstract
    Rationale: Klf2 is critical to establish and maintain endothelial integrity. Objective:Therefore, determining upstream and downstream mediators of Klf2 would lead to alternative therapeutic targets in cardiovascular disease management. Methods and Results: Here we identify Desert Hedgehog (Dhh) as a downstream effector of Klf2, whose expression in endothelial cells (ECs) is upregulated by shear stress and decreased by inflammatory cytokines. Consequently, we show that Dhh knock down in ECs promotes endothelial permeability and EC activation and that Dhh agonist prevents TNFα or glucose-induced EC dysfunction. Moreover, we demonstrate that human critical limb ischemia (CLI), a pathological condition linked to diabetes and inflammation, is associated to major EC dysfunction. By recreating a complex model of CLI in diabetic mice, we found that Dhh-signaling agonist significantly improved EC function without promoting angiogenesis, which subsequently improved muscle perfusion. Conclusions: Restoring EC functi...
    AAV8-mediated overexpression of mPCSK9 in liver differs between male and female mice
    Lemlem Brook, Journal of Cellular Biochemistry - 2018
    Abstract
    Glioblastoma (GBM) is the most common malignant brain tumor and is associated with a poor prognosis, with most patients living less than a year after diagnosis. Given that GBM nearly always recurs after conventional treatments, there is an urgent need to identify novel molecular targets. Hairless (HR) is a nuclear factor enriched in the skin and has been previously implicated in hair cycling. HR is also highly expressed in the brain, but its significance is unknown. We found that human hairless gene (HR) expression is significantly decreased in all GBM subtypes compared with normal brain tissue and is predictive of prognosis, which suggests that loss of HR expression can contribute to GBM pathogenesis. HR was recently discovered to bind to and regulate p53 responsive elements, and thus we hypothesized that HR may have a tumor suppressive function in GBM by modulating p53 target gene expression. We found that HR indeed regulates p53 target genes, including those implicated in cell cycle progression and apoptosis in the GBM-derived U87 cell line, and restoring HR expression triggered G2/M arrest and apoptosis. An analysis of sequenced genomes from patients with GBM revealed 10 HR somatic mutations in patients with glioma, two of which are located in the histone demethylase domain of HR. These two mutations, P996S and K1004N, were reconstructed and found to have impaired p53 transactivating properties. Collectively, the results of our study suggest that HR has tumor suppressive functions in GBM, which may be clinically relevant and a potential avenue for therapeutic intervention.
    AAV8-mediated overexpression of mPCSK9 in liver differs between male and female mice
    Aimee E. Vozenilek, Atherosclerosis - 2018
    Abstract
    Abstrat Background and aims The recombinant adeno-associated viral vector serotype 8 expressing the gain-of-function mutation of mouse proprotein convertase subtilisin/kexin type 9 (AAV8- PCSK9) is a new model for the induction of hypercholesterolemia. AAV8 preferentially infects hepatocytes and the incorporated liver-specific promoter should ensure expression of PCSK9 in the liver. Since tissue distribution of AAVs can differ between male and female mice, we investigated the differences in PCSK9 expression and hypercholesterolemia development between male and female mice using the AAV8-PCSK9 model. Methods Male and female C57BL/6 mice were injected with either a low-dose or high-dose of AAV8-PCSK9 and fed a high-fat diet. Plasma lipid levels were evaluated as a measure of the induction of hypercholesterolemia. Results Injection of mice with low dose AAV8-PCSK9 dramatically elevated both serum PCSK9 and cholesterol levels in male but not female mice. Increasing the dose of AAV8-PCSK9 threefold in female mice rescued the hypercholesterolemia phenotype but did not result in full restoration of AAV8-PCSK9 transduction of livers in female mice compared to the low-dose male mice. Our data demonstrate female mice respond differently to AAV8-PCSK9 injection compared to male mice. Conclusions These differences do not hinder the use of female mice when AAV8-PCSK9 doses are taken into consideration. However, localization to and production of AAV8-PCSK9 in organs besides the liver in mice may introduce confounding factors into studies and should be considered during experimental design.
    Genetic diversity of Hepatitis C Virus in Pakistan using Next Generation Sequencing
    Sana Saleem, Journal of Clinical Virology - 2018
    Abstract
    Background In Pakistan, HCV disease is considered a major public health issue with about 10–17 million people suffering with this infection and rate is increasing every day without any hindrance. The currently available Pyrosequencing approach used to analyze complex viral genomes as it can determine minor variants. It is crucial to understand viral evolution and quasispecies diversity in complex viral strains. Objectives To assess genetic diversity in patients with HCV using Next Generation Sequencing (NGS) and compare nucleotide diversity of genotype 3a with respect to other genotypes. Study design Intra-host viral diversity of HCV was determined using NGS from 13 chronically HCV infected individuals. NGS of three different regions (E2 (HVR1), NS3 and NS5B) of HCV-3a allowed for a comprehensive analysis of the viral population. Result Phylogenetic analysis of different HCV genes revealed great variability within the Pakistani population. The average nucleotide diversity for HVR1, NS3 and NS5B was 0.029, 0.011 and 0.010 respectively. Conclusion Our findings clearly indicate that patient-2 greater quasispecies heterogeneity than other patients of same genotype-3a using phylogenetic and one step network analyses. Initially phylogenetic analysis of these three genes showed that genotype 3a samples have greater genetic diversity. However, no significant difference was determined when nucleotide variability of genotype 3a compared with other genotypes (1a, 1b, 2a & 4a).
    Sperm capacitation is associated with phosphorylation of the testis-specific radial spoke protein Rsph6a
    Bidur Paudel, Biology of Reproduction - 2018
    Abstract
    A sequence corresponding to the N-terminal domain of the radial spoke protein Rsph6 was found phosphorylated in capacitated sperm. Rsph6 expression is a testis-
    Localization of the 1,25-dihydroxyvitamin D-mediated response in the intestines of mice
    Carmen J. Reynolds, The Journal of Steroid Biochemistry and Molecular Biology - 2018
    Abstract
    1,25-Dihydroxyvitamin D3 (1,25(OH)2D) elicits a transcriptional response in the intestines. Assessments of this response are often derived from crude tissue homogenates and eliminate the ability to discriminate among different cell types. Here, we used an RNA in situ hybridization assay, RNAScope (Advanced Cell Diagnostics, Newark, CA), to identify the cells in the intestine that respond to 1,25(OH)2D with expression of cytochrome P450 family 24 subfamily A member 1 (Cyp24a1) mRNA. Mice were gavaged with a single bolus dose of 1,25(OH)2D to target the duodenum or a glucuronic acid conjugate of 1,25(OH)2D, β-G-1,25(OH)2D, to target the colon. QRT-PCR analysis of Cyp24a1 mRNA verified that the 1,25(OH)2D-induced responses were present. RNAScope revealed that the mRNA response present after six hours is limited to mature enterocytes exposed to the intestinal lumen in both the duodenum and colon. No detectable expression was observed in goblet cells, lamina propria, muscularis mucosa muscle, submucosa and submucosal lymphoid follicles, or tunica muscularis. Our findings have identified epithelial enterocytes to be the intestinal targets for 1,25(OH)2D in both the duodenum and colon.
    Loss-of-function mutations in CARD14 are associated with a severe variant of atopic dermatitis
    Alon Peled, Journal of Allergy and Clinical Immunology - 2018
    Abstract
    Background Atopic dermatitis (AD) is a highly prevalent chronic inflammatory skin disease which is known to be, at least in part, genetically determined. Mutations in CARD14 have been shown to result in various forms of psoriasis and related disorders. Objective We aimed to identify rare DNA variants conferring a significant risk for AD through genetic and functional studies in a cohort of patients affected with severe atopic dermatitis. Methods Whole exome and direct gene sequencing, immunohistochemistry, real-time PCR, ELISA and functional assays in human keratinocytes were used. Results In a cohort of individuals referred with severe atopic dermatitis, DNA sequencing revealed in 4 patients two rare heterozygous missense mutations in CARD14 encoding the Caspase Recruitment Domain-Containing Protein 14, a major regulator of NF-κB. A dual luciferase reporter assay demonstrated that both mutations exert a dominant loss-of-function effect and result in decreased NF-κB signaling. Accordingly, immunohistochemistry staining showed decreased expression of CARD14 in patient skin as well as decreased levels of activated p65, a surrogate marker for NF-κB activity. CARD14-deficient or mutant-expressing keratinocytes displayed abnormal secretion of key mediators of innate immunity. Conclusions While dominant gain-of-function mutations in CARD14 are associated with psoriasis and related diseases, loss-of-function mutations in the same gene are associated with a severe variant of atopic dermatitis.
    Tissue-specific gene regulation corresponds with seasonal plasticity in female testosterone
    Alexandra B. Bentz, General and Comparative Endocrinology - 2018
    Abstract
    Testosterone (T) is a sex steroid hormone that often varies seasonally and mediates trade-offs between territorial aggression and parental care. Prior work has provided key insights into the ‘top-down’ hypothalamic control of this seasonal plasticity in T, yet mechanisms acting outside of the brain may also influence circulating T levels. We hypothesized that peripheral mechanisms may be especially critical for females, because peripheral regulation may mitigate the costs of systemically elevated T. Here, we begin to test this hypothesis using a seasonal comparative approach, measuring gene expression in peripheral tissues in tree swallows (Tachycineta bicolor), a songbird with intense female-female competition and T-mediated aggression. We focused on the gonad and liver for their role in T production and metabolism, respectively, and we contrasted females captured during territory establishment versus incubation. During territory establishment, when T levels are highest, we found elevated gene expression of the hepatic steroid metabolizing enzyme CYP2C19 along with several ovarian steroidogenic enzymes, including the androgenic 5α-reductase. Despite these seasonal changes in gene expression along the steroidogenic pathway, we did not observe seasonal changes in sensitivity to upstream signals, measured as ovarian mRNA abundance of luteinizing hormone receptor. Together, these data suggest that differential regulation of steroidogenic gene expression in the ovary is a potentially major contributor to seasonal changes in T levels in females. Furthermore, these data provide a unique and organismal glimpse into tissue-specific gene regulation and its potential role in hormonal plasticity in females.
    Potent in vivo lung cancer Wnt signaling inhibition via cyclodextrin-LGK974 inclusion complexes
    Pedro P. G. Guimaraes, Journal of Controlled Release - 2018
    Abstract
    Activation of the Wnt signaling pathway promotes lung cancer progression and contributes to poor patient prognosis. The porcupine inhibitor LGK974, a novel orally bioavailable cancer therapeutic in Phase I clinical trials, induces potent Wnt inhibition leading to suppressed growth and progression of multiple types of cancers. The clinical use of LGK974, however, is limited in part due to its low solubility and high toxicity in tissues that rely on Wnt signaling for normal homeostasis. Here, we report the use of host-guest chemistry to enhance solubility and bioavailability of LGK974 in mice through complexation with cyclodextrins (CD). We assessed the effects of these complexes to inhibit Wnt signaling in lung adenocarcinomas that are typically driven by overactive Wnt signaling. 2D H1 NMR confirmed host-guest complexation of CDs with LGK974. CD:LGK974 complexes significantly decreased the expression of Wnt target genes both in vitro and in vivo. Further, CD:LGK974 complexes increased the bioavailability upon oral administration in mice compared to free LGK974. In a mouse lung cancer allograft model, CD:LGK974 complexes induced potent Wnt signaling inhibition with reduced intestinal toxicity compared to administration of free drug. Collectively, the development of these complexes enables safer and repeated oral or parenteral administration of porcupine inhibitors, which hold promise for the treatment of multiple types of malignancies.
    Alterations of EDEM1 functions enhance ATF6 pro-survival signaling
    Alexandra Papaioannou, The FEBS Journal - 2018
    Abstract
    Activating transcription factor 6 alpha (referred to as ATF6 hereafter) is an endoplasmic reticulum (ER)-resident glycoprotein and one of the 3 sensors of the unfolded protein response (UPR). Upon ER stress, ATF6 is exported to the Golgi complex where it is cleaved by the S1P and S2P proteases thus releasing ATF6 cytosolic fragment and leading to the transcription of ATF6 target genes. In this study, we performed a phenotypic small interfering RNA (siRNA) screening to better characterize the ER mechanisms involved in ATF6 activation upon ER stress. This revealed that silencing of ER-degradation enhancing alpha-mannosidase-like protein-1 (EDEM1) increased the bioavailability of ER stress-induced ATF6 export to the Golgi complex through the stabilization of the natively unstable ATF6 protein. Moreover, we characterized a somatic variant of EDEM1 (N198I) found in hepatocellular carcinoma that alters ATF6 signaling and might provide a selective advantage to the transforming cells. Hence, our work confirms the natively unstable nature of ATF6 and links this property to potentially associated pro-oncogenic functions. This article is protected by copyright. All rights reserved.
    Expression of a hyperthermophilic endoglucanase in hybrid poplar modifies the plant cell wall and enhances digestibility
    Yao Xiao, Biotechnology for Biofuels - 2018
    Abstract
    Expression of glycosyl hydrolases in lignocellulosic biomass has been proposed as an alternative to improve efficiency of cellulosic ethanol production. In planta production of hyperthermophilic hydrolytic enzymes could prevent the detrimental effects often seen resulting from the expression of recombinant mesophilic enzymes to plant hosts. Utilizing lignocellulosic feedstocks to produce hyperthermophilic hydrolases provides additional benefits for ethanol production in the way of transgenic feedstocks serving as both enzyme providers and cellulosic substrates.
    Endocrine-immune signaling as a predictor of survival: A prospective study in developing songbird chicks
    Emily E. Virgin, General and Comparative Endocrinology - 2018
    Abstract
    Immune function varies with an animal’s endocrine physiology and energy reserves, as well as its abiotic and biotic environment. This context-dependency is thought to relate to adaptive trade-off resolution that varies from one context to the next; however, it is less clear how state- and environmentally-dependent differences in endocrine-immune signaling relate to survival in natural populations. We begin to address this question in a prospective study on a free-living passerine bird, the tree swallow (Tachycineta bicolor), by capitalizing upon naturally-occurring variation in ectoparasitism in 12-day old chicks. We measured body mass, hematological gene expression of the pro-inflammatory cytokine interleukin-6 (IL-6) as well as corticosterone (CORT) secretion at baseline and in response to 30 min of handling. We found that chicks with ectoparasites had smaller body mass and higher levels of IL-6 gene expression at this critical stage of post-natal growth and development. Mass and IL-6 were positively correlated, but only among parasitized chicks, suggesting that larger chicks mount stronger immune responses when necessary, i.e. in the presence of ectoparasites that are known to induce inflammation. IL-6 mRNA expression was negatively correlated with stress-induced CORT levels, suggesting that this proxy of inflammation may be co-regulated with or coordinated by glucocorticoids. More importantly, these endocrine-immune parameters predicted survival to fledging, which was positively associated with IL-6 mRNA abundance and, to a lesser degree, CORT reactivity. These results suggest a link between endocrine-immune interactions and performance in nature, and as a consequence, they shed light on the potentially adaptive, context-dependent interplay between body mass, immunity, and endocrine physiology during development.
    time-ChIP: A Method to Determine Long-Term Locus-Specific Nucleosome Inheritance
    Wojciech Siwek, Histone Variants - 2018
    Abstract
    Understanding chromatin dynamics is essential to define the contribution of chromatin to heritable gene silencing and the long-term maintenance of gene expression. Here we present a detailed protocol for time-ChIP, a novel method to measure histone turnover at high resolution across long timescales. This method is based on the SNAP-tag, a self-labeling enzyme that can be pulse labeled with small molecules in cells. Upon pulse biotinylation of a cohort of SNAP-tagged histones we can determine their abundance and fate across a chase period using a biotin-specific chromatin pulldown followed by DNA sequencing or quantitative PCR. This method is unique in its ability to trace the long-term fate of a chromatin bound histone pool, genome wide. In addition to a step by step protocol, we outline advantages and limitations of the method in relation to other existing techniques. time-ChIP can define regions of high and low histone turnover and identify the location of pools of long lived histones.
    Exploiting the impact of the secretome of MSCs isolated from different tissue sources on neuronal differentiation and axonal growth
    Rita C. Assunção-Silva, Biochimie - 2018
    Abstract
    Cell transplantation free-based therapies using Mesenchymal stem cell (MSC) secretome have recently been presented as a possible for CNS related disorders. MSC secretome is rich in several bio-factors that act synergically towards the repair of damaged tissues, thus making it an ideal candidate for regenerative applications. Great effort is currently being made to map the molecules that compose the MSC secretome. Previous proteomic characterization of the secretome (in the form of conditioned media - CM) of MSCs derived from adipose tissue (ASC), bone-marrow (BMSC) and umbilical cord (HUCPVC) was performed by our group, where proteins relevant for neuroprotection, neurogenic, neurodifferentiation, axon guidance and growth functions were identified. Moreover, we have found significant differences among the expression of several molecules, which may indicate that their therapeutic outcome might be distinct. Having this in mind, in the present study, the neuroregulatory potential of ASC, BMSC and HUCPVC CM in promoting neurodifferentiation and axonal outgrowth was tested in vitro, using human telencephalon neuroprogenitor cells and dorsal root ganglion explants, respectively. The CM from the three MSC populations induced neuronal differentiation from human neural progenitor cells, as well as neurite outgrowth from dorsal root ganglion explants. Moreover, all the MSC populations promoted the same extent of neurodifferentiation, while ASC CM demonstrated higher potential in promoting axonal growth.
    Bifidobacterium pseudolongum in the Ceca of Rats Fed Hi-Maize Starch Has Characteristics of a Keystone Species in Bifidobacterial Blooms
    Manuela Centanni, Applied and Environmental Microbiology - 2018
    Abstract
    Starches resistant to mammalian digestion are present in foods and pass to the large bowel, where they may be degraded and fermented by the microbiota. Increases in relative abundances of bifidobacteria (blooms) have been reported in rats whose diet was supplemented with Hi-Maize resistant starch. We determined that the bifidobacterial species present in the rat cecum under these circumstances mostly belonged to Bifidobacterium animalis. However, cultures of B. animalis isolated from the rats failed to degrade Hi-Maize starch to any extent. In contrast, Bifidobacterium pseudolongum also detected in the rat microbiota had high starch-degrading ability. Transcriptional comparisons showed increased expression of a type 1 pullulanase, alpha-amylase, and glycogen debranching enzyme by B. pseudolongum when cultured in medium containing Hi-Maize starch. Maltose was released into the culture medium, and B. animalis cultures had shorter doubling times in maltose medium than did B. pseudolongum. Thus, B. pseudolongum, which was present at a consistently low abundance in the microbiota, but which has extensive enzymatic capacity to degrade resistant starch, showed the attributes of a keystone species associated with the bifidobacterial bloom. IMPORTANCE This study addresses the microbiology and function of a natural ecosystem (the rat gut) using DNA-based observations and in vitro experimentation. The microbial community of the large bowel of animals, including humans, has been studied extensively through the use of high-throughput DNA sequencing methods and advanced bioinformatics analysis. These studies reveal the compositions and genetic capacities of microbiotas but not the intricacies of how microbial communities function. Our work, combining DNA sequence analysis and laboratory experiments with cultured strains of bacteria, revealed that the increased abundance of bifidobacteria in the rat gut, induced by feeding indigestible starch, involved a species that cannot itself degrade the starch (Bifidobacterium animalis) but cohabits with a species that can (Bifidobacterium pseudolongum). B. pseudolongum has the characteristics of a keystone species in the community because it had low abundance but high ability to perform a critical function, the hydrolysis of resistant starch.
    Increased Alternative Splicing as a Host Response to Edwardsiella ictaluri Infection in Catfish
    Suxu Tan, Marine Biotechnology - 2018
    Abstract
    Alternative splicing is the process of generating multiple transcripts from a single pre-mRNA used by eukaryotes to regulate gene expression and increase proteomic complexity. Although alternative splicing profiles have been well studied in mammalian species, they have not been well studied in aquatic species, especially after biotic stresses. In the present study, genomic information and RNA-Seq datasets were utilized to characterize alternative splicing profiles and their induced changes after bacterial infection with Edwardsiella ictaluri in channel catfish (Ictalurus punctatus). A total of 27,476 alternative splicing events, derived from 9694 genes, were identified in channel catfish. Exon skipping was the most abundant while mutually exclusive exon was the least abundant type of alternative splicing. Alternative splicing was greatly induced by E. ictaluri infection with 21.9% increase in alternative splicing events. Interestingly, genes involved in RNA binding and RNA splicing themselves were significantly enriched in differentially alternatively spliced genes after infection. Sequence analyses of splice variants of a representative alternatively spliced gene, splicing factor srsf2, revealed that certain spliced transcripts may undergo nonsense-mediated decay (NMD), suggesting functional significance of the induced alternative splicing. Although statistical analysis was not possible with such large datasets, results from quantitative real-time PCR from representative differential alternative splicing events provided general validation of the bacterial infection-induced alternative splicing. This is the first comprehensive study of alternative splicing and its changes in response to bacterial infection in fish species, providing insights into the molecular mechanisms of host responses to biotic stresses.
    Segregation of dopamine and glutamate release sites in dopamine neuron axons: regulation by striatal target cells
    Guillaume M. Fortin, The FASEB Journal - 2018
    Abstract
    Dopamine (DA) is a key regulator of circuits controlling movement and motivation. A subset of midbrain DA neurons has been shown to express the vesicular glutamate transporter (VGLUT)2, underlying their capacity for glutamate release. Glutamate release is found mainly by DA neurons of the ventral tegmental area (VTA) and can be detected at terminals contacting ventral, but not dorsal, striatal neurons, suggesting the possibility that target-derived signals regulate the neurotransmitter phenotype of DA neurons. Whether glutamate can be released from the same terminals that release DA or from a special subset of axon terminals is unclear. Here, we provide in vitro and in vivo data supporting the hypothesis that DA and glutamate-releasing terminals in mice are mostly segregated and that striatal neurons regulate the cophenotype of midbrain DA neurons and the segregation of release sites. Our work unveils a fundamental feature of dual neurotransmission and plasticity of the DA system.—Fortin, G. M., Ducrot, C., Giguère, N., Kouwenhoven, W. M., Bourque, M.-J., Pacelli, C., Varaschin, R. K., Brill, M., Singh, S., Wiseman, P. W., Trudeau, L.-E. Segregation of dopamine and glutamate release sites in dopamine neuron axons: regulation by striatal target cells.
    Differentiation of the granulosa layer from hen prehierarchal follicles associated with follicle stimulating hormone receptor signaling
    Dongwon Kim, Molecular Reproduction and Development - 2018
    Abstract
    Recruitment of a single follicle into the preovulatory hierarchy of the domestic hen ovary occurs from a small cohort of prehierarchal follicles measuring 6-8 mm in diameter. We have previously reported that granulosa cells (GC) collected from prehierarchal follicles express highest levels of membrane-localized follicle-stimulating hormone receptor (FSHR) during follicle development, yet fail to initiate signaling via cAMP following short-term incubation with FSH. Consequently, GC from prehierarchal follicles remain in an undifferentiated state and lack the capacity for steroidogenesis due to a deficiency of cAMP-dependent STAR protein and CYP11A1 gene expression. The present studies investigate FSH responsiveness in GC before and after the transition from undifferentiated to a differentiated state at follicle recruitment. Prior to recruitment focus is directed towards the inhibition of FSHR signaling by β-ARRESTIN (βARR). Specifically, knockdown of βARR mRNA in cultured, undifferentiated GC using small interfering RNA (siRNA) facilitated FSH-induced cAMP formation, STAR expression and progesterone production. Furthermore, over-expression of bovine βARR1 and G PROTEIN-COUPLED RECEPTOR KINASE2 in actively differentiating GC significantly decreased cAMP accumulation and progesterone production following a challenge with FSH. We propose that a βARR-mediated mechanism maintains FSHR unresponsiveness in undifferentiated GC from prehierarchal follicles, and as a result prevents GC differentiation until the time of follicle recruitment. This article is protected by copyright. All rights reserved.
    Thymoquinone inhibits cell proliferation, migration, and invasion by regulating the elongation factor 2 kinase (eEF-2K) signaling axis in triple-negative breast cancer
    Nashwa Kabil, Breast Cancer Research and Treatment - 2018
    Abstract
    Background/purposeTriple-negative breast cancer (TNBC) is the most aggressive and chemoresistant subtype of breast cancer. Therefore, new molecular targets and treatments need to be developed to improve poor patient prognosis and survival. We have previously shown that eukaryotic elongation factor 2 kinase (eEF-2K) is highly expressed in TNBC cells, is associated with poor patient survival and prognosis, and promotes cell proliferation, migration, and invasion. In vivo targeting of eEF-2K significantly reduces the tumor growth of orthotopic TNBC xenograft mouse models, suggesting that eEF-2K may serve as a potential novel therapeutic target.Methods/resultsIn the current study, we identified thymoquinone (TQ), an active ingredient of Nigella sativa, as a potential safe and effective eEF-2K inhibitor in TNBC. We demonstrated for the first time that TQ inhibits the protein and mRNA expression of eEF-2K, as well as the clinically relevant downstream targets, including Src/FAK and Akt, and induces the tumor suppressor miR-603, in response to NF-kB inhibition. This effect was associated with a significant decrease in the proliferation, colony formation, migration, and invasion of TNBC cells. Furthermore, systemic in vivo injection of TQ (20 and 100 mg/kg) significantly reduced the growth of MDA-MB-231 tumors and inhibited the eEF-2K expression in an orthotopic tumor model in mice.ConclusionOur study provides first evidence that TQ treatment inhibits cell proliferation, migration/invasion, and tumor growth, in part through the inhibition of eEF-2K signaling in TNBC. Thus, our findings suggest that systemic TQ treatment may be used as a targeted therapeutic strategy for the inhibition of eEF-2K in TNBC tumor growth and progression.
    Exercise Preconditioning Diminishes Skeletal Muscle Atrophy after Hindlimb Suspension in Mice*
    Nicholas T. Theilen, Journal of Applied Physiology - 2018
    Abstract
    The aim of the present study was to investigate if short-term, concurrent exercise training prior to hindlimb suspension (HLS) prevents or diminishes both soleus and gastrocnemius atrophy and to analyze if changes in mitochondrial molecular markers were associated. Male C57BL/6 (WT) mice (12-14 weeks old) were assigned to control, 7-day HLS (HLS), 2-weeks exercise training prior to 7-day HLS (Ex+HLS), and 2-weeks exercise training (Ex) groups. HLS resulted in a 27.1% and 21.5% decrease in soleus and gastrocnemius muscle weight to bodyweight ratio, respectively, in WT mice. Exercise training prior to HLS resulted in a 5.6% and 8.1% decrease in soleus and gastrocnemius weight to bodyweight ratio, respectively. Exercise increased mitochondrial biogenesis and function associated markers, slow myosin heavy chain (SMHC) expression, and reduced the fiber-type transitioning marker myosin heavy chain 4 (Myh4). Ex+HLS revealed decreased reactive oxygen species (ROS) and oxidative stress compared to HLS. Our data indicated the time prior to an atrophic setting, particularly caused by muscle unloading, may be a useful period to intervene short-term, progressive exercise training to prevent skeletal muscle atrophy and is associated with mitochondrial biogenesis, function, and redox balance.
    In Silico Analyses of Rice Thionin genes and the Antimicrobial Activity of OsTHION15 against Phytopathogens
    Krissana Boonpa, Phytopathology - 2018
    Abstract
    Thionins are a family of antimicrobial peptides. We performed in silico expression analyses of the 44 rice (Oryza sativa L.) thionins (OsTHIONs). Modulated expression levels of OsTHIONs under different treatments suggest their involvement in many processes, including biotic, abiotic and nutritional stress responses, and in hormone signaling. OsTHION15 (LOC_Os06g32600) was selected for further characterization based on several in silico analyses. OsTHION15 in O. sativa L. ssp. indica ‘KDML 105’ was expressed in all of the tissues/organs examined, including germinating seeds, leaves and roots of seedlings and mature plants, and inflorescences. To investigate the antimicrobial activity of OsTHION15, we produced a recombinant peptide in Escherichia coli Rosetta-gami (DE3). The recombinant OsTHION15 exhibited inhibitory activities toward rice pathogenic bacteria, such as Xanthomonas oryzae pv. oryzae and Pectobacterium carotovorum pv. atroseptica, with minimum inhibitory concentrations of 112.6 and 14.1 µg ml-1, respectively. A significant hyphal growth inhibition was also observed towards Fusarium oxysporum ssp. cubense and Helminthosporium oryzae. In addition, we demonstrated the in planta antibacterial activity of this peptide in Nicotiana benthamiana against Xanthomonas campestris pv. glycines. These activities suggest the possible application of OsTHION15 in plant disease control.
    Pax-5 Inhibits NF-κB Activity in Breast Cancer Cells Through IKKε and miRNA-155 Effectors
    Jason Harquail, Journal of Mammary Gland Biology and Neoplasia - 2018
    Abstract
    Pax-5, an essential transcription factor in B cell development, is aberrantly expressed in various B cell cancer lesions and solid tumors such as breast carcinoma. We have recently shown that Pax-5 regulates NF-κB activity which lead to the modulation of breast cancer phenotypic features (EMT-MET). NF-κB is known as a central mediator in inflammation, stress response as well as being a gatekeeper of pro-tumorigenic activity. However, little is known as to how Pax-5 affects this modulation. We thus turned our attention to microRNAs as potential regulatory effectors. In this study, we set out to elucidate the regulatory network between differential Pax-5 expression and NF-κB activity which dictate breast cancer malignancy. Through next-generation sequencing (NGS) of breast cancer cells conditionally expressing Pax-5, we profile significantly upregulated microRNAs; including microRNA-155, a known regulator of pathological processes and suppressor of malignant growth. Through the conditional expression of microRNA-155 in breast cancer models, we identify and validate IKKε (IKBKE) as a downstream target and an essential effector of Pax-5-mediated suppression of NF-κB signaling. Using rescue experiments, we also confirm that Pax-5 modulates NF-κB activity via IKKε downregulation. Interestingly, we also show that microRNA-155, in turn, supresses Pax-5 expression, indicative of an auto-regulatory feedback loop. Altogether, we demonstrate that Pax-5 inhibits NF-κB signalling through the regulation of microRNA-155 and its downstream target IKKε. The elucidation of this signaling network is relevant as Pax-5 and NF-κB are potent transcriptional regulators of breast cancer aggressivity. In addition, IKKε is relevant oncogene aberrantly expressed in 30% of breast carcinomas. Further insight into the regulatory pathways of breast cancer progression will eventually identify strategic therapeutic and prognostic targets to improve cancer patient outcome.
    The effects of electronic cigarette vapor on placental trophoblast cell function: Short title : E-cigarette and trophoblast function
    Sergio Raez-Villanueva, Reproductive Toxicology - 2018
    Abstract
    Despite evidence that maternal smoking is associated with numerous adverse outcomes, 10-35% of women still smoke during pregnancy. Recently, many smokers have turned to electronic cigarettes (e-cigarettes) as a smoking cessation tool. However, there is considerable uncertainty regarding their safety for use during pregnancy. The goal of this study was to examine the effects of e-cigarette vapor on placental trophoblast function. HTR-8/SVneo cells were exposed to unflavored e-cigarette vapor-conditioned media with and without nicotine to assess cell viability, proliferation, migration (wound healing assay), invasion (transwell extracellular matrix invasion assay), and tube formation, a surrogate for angiogenesis. While there was no effect on cell viability, proliferation or migration (all p > 0.05), e-cigarette conditioned media significantly reduced trophoblast invasion and tube formation; these effects could not be solely attributed to the presence of nicotine. These results suggest that an evaluation of the safety of e-cigarette use during pregnancy is urgently required.
    Post-exposure effects of the piscicide 3-trifluoromethyl-4-nitrophenol (TFM) on the stress response and liver metabolic capacity in rainbow trout (Oncorhynchus mykiss)
    Oana Birceanu, PLOS ONE - 2018
    Abstract
    The piscicide 3-trifluoromethyl-4-nitrophenol (TFM) has been used to control invasive sea lamprey (Petromyzon marinus) populations in the Great Lakes for almost 60 years. Applied to rivers and streams containing larval lampreys, TFM seldom harms non-target fishes, but the effects of sub-lethal treatments on fish physiology are not well understood. We examined the effects of 9 h exposure to TFM on the stress axis and liver metabolic capacity of rainbow trout (Oncorhynchus mykiss) using in vivo and in vitro approaches. The fish that had been acutely exposed to TFM in vivo had increased plasma cortisol levels at 12 h post-treatment, but TFM exposure did not interfere with in vitro cortisol production in head kidney preparations. Subjecting trout to an acute handling stressor 12 h post-TFM exposure resulted in a relative attenuation of the plasma cortisol and glucose response compared to pre-stress levels. We conclude that routine TFM treatments can lead to elevations of plasma cortisol following exposure, plus a relative dampening of the stress response in rainbow trout, with high cortisol levels lasting at least 12 h post-treatment. Since the ability of the fish to produce cortisol and the liver metabolic capacity were not compromised following TFM exposure, it is likely that their ability to cope with other stressors is not altered in the long-term.
    Antimicrobial peptide expression in swine granulosa cells in response to lipopolysaccharide
    Xiaofeng Sun, Theriogenology - 2018
    Abstract
    Antimicrobial peptides (AMP) are host defense peptides present in all species examined. The objective of the current study was to characterize the expression of a group of antimicrobial peptides in ovarian cells, and to investigate their expression response to pathogen ligands. It was found that while PG1 transcript was not detected in the ovary, the expression of BD2 is the highest in small follicle derived granulosa cells (SGC), and its expression decreases during follicular development to large follicle stage (LGC; p < 0.05). The expression of BD2 in cumulus cells also decreased from GV to MII stage of oocyte maturation. ANG4 expression increased in granulosa cells during follicular development from SGC to LGC stage (p < 0.05), although no significant difference was observed in cumulus cells from different stages of oocyte maturation. We further examined AMP expression in follicle cells treated with different toll-like receptor (TLR) ligands which mimic pathogen exposure in the ovary. Of the four TLR ligands examined, lipopolysaccharide (LPS) exposure resulted in a 11.5 fold increase of BD2 expression, and a significant decrease of LYZ in LGC. A similar response pattern in BD2 and LYZ expression was also observed in SGC. These responses of AMP expression to LPS are associated with increased TLR4 signaling pathway component in mRNA and protein level, such as MyD88 and NFkB, and pro-inflammatory cytokines/chemokines, such as IL-6, TNFα and IL-8 (p < 0.05). Our data suggest that AMPs may play a role in innate defense as well as other physiological functions during ovarian follicular development and oocyte maturation.
    Effect of Tim23 Knockdown in vivo on Mitochondrial Protein Import and Retrograde Signaling to the UPRmt in Muscle
    Ashley N Oliveira, American Journal of Physiology-Cell Physiology - 2018
    Abstract
    The mitochondrial unfolded protein response (UPRmt) is a protein quality control mechanism that strives to achieve proteostasis in the face of misfolded proteins. Due to the reliance of mitochondria on both the nuclear and mitochondrial genomes, a perturbation of the coordination of these genomes results in a mito-nuclear imbalance in which holoenzymes are unable to assume mature stoichiometry and thereby activates the UPRmt. Thus, we sought to perturb this genomic coordination by using a systemic anti-sense oligonucleotide (in-vivo Morpholino) targeted to Tim23, the major channel of the inner membrane. This resulted in a 40% reduction in Tim23 protein content, a 32% decrease in matrix-destined protein import, and a trend to elevate ROS emission under maximal respiration conditions. This import defect activated the CHOP-branch of the UPRmt, as evident from increases in ClpP and cpn10, but not the ATF5 arm. Thus, in the face of proteotoxic stress, CHOP and ATF5 could be activated independently to regain proteostasis. Our second aim was to investigate the role of proteolytically-derived peptides in mediating retrograde signaling. Peptides released from the mitochondrion following basal proteolysis were isolated and incubated with import reactions. Dose- and time-dependent effect of peptides on protein import was observed. Our data suggest that mitochondrial proteolytic byproducts exert an inhibitory effect on protein import, possibly to reduce excessive protein import as a potential negative feedback mechanism. The inhibition of import into the organelle also serves a retrograde function, possibly via ROS emission, to modify nuclear gene expression and ultimately improve folding capacity.
    HISTONE DEACETYLASE 19 and the flowering time gene FD maintain reproductive meristem identity in an age-dependent manner
    Sasha R. Gorham, Journal of Experimental Botany - 2018
    Abstract
    The shoot apical meristem (SAM) undergoes developmental transitions that include a shift from vegetative to reproductive growth. This transition is triggered by flowering time genes, which up-regulate floral meristem (FM) identity genes that, in turn, control flower development by activating floral organ identity genes. This cascade of transcriptional activation is refined by repression mechanisms that temporally and spatially restrict gene expression to ensure proper development. Here, we demonstrate that HISTONE DEACETYLASE 19 (HDA19) maintains the identity of the reproductive SAM, or inflorescence meristem (IM), late in Arabidopsis thaliana development. At late stages of growth, hda19 IMs display a striking patterning defect characterized by ectopic expression of floral organ identity genes and the replacement of flowers with individual stamenoid organs. We further show that the flowering time gene FD has a specific function in this regulatory process, as fd hastens the emergence of these patterning defects in hda19 growth. Our work therefore identifies a new role for FD in reproductive patterning, as FD regulates IM function together with HDA19 in an age-dependent fashion. To effect these abnormalities, hda19 and fd may accentuate the weakening of transcriptional repression that occurs naturally with reproductive meristem proliferation.
    Rnd3/RhoE expression is regulated by G-actin through MKL1-SRF signaling pathway
    Léo Piquet, Experimental Cell Research - 2018
    Abstract
    Rnd3/RhoE is an atypical member of the Rho family of small GTPases, devoid of intrinsic GTP hydrolytic activity and a general modulator of important cellular processes such as migration and proliferation. Here, we show that Rnd3 is a target of the transcription factor SRF and its co-activator MKL1. The MKL1-SRF pathway assures the translation of physical forces into a transcriptional response. Rho GTPases can modulate the activity of this mechanotransduction pathway through actin cytoskeleton regulation, and many MKL1-SRF targets are involved in the regulation of actin. We found that Rnd3 expression is altered by G-actin signaling and sensitive to actin-targeting drugs and MKL1 mutants. We further characterized a consensus SRF binding site in the Rnd3 promoter. We found that MKL1-SRF modulation regulates Rnd3 promoter activity and Rnd3 expression can affect MKL1-SRF pathway activity in return. We demonstrated that this novel MKL1-SRF target is required in mechanosensitive mechanisms such as cell spreading and spheroid formation. Thus, Rnd3 is a MKL1-SRF target that plays a key role in the feedback loop described between the MKL1-SRF pathway and the organization of the actin cytoskeleton.
    Improved fatty acid profiles in seeds of Camelina sativa by artificial microRNA mediated FATB gene suppression
    Mehmet E. Ozseyhan, Biochemical and Biophysical Research Communications - 2018
    Abstract
    The fatty acid profile of plant oils determines their quality and uses. Saturated fatty acids are often not desirable from the standpoints of nutrition and some industrial applications. Camelina sativa is a re-emerged oilseed crop, however its oil needs to be improved to meet different application requirements. In this study, saturated fatty acids were greatly reduced by down-regulating genes encoding the fatty acyl-ACP thioesterases (FATB). An artificial microRNA (amiFATB) was created by replacing a microRNA sequence in the camelina Csa-miR159a gene with a FATB gene specific sequence. Seed-specific expression of amiFATB caused a 45% reduction of palmitic acid (16:0) and a 38% reduction of stearic acid (18:0) compared to wildtype seeds. The total saturated fatty acid content was decreased by 35% from 14.6% to 9.4% of total fatty acids. When amiFATB was expressed in a high-oleic acid transgenic line, it caused further increased oleic acid content. This work demonstrates that the FATB genes in camelina can be effectively knocked down by an artificial microRNA targeting gene-specific sequences, thus provides an additional tool to improve seed oils for desired properties.
    Mechanical load increase–induced changes in cytoskeletal structure and cellular barrier function in human cerebral endothelial cells
    Dongjoo Kim, Biotechnology and Bioengineering - 2018
    Abstract
    Globally, approximately a billion patients are estimated to suffer from neurological disorders. Although there are many therapeutic candidates for the central nervous system, treatment of brain disorders is restricted by the blood–brain barrier (BBB), which is a highly selective membrane that protects the brain from exogenous substances. This study was undertaken to develop a novel strategy to overcome the BBB and improve the efficiency of drug delivery to the brain by mechanical load increase using hypergravity. Human cerebral microvascular endothelial cells were exposed three times to 20 min hypergravity (10g), with a 20-min rest period between each exposure. The applied hypergravity reversibly decreased the cellular metabolic activity and increased the permeation rate of fluorescein sodium salt, fluorescein isothiocyanate–labeled dextran (FD-4), and fluorescein-labeled jacalin. Following the exposure to hypergravity, we also observed structural changes of the cytoskeleton and tight junctions, and an alteration in the expression levels of related genes. These results indicate that increased mechanical load due to the applied hypergravity affects the cytoskeletal arrangement and tight junctions, thereby weakening the cell barrier function and enhancing the permeability of the paracellular pathway. Thus, the mechanical load increase by hypergravity has the potential of being used as a novel strategy to overcome the BBB for brain drug delivery.
    The ATP-stimulated translocation promoter (ASTP) activity of glycerol kinase plays central role in adipogenesis
    Lilly S. Parr, Molecular Genetics and Metabolism - 2018
    Abstract
    Glycerol kinase (GK) is a multifunctional enzyme located at the interface of carbohydrate and fat metabolism. It contributes to both central carbon metabolism and adipogenesis; specifically, through its role as the ATP-stimulated translocation promoter (ASTP). GK overexpression leads to increased ASTP activity and increased fat storage in H4IIE cells. We performed metabolic flux analysis in human GK-overexpressing H4IIE cells and found that overexpressing cells had significantly altered fluxes through central carbon and lipid metabolism including increased flux through the pentose phosphate pathway and increased production of lipids. We also observed an equal contribution of glycerol to carbohydrate metabolism in all cell lines, suggesting that GK's alternate functions rather than its enzymatic function are important for these processes. To further elucidate the contributions of the enzymatic (phosphorylation) and alternative (ASTP) functions of GK in adipogenesis, we performed experiments on mammalian GK and E. coli GK. We determined that the ASTP function of GK (which is absent in E. coli GK) plays a greater role than the enzymatic activity in these processes. These studies further emphasize GK's diverse functionality and provides fundamental insights into the multiple protein functions of glycerol kinase.
    IL-36 and IL-1/IL-17 Drive Immunity to Oral Candidiasis via Parallel Mechanisms
    Akash H. Verma, The Journal of Immunology - 2018
    Abstract
    Protection against microbial infection by the induction of inflammation is a key function of the IL-1 superfamily, including both classical IL-1 and the new IL-36 cytokine families. Candida albicans is a frequent human fungal pathogen causing mucosal infections. Although the initiators and effectors important in protective host responses to C. albicans are well described, the key players in driving these responses remain poorly defined. Recent work has identified a central role played by IL-1 in inducing innate Type-17 immune responses to clear C. albicans infections. Despite this, lack of IL-1 signaling does not result in complete loss of immunity, indicating that there are other factors involved in mediating protection to this fungus. In this study, we identify IL-36 cytokines as a new player in these responses. We show that C. albicans infection of the oral mucosa induces the production of IL-36. As with IL-1α/β, induction of epithelial IL-36 depends on the hypha-associated peptide toxin Candidalysin. Epithelial IL-36 gene expression requires p38-MAPK/c-Fos, NF-κB, and PI3K signaling and is regulated by the MAPK phosphatase MKP1. Oral candidiasis in IL-36R−/− mice shows increased fungal burdens and reduced IL-23 gene expression, indicating a key role played by IL-36 and IL-23 in innate protective responses to this fungus. Strikingly, we observed no impact on gene expression of IL-17 or IL-17–dependent genes, indicating that this protection occurs via an alternative pathway to IL-1–driven immunity. Thus, IL-1 and IL-36 represent parallel epithelial cell–driven protective pathways in immunity to oral C. albicans infection.
    Molecular cloning and characterization of a sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) from Y-organs of the blue crab (Callinectes sapidus)
    Megan E. Roegner, Gene - 2018
    Abstract
    Existing data indicate that a Ca2+ signal stimulates ecdysteroid hormone production by crustacean molting glands (Y-organs). Ca2+ signaling is dependent on a tightly regulated Ca2+ gradient, with intracellular free Ca2+ maintained at a low basal level (typically sub-micromolar). This is achieved through the action of proteins intrinsic to the plasma membrane and the membranes of organelles. One such protein, the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), pumps Ca2+ from cytosol to the lumen of the endoplasmic reticulum. As a step toward understanding Ca2+-mediated regulation of ecdysteroidogenesis, we have begun investigating Ca2+ transport proteins in Y-organs. In studies reported here, we used a PCR-based strategy to clone from Y-organs of the blue crab (Callinectes sapidus) a cDNA encoding a putative SERCA protein. The cloned Cas-SERCA cDNA (3806 bp) includes a 3057-bp open reading frame that encodes a 1019-residue protein (Cas-SERCA). The conceptually translated protein has a predicted molecular mass of 111.42 × 103 and contains all signature domains of an authentic SERCA, including ten transmembrane domains and a phosphorylation site at aspartate 351. A homology model of Cas-SERCA closely resembles models of related SERCA proteins. Phylogenetic analysis shows Cas-SERCA clusters with SERCA proteins from other arthropods. An assessment of tissue distribution indicates the Cas-SERCA transcript is widely distributed across tissues. Studies using quantitative PCR showed Cas-SERCA transcript abundance increased significantly in Y-organs activated by eyestalk ablation, a pattern consistent with the hypothesis that Cas-SERCA functions to maintain Ca2+ homeostasis in Y-organs.
    Accelerated neural differentiation of mouse embryonic stem cells on aligned GYIGSR-functionalized nanofibers
    Elena A. Silantyeva, Acta Biomaterialia - 2018
    Abstract
    Substrates for embryonic stem cell culture are typified by poorly defined xenogenic, whole proteins or cellular components that are difficult and expensive to generate, characterize, and recapitulate. Herein, the generation of well-defined scaffolds of Gly-Tyr-Ile-Gly-Ser-Arg (GYIGSR) peptide-functionalized poly(ε-caprolactone) (PCL) aligned nanofibers are used to accelerate the neural lineage commitment and differentiation of D3 mouse embryonic stem cells (mESCs). Gene expression trends and immunocytochemistry analysis were similar to laminin-coated glass, and indicated an earlier differentiation progression than D3 mESCs on laminin. Further, GYIGSR-functionalized nanofiber substrates yielded an increased gene expression of Sox1, a neural progenitor cell marker, and Tubb3, Cdh2, Syp, neuronal cell markers, at early time points. In addition, guidance of neurites was found to parallel the fiber direction. We demonstrate the fabrication of a well-defined, xeno-free functional nanofiber scaffold and demonstrates its use as a surrogate for xenogenic and complex matrixes currently used for the neural differentiation of stem cells ex vivo. Statement of Significance In this paper, we report the use of GYIGSR-functionalized poly(ε-caprolactone) aligned nanofibers as a tool to accelerate the neural lineage commitment and differentiation of D3 mouse embryonic stem cells. The results indicate that functional nanofiber substrates promote faster differentiation than laminin coated substrates. The data suggest that aligned nanofibers and post-electrospinning surface modification with bioactive species can be combined to produce translationally relevant xeno-free substrates for stem cell therapy. Future development efforts are focused on additional bioactive species that are able to function as surrogates for other xenogenic factors found in differentiation media.
    Leaderless mRNAs are circularized in Chlamydomonas reinhardtii mitochondria
    A. Bruce Cahoon, Current Genetics - 2018
    Abstract
    The mitochondrial genome of Chlamydomonas reinhardtii encodes eight protein coding genes transcribed on two polycistronic primary transcripts. The mRNAs are endonucleolytically cleaved from these transcripts directly upstream of their AUG start codons, creating leaderless mRNAs with 3′ untranslated regions (UTR) comprised of most or all of their downstream intergenic regions. In this report, we provide evidence that these processed linear mRNAs are circularized, which places the 3′ UTR upstream of the 5′ start codon, creating a leader sequence ex post facto. The circular mRNAs were found to be ribosome associate by polysome profiling experiments suggesting they are translated. Sequencing of the 3′–5′ junctions of the circularized mRNAs found the intra-molecular ligations occurred between fully processed 5′ ends (the start AUG) and a variable 3′ terminus. For five genes (cob, cox, nd2, nd4, and nd6), some of the 3′ ends maintained an oligonucleotide addition during ligation, and for two of them, cob and nd6, these 3′ termini were the most commonly recovered sequence. Previous reports have shown that after cleavage, three untemplated oligonucleotide additions may occur on the 3′ termini of these mRNAs—adenylation, uridylylation, or cytidylation. These results suggest oligo(U) and oligo(C) additions may be part of the maturation process since they are maintained in the circular mRNAs. Circular RNAs occur in organisms across the biological spectrum, but their purpose in some systems, such as organelles (mitochondria and chloroplasts) is unclear. We hypothesize, that in C. reinhardtii mitochondria it may create a leader sequence to facilitate translation initiation, which may negate the need for an alternative translation initiation mechanism in this system, as previously speculated. In addition, circularization may play a protective role against exonucleases, and/or increase translational productivity.
    The Yeast Three-Hybrid System for Screening RNA-Binding Proteins in Plants
    Sung Ki Cho, Two-Hybrid Systems - 2018
    Abstract
    Yeast-hybrid methods have been successfully applied for screening interacting partners of DNAs or proteins. A yeast-based method, the yeast three-hybrid system, using a chimeric protein of a DNA-binding domain (LexA or GAL4BD) with a protein (MS2 coat protein or HIV Rev. M10) having a hybrid RNA at the 3′ end of a target RNA sequence, has been developed for screening RNA-binding proteins. When the target RNA interacts with RNA-binding proteins fused with an activation domain (AD), yeast cells having all the interacting components can survive on selection media, and interacting reporters, HIS3 and LacZ, are activated. Based on this selection, interaction can be easily monitored and detected by simple biochemical assays. The in vivo screening strategy has been widely applied for characterizing and evaluating specific interactions between target RNAs and RNA-binding proteins. Here, we describe a library screening strategy for isolating RNA-binding proteins of select target RNAs using the yeast three-hybrid method. We also describe strategies to verify binding specificity using both a yeast-dependent reporter system and a yeast-independent method, in vivo RNA immunoprecipitation (RIP).
    PAK1 regulates ATXN1 levels providing an opportunity to modify its toxicity in spinocerebellar ataxia type 1
    Vitaliy V. Bondar, Human Molecular Genetics - 2018
    Abstract
    Spinocerebellar ataxia type 1 (SCA1) is caused by the expansion of a trinucleotide repeat that encodes a polyglutamine tract in ataxin-1 (ATXN1). The expanded polyglutamine in ATXN1 increases the protein’s stability and results in its accumulation and toxicity. Previous studies have demonstrated that decreasing ATXN1 levels ameliorates SCA1 phenotypes and pathology in mouse models. We rationalized that reducing ATXN1 levels through pharmacological inhibition of its modulators could provide a therapeutic avenue for SCA1. Here, through a forward genetic screen in Drosophila we identified, p21-activated kinase 3 (Pak3) as a modulator of ATXN1 levels. Loss-of-function of fly Pak3 or Pak1, whose mammalian homologs belong to Group I of PAK proteins, reduces ATXN1 levels, and accordingly, improves disease pathology in a Drosophila model of SCA1. Knockdown of PAK1 potently reduces ATXN1 levels in mammalian cells independent of the well-characterized S776 phosphorylation site (known to stabilize ATXN1) thus revealing a novel molecular pathway that regulates ATXN1 levels. Furthermore, pharmacological inhibition of PAKs decreases ATXN1 levels in a mouse model of SCA1. To explore the potential of using PAK inhibitors in combination therapy, we combined the pharmacological inhibition of PAK with MSK1, a previously identified modulator of ATXN1, and examined their effects on ATXN1 levels. We found that inhibition of both pathways results in an additive decrease in ATXN1 levels. Together, this study identifies PAK signaling as a distinct molecular pathway that regulates ATXN1 levels and presents a promising opportunity to pursue for developing potential therapeutics for SCA1.
    The distribution and detection of grapevine red blotch virus in its host depends on time of sampling and tissue type
    Felicia J Setiono, Plant Disease - 2018
    Abstract
    Grapevine red blotch virus (GRBV) is the causal agent of grapevine red blotch, an emerging disease that affects cultivated grapevine such as Vitis vinifera. The ability to detect viruses in grapevine is often hindered by low virus titers compounded by a variable distribution in the plant and seasonal variations. In order to examine these two variables in relation to GRBV, we developed a quantitative qPCR method that incorporates both internal and external references to enhance assay robustness. In greenhouse-grown vines infected with GRBV, qPCR identified highest virus titers in the petioles of fully expanded leaves and significantly reduced levels of virus in the shoot extremities. In vineyard-grown vines infected with GRBV, the virus titer in July and October 2016 followed a similar pattern to that found for the greenhouse-grown plants, but most strikingly close to half (44%) of the samples analyzed in June 2015 tested negative for infection. The technique presented and results obtained highlight the variability of virus distribution in its host and provide a useful guide for selecting the best tissues for optimal GRBV diagnosis.
    Porcine circovirus 2 infection induces IFNβ expression through increased expression of genes involved in RIG-I and IRF7 signaling pathways
    Cheryl M. T.Dvorak, Virus Research - 2018
    Abstract
    Porcine circovirus-associated disease (PCVAD), caused by porcine circovirus 2 (PCV2), is characterized by a highly variable pathogenesis that is manifested by various disease syndromes and includes immune evasion. Hence, even though PCVAD is effectively controlled by vaccination, pigs and farms remain infected so that continued vaccination is necessary to control disease. We investigated the molecular interactions of PCV2 and its permissive VR1BL host cell for gene expression signatures that could provide insight into mechanisms leading towards disease. Molecular pathways involved in the innate immune response to PCV2 infection were examined to identify changes in gene expression associated with productive infection of VR1BL cells. RNA profiling from infected and uninfected cells showed that 139 genes were induced by infection and 43 genes were down-regulated, using a p value <0.05 and an absolute fold-change difference>2. A strong type 1 interferon response, including an increase in genes involved in the RIG-I/MDA5 pathway and downstream interferon induced genes, was observed. Key regulators involved in PCV2 infection were identified as IFNβ, DDX58 (RIG-I), and IRF7. PCV2 infection induces a strong interferon response which unexpectedly facilitates viral gene expression, perhaps due to the presence of an interferon-sensitive response element in the viral promoter. The findings suggest that PCV2 interventions that attenuate type 1 interferon responses at the cellular level might enhance immunity and eliminate persistent infection.
    Molecular network, pathway, and functional analysis of time-dependent gene changes related to cathepsin G exposure in neonatal rat cardiomyocytes
    Sanket Kumar Shukla, Gene - 2018
    Abstract
    The molecular pathways activated in response to acute cathepsin G (CG) exposure, as well as the mechanisms involved in activation of signaling pathways that culminate in myocyte detachment and apoptosis remain unclear. This study aimed to determine the changes in gene expression patterns associated with time dependent CG exposure to neonatal rat cardiomyocytes (NRCMs). Microarray analysis revealed a total of 451, 572 and 1127 differentially expressed genes after CG exposure at 1, 4 and 8 h respectively. A total of 54 overlapped genes at each time point were mapped by Ingenuity Pathway Analysis (IPA). The top up-regulated genes included Hamp, SMAD6, NR4A1, FOSL2, ID3 and SLAMF7, and down-regulated genes included CYR61, GDF6, Olr640, Vom2r36, DUSP6 and MMP20. Our data suggest that there are multiple deregulated pathways associated with cardiomyocyte death after CG exposure, including JAK/Stat signaling, IL-9 signaling and Nur77 signaling. In addition, we also generated the molecular network of expressed gene and found most of the molecules were connected to ERK1/2, caspase, BCR (complex) and Cyclins. Our study reveals the ability to assess time-dependent changes in gene expression patterns in NRCMs associated with CG exposure. The global gene expression profiles may provide insight into the cellular mechanism that regulates CG dependent myocyte apoptosis. In future, the pathways important in CG response, as well as the genes found to be differentially expressed might represent the therapeutic targets for myocyte survival in heart failure.
    Treatment of limb wounds of horses with orf virus IL-10 and VEGF-E accelerates resolution of exuberant granulation tissue, but does not prevent its development
    Lyn M. Wise, PLOS ONE - 2018
    Abstract
    Bandaging of limb wounds in horses leads to formation of exuberant granulation tissue (EGT) that retards healing due to protracted inflammation, aberrant vascularisation and delayed epithelialisation. EGT is not observed if wounds are left undressed or when wounds are on the body. A previous study showed that short-term administration of proteins derived from orf virus dampened inflammation and promoted epithelialisation of open wounds in horses. Here, we investigated the impact of orf virus interleukin-10 and vascular endothelial growth factor-E on the development and resolution of EGT. Excisional wounds were created on the forelimb of four horses, and bandages were maintained until full healing to induce EGT formation. Matching body wounds were created to ensure EGT was limited to the limb, and to differentiate the effects of the viral proteins on normal healing and on EGT formation. Viral proteins or the hydrogel vehicle control were administered topically to site-matched wounds at day 1, with repeat administration at day 8. Wound healing and EGT formation were monitored macroscopically. Wound margin samples were harvested at 2, 7 and 14 days, and at full healing, with histology used to observe epithelialisation, immunofluorescence used to detect inflammatory cells, angiogenesis and cell death, and qPCR to measure expression of genes regulating inflammation and angiogenesis. Limb wounds developed EGT, and exhibited slower healing than body wounds. Viral protein treatment did not accelerate healing at either location nor limit EGT formation in limb wounds. Treatment of limb wounds did however increase epithelialisation and angiogenesis, without dampening inflammatory cell infiltration or gene expression. The healed wounds also had less occlusion and death of blood vessels and fewer epidermal rete ridges following viral protein treatment. These findings indicate that the viral protein treatment does not suppress wound inflammation or EGT formation, but does promote vascular and epidermal repair and EGT resolution.
    Association of residual feed intake with abundance of ruminal bacteria and biopolymer hydrolyzing enzyme activities during the peripartal period and early lactation in Holstein dairy cows
    Ahmed A. Elolimy, Journal of Animal Science and Biotechnology - 2018
    Abstract
    Residual feed intake (RFI) in dairy cattle typically calculated at peak lactation is a measure of feed efficiency independent of milk production level. The objective of this study was to evaluate differences in ruminal bacteria, biopolymer hydrolyzing enzyme activities, and overall performance between the most- and the least-efficient dairy cows during the peripartal period. Twenty multiparous Holstein dairy cows with daily ad libitum access to a total mixed ration from d − 10 to d 60 relative to the calving date were used. Cows were classified into most-efficient (i.e. with low RFI, n = 10) and least-efficient (i.e. with high RFI, n = 10) based on a linear regression model involving dry matter intake (DMI), fat-corrected milk (FCM), changes in body weight (BW), and metabolic BW.
    Different adaptive strategies in E. coli populations evolving under macronutrient limitation and metal ion limitation
    Omar M. Warsi, BMC Evolutionary Biology - 2018
    Abstract
    Adaptive responses to nutrient limitation involve mutations that increase the efficiency of usage or uptake of the limiting nutrient. However, starvation of different nutrients has contrasting effects on physiology, resulting in different evolutionary responses. Most studies performed to understand these evolutionary responses have focused only on macronutrient limitation. Hence our understanding of adaptation under limitation of other forms of nutrients is limited. In this study, we compared the evolutionary response in populations evolving under growth-limiting conditions for a macronutrient and a major cation.
    Cocoa procyanidins modulate transcriptional pathways linked to inflammation and metabolism in human dendritic cells
    Helene L. E. Midttun, Food & Function - 2018
    Abstract
    Foods rich in polyphenols such as procyanidins (PC) have been proposed to have anti-inflammatory properties, and we have previously reported inhibition of lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in human dendritic cells (DCs) by PC derived from cocoa. To explore the mechanistic basis of this inhibition, here we conducted transcriptomic analysis on DCs cultured with either LPS or LPS combined with oligomeric cocoa PC. Procyanidins suppressed a number of genes encoding cytokines and chemokines such as CXCL1, but also genes involved in the cGMP pathway such as GUCY1A3 (encoding guanylate cyclase soluble subunit alpha-3). Upregulated genes were involved in diverse metabolic pathways, but notably two of the four most upregulated genes (NMB, encoding neuromedin B and ADCY3, encoding adenyl cyclase type 3) were involved in the cAMP signalling pathway. Gene-set enrichment analysis demonstrated that upregulated gene pathways were primarily involved in nutrient transport, carbohydrate metabolism and lysosome function, whereas down-regulated gene pathways involved cell cycle, signal transduction and gene transcription, as well as immune function. qPCR analysis verified differential expression of GUCY1A3, ADCY3, NMB as well as a number of other genes, and marked suppression of LPS-induced CXCL1 and IL-23 protein secretion was also observed. Thus, our results confirm a marked anti-inflammatory effect of PC in human DCs, which may be related mechanistically to second-messenger function and metabolic activity. Our results provide a foundation to further investigate metabolic pathways altered by PC during intestinal inflammation, and further encourage investigation of the health-promoting potential of PC-rich functional foods.
    The Phenotypic Effects of Exosomes Secreted from Distinct Cellular Sources: a Comparative Study Based on miRNA Composition
    Scott Ferguson, The AAPS Journal - 2018
    Abstract
    Exosomes are nano-sized vesicles composed of lipids, proteins, and nucleic acids. Their molecular landscape is diverse, and exosomes derived from different cell types have distinct biological activities. Since exosomes are now being utilized as delivery vehicles for exogenous therapeutic cargoes, their intrinsic properties and biological effects must be understood. We performed miRNA profiling and found substantial differences in the miRNA landscape of prostate cancer (PC3) and human embryonic kidney (HEK) 293 exosomes with little correlation in abundance of common miRNAs (R2 = 0.16). Using a systems-level bioinformatics approach, the most abundant miRNAs in PC3 exosomes but not HEK exosomes were predicted to significantly modulate integrin signaling, with integrin-β3 loss inducing macrophage M2 polarization. PC3 but not HEK exosomes downregulated integrin-β3 expression levels by 70%. There was a dose-dependent polarization of RAW 264.7 macrophages toward an M2 phenotype when treated with PC3-derived exosomes but not HEK-derived exosomes. Conversely, HEK exosomes, widely utilized as delivery vehicles, were predicted to target cadherin signaling, with experimental validation showing a significant increase in the migratory potential of MCF7 breast cancer cells treated with HEK exosomes. Even widely utilized exosomes are unlikely to be inert, and their intrinsic activity ought to be assessed before therapeutic deployment.
    Characterization of Chlamydial Rho and the Role of Rho-Mediated Transcriptional Polarity during Interferon-gamma-mediated Tryptophan Limitation
    Scot P. Ouellette, Infection and Immunity - 2018
    Abstract
    As an obligate intracellular, developmentally regulated bacterium, Chlamydia is sensitive to amino acid fluctuations within its host cell. When human epithelial cells are treated with the cytokine interferon-γ (IFNγ), the tryptophan (trp)-degrading enzyme, indoleamine-2,3-dioxygenase, is induced. Chlamydiae within such cells are starved for trp and enter a state of so-called persistence. Chlamydia lacks the stringent response used by many eubacteria to respond to this stress. Unusually, chlamydial transcription is globally elevated during trp starvation with transcripts for trp-codon containing genes disproportionately increased. Yet, the presence of trp codons destabilized 3′ ends of transcripts in operons or large genes. We initially hypothesized that ribosome stalling on trp codons rendered the 3′ ends sensitive to ribonuclease activity. The half-life of chlamydial transcripts containing different numbers of trp codons was thus measured in untreated and IFNγ-treated infected cells to determine whether trp codons influenced the stability of transcripts. However, no effect of trp codon content was detected. Therefore, we investigated whether Rho-dependent transcription termination could play a role in mediating transcript instability. Rho is expressed as a mid-cycle gene product, interacts with itself as predicted, and is present in all chlamydial species. Inhibition of Rho via the Rho-specific antibiotic, bicyclomycin, as well as overexpression of Rho are detrimental to chlamydiae. Finally, when we measured transcript abundance 3′ to trp codons in the presence of bicyclomycin, we observed that transcript abundance increased. These data are the first to demonstrate the importance of Rho in Chlamydia and the role of Rho-dependent transcription polarity during persistence.
    Probiotics Strains Modulate Gut Microbiota and Lipid Metabolism in Mule Ducks
    Maxime Even, The Open Microbiology Journal - 2018
    Abstract
    Probiotics Strains Modulate Gut Microbiota and Lipid Metabolism in Mule Ducks
    The influence of oxygen and methane on nitrogen fixation in subarctic
    Martine A. R. Kox, AMB Express - 2018
    Abstract
    Biological nitrogen fixation is an important source of bioavailable nitrogen in Sphagnum dominated peatlands. Sphagnum mosses harbor a diverse microbiome including nitrogen-fixing and methane (CH4) oxidizing bacteria. The inhibitory effect of oxygen on microbial nitrogen fixation is documented for many bacteria. However, the role of nitrogen-fixing methanotrophs in nitrogen supply to Sphagnum peat mosses is not well explored. Here, we investigated the role of both oxygen and methane on nitrogen fixation in subarctic Sphagnum peat mosses. Five species of Sphagnum mosses were sampled from two mesotrophic and three oligotrophic sites within the Lakkasuo peatland in Orivesi, central Finland. Mosses were incubated under either ambient or low oxygen conditions in the presence or absence of methane. Stable isotope activity assays revealed considerable nitrogen-fixing and methane-assimilating rates at all sites (1.4 ± 0.2 µmol 15N–N2 g−1 DW day−1 and 12.0 ± 1.1 µmol 13C–CH4 g−1 DW day−1, respectively). Addition of methane did not stimulate incorporation of 15N-nitrogen into biomass, whereas oxygen depletion increased the activity of the nitrogen-fixing community. Analysis of the 16S rRNA genes at the bacterial community level showed a very diverse microbiome that was dominated by Alphaproteobacteria in all sites. Bona fide methane-oxidizing taxa were not very abundant (relative abundance less than 0.1%). Based on our results we conclude that methanotrophs did not contribute significantly to nitrogen fixation in the investigated peatlands.
    Orsay δ protein is required for non-lytic viral egress
    Wang Yuan, Journal of Virology - 2018
    Abstract
    Non-enveloped gastrointestinal viruses such as human rotavirus can exit infected cells from the apical surface without cell lysis. The mechanism of such non-lytic exit is poorly understood. The non-enveloped Orsay virus is an RNA virus infecting the intestine cells of the nematode Caenorhabditis elegans. Dye staining results suggested that Orsay exits from the intestine of infected worms in a non-lytic manner. Therefore, the Orsay-C. elegans system provides an excellent in vivo model to study viral exit. The Orsay genome encodes three proteins: RNA-dependent RNA polymerase, capsid protein (CP), and a nonstructural protein δ. δ can also be expressed as a structural CP-δ fusion. We generated an ATG-to-CTG mutant virus that had normal CP-δ fusion but could not produce free δ due to lack of the start codon. This mutant virus showed a viral exit defect without obvious phenotypes in other steps of viral infection, suggesting that δ is involved in viral exit. Ectopically expressed free δ localized near the apical membrane of intestine cells in C. elegans and co-localized with ACT-5, an intestine-specific actin that is a component of the terminal web. Orsay infection rearranged ACT-5 apical localization. Reduction of ACT-5 level via RNAi significantly exacerbated the viral exit defect of the δ mutant virus, suggesting that δ and ACT-5 functionally interact to promote Orsay exit. Together, these data support a model that the viral δ protein interacts with the actin network at the apical side of host intestine cells to mediate polarized, non-lytic egress of the Orsay virus. Importance An important step of the viral life cycle is how viruses exit from host cells to spread to other cells. Certain non-enveloped viruses can exit cultured cells in non-lytic ways, however, such non-lytic exit has not been demonstrated in vivo. In addition, it is not clear how such non-lytic exit is achieved mechanistically in vivo. Orsay is a non-enveloped RNA virus that infects the intestine cells of the nematode C. elegans. It is currently the only virus known to naturally infect C. elegans. Using this in vivo model, we show that the δ protein encoded by Orsay facilitates the non-lytic exit of the virus, possibly by interacting with host actin on the apical side of the worm intestine cells.
    The Alternative NF-κB Pathway in Regulatory T Cell Homeostasis and Suppressive Function
    Yenkel Grinberg-Bleyer, The Journal of Immunology - 2018
    Abstract
    CD4+Foxp3+ regulatory T cells (Tregs) are essential regulators of immune responses. Perturbation of Treg homeostasis or function can lead to uncontrolled inflammation and autoimmunity. Therefore, understanding the molecular mechanisms involved in Treg biology remains an active area of investigation. It has been shown previously that the NF-κB family of transcription factors, in particular, the canonical pathway subunits, c-Rel and p65, are crucial for the development, maintenance, and function of Tregs. However, the role of the alternative NF-κB pathway components, p100 and RelB, in Treg biology remains unclear. In this article, we show that conditional deletion of the p100 gene, nfkb2, in Tregs, resulted in massive inflammation because of impaired suppressive function of nfkb2-deficient Tregs. Surprisingly, mice lacking RelB in Tregs did not exhibit the same phenotype. Instead, deletion of both relb and nfkb2 rescued the inflammatory phenotype, demonstrating an essential role for p100 as an inhibitor of RelB in Tregs. Our data therefore illustrate a new role for the alternative NF-κB signaling pathway in Tregs that has implications for the understanding of molecular pathways driving tolerance and immunity.
    LasΔ5315 Effector Induces Extreme Starch Accumulation and Chlorosis as Ca. Liberibacter asiaticus Infection in Nicotiana benthamiana
    Marco Pitino, Frontiers in Plant Science - 2018
    Abstract
    Huanglongbing (HLB), a destructive plant bacterial disease, severely impedes worldwide citrus production. HLB is associated with a phloem-limited α-proteobacterium, Candidatus Liberibacter asiaticus (Las). Las infection causes yellow shoots and blotchy mottle on leaves and is associated with excessive starch accumulation. However, the mechanisms underlying the starch accumulation remain unknown. We previously showed that the Las5315mp effector induced callose deposition and cell death in Nicotiana benthamiana. In this study, we demonstrated that Las can experimentally infect N. benthamiana via dodder transmission. Furthermore, we revealed another key function of the Las5315 effector by demonstrating that transient expression of the truncated form of the effector, LasΔ5315, induced excessive starch accumulation by 6 fold after 8 dpi in N. benthamiana after removal of the chloroplast transit peptide from the Las5315mp. The induction mechanisms of LasΔ5315 in N. benthamiana were attributed to the up-regulation of ADP-glucose pyrophosphorylase, granule-bound starch synthase, soluble starch synthase, and starch branching enzyme for increasing starch production, and to the significant down-regulation of the starch degradation enzymes: alpha-glucosidase, alpha-amylase, and glycosyl hydrolase for decreasing starch degradation. This is the first report that Las can infect the model plant N. benthamiana. Using this model plant, we demonstrated that the LasΔ5315 effector caused the most prominent HLB symptoms, starch accumulation and chlorosis as Las infection in N. benthamiana. Altogether the Las 5315 effector is critical for Las pathogenesis, and therefore, an important target for interference.
    Development-related aberrations in Kv1.1 α-subunit exert disruptive effects on bioelectrical activities of neurons in a mouse model of fragile X syndrome
    Pingping Zhu, Progress in Neuro-Psychopharmacology and Biological Psychiatry - 2018
    Abstract
    Kv1.1, a Shaker homologue potassium channel, plays a critical role in homeostatic regulation of neuronal excitability. Aberrations in the functional properties of Kv1.1 have been implicated in several neurological disorders featured by neuronal hyperexcitability. Fragile X syndrome (FXS), the most common form of inherited mental retardation, is characterized by hyperexcitability in neural network and intrinsic membrane properties. The Kv1.1 channel provides an intriguing mechanistic candidate for FXS. We investigated the development-related expression pattern of the Kv1.1 α-subunit by using a Fmr1 knockout (KO) mouse model of FXS. Markedly decreased protein expression of Kv1.1 was found in neonatal and adult stages when compared to age-matched wild-type (WT) mice. Immunohistochemical investigations supported the delayed development-related increases in Kv1.1 expression, especially in CA3 pyramidal neurons. By applying a Kv1.1-specific blocker, dendrotoxin-κ (DTX-κ), we isolated the Kv1.1-mediated currents in the CA3 pyramidal neurons. The isolated DTX-κ-sensitive current of neurons from KO mice exhibited decreased amplitude, lower threshold of activation, and faster recovery from inactivation. The equivalent reduction in potassium current in the WT neurons following application of the appropriate amount of DTX-κ reproduced the enhanced firing abilities of KO neurons, suggesting the Kv1.1 channel as a critical contributor to the hyperexcitability of KO neurons. The role of Kv1.1 in controlling neuronal discharges was further supported by the parallel developmental trajectories of Kv1.1 expression, current amplitude, and discharge impacts, with a significant correlation between the amplitude of Kv1.1-mediated currents and Kv1.1-blocking-induced firing enhancement. These data suggest that the expression of the Kv1.1 α-subunit has a profound pathological relevance to hyperexcitability in FXS, as well as implications for normal development, maintenance, and control of neuronal activities.
    The effects of feeding mixed tocopherol oil on whole-blood respiratory burst and neutrophil immunometabolic-related gene expression in lactating dairy cows
    Y. Qu, Journal of Dairy Science - 2018
    Abstract
    The 4 major tocopherol isoforms differ in their biochemical reactivity and cellular effects due to basic chemical structural differences. Alpha-tocopherol has been well studied regarding effects on bovine polymorphonuclear leukocyte (PMN) function and its involvement in respiratory burst. However, no studies to date have identified the effects of supplementing a mixed tocopherol oil (Tmix) particularly enriched in non-α tocopherol isoforms (i.e., γ- and δ-isoforms) on fundamental immunometabolic changes in dairy cows. Therefore, the objectives of this study were to determine whether short-term feeding of vegetable oil–derived Tmix alters specific biomarkers of metabolism, whole-blood leukocyte populations, respiratory burst, immunometabolic-related gene expression of PMN, or gene expression of isolated PMN when challenged with lipopolysaccharides (LPS). Clinically healthy multiparous lactating Holstein cows (n = 12; 179 ± 17 d in milk, 40.65 ± 3.68 kg of milk yield) were fed Tmix (620 g/d) for 7 consecutive days. Jugular blood (EDTA anticoagulant) was collected from all cows on d 0 before treatment initiation and again on d 7 after Tmix feeding. Total stimulated respiratory burst activity (RBA) and leukocyte populations were assessed in whole blood, and tocopherol isoform concentrations, metabolites, and hormones were measured in plasma. For gene expression analysis, isolated PMN from cows before and after Tmix feeding were incubated with LPS at a final concentration of either 0.0 or 1.5 µg/mL. Feeding of Tmix for 7 d increased the concentrations of α- and γ-tocopherol. The Tmix did not alter plasma insulin but decreased cholesterol. The Tmix did not alter whole-blood RBA or the leukocyte populations. The LPS challenge increased the expression of proinflammatory genes TNFA and IL6. However, Tmix treatment did not alter the patterns of LPS-affected expression of genes (e.g., TNFA, ITGB2, PPARA, and RXRA) associated with the immune or metabolic response. In conclusion, short-term feeding of Tmix may have no negative effect on animal health as Tmix increased α- and γ-tocopherol concentrations in blood and did not impair whole-blood RBA or alter leukocyte populations. The data provide further support that the α- and γ-tocopherol isoforms do not interfere with normal immune or metabolic function.
    Na+/H+ Exchanger Isoform 1-Induced Osteopontin Expression Facilitates Cardiac Hypertrophy Through p90 Ribosomal S6 Kinase
    Nabeel Abdulrahman, Physiological Genomics - 2018
    Abstract
    Cardiovascular diseases are the leading cause of death worldwide. One in three cases of heart failure is due to dilated cardiomyopathy. The Na+/H+ exchanger isoform 1 (NHE1), a multifunctional protein and the key pH regulator in the heart, has been demonstrated to be increased in this condition. We have previously demonstrated that elevated NHE1 activity induced cardiac hypertrophy in vivo. Furthermore, the overexpression of active NHE1 elicited modulation of gene expression in cardiomyocytes including an upregulation of myocardial osteopontin (OPN) expression. To determine the role of OPN in inducing NHE1 mediated cardiomyocyte hypertrophy, a double transgenic mouse expressing active NHE1 and OPN knock-out were generated and assessed by echocardiography and the cardiac phenotype. Our studies showed that hearts expressing active NHE1 exhibited cardiac remodeling indicated by increased systolic and diastolic left ventricular internal diameter and increased ventricular volume. Moreover, these hearts demonstrated impaired function with decreased fractional shortening and ejection fraction. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) mRNA was upregulated, and there was an increase in heart cell cross sectional area confirming the cardiac hypertrophic effect. Moreover, NHE1 transgenic mice also showed increased collagen deposition, upregulation of CD44 and phosphorylation of p90 ribosomal s6 kinase (RSK), effects that were regressed in OPN knock-out mice. In conclusion, we developed an interesting comparative model of active NHE1 transgenic mouse lines which express a dilated hypertrophic phenotype expressing CD44 and phosphorylated RSK, effects which are regressed in absence of OPN.
    Inhibition of tomato fruit ripening by 1-MCP, wortmannin and hexanal is associated with a decrease in transcript levels of phospholipase D and other ripening related genes
    Mohd Sabri Pak Dek, Postharvest Biology and Technology - 2018
    Abstract
    Membrane deterioration is an inherent aspect of the advancement in senescence and loss in fruit quality during storage. Postharvest technologies used for extending shelf life and quality are targeted to reduce membrane damage through downregulating or blocking ethylene action. In this study, mature green tomato fruit were treated with inhibitors of ethylene receptor (ETR), phosphatidylinositol 3-kinase (PI3K) and phospholipase D (PLD), all recognized to be targets of regulation of fruit ripening. The inhibitors used included 1-methylcyclopropene (1-MCP, an ethylene receptor blocker), wortmannin (an inhibitor of PI3K), and hexanal (a PLD inhibitor). Fruit were treated at optimal levels of the inhibitors and were stored at 21 °C for 10 days. Color development was strongly delayed in wortmannin treated tomatoes just as in 1-MCP treated fruit; while, changes in respiration, firmness and ethylene evolution were very similar to that of control fruit. Hexanal delayed the initiation of these changes; while 1-MCP and wortmannin blocked the ripening process. Changes in expression levels of key genes involved in ethylene signalling, phosphoinositide metabolism, and lycopene synthesis that occurred in response to inhibitors, suggested potential roles for PI3K and PLD in ethylene signalling. Furthermore, fruit treated with all the three inhibitors showed a marked reduction in PLD transcript levels; suggesting that, regulation of PLD gene expression is a common critical regulatory point that regulates ripening. Lowered PLD levels may reduce membrane lipid catabolism and the generation of phosphatidic acid (PA), an intermediate in ethylene signalling regulation through downstream components.
    Inhibition of fibroblast growth factor 23 (FGF23) signaling rescues renal anemia
    Rafiou Agoro, The FASEB Journal - 2018
    Abstract
    Severe anemia and iron deficiency are common complications in chronic kidney disease. The cause of renal anemia is multifactorial and includes decreased erythropoietin (Epo) production, iron deficiency, and inflammation, and it is currently treated with injections of synthetic Epo. However, the use of recombinant Epo has several adverse effects. We previously reported that high fibroblast growth factor 23 (FGF23) levels in mice are associated with decreased red blood cell production, whereas genetic inactivation of Fgf23 results in expansion of the erythroid lineage. The present study is the first to show that high FGF23 levels in a mouse model of renal failure contribute to renal anemia, and inhibiting FGF23 signaling stimulates erythropoiesis and abolishes anemia and iron deficiency. Moreover, we show that inhibition of FGF23 signaling significantly decreases erythroid cell apoptosis and influences the commitment of hematopoietic stem cells toward the erythroid linage. Furthermore, we show that blocking FGF23 signaling attenuates inflammation, resulting in increased serum iron and ferritin levels. Our data clearly demonstrate that elevated FGF23 is a causative factor in the development of renal anemia and iron deficiency, and importantly, blocking FGF23 signaling represents a novel approach to stimulate erythropoiesis and possibly improve survival for millions of chronic kidney disease patients worldwide.—Agoro, R., Montagna, A., Goetz, R., Aligbe, O., Singh, G., Coe, L. M., Mohammadi, M., Rivella, S., Sitara, D. Inhibition of fibroblast growth factor 23 (FGF23) signaling rescues renal anemia.
    Blood Glutamate Scavenger as a novel neuroprotective treatment in spinal cord injury
    Yona Goldshmit, Journal of Neurotrauma - 2018
    Abstract
    Neurotrauma causes immediate elevation of extracellular glutamate levels, which creates excitotoxicity and facilitates inflammation, glial scar formation and consequently neuronal death. Finding factors that reduce the inflammatory response, glial scar formation and increase neuronal survival and neurite outgrowth, are of major importance for improving the outcome after spinal cord injury (SCI). In the present study, we evaluated a new treatment aiming to remove CNS glutamate into the systemic blood circulation by intravenous administration of blood glutamate scavengers (BGS) such as recombinant enzyme glutamate-oxaloacetate transaminase (rGOT1) and its co-substrate. In this study we induced in mice a spinal cord injury (hemisection), and one-hour post injury started administering BGS treatment for five consecutive days. The treatment reduced the expression levels of p-p38, which regulates apoptosis and increased the expression of p-Akt, which mediates cell survival. Moreover, this treatment decreased pro-inflammatory cytokine expression and microglia activation, reduced astrocytes’ reactivity and facilitated expression of radial glia markers such as Pax6 and nestin. BGS treatment increased the survival of neurons at lesion site and enabled axonal regeneration into the injury site. These effects were correlated with improved functional recovery of the left paretic hindlimb. Thus, early pharmacological intervention with BGS following SCI may be neuroprotective and create a pro-regenerative environment by modulating glia cell response. In light of our results, the availability of the method to remove excess glutamate from CNS without the need to deliver drugs across the blood-brain barrier (BBB) and with minimal or no adverse effects may provide a major therapeutic asset.
    Safety Analysis of Leishmania Vaccine Used in a Randomized Canine Vaccine/Immunotherapy Trial
    Mandy Larson, ASTMH - 2018
    Abstract
    In Leishmania infantum–endemic countries, controlling infection within dogs, the domestic reservoir, is critical to public health. There is a need for safe vaccines that prevent canine progression with disease and transmission to others. Protective vaccination against Leishmania requires mounting a strong, inflammatory, Type 1 response. Three commercially available canine vaccines on the global veterinary market use saponin or inflammatory antigen components (Letifend) as a strong pro-inflammatory adjuvant. There is very little information detailing safety of saponin as an adjuvant in field trials. Safety analyses for the use of vaccine as an immunotherapeutic in asymptomatically infected animals are completely lacking. Leishmania infantum, the causative agent of canine leishmaniasis, is enzootic within U.S. hunting hounds. We assessed the safety of LeishTec® after use in dogs from two different clinical states: 1) without clinical signs and tested negative on polymerase chain reaction and serology or 2) without clinical signs and positive for at least one Leishmania diagnostic test. Vaccine safety was assessed after all three vaccinations to quantify the number and severity of adverse events. Vaccinated animals had an adverse event rate of 3.09%, whereas placebo animals had 0.68%. Receiving vaccine was correlated with the occurrence of mild, site-specific, reactions. Occurrence of severe adverse events was not associated with having received vaccine. Infected, asymptomatic animals did not have a higher rate of adverse events. Use of vaccination is, therefore, likely to be safe in infected, asymptomatic animals.
    Altered DNA methylation is associated with aberrant gene expression in parenchymal but not airway fibroblasts isolated from individuals with COPD
    Rachel L. Clifford, Clinical Epigenetics - 2018
    Abstract
    Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease of the lungs that is currently the fourth leading cause of death worldwide. Genetic factors account for only a small amount of COPD risk, but epigenetic mechanisms, including DNA methylation, have the potential to mediate the interactions between an individual’s genetics and environmental exposure. DNA methylation is highly cell type-specific, and individual cell type studies of DNA methylation in COPD are sparse. Fibroblasts are present within the airway and parenchyma of the lung and contribute to the aberrant deposition of extracellular matrix in COPD. No assessment or comparison of genome-wide DNA methylation profiles in the airway and parenchymal fibroblasts from individuals with and without COPD has been undertaken. These data provide valuable insight into the molecular mechanisms contributing to COPD and the differing pathologies of small airways disease and emphysema in COPD.
    Polycyclic aromatic hydrocarbon exposure in seaside sparrows (Ammodramus maritimus) following the 2010 Deepwater Horizon oil spill
    Anna A. Perez-Umphrey, Science of The Total Environment - 2018
    Abstract
    The seaside sparrow (Ammodramus maritimus) is an abundant and permanent resident of coastal salt marshes impacted by the 2010 BP Deepwater Horizon oil spill. Such terrestrial species are often overlooked in the aftermath of marine spills, despite the potential for long-term oil exposure. We sampled the livers of seaside sparrows residing in oiled and unoiled sites from 2011 to 2014 and quantified expression of cytochrome p450 1A (CYP1A), a gene involved in the metabolism of polycyclic aromatic hydrocarbons (PAHs). In August 2011, CYP1A expression was markedly higher in birds from an oiled site compared to an unoiled site, but differences had disappeared by June 2012. In June 2013, CYP1A expression was elevated compared to 2012 levels on all sites, including those collected from sites that had not been directly oiled during the spill. This rise in CYP1A expression was possibly due to Hurricane Isaac, which made landfall near our sites between the 2012 and 2013 sampling periods. CYP1A expression was significantly attenuated again in June 2014. We also collected sediment samples from the same marshes for a total concentration analysis of PAHs. The PAH concentrations in sediment samples exhibited a similar pattern to the CYP1A data, supporting the link between marsh PAHs and bird CYP1A expression. These results indicate that contamination from marine oil spills can immediately extend to terrestrial ecosystems, and that storms, weather, or other factors may influence subsequent spatial and temporal oil exposure for several additional years.
    Steroid Receptor Coactivator-1 Knockdown Decreases Synaptic Plasticity and Impairs Spatial Memory in the Hippocampus of Mice
    Chen Bian, Neuroscience - 2018
    Abstract
    Steroids have been demonstrated to play profound roles in the regulation of hippocampal function by acting on their receptors, which need coactivators for their transcriptional activities. Previous studies have shown that steroid receptor coactivator-1 (SRC-1) is the predominant coactivator in the hippocampus, but its exact role and the underlying mechanisms remain unclear. In this study, we constructed SRC-1 RNA interference (RNAi) lentiviruses, injected them into the hippocampus of male mice, and then examined the changes in the expression of selected synaptic proteins, CA1 synapse density, postsynaptic density (PSD) thickness, and in vivo long-term potentiation (LTP). Spatial learning and memory behavior changes were investigated using the Morris water maze. We then transfected the lentiviruses into cultured hippocampal cells and examined the changes in synaptic protein and phospho-cyclic AMP response element-binding protein (pCREB) expression. The in vivo results showed that SRC-1 knockdown significantly decreased the expression of synaptic proteins and CA1 synapse density as well as PSD thickness; SRC-1 knockdown also significantly impaired in vivo LTP and disrupted spatial learning and memory. The in vitro results showed that while the expression of synaptic proteins was significantly decreased by SRC-1 knockdown, pCREB expression was also significantly decreased. The above results suggest a pivotal role of SRC-1 in the regulation of hippocampal synaptic plasticity and spatial learning and memory, strongly indicating SRC-1 may serve as a novel therapeutic target for hippocampus-dependent memory disorders.
    Inhibition of pMAPK14 Overcomes Resistance to Sorafenib in Hepatoma Cells with Hepatitis B Virus
    Dvora Witt-Kehati, Translational Oncology - 2018
    Abstract
    Hepatitis B virus (HBV) targets the liver and is a major driver for liver cancer. Clinical data suggest that HBV infection is associated with reduced response to treatment with the multi-kinase inhibitor sorafenib, the first available molecularly targeted anti-hepatocellular carcinoma (HCC) drug. Given that Raf is one of the major targets of sorafenib, we investigated the activation state of the Raf-Mek-Erk pathway in the presence of HBV and in response to sorafenib. Here we show that hepatoma cells with replicating HBV are less susceptible to sorafenib inhibitory effect as compared to cells in which HBV expression is suppressed. However, although HBV replication is associated with increased level of pErk, its blockade only modestly augments sorafenib effect. In contrast, the phosphorylated form of the pro-oncogenic Mitogen-Activated Protein Kinase 14 (pMAPK14), a protein kinase that was recently linked to sorafenib resistance, is induced in sorafenib-treated hepatoma cells in association with HBV X protein expression. Knocking down pMAPK14 results in augmentation of the therapeutic efficacy of sorafenib and largely alleviates resistance to sorafenib in the presence of HBV. Thus, this study suggests that HBV promotes HCC resistance to sorafenib. Combining pMAPK14 inhibitors with sorafenib may be beneficial in patients with HBV-associated HCC.
    Implant delivering hydroxychloroquine attenuates vaginal T lymphocyte activation and inflammation
    Yufei Chen, Journal of Controlled Release - 2018
    Abstract
    Evidence suggests that women who are naturally resistant to HIV infection exhibit low baseline immune activation at the female genital tract (FGT). This “immune quiescent” state is associated with lower expression of T-cell activation markers, reduced levels of gene transcription and pro-inflammatory cytokine or chemokine production involved in HIV infection while maintaining an intact immune response against pathogens. Therefore, if this unique immune quiescent state can be pharmacologically induced locally, it will provide an excellent women-oriented strategy against HIV infection To our knowledge, this is the first research article evaluating in vivo, an innovative trackable implant that can provide controlled delivery of hydroxychloroquine (HCQ) to successfully attenuate vaginal T lymphocyte activation and inflammation in a rabbit model as a potential strategy to induce an “immune quiescent” state within the FGT for the prevention of HIV infection. This biocompatible implant can deliver HCQ above therapeutic concentrations in a controlled manner, reduce submucosal immune cell recruitment, improve mucosal epithelium integrity, decrease protein and gene expression of T-cell activation markers, and attenuate the induction of key pro-inflammatory mediators. Our results suggest that microbicides designed to maintain a low level of immune activation at the FGT may offer a promising new strategy for reducing HIV infection.
    Effect of contractile activity on PGC-1α transcription in young and aged skeletal muscle
    Heather N. Carter, Journal of Applied Physiology - 2018
    Abstract
    Mitochondrial impairments are often noted in aged skeletal muscle. The transcriptional coactivator PGC-1α is integral to maintaining mitochondria, and its expression declines in aged muscle. It remains unknown whether this is due to a transcriptional deficit during aging. Our study examined PGC-1α transcription in muscle from young and old F344BN rats. Using a rat PGC-1α promoter-reporter construct, we found that PGC-1α transcription was reduced by ~65% in aged TA muscle, accompanied by decreases in PGC-1α mRNA and transcript stability. Altered expression patterns in PGC-1α transcription regulatory factors, including Nrf2, USF1, ATF2 and YY1, were noted in aged muscle. Acute contractile activity (CA) followed by recovery was employed to examine whether PGC-1α transcription could be activated in aged muscle similar to that observed in young muscle. AMPK and p38 signaling was attenuated in aged muscle. CA evoked an upregulation of PGC-1α transcription in both young and aged groups, while mRNAs encoding PGC-1α and COX IV were induced during the recovery period. Global DNA methylation, an inhibitory event for transcription, was enhanced in aged muscle, likely a result of elevated methyltransferase enzyme Dnmt3b in aged muscle. Successive bouts of CA for 7 days to evaluate longer-term consequences resulted in a rescue of PGC-1α and downstream mRNAs in aged muscle. Our data indicate that diminished mitochondria in aged muscle is partly due to a deficit in PGC-1α transcription, a result of attenuated upstream signaling. Contractile activity is an appropriate countermeasure to restore PGC-1α expression and mitochondrial content in aged muscle.
    Overexpression of miR169o, an Overlapping MicroRNA in Response to Both Nitrogen Limitation and Bacterial Infection, Promotes Nitrogen Use Efficiency and Susceptibility to Bacterial Blight in Rice
    Chao Yu, Plant and Cell Physiology - 2018
    Abstract
    Limiting nitrogen (N) supply contributes to improved resistance to bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) in susceptible rice (Oryza sativa). To understand the regulatory roles of microRNAs (miRNAs) in this phenomenon, 63 differentially expressed overlapping miRNAs in response to Xoo infection and N limitation stress in rice were identified through deep RNA sequencing and stem–loop quantitative real-time PCR. Among these, miR169o was further assessed as a typical overlapping miRNA through the overexpression of the miR169o primary gene. Osa-miR169o-OX plants were taller, and had more biomass accumulation with significantly increased nitrate and total amino acid contents in roots than the wild type (WT). Transcript level assays showed that under different N supply conditions, miR169o oppositely regulated NRT2, and this is reduced under normal N supply conditions but remarkably induced under N-limiting stress. On the other hand, osa-miR169o-OX plants also displayed increased disease lesion lengths and reduced transcriptional levels of defense gene (PR1b, PR10a, PR10b and PAL) compared with the WT after inoculation with Xoo. In addition, miR169o impeded Xoo-mediated NRT transcription. Therefore, the overlapping miR169o contributes to increase N use efficiency and negatively regulates the resistance to BB in rice. Consistently, transient expression of NF-YA genes in rice protoplasts promoted the transcripts of PR genes and NRT2 genes, while it reduced the transcripts of NRT1 genes. Our results provide novel and additional insights into the co ordinated regulatory mechanisms of cross-talk between Xoo infection and N deficiency responses in rice.
    Continuous Exposure to Simulated Hypergravity-Induced Changes in Proliferation, Morphology, and Gene Expression of Human Tendon Cells
    Raquel Costa-Almeida, Stem Cells and Development - 2018
    Abstract
    Gravity influences physical and biological processes, especially during development and homeostasis of several tissues in the human body. Studies under altered gravity have been receiving great attention toward a better understanding of microgravity-, hypogravity (<1 g)-, or hypergravity (>1 g)-induced alterations. In this work, the influence of simulated hypergravity over human tendon-derived cells (hTDCs) was studied at 5, 10, 15, and 20 g for 4 or 16 h, using a large diameter centrifuge. Main results showed that 16 h of simulated hypergravity limited cell proliferation. Cell area was higher in hTDCs cultured at 5, 10, and 15 g for 16 h, in comparison to 1 g control. Actin filaments were more pronounced in hTDCs cultured at 5 and 10 g for 16 h. Focal adhesion kinase (FAK) was mainly expressed in focal adhesion sites upon hypergravity stimulation, in comparison to perinuclear localization in control cells after 16 h; and FAK number/cell increased with increasing g-levels. A tendency toward an upregulation of tenogenic markers was observed; scleraxis (SCX), tenascin C (TNC), collagen type III (COL3A1), and decorin (DCN) were significantly upregulated in hTDCs cultured at 15 g and COL3A1 and DCN were significantly upregulated in hTDCs cultured at 20 g. Overall, simulated hypergravity affected the behavior of hTDCs, with more pronounced effects in the long-term period (16 h) of stimulation.
    High-fat diet modifies expression of hepatic cellular senescence gene p16(INK4a) through chromatin modifications in adult male rats
    Xiyuan Zhang, Genes & Nutrition - 2018
    Abstract
    Liver is the crucial organ as a hub for metabolic reactions. p16(INK4a) is a well-established cyclin-dependent kinase (CDK) inhibitor that plays important role in the molecular pathways of senescence, which lead to irreversible cell cycle arrest with secretion of proinflammatory cytokines and mitochondrial dysfunction. This study tested the hypothesis that cellular senescence regulated by p16(INK4a) is associated with high-fat diet in adult male rats.
    High calcium, phosphate and calcitriol supplementation leads to an osteocyte-like phenotype in calcified vessels and bone mineralisation defect in uremic rats
    Sarah-Kim Bisson, Journal of Bone and Mineral Metabolism - 2018
    Abstract
    A link between vascular calcification and bone anomalies has been suggested in chronic kidney disease (CKD) patients with low bone turnover disease. We investigated the vascular expression of osteocyte markers in relation to bone microarchitecture and mineralization defects in a model of low bone turnover CKD rats with vascular calcification. CKD with vascular calcification was induced by 5/6 nephrectomy followed by high calcium and phosphate diet, and vitamin D supplementation (Ca/P/VitD). CKD + Ca/P/VitD group (n = 12) was compared to CKD + normal diet (n = 12), control + normal diet (n = 8) and control + Ca/P/VitD supplementation (n = 8). At week 6, tibia, femurs and the thoracic aorta were analysed by Micro-Ct, histomorphometry and for expression of osteocyte markers. High Ca/P/VitD treatment induced vascular calcification only in CKD rats, suppressed serum parathyroid hormone levels and led to higher sclerostin, DKK1 and FGF23 serum levels. Expression of sclerostin, DKK1 and DMP1 but not FGF23 were increased in calcified vessels from CKD + Ca/P/VitD rats. Despite low parathyroid hormone levels, tibia bone cortical thickness was significantly lower in CKD + Ca/P/VitD rats as compared to control rats fed a normal diet, which is likely the result of radial growth impairment. Finally, Ca/P/VitD treatment in CKD rats induced a bone mineralization defect, which is likely explained by the high calcitriol dose. In conclusion, Ca/P/VitD supplementation in CKD rats induces expression of osteocyte markers in vessels and bone mineralisation anomalies. Further studies should evaluate the mechanisms of high dose calcitriol-induced bone mineralisation defects in CKD.
    The arginine methyltransferase CARM1 represses p300•ACT•CREMτ activity and is required for spermiogenesis
    Jianqiang Bao, Nucleic Acids Research - 2018
    Abstract
    CARM1 is a protein arginine methyltransferase (PRMT) that has been firmly implicated in transcriptional regulation. However, the molecular mechanisms by which CARM1 orchestrates transcriptional regulation are not fully understood, especially in a tissue-specific context. We found that Carm1 is highly expressed in the mouse testis and localizes to the nucleus in spermatids, suggesting an important role for Carm1 in spermiogenesis. Using a germline-specific conditional Carm1 knockout mouse model, we found that it is essential for the late stages of haploid germ cell development. Loss of Carm1 led to a low sperm count and deformed sperm heads that can be attributed to defective elongation of round spermatids. RNA-seq analysis of Carm1-null spermatids revealed that the deregulated genes fell into similar categories as those impacted by p300-loss, thus providing a link between Carm1 and p300. Importantly, p300 has long been known to be a major Carm1 substrate. We found that CREMτ, a key testis-specific transcription factor, associates with p300 through its activator, ACT, and that this interaction is negatively regulated by the methylation of p300 by Carm1. Thus, high nuclear Carm1 levels negatively impact the p300•ACT•CREMτ axis during late stages of spermiogenesis.
    In vitro bioassessment of the immunomodulatory activity of Saccharomyces cerevisiae components using bovine macrophages and Mycobacterium avium ssp. paratuberculosis
    Z. Li, Journal of Dairy Science - 2018
    Abstract
    The yeast Saccharomyces cerevisiae and its components are used for the prevention and treatment of enteric disease in different species; therefore, they may also be useful for preventing Johne's disease, a chronic inflammatory bowel disease of ruminants caused by Mycobacterium avium ssp. paratuberculosis (MAP). The objective of this study was to identify potential immunomodulatory S. cerevisiae components using a bovine macrophage cell line (BOMAC). The BOMAC phagocytic activity, reactive oxygen species production, and immune-related gene (IL6, IL10, IL12p40, IL13, IL23), transforming growth factor β, ARG1, CASP1, and inducible nitric oxide synthase expression were investigated when BOMAC were cocultured with cell wall components from 4 different strains (A, B, C, and D) and 2 forms of dead yeast from strain A. The BOMAC phagocytosis of mCherry-labeled MAP was concentration-dependently attenuated when BOMAC were cocultured with yeast components for 6 h. Each yeast derivative also induced a concentration-dependent increase in BOMAC reactive oxygen species production after a 6-h exposure. In addition, BOMAC mRNA expression of the immune-related genes was investigated after 6 and 24 h of exposure to yeast components. All yeast components were found to regulate the immunomodulatory genes of BOMAC; however, the response varied among components and over time. The in vitro bioassessment studies reported here suggest that dead yeast and its cell wall components may be useful for modulating macrophage function before or during MAP infection.
    Neuropilin-2 regulates airway inflammatory responses to inhaled lipopolysaccharide
    Robert M. Immormino, American Journal of Physiology-Lung Cellular and Molecular Physiology - 2018
    Abstract
    Neuropilins are multifunctional receptors that play important roles in immune regulation. Neuropilin-2 (NRP2) is expressed in the lungs, but whether it regulates airway immune responses is unknown. Here, we report that Nrp2 is weakly expressed by alveolar macrophages (AM) in the steady-state, but is dramatically up-regulated following in vivo lipopolysaccharide (LPS) inhalation. Ex vivo treatment of human AM with LPS also increased NRP2 mRNA expression and cell-surface display of NRP2 protein. LPS-induced Nrp2 expression in AM was dependent upon the MyD88 signaling pathway and the transcription factor nuclear factor kappa B (NFκB). In addition to up-regulating display of NRP2 on the cell membrane, inhaled LPS also triggered AM to release soluble NRP2 into the airways. Finally, myeloid-specific ablation of NRP2 resulted in increased expression of the chemokine Ccl2 in the lungs and prolonged leukocyte infiltration in the airways following LPS inhalation. These findings suggest that NRP2 expression by AM regulates LPS-induced inflammatory cell recruitment to the airways, and reveal a novel role for NRP2 during innate immune responses in the lungs.
    Genome-wide expression analysis suggests a role for jasmonates in the resistance to blue mold in apple
    Masoud Ahmadi-Afzadi, Plant Growth Regulation - 2018
    Abstract
    Blue mold, caused by the necrotrophic fungal pathogen Penicillium expansum, causes serious postharvest losses in apple, and threatens human health through production of the potent mycotoxin patulin. Recent studies indicate a quantitative control of resistance against this disease in apple cultivars. A whole genome apple microarray covering 60k transcripts was used to identify gene(s) that appear to be differentially regulated between resistant and susceptible cultivars in P. expansum-infected fruits. A number of potential candidates was encountered among defense- and oxidative stress-related genes, cell wall modification and lignification genes, and genes related to localization and transport. Induction of one cell wall-related gene and three genes involved in the ‘down-stream’ flavonoid biosynthesis pathway, demonstrates the fundamental role of the cell wall as an important barrier, and suggests that fruit flavonoids are involved in the resistance to blue mold. Moreover, exogenous application of the plant hormone methyl jasmonate (MeJA) reduced the symptoms resulting from inoculating apples with P. expansum. This is the first report linking MeJA and activation of cell wall and flavonoid pathway genes to resistance against blue mold in a study comparing different cultivars of domesticated apple. Our results provide an initial categorization of genes that are potentially involved in the resistance mechanism, and should be useful for developing tools for gene marker-assisted breeding of apple cultivars with an improved resistance to blue mold.
    Phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) in mammary tissue of Holstein cows during the periparturient period is associated with mRNA abundance of antioxidant gene networks
    L. Q. Han, Journal of Dairy Science - 2018
    Abstract
    Changes in the production of reactive oxygen species in the mammary gland of dairy cows during the periparturient period could lead to oxidative stress and potentially impair mammary function. Phosphorylation of the transcription factor nuclear factor erythroid 2-like 2 (NFE2L2), also known as nuclear factor-E2-related factor 2, controls mRNA abundance of genes encoding antioxidant proteins and enzymes. The hypothesis was that NFE2L2 phosphorylation status and target gene mRNA abundance in the mammary gland of dairy cows is altered around parturition. Total NFE2L2 protein, phosphorylated protein (p-NFE2L2), and ratio of p-NFE2L2 to NFE2L2 along with mRNA abundance of 24 genes related to the NFE2L2 signaling pathway, apoptosis, and cell proliferation were measured in mammary tissue samples from Holstein cows at −30, 1, 15, and 30 d relative to parturition. Although total NFE2L2 protein abundance did not differ, p-NFE2L2 and p-NFE2L2-to-NFE2L2 ratio were greater after parturition. The upregulation of DNA damage inducible transcript 3 (DDIT3) postpartum indicated a localized oxidative stress state. Among genes evaluated, thioredoxin (TXN), glutathione peroxidase 1 (GPX1), and glutathione S-transferase mu 1 (GSTM1) had the highest (37.1, 15.1, and 4.8% of total mRNA measured, respectively) abundance. The mRNA abundance of various target genes with detoxifying enzymatic functions and free radical scavenging activities [glutamate-cysteine ligase catalytic subunit (GCLC); glutathione reductase (GSR); ferrochelatase (FECH); TXN; thioredoxin reductase 1 (TXNRD1); and NAD(P)H quinone dehydrogenase 1 (NQO1)] were consistently upregulated (linear effect of time) as parturition approached and lactation began. Among the transcription regulators, NFE2L2 had the highest mRNA abundance (7.3% of total mRNA measured). Abundance of NFE2L2 and other transcription factors [nuclear factor kappa B subunit 1 (NFKB1), retinoid X receptor α (RXRA), and mitogen-activated protein kinase 14 (MAPK14)] were upregulated (linear effect of time) from −30 d to 30 d relative to parturition. Overall, NFE2L2 phosphorylation and downstream signaling leading to postpartal upregulation of genes associated with oxidative stress and inflammation in the mammary gland seem to be key components of normal cellular function to maintain proper redox homeostasis. However, if the longitudinal increases in mRNA and protein abundance of these antioxidant mechanisms are a reflection of cellular oxidative stress, then the likelihood of protein and DNA damage would be greater and might be one factor compromising cell viability and potentially lactation persistency. The actual cues coordinating these molecular responses remain to be determined.
    Combined analytical approaches to define biodistribution and biological activity of semi-synthetic berberrubine, the active metabolite of natural berberine
    Emanuele Porru, Analytical and Bioanalytical Chemistry - 2018
    Abstract
    Berberine (BBR) is a natural alkaloid obtained from Berberis species plants, known for its protective effects against several diseases. Among the primary BBR metabolites, berberrubine (M1) showed the highest plasma concentration but few and conflicting data are available regarding its concentration in biological fluids related to its new potential activity on vascular cells. A combined analytical approach was applied to study biodistribution of M1 in comparison with BBR. The optimization of sample clean-up combined with a fully validated HPLC-ESI-MS/MS tailored for M1 allows sufficient detectability and accuracy to be reached in the different studied organs even when administered at low dose, comparable to that assumed by human. A predictive human vascular endothelial cell-based assay to measure intracellular xanthine oxidase has been developed and applied to study unexplored activities of M1 alongside other common activities. Results showed that oral M1 treatment exhibits higher plasma levels than BBR, reaching maximum concentration 400-fold higher than BBR (204 vs 0.5 ng/mL); moreover, M1 exhibits higher concentrations than BBR also in all the biological compartments analyzed. Noteworthy, the two compounds follow two different excretion routes: M1 through urine, while BBR through feces. In vitro studies demonstrated that M1 inhibited intracellular xanthine oxidase activity, one of the major sources of reactive oxygen species in vasculature, with an IC50 = 9.90 ± 0.01 μg/mL and reduced the expression of the inflammatory marker ICAM-1. These peculiar characteristics allow new perspectives to be opened up for the direct use of M1 instead of BBR in endothelial dysfunction treatment.
    Teriparatide (human PTH1–34) compensates for impaired fracture healing in COX-2 deficient mice
    Kiminori Yukata, Bone - 2018
    Abstract
    Genetic ablation of cyclooxygenase-2 (COX-2) in mice is known to impair fracture healing. To determine if teriparatide (human PTH1–34) can promote healing of Cox-2-deficient fractures, we performed detailed in vivo analyses using a murine stabilized tibia fracture model. Periosteal progenitor cell proliferation as well as bony callus formation was markedly reduced in Cox-2−/− mice at day 10 post-fracture. Remarkably, intermittent PTH1–34 administration increased proliferation of periosteal progenitor cells, restored callus formation on day 7, and enhanced bone formation on days 10, 14 and 21 in Cox-2-deficient mice. PTH1–34 also increased biomechanical torsional properties at days 10 or 14 in all genotypes, consistent with enhanced bony callus formation by radiologic examinations. To determine the effects of intermittent PTH1–34 for callus remodeling, TRAP staining was performed. Intermittent PTH1–34 treatment increased the number of TRAP positive cells per total callus area on day 21 in Cox-2−/− fractures. Taken together, the present findings indicate that intermittent PTH1–34 treatment could compensate for COX-2 deficiency and improve impaired fracture healing in Cox-2-deficient mice.
    Histamine H3 Receptors Decrease Dopamine Release in the Ventral Striatum by Reducing the Activity of Striatal Cholinergic Interneurons
    Rafael Koerich Varaschin, Neuroscience - 2018
    Abstract
    Histamine H3 receptors are widely distributed Gi-coupled receptors whose activation reduces neuronal activity and inhibits release of numerous neurotransmitters. Although these receptors are abundantly expressed in the striatum, their modulatory role on activity-dependent dopamine release is not well understood. Here, we observed that histamine H3 receptor activation indirectly diminishes dopamine overflow in the ventral striatum by reducing cholinergic interneuron activity. Acute brain slices from C57BL/6 or channelrhodopsin-2-transfected DAT-cre mice were obtained, and dopamine transients evoked either electrically or optogenetically were measured by fast-scan cyclic voltammetry. The H3 agonist α-methylhistamine significantly reduced electrically- evoked dopamine overflow, an effect blocked by the nicotinic acetylcholine receptor antagonist dihydro-β-erythroidine, suggesting involvement of cholinergic interneurons. None of the drug treatments targeting H3 receptors affected optogenetically evoked dopamine overflow, indicating that direct H3-modulation of dopaminergic axons is unlikely. Next, we used qPCR and confirmed the expression of histamine H3 receptor mRNA in cholinergic interneurons, both in ventral and dorsal striatum. Activation of H3 receptors by α-methylhistamine reduced spontaneous firing of cholinergic interneurons in the ventral, but not in the dorsal striatum. Resting membrane potential and number of spontaneous action potentials in ventral-striatal cholinergic interneurons were significantly reduced by α-methylhistamine. Acetylcholine release from isolated striatal synaptosomes, however, was not altered by α-methylhistamine. Together, these results indicate that histamine H3 receptors are important modulators of dopamine release, specifically in the ventral striatum, and that they do so by decreasing the firing rate of cholinergic neurons and, consequently, reducing cholinergic tone on dopaminergic axons.
    Endogenous Sonic Hedgehog limits inflammation and angiogenesis in the ischaemic skeletal muscle of mice
    Caroline Caradu, Cardiovascular Research - 2018
    Abstract
    AimsHedgehog (Hh) signalling has been shown to be re-activated in ischaemic tissues and participate in ischaemia-induced angiogenesis. Sonic Hedgehog (Shh) is upregulated by more than 80-fold in the ischaemic skeletal muscle, however its specific role in ischaemia-induced angiogenesis has not yet been fully investigated.The purpose of the present study was to investigate the role of endogenous Shh in ischaemia-induced angiogenesis.Methods and resultsTo this aim, we used inducible Shh knock-out (KO) mice and unexpectedly found that capillary density was significantly increased in re-generating muscle of Shh deficient mice 5 days after hind limb ischaemia was induced, demonstrating that endogenous Shh does not promote angiogenesis but more likely limits it. Myosin and MyoD expression were equivalent in Shh deficient mice and control mice, indicating that endogenous Shh is not required for ischaemia-induced myogenesis. Additionally, we observed a significant increase in macrophage infiltration in the ischaemic muscle of Shh deficient mice. Our data indicate that this was due to an increase in chemokine expression by myoblasts in the setting of impaired Hh signalling, using tissue specific Smoothened conditional KO mice. The increased macrophage infiltration in mice deficient for Hh signalling in myocytes was associated with increased VEGFA expression and a transiently increased angiogenesis, demonstrating that Shh limits inflammation and angiogenesis indirectly by signalling to myocytes.ConclusionAlthough ectopic administration of Shh has previously been shown to promote ischaemia-induced angiogenesis, the present study reveals that endogenous Shh does not promote ischaemia-induced angiogenesis. On the contrary, the absence of Shh leads to aberrant ischaemic tissue inflammation and a transiently increased angiogenesis.
    Pituitary adenylate-cyclase activating-polypeptide (PACAP) signaling in the prefrontal cortex modulates cued fear learning, but not spatial working memory, in female rats
    Adam J. Kirry, Neuropharmacology - 2018
    Abstract
    A genetic polymorphism within the gene encoding the pituitary adenylate-cyclase activating polypeptide (PACAP) receptor type I (PAC1R) has recently been associated with hyper-reactivity to threat-related cues in women, but not men, with post-traumatic stress disorder (PTSD). PACAP is a highly conserved peptide, whose role in mediating adaptive physiological stress responses is well established. Far less is understood about the contribution of PACAP signaling in emotional learning and memory, particularly the encoding of fear to discrete cues. Moreover, a neurobiological substrate that may account for the observed link between PAC1R and PTSD in women, but not men, has yet to be identified. Sex differences in PACAP signaling during emotional learning could provide novel targets for the treatment of PTSD. Here we investigated the contribution of PAC1R signaling within the prefrontal cortex to the acquisition of cued fear in female and male rats. We used a variant of fear conditioning called trace fear conditioning, which requires sustained attention to fear cues and depends on working-memory like neuronal activity within the prefrontal cortex. We found that cued fear learning, but not spatial working memory, was impaired by administration of a PAC1R antagonist directly into the prelimbic area of the prefrontal cortex. This effect was specific to females. We also found that levels of mRNA for the PAC1R receptor in the prelimbic cortex were greater in females compared with males, and were highest during and immediately following the proestrus stage of the estrous cycle. Together, these results demonstrate a sex-specific role of PAC1R signaling in learning about threat-related cues.
    Matrix Metalloproteinase 12 (MMP-12) Promotes Tumor Propagation in the Lung
    Ezra Ella, The Journal of Thoracic and Cardiovascular Surgery - 2018
    Abstract
    Objective Past studies are inconsistent with regard to the role of MMP-12 in lung tumorigenesis. This is due, in part, to differential tumorigenesis based on tumor- versus immune-derived MMP-12 expression. Our study aims to thoroughly dissect the role of MMP-12 in lung tumorigenesis. Methods We tested MMP-12 expression, and association with prognosis using a tissue-array and a published NSCLC gene expression database. In addition, we characterized the contribution of MMP-12 to tumor propagation in the lung using a series of in vitro and in vivo studies. Results Tumor cells of a diverse set of human lung-cancers stained positive for MMP-12 and high MMP-12 mRNA levels in the tumor were associated with reduced survival. The lung microenvironment stimulated endogenous production of MMP-12 in lung-cancer cells (H460, LLC). In-vitro MMP-12 KO LLC and LLC cells had the same proliferation rate but LLC showed increased invasiveness. In-vivo, deficiency of MMP-12 in LLC cells -- but not in the host -- reduced tumor growth and invasiveness. Conclusion We suggest that tumor-cell-derived MMP-12 promotes tumor propagation in the lung and that in the context of pulmonary malignancies MMP-12 should further be tested as a potential novel therapeutic target.
    Fibroblast Growth Factor-2 and Transforming Growth Factor-beta1 Oppositely Regulate miR-221 that Targets Thrombospondin-1 in Bovine Luteal Endothelial Cells
    Svetlana Farberov, Biology of Reproduction - 2018
    Abstract
    Thrombospondin-1 (THBS1) is an important mediator of corpus luteum (CL) regression. Highly induced during luteolysis, it acts as a natural anti-angiogenic, proapoptotic compound. THBS1 expression is regulated in bovine luteal endothelial cells (LECs) by fibroblast growth factor-2 (FGF2) and transforming growth factor-beta1 (TGFB1) acting in an opposite manner. In this study we sought to identify specific microRNAs (miRNAs) targeting THBS1 and investigate their possible involvement in FGF2 and TGFB1-mediated THBS1 expression. Several miRNAs predicted to target THBS1 mRNA (miR-1, miR-18a, miR-144, miR-194, and miR-221) were experimentally tested. Of these, miR-221 was shown to efficiently target THBS1 expression and function in LECs. We found that this miRNA is highly expressed in luteal cells and in mid-cycle CL. Consistent with the inhibition of THBS1 function, miR-221 also reduced SERPINE1 in LECs and promoted angiogenic characteristics of LECs. Plasminogen activator inhibitor-1 (PAI-1), the gene product of SERPINE1, inhibited cell adhesion, suggesting that PAI-1, like THBS1, has anti-angiogenic properties. Importantly, FGF2, which negatively regulates THBS1, elevates miR-221. Conversely, TGFB1 that stimulates THBS1, significantly reduces miR-221. Furthermore, FGF2 enhances the suppression of THBS1 caused by miR-221 mimic, and prevents the increase in THBS1 induced by miR-221 inhibitor. In contrast, TGFB1 reverses the inhibitory effect of miR-221 mimic on THBS1, and enhances the upregulation of THBS1 induced by miR-221 inhibitor. These data support the contention that FGF2 and TGFB1 modulate THBS1 via miR-221. These in vitro data propose that dynamic regulation of miR-221 throughout the cycle, affecting THBS1 and SERPINE1, can modulate vascular function in the CL.
    Crosstalk between adipose stem cells and tendon cells reveals a temporal regulation of tenogenesis by matrix deposition and remodeling
    Raquel Costa-Almeida, Journal of Cellular Physiology - 2018
    Abstract
    Tendon injuries constitute an unmet clinical challenge owing to the limited intrinsic regenerative ability of this tissue. Cell-based therapies aim at improving tendon healing through the delicate orchestration of tissue rebuilding and regain of function. Hence, human adipose-derived stem cells (hASCs) have been proposed as a promising cell source for boosting tendon regeneration. In this work, we investigated the influence of hASCs on native human tendon-derived cells (hTDCs) through the establishment of a direct contact co-culture system. Results demonstrated that direct interactions between these cell types resulted in controlled proliferation and spontaneous cell elongation. ECM-related genes, particularly COL1A1 and TNC, and genes involved in ECM remodeling, such as MMP1, MMP2, MMP3 and TIMP1, were expressed in co-cultures in a temporally regulated manner. In addition, deposition of collagen type I was accelerated in co-cultures systems and favored over the production of collagen type III, resulting in an enhanced COL1/COL3 ratio as soon as 7 days. In conclusion, hASCs seem to be good candidates in modulating the behavior of native tendon cells, particularly through a balanced process of ECM synthesis and degradation. This article is protected by copyright. All rights reserved
    Identification of Estrogen-Related Receptor Alpha Agonists in the Tox21 Compound Library
    Caitlin Lynch, Endocrinology - 2018
    Abstract
    The estrogen-related receptor alpha (ERRα) is an orphan nuclear receptor (NR) that plays a role in energy homeostasis and controls mitochondrial oxidative respiration. Increased expression of ERRα in certain ovarian, breast, and colon cancers has a negative prognosis, indicating an important role for ERRα in cancer progression. An interaction between ERRα and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) has also recently been shown to regulate an enzyme in the β-oxidation of free fatty acids, thereby suggesting that ERRα plays an important role in obesity and type 2 diabetes. Therefore, it would be prudent to identify compounds that can act as activators of ERRα. In this study, we screened ∼10,000 (8,311 unique) compounds, known as the Tox21 10K collection, to identify agonists of ERRα. We performed this screen using two stably transfected HEK 293 cell lines, one with the ERRα-reporter alone and the other with both ERRα-reporter and PGC-1α expression vectors. After the primary screening, we identified more than five agonist clusters based on compound structural similarity analysis (e.g., statins). By examining the activities of the confirmed ERRα modulators in other Tox21 NR assays, eliminating those with promiscuous NR activity, and performing follow-up assays (e.g., siRNA knockdown) we identified compounds that might act as endocrine disrupters through effects on ERRα signaling. This study is the first comprehensive analysis in discovering potential endocrine disrupters which affect the ERRα signaling pathway.
    Expression of heterologous non-oxidative pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli methylotroph
    R. Kyle Bennett, Metabolic Engineering - 2018
    Abstract
    Synthetic methylotrophy aims to develop non-native methylotrophic microorganisms to utilize methane or methanol to produce chemicals and biofuels. We report two complimentary strategies to further engineer a previously engineered methylotrophic E. coli strain for improved methanol utilization. First, we demonstrate improved methanol assimilation in the presence of small amounts of yeast extract by expressing the non-oxidative pentose phosphate pathway (PPP) from Bacillus methanolicus. Second, we demonstrate improved co-utilization of methanol and glucose by deleting the phosphoglucose isomerase gene (pgi), which rerouted glucose carbon flux through the oxidative PPP. Both strategies led to significant improvements in methanol assimilation as determined by 13C-labeling in intracellular metabolites. Introduction of an acetone-formation pathway in the pgi-deficient methylotrophic E. coli strain led to improved methanol utilization and acetone titers during glucose fed-batch fermentation.
    The effect of within-instar development on tracheal diameter and hypoxia-inducible factors α and β in the tobacco hornworm, Manduca sexta
    Taylor A. Lundquist, Journal of Insect Physiology - 2018
    Abstract
    As insects grow within an instar, body mass increases, often more than doubling. The increase in mass causes an increase in metabolic rate and hence oxygen demand. However, the insect tracheal system is hypothesized to increase only after molting and may be compressed as tissues grow within an instar. The increase in oxygen demand in the face of a potentially fixed or decreasing supply could result in hypoxia as insects near the end of an instar. To test these hypotheses, we first used synchrotron X-ray imaging to determine how diameters of large tracheae change within an instar and after molting to the next instar in the tobacco hornworm, Manduca sexta. Large tracheae did not increase in diameter within the first, second, third, and fourth instars, but increased upon molting. To determine if insects are hypoxic at the end of instars, we used the presence of hypoxia-inducible factors (HIFs) as an index. HIF-α and HIF-β dimerize in hypoxia and act as a transcription factor that turns on genes that will increase oxygen delivery. We sequenced both of these genes and measured their mRNA levels at the beginning and end of each larval instar. Finally, we obtained an antibody to HIF-α and measured protein expression during the same time. Both mRNA and protein levels of HIFs were increased at the end of most instars. These data support the hypothesis that some insects may experience hypoxia at the end of an instar, which could be a signal for molting. Summary Statement As caterpillars grow within an instar, major tracheae do not increase in size, while metabolic demand increases. At the same life stages, caterpillars increased expression of hypoxia inducible factors, suggesting that they become hypoxic near the end of an instar.
    Effects of a novel microtubule-depolymerizer on pro-inflammatory signaling in RAW264.7 macrophages
    Samuel P. Gilmore, Chemico-Biological Interactions - 2018
    Abstract
    The Nuclear Factor-kappa B (NF-κB) pathway is vital for immune system regulation and pro-inflammatory signaling. Many inflammatory disorders and diseases, including cancer, are linked to dysregulation of NF-κB signaling. When macrophages recognize the presence of a pathogen, the signaling pathway is activated, resulting in the nuclear translocation of the transcription factor, NF-κB, to turn on pro-inflammatory genes. Here, we demonstrate the effects of a novel microtubule depolymerizer, NT-07-16, a polysubstituted pyrrole compound, on this process. Treatment with NT-07-16 decreased the production of pro-inflammatory cytokines in RAW264.7 mouse macrophages. It appears that the reduction in pro-inflammatory mediators produced by the macrophages after exposure to NT-07-16 may be due to activities upstream of the translocation of NF-κB into the nucleus. NF-κB translocation occurs after its inhibitory protein, IκB-α is phosphorylated which signals for its degradation releasing NF-κB so it is free to move into the nucleus. Previous studies from other laboratories indicate that these processes are associated with the microtubule network. Our results show that exposure to the microtubule-depolymerizer, NT-07-16 reduces the phosphorylation of IκB-α and also decreases the association of NF-κB with tubulin which may affect the ability of NF-κB to translocate into the nucleus. Therefore, the anti-inflammatory activity of NT-07-16 may be explained, at least in part, by alterations in these steps in the NF-κB signaling pathway leading to less NF-κB entering the nucleus and reducing the production of pro-inflammatory mediators by the activated macrophages.
    Nlrp12 Mediates Adverse Neutrophil Recruitment during Influenza Virus Infection
    Emma E. Hornick, The Journal of Immunology - 2018
    Abstract
    Exaggerated inflammatory responses during influenza A virus (IAV) infection are typically associated with severe disease. Neutrophils are among the immune cells that can drive this excessive and detrimental inflammation. In moderation, however, neutrophils are necessary for optimal viral control. In this study, we explore the role of the nucleotide-binding domain leucine-rich repeat containing receptor family member Nlrp12 in modulating neutrophilic responses during lethal IAV infection. Nlrp12−/− mice are protected from lethality during IAV infection and show decreased vascular permeability, fewer pulmonary neutrophils, and a reduction in levels of neutrophil chemoattractant CXCL1 in their lungs compared with wild-type mice. Nlrp12−/− neutrophils and dendritic cells within the IAV-infected lungs produce less CXCL1 than their wild-type counterparts. Decreased CXCL1 production by Nlrp12−/− dendritic cells was not due to a difference in CXCL1 protein stability, but instead to a decrease in Cxcl1 mRNA stability. Together, these data demonstrate a previously unappreciated role for Nlrp12 in exacerbating the pathogenesis of IAV infection through the regulation of CXCL1-mediated neutrophilic responses.
    BDNF Function in Long-Term Synaptic Plasticity in the Dentate Gyrus In Vivo: Methods for Local Drug Delivery and Biochemical Analysis of Translation
    Debabrata Panja, SpringerLink - 2018
    Abstract
    Neurotrophins are essential for multiple aspects of neuronal development and to important functions like synaptic plasticity. Brain-derived neurotrophic factor (BDNF) is a critical activity-dependent modulator of gene expression which regulates both transcription and translation. BDNF is crucial in the maintenance of long-term potentiation (LTP) at synapses and regulates protein synthesis at the dendritic and synaptic level. To elucidate the mechanisms operating in the hippocampal dentate gyrus region, in vivo electrophysiology and pharmacology is combined with analysis of signaling pathways and protein synthesis. Here, we present methods for the analysis of translation initiation, polysome formation, and translational efficiency in the context of LTP consolidation in live rodents.
    Allelic variants of the aryl hydrocarbon receptor differentially influence UVB-mediated skin inflammatory responses in SKH1 mice
    Kayla J. Smith, Toxicology - 2017
    Abstract
    The mouse strain SKH1 is widely used in skin research due to its hairless phenotype and intact immune system. Due to the complex nature of aryl hydrocarbon receptor (AHR) function in the skin, the development of additional in vivo models is necessary to study its role in cutaneous homeostasis and pathology. Variants of the Ah allele, exist among different mouse strains. The Ahb−1 and Ahd alleles express high and low affinity ligand binding forms of the AHR, respectively. The outbred SKH1 mice express the Ahb−2 and/or Ahd alleles. SKH1 mice were crossed with C57BL/6J mice, which harbor the Ahb−1 allele, to create useful models for studying endogenous AHR function. SKH1 mice were bred to be homozygous for either the Ahb−1 or Ahd allele to establish strains for use in comparative studies of the effects of differential ligand-mediated activation through gene expression changes upon UVB exposure. Ahb−1 or Ahd allelic status was confirmed by DNA sequence analysis. We tested the hypothesis that SKH1-Ahb−1 mice would display enhanced inflammatory signaling upon UVB exposure compared to SKH1-Ahd mice. Differential basal AHR activation between the strains was determined by assessing Cyp1a1 expression levels in the small intestine, liver, and skin of the SKH1-Ahb−1 mice compared to SKH1-Ahd mice. To determine whether SKH1-Ahb−1 mice are more prone to a pro-inflammatory phenotype in response to UVB, gene expression of inflammatory mediators was analyzed. SKH1-Ahb−1 mice expressed enhanced gene expression of the chemotactic factors Cxcl5, Cxcl1, and Ccl20, as well as the inflammatory signaling factors S100a9 and Ptgs2, compared to SKH1-Ahd mice in skin. These data supports a role for AHR activation and enhanced inflammatory signaling in skin.
    Uptake and biological responses in land snail Cornu aspersum exposed to vaporized CdCl2
    L. Sturba, Ecotoxicology and Environmental Safety - 2017
    Abstract
    The uptake of Cd and some biomarkers of exposure and effects have been investigated in specimens of land snail Cornu aspersum exposed to vaporized CdCl2 (10mg/L) for 7 days. The Cd levels quantified in snail's whole bodies confirmed Cd bioavailability trough vaporization and an higher accumulation in the midgut gland compared to the foot. Biological responses investigated showed a reduction of destabilization time of lysosomal membranes (NRRT) in hemocytes and an induction of catalase activities (CAT) in midgut gland. A further evidence of CdCl2 vaporized exposure was given by an increase in MT protein content as well as induction of Cd-MT gene expression, highlighting the central role of the midgut gland in Cd detoxification. These biomarkers can thus be considered as sensitive tools for the assessment of Cd contamination in the air using land snails as bioindicators. No changes in of GST activity and MDA were observed. From the overall results, the land snail, C. aspersum, could be used as good bioindicator of air quality for pollution monitoring purposes having shown clear signs of exposure and effects due Cd exposure by air.
    Quantifying mitochondrial DNA copy number using robust regression to interpret real time PCR results
    Paulo Refinetti, BMC Research Notes - 2017
    Abstract
    Real time PCR (rtPCR) is a quantitative assay to determine the relative DNA copy number in a sample versus a reference. The $$\Delta C_T$$ Δ C T method is the standard for the analysis of the output data generated by an rtPCR experiment. We developed an alternative based on fitting a robust regression to the rtPCR signal. This new data analysis tool reduces potential biases and does not require all of the compared DNA fragments to have the same PCR efficiency.
    Abnormal Microglia and Enhanced Inflammation-Related Gene Transcription in Mice with Conditional Deletion of Ctcf in Camk2a-Cre-Expressing Neurons. | Journal of Neuroscience
    Bryan E.McGill, Journal of Neuroscience - 2017
    Abstract
    CCCTC-binding factor (CTCF) is an 11 zinc finger DNA-binding domain protein that regulates gene expression by modifying three dimensional chromatin structure. Human mutations in CTCF cause intellectual disability and autistic features. Knocking out Ctcf in mouse embryonic neurons is lethal by neonatal age, but the effects of CTCF deficiency in postnatal neurons are less well studied. We knocked out Ctcf postnatally in glutamatergic forebrain neurons under the control of Camk2a-Cre. CtcfloxP/loxP;Camk2a-Cre+ (Ctcf CKO) mice of both sexes were viable and exhibited profound deficits in spatial learning/memory, impaired motor co-ordination, and decreased sociability by 4 months of age. Ctcf CKO mice also had reduced dendritic spine density in the hippocampus and cerebral cortex. Microarray analysis of mRNA from Ctcf CKO mouse hippocampus identified increased transcription of inflammation-related genes linked to microglia. Separate microarray analysis of mRNA isolated specifically from Ctcf CKO mouse hippocampal neurons by ribosomal affinity purification identified upregulation of chemokine signaling genes, suggesting crosstalk between neurons and microglia in Ctcf CKO hippocampus. Finally, we found that microglia in Ctcf CKO mouse hippocampus had abnormal morphology by Sholl analysis and increased immunostaining for CD68, a marker of microglial activation. Our findings confirm that Ctcf knockout in postnatal neurons causes a neurobehavioral phenotype in mice, and we provide novel evidence that CTCF depletion leads to overexpression of inflammation-related genes and microglial dysfunction. SIGNIFICANCE STATEMENT CCCTC-binding factor (CTCF) is a DNA-binding protein that organizes nuclear chromatin topology. Mutations in CTCF cause intellectual disability and autistic features in humans. CTCF deficiency in embryonic neurons is lethal in mice, but mice with postnatal CTCF depletion are less well studied. We find that mice lacking Ctcf in Camk2a-expressing neurons (Ctcf CKO mice) have: spatial learning/memory deficits, impaired fine-motor skills, subtly altered social interactions, and decreased dendritic spine density. We uniquely demonstrate that Ctcf CKO mice overexpress inflammation-related genes in the brain, and have microglia with abnormal morphology that label positive for CD68, a marker of microglial activation. Our findings suggest that inflammation and dysfunctional neuron-microglia interactions are factors in the pathology of CTCF deficiency.
    Effects of EPSPS Copy Number Variation (CNV) and Glyphosate Application on the Aromatic and Branched Chain Amino Acid Synthesis Pathways in Amaranthus palmeri
    Manuel Fernández-Escalada, Frontiers in Plant Science - 2017
    Abstract
    A key enzyme of the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19), is the known target of the widely used herbicide glyphosate. Glyphosate resistance in Amaranthus palmeri, one of the most troublesome weeds in agriculture, has evolved through increased EPSPS gene copy number. The aim of this work was to study the pleiotropic effects of (i) EPSPS increased transcript abundance due to gene copy number variation (CNV) and of (ii) glyphosate application on the aromatic amino acid (AAA) and branched chain amino acid (BCAA) synthesis pathways. Hydroponically grown glyphosate sensitive (GS) and glyphosate resistant (GR) plants were treated with glyphosate three days after treatment. In absence of glyphosate treatment, high EPSPS gene copy number had only a subtle effect on transcriptional regulation of AAA and BCAA pathway genes. In contrast, glyphosate treatment provoked a general accumulation of the transcripts corresponding to genes of the AAA pathway leading to synthesis of chorismate in both GS and GR. After chorismate, anthranilate synthase transcript abundance was higher while chorismate mutase transcription showed a small decrease in GR and remained stable in GS, suggesting a regulatory branch point in the pathway that favors synthesis towards tryptophan over phenylalanine and tyrosine after glyphosate treatment. This was confirmed by studying enzyme activities in vitro and amino acid analysis. Importantly, this upregulation was glyphosate dose dependent and was observed similarly in both GS and GR populations. Glyphosate treatment also had a slight effect on the expression of BCAA genes but no general effect on the pathway could be observed. Taken together, our observations suggest that the high copy number variation of EPSPS in A. palmeri GR populations has no major pleiotropic effect on the expression of AAA biosynthetic genes, even in response to glyphosate treatment. This finding supports the idea that the fitness cost associated with EPSPS CNV in A. palmeri may be limited.
    miR-25 Tough Decoy enhances cardiac function in heart failure
    Dongtak Jeong, Molecular Therapy - 2017
    Abstract
    microRNAs are promising therapeutic targets since their inhibition has the potential to normalize gene expression in diseased states. Recently, our group found that miR-25 is a key SERCA2a regulating microRNA, and we showed that multiple injections of antagomiRs against miR-25 enhances cardiac contractility and function through SERCA2a restoration in a murine heart failure model. However, for clinical application, a more stable suppressor of miR-25 would be desirable. Tough Decoy inhibitors are emerging as a highly effective method for microRNA inhibition due to their resistance to endonucleolytic degradation, high miRNA binding affinity, and efficient delivery. We generated a miR-25 Tough Decoy inhibitor and subcloned it into a cardiotropic AAV9 vector to evaluate its efficacy. The AAV9 Tough Decoy showed selective inhibition of miR-25 in vitro cardiomyoblast culture. In vivo, AAV9-miR25 Tough Decoy delivered to the murine pressure-overload heart failure model selectively decreased expression of miR- 25, increased levels of SERCA2a protein and ameliorated cardiac dysfunction and fibrosis. Our data indicate that miR-25 Tough Decoy is an effective long-term suppressor of miR-25 and a promising therapeutic candidate to treat heart failure.
    Leishmania-derived trimannose modulates inflammatory response to significantly reduce Leishmania (L.) major-induced lesions
    Tara L. Grinnage-Pulley, Infection and Immunity - 2017
    Abstract
    Leishmania lipophosphoglycan (LPG) is a key virulence factor, initiating inflammation resulting in cutaneous lesions. LPG is capped by various oligosaccharides. How these glycans are recognized and how they alter the course of Leishmania infection is poorly understood. Previous studies synthesized α-1,2-trimannose cap sugars on latex beads demonstrated that C57BL/6 mice co-inoculated with L. major and trimannose-coated beads produced significantly higher levels of IL-12 p40 and other pro-inflammatory, type 1 cytokines compared L. major infection alone within the first 48 h of infection. However, as L. major infection typically progress over weeks to months, the role of trimannose in altering disease progression over the course of infection was unknown. Wild-type mice were inoculated with either trimannose or carrier (uncoated) beads, infected with L. major alone, co-inoculated with carrier beads and L. major, or co-inoculated with trimannose beads and L. major. Trimannose treatment of L. major-infected mice decreased parasite load and significantly decreased lesion size at 14 days post infection (pi) compared to non-treated, infected mice. Infected, trimannose-treated mice had decreased IL-12p40 and IL-10 secretion and increased IFN-γ at 14 days pi. Mice lacking the ability to detect trimannose, mannose-receptor deleted mice (MR-/-), when treated with trimannose beads and infected with L. major did not have decreased lesion size. Leishmania-derived trimannose represents a novel immunomodulator that provides early type 1-skewed cytokine production to control parasite load and alter the course of cutaneous leishmaniasis.
    EXERSOMES, METHODS OF PRODUCING AND METHOD OF USING
    Mark Tarnopolsky, EXERKINE CORPORATION (Hamilton, CA) - 2017
    Abstract
    An exosome pellet or physiological solution comprising resuspended exosomes is provided. The exosomes are essentially free from undesirable particles having a diameter less than 20 nm or greater than 140 nm, and the exosomes comprise one or more metabolic products. The exosomes may be used to induce mitochondrial biogenesis, increase thermogenesis (browning) of subcutaneous white adipose tissue, and/or mediate other systemic effects of exercise in a mammal.
    Exercise induces TFEB expression and activity in skeletal muscle in a PGC-1α-dependent manner
    Avigail T. Erlich, American Journal of Physiology - Cell Physiology - 2017
    Abstract
    The mitochondrial network in muscle is controlled by the opposing processes of mitochondrial biogenesis and mitophagy. The coactivator PGC-1α regulates biogenesis, while the transcription of mitophagy-related genes is controlled by transcription factor EB (TFEB). PGC-1α activation is induced with exercise, however the effect of exercise on TFEB is not fully known. We investigated the interplay between PGC-1α and TFEB on mitochondria in response to acute contractile activity in C2C12 myotubes, and following exercise in WT and PGC-1α KO mice. TFEB nuclear localization was increased by 1.6-fold following 2 hours of acute myotube contractile activity. TFEB transcription and LC3 localization to mitochondria were also simultaneously increased by 2-3-fold. Viral overexpression of TFEB increased PGC-1α and COXIV gene expression. In WT mice, TFEB translocation to the nucleus increased 2.4-fold in response to acute exercise, while TFEB transcription, assessed through the electroporation of a TFEB promoter construct, was elevated by 4-fold. These exercise effects were dependent on the presence of PGC-1α. Our data suggest that acute exercise provokes TFEB expression and activation both in vitro and in vivo, in a PGC-1α-dependent manner. Our results indicate that TFEB, along with PGC-1α, are important regulators of mitochondrial biogenesis in muscle as a result of exercise.
    TIEG and estrogen modulate SOST expression in the murine skeleton
    Malayannan Subramaniam, Journal of Cellular Physiology - 2017
    Abstract
    TIEG knockout (KO) mice exhibit a female-specific osteopenic phenotype and altered expression of TIEG in humans is associated with osteoporosis. Gene expression profiling studies identified sclerostin as one of the most highly up-regulated transcripts in the long bones of TIEG KO mice relative to WT littermates suggesting that TIEG may regulate SOST expression. TIEG was shown to substantially suppress SOST promoter activity and the regulatory elements through which TIEG functions were identified using promoter deletion and chromatin immunoprecipitation assays. Knockdown of TIEG in IDG-SW3 osteocyte cells using shRNA and CRISPR-Cas9 technology resulted in increased SOST expression and delayed mineralization, mimicking the results obtained from TIEG KO mouse bones. Given that TIEG is an estrogen regulated gene, and since changes in the hormonal milieu affect SOST expression, we performed ovariectomy (OVX) and estrogen replacement therapy (ERT) studies in WT and TIEG KO mice followed by miRNA and mRNA sequencing of cortical and trabecular compartments of femurs. SOST expression levels were considerably higher in cortical bone compared to trabecular bone. In cortical bone, SOST expression was increased following OVX only in WT mice and was suppressed following ERT in both genotypes. In contrast, SOST expression in trabecular bone was decreased following OVX and significantly increased following ERT. Interestingly, a number of miRNAs that are predicted to target sclerostin exhibited inverse expression levels in response to OVX and ERT. These data implicate important roles for TIEG and estrogen-regulated miRNAs in modulating SOST expression in bone. This article is protected by copyright. All rights reserved
    A comparative evaluation of crowding stress on muscle HSP90 and myostatin expression in salmonids
    Nicholas J. Galt, Aquaculture - 2017
    Abstract
    Stress is a major factor that contributes to poor production and animal welfare concerns in aquaculture. As such, a thorough understanding of mechanisms involved in the stress response is imperative to developing strategies to mitigate the negative side effects of stressors, including the impact of high stocking densities on growth. The purpose of this study was to determine how the muscle growth inhibitor, myostatin, and the stress-responsive gene HSP90 are regulated in response to crowding stress in rainbow trout (Oncorhynchus mykiss), cutthroat trout (Oncorhynchus clarki), brook trout (Salvelinus fontinalis), and Atlantic salmon (Salmo salar). All species exhibited higher cortisol and glucose levels following the handling stress, indicating physiological response to the treatment. Additionally, all species, except rainbow trout, exhibited higher HSP90 levels in muscle after a 48h crowding stress. Crowding stress resulted in a decrease of myostatin-1a in brook trout white muscle but not red muscle, while, myostatin-1a and -2a levels increased in white muscle and myostatin-1b levels increased in red muscle in Atlantic salmon. In rainbow trout, no significant changes were detected in either muscle type, but myostatin-1a was upregulated in both white and red skeletal muscle in the closely related cutthroat trout. The variation in response to crowding suggests a complex and species-specific interaction between stress and the muscle gene regulation in these salmonids. Only Atlantic salmon and cutthroat trout exhibited increased muscle myostatin transcription, and also exhibited the largest increase in circulating glucose in response to crowding. These results suggest that species-specific farming practices should be carefully examined in order to optimize low stress culture conditions.
    T regulatory cell induced Foxp3 binds the IL2, IFNγ, and TNFα promoters in virus-specific CD8+ T cells from feline immunodeficiency virus infected cats.
    Yan Annie Wang, AIDS Research and Human Retroviruses - 2017
    Abstract
    Polyfunctional CD8+ T cells play a critical role in controlling viremia during AIDS lentiviral infections. However, for most HIV infected individuals, virus-specific CD8+ T cells exhibit loss of polyfunctionality including loss of IL2, TNFα, and IFNγ. Using the feline immunodeficiency virus (FIV) model for AIDS lentiviral persistence, our laboratory has demonstrated that FIV-activated Treg cells target CD8+ T cells, leading to a reduction in IL2 and IFNγ production. Further, we have demonstrated that Treg cells induce expression of the repressive transcription factor, Foxp3 in CD8+ T cells. Based upon these findings, we asked if Treg-induced Foxp3 could bind to the IL2, TNFα, and IFNγ promoter regions in virus-specific CD8+ T cells. Following coculture with autologous Treg cells, we demonstrated decreased mRNA levels of IL2 and IFNγ at weeks 4 and 8 post-infection and decreased TNFα at week 4 post infection in virus-specific CD8+ T cells. We also clearly demonstrated Treg cell induced Foxp3 expression in virus-specific CD8+ T cells at weeks 1, 4, and 8 post-infection. Finally, we documented Foxp3 binding to the IL2, TNFα and IFNγ promoters at 8 weeks and 6 months post-infection in virus-specific CD8+ T cells following Treg cell coculture. In summary, the results here clearly demonstrate that Foxp3 inhibits IL2, TNFα and IFNγ transcription by binding to their promoter regions in lentivirus-specific CD8+ T cells. We believe this is the first description of this process during the course of AIDS lentiviral infection.
    Genome-wide binding analysis of AtGNC and AtCGA1 demonstrates their cross-regulation and common and specific functions
    Zhenhua Xu, Plant Direct - 2017
    Abstract
    GATA transcription factors are involved in multiple processes in plant growth and development. Two GATA factors, NITRATE-INDUCIBLE, CARBON METABOLISM-INVOLVED (GNC) and CYTOKININ-RESPONSIVE GATA FACTOR 1 (CGA1, also named GNL), are important regulators in greening, flowering, senescence, and hormone signaling. However, their direct target genes related to these biological processes are poorly characterized. Here, GNC and CGA1 are shown to be transcription activators and by using chromatin immunoprecipitation sequencing (ChIP-seq), 1475 and 638 genes are identified to be associated with GNC and CGA1 binding, respectively. Enrichment of diverse motifs in the peak binding regions for GNC and CGA1 suggests the possibility that these two transcription factors also interact with other transcription factors and in addition genes coding for DNA-binding proteins are highly enriched among GNC- and CGA1-associated genes. Despite the fact that these two GATA factors are known to share a large portion of co-expressed genes, our analysis revealed a low percentage of overlapping binding-associated genes for these two homologues. This suggests a possible cross-regulation between these, which is verified using ChIP-qPCR. The common and specific biological processes regulated by GNC and CGA1 also support this notion. Functional analysis of the binding-associated genes revealed that those encoding transcription factors, E3 ligase, as well as genes with roles in plant development are highly enriched, indicating that GNC and CGA1 mediate complex genetic networks in regulating different aspects of plant growth and development.
    A newly distal hereditary motor neuropathy caused by a rare AIFM1 mutation
    Paula Sancho, neurogenetics - 2017
    Abstract
    In two siblings, who suffer from an early childhood-onset axonal polyneuropathy with exclusive involvement of motor fibers, the c.629T>C (p.F210S) mutation was identified in the X-linked AIFM1 gene, which encodes for the apoptosis-inducing factor (AIF). The mutation was predicted as deleterious, according to in silico analysis. A decreased expression of the AIF protein, altered cellular morphology, and a fragmented mitochondrial network were observed in the proband’s fibroblasts. This new form of motor neuropathy expands the phenotypic spectrum of AIFM1 mutations and therefore, the AIFM1 gene should be considered in the diagnosis of hereditary motor neuropathies.
    Loop-mediated isothermal DNA amplification for asymptomatic malaria detection in challenging field settings: Technical performance and pilot implementation in the Peruvian Amazon
    Elisa Serra-Casas, PLOS ONE - 2017
    Abstract
    Background Loop-mediated isothermal DNA amplification (LAMP) methodology offers an opportunity for point-of-care (POC) molecular detection of asymptomatic malaria infections. However, there is still little evidence on the feasibility of implementing this technique for population screenings in isolated field settings. Methods Overall, we recruited 1167 individuals from terrestrial (‘road’) and hydric (‘riverine’) communities of the Peruvian Amazon for a cross-sectional survey to detect asymptomatic malaria infections. The technical performance of LAMP was evaluated in a subgroup of 503 samples, using real-time Polymerase Chain Reaction (qPCR) as reference standard. The operational feasibility of introducing LAMP testing in the mobile screening campaigns was assessed based on field-suitability parameters, along with a pilot POC-LAMP assay in a riverine community without laboratory infrastructure. Results LAMP had a sensitivity of 91.8% (87.7–94.9) and specificity of 91.9% (87.8–95.0), and the overall accuracy was significantly better among samples collected during road screenings than riverine communities (p≤0.004). LAMP-based diagnostic strategy was successfully implemented within the field-team logistics and the POC-LAMP pilot in the riverine community allowed for a reduction in the turnaround time for case management, from 12–24 hours to less than 5 hours. Specimens with haemolytic appearance were regularly observed in riverine screenings and could help explaining the hindered performance/interpretation of the LAMP reaction in these communities. Conclusions LAMP-based molecular malaria diagnosis can be deployed outside of reference laboratories, providing similar performance as qPCR. However, scale-up in remote field settings such as riverine communities needs to consider a number of logistical challenges (e.g. environmental conditions, labour-intensiveness in large population screenings) that can influence its optimal implementation.
    Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription
    Nicolás Cifuentes-Muñoz, Journal of Virology - 2017
    Abstract
    Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease to all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 Kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positive-sense RNA (+RNA) and genomic RNA (vRNA). Time-course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times post-infection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with translation of viral proteins closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time-course RT-qPCR analysis suggested that coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications. IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatio-temporal analysis of HMPV replication and transcription in bronchial epithelial-derived immortal cells was performed. HMPV was shown to induce formation of large cytoplasmic granules, named inclusion bodies, for genome replication and transcription. Unlike other cytoplasmic structures such as stress granules and P-bodies, inclusion bodies are exclusively present in infected cells and contain HMPV RNA and proteins to more efficiently transcribe and replicate the viral genome. Though nuanced, inclusion body formation corresponds to a more generalized strategy used by different viruses, including filoviruses and rhabdoviruses, for genome transcription and replication. Thus, understanding inclusion body formation is crucial for the discovery of innovative therapeutic targets.
    Solar thermotherapy reduces the titer of Candidatus Liberibacter asiaticus and enhances canopy growth by altering gene expression profiles in HLB-affected citrus plants
    Melissa M Doud, Horticulture Research - 2017
    Abstract
    Huanglongbing (HLB), a systemic and destructive disease of citrus, is associated with ‘Candidatus Liberibacter asiaticus’ (Las) in the United States. Our earlier work has shown that Las bacteria were significantly reduced or eliminated when potted HLB-affected citrus were continuously exposed to high temperatures of 40 to 42 °C for a minimum of 48 h. To determine the feasibility and effectiveness of solar thermotherapy in the field, portable plastic enclosures were placed over commercial and residential citrus, exposing trees to high temperatures through solarization. Within 3–6 weeks after treatment, most trees responded with vigorous new growth. Las titer in new growth was greatly reduced for 18–36 months after treatment. Unlike with potted trees, exposure to high heat did not eradicate the Las population under field conditions. This may be attributed to reduced temperatures at night in the field compared to continuous high temperature exposure that can be maintained in growth chambers, and the failure to achieve therapeutic temperatures in the root zone. Despite the presence of Las in heat-treated commercial citrus, many trees produced abundant flush and grew vigorously for 2 to 3 years after treatment. Transcriptome analysis comparing healthy trees to HLB-affected citrus both before and after heat treatment demonstrated that post-treatment transcriptional expression patterns more closely resembled the expression patterns of healthy controls for most differentially expressed genes and that genes involved with plant-bacterium interactions are upregulated after heat treatment. Overall, these results indicate that solar thermotherapy can be an effective component of an integrated control strategy for citrus HLB.
    Protective role of Indoleamine 2,3 dioxygenase in Respiratory Syncytial Virus associated immune response in airway epithelial cells
    Devi Rajan, Virology - 2017
    Abstract
    RSV is a major cause of severe lower respiratory infection in infants and young children. With no vaccine yet available, it is important to clarify mechanisms of disease pathogenesis. Since indoleamine-2,3-dioxygenase (IDO) is an immunomodulatory enzyme and is upregulated with RSV infection, we studied it in vivo during infection of BALB/c mice and in vitro in A549 cells. RSV infection upregulated IDO transcripts in vivo and in vitro. IDO siRNA decreased IDO transcripts ~2 fold compared to control siRNA after RSV infection but this decrease did not affect RSV replication. In the presence of IFN-γ, siRNA-induced a decrease in IDO expression that was associated with an increase in virus replication and increased levels of IL-6, IL-8, CXCL10 and CCL4. Thus, our results show IDO is upregulated with RSV infection and this upregulation likely participates with IFN-γ in inhibition of virus replication and suppression of some host cell responses to infection.
    High environmental ammonia exposure has developmental-stage specific and long-term consequences on the cortisol stress response in zebrafish
    Tegan A. Williams, General and Comparative Endocrinology - 2017
    Abstract
    The capacity for early life environmental stressors to induce programming effects on the endocrine stress response in fish is largely unknown. In this study we determined the effects of high environmental ammonia (HEA) exposure on the stress response in larval zebrafish, assessed the tolerance of embryonic and larval stages to HEA, and evaluated whether early life HEA exposure has long-term consequences on the cortisol response to a novel stressor. Exposure to 500–2000μM NH4Cl for 16h did not affect the gene expression of corticotropin-releasing factor (CRF) system components in 1day post-fertilization (dpf) embryos, but differentially increased crfa, crfb and CRF binding protein (crfbp) expression and stimulated both dose- and time-dependent increases in the whole body cortisol of 5dpf larvae. Pre-acclimation to HEA at 1dpf did not affect the cortisol response to a subsequent NH4Cl exposure at 5dpf. In contrast, pre-acclimation to HEA at 5dpf caused a small but significant reduction in the cortisol response to a second NH4Cl exposure at 10dpf. While continuous exposure to 500–2000μM NH4Cl between 0 and 5dpf had a modest effect on mean survival time, exposure to 400–1000μM NH4Cl between 10 and 14dpf decreased mean survival time in a dose-dependent manner. Moreover, pre-acclimation to HEA at 5dpf significantly decreased the risk of mortality to continuous NH4Cl exposure between 10 and 14dpf. Finally, while HEA at 1dpf did not affect the cortisol stress response to a novel vortex stressor at 5dpf, the same HEA treatment at 5dpf abolished vortex stressor-induced increases in whole body cortisol at 10 and 60dpf. Together these results show that the impact of HEA on the cortisol stress response during development is life-stage specific and closely linked to ammonia tolerance. Further, we demonstrate that HEA exposure at the larval stage can have persistent effects on the capacity to respond to stressors in later life.
    H3K27 Methylation Dynamics during CD4 T Cell Activation: Regulation of JAK/STAT and IL12RB2 Expression by JMJD3
    Sarah A. LaMere, The Journal of Immunology - 2017
    Abstract
    The changes to the epigenetic landscape in response to Ag during CD4 T cell activation have not been well characterized. Although CD4 T cell subsets have been mapped globally for numerous epigenetic marks, little has been done to study their dynamics early after activation. We have studied changes to promoter H3K27me3 during activation of human naive and memory CD4 T cells. Our results show that these changes occur relatively early (1 d) after activation of naive and memory cells and that demethylation is the predominant change to H3K27me3 at this time point, reinforcing high expression of target genes. Additionally, inhibition of the H3K27 demethylase JMJD3 in naive CD4 T cells demonstrates how critically important molecules required for T cell differentiation, such as JAK2 and IL12RB2, are regulated by H3K27me3. Our results show that H3K27me3 is a dynamic and important epigenetic modification during CD4 T cell activation and that JMJD3-driven H3K27 demethylation is critical for CD4 T cell function.
    Sphingosine Kinase 1 expression in Peritoneal Macrophages is required for Colon Carcinogenesis
    Hideki Furuya, Carcinogenesis - 2017
    Abstract
    Accumulating evidence suggests that the sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate (S1P) pathway plays a pivotal role in colon carcinogenesis. Our previous studies indicate that the SphK1/S1P pathway mediates colon carcinogenesis at least by regulating COX-2 expression and prostaglandin E2 (PGE2) production. However, the mechanisms by which this pathway regulates colon carcinogenesis are still unclear. First, we show that SphK1 deficient mice significantly attenuated azoxymethane (AOM)-induced colon carcinogenesis as measured by colon tumor incidence, multiplicity, and volume. We found that AOM activates peritoneal macrophages to induce SphK1, COX-2, and TNF-α expression in WT mice. Interestingly, SphK1 KO mice revealed significant reduction of COX-2 and TNF-α expression from AOM-activated peritoneal macrophages, suggesting that SphK1 regulates COX-2 and TNF-α expression in peritoneal macrophages. We found that inoculation of WT peritoneal macrophages restored the carcinogenic effect of AOM in Sphk1 KO mice as measured by aberrant crypt foci (ACF) formation, preneoplastic lesions of colon cancer. In addition, downregulation of SphK1 only in peritoneal macrophage by shRNA reduced the number of ACF per colon induced by AOM. Intraperitoneal injection of sphingolipids demonstrates that S1P enhanced AOM-induced ACF formation, while ceramide inhibited. Finally, we show that SphK inhibitor SKI-II significantly reduced the number of ACF per colon. These results suggest that SphK1 expression plays a pivotal role in the early stages of colon carcinogenesis through regulating COX-2 and TNF-α expression from activated peritoneal macrophages.
    Short chain fatty acids ameliorate immune-mediated uveitis partially by altering migration of lymphocytes from the intestine
    Yukiko K. Nakamura, Scientific Reports - 2017
    Abstract
    Short chain fatty acids (SCFA) are metabolites of intestinal bacteria resulting from fermentation of dietary fiber. SCFA are protective in various animal models of inflammatory disease. We investigated the effects of exogenous administration of SFCAs, particularly propionate, on uveitis using an inducible model of experimental autoimmune uveitis (EAU). Oral SCFA administration attenuated uveitis severity in a mouse strain-dependent manner through regulatory T cell induction among lymphocytes in the intestinal lamina propria (LPL) and cervical lymph nodes (CLN). SCFA also suppressed effector T cell induction in the CLN and mesenteric lymph nodes (MLN). Alterations in intestinal morphology and gene expression demonstrated in the EAU model prior to the onset of uveitis were blunted by oral SCFA administration. Using a Kaede transgenic mouse, we demonstrated enhanced leukocyte trafficking between the intestine and the eye in EAU. Propionate suppressed T effector cell migration between the intestine and the spleen in EAU Kaede mice. In conclusion, our findings support exogenous administration of SCFAs as a potential treatment strategy for uveitis through the stabilization of subclinical intestinal alterations that occur in inflammatory diseases including uveitis, as well as prevention of trafficking of leukocytes between the gastrointestinal tract and extra-intestinal tissues.
    Maternal provision of transformer-2 is required for female development and embryo viability in the wasp Nasonia vitripennis
    Elzemiek Geuverink, Insect Biochemistry and Molecular Biology - 2017
    Abstract
    In insect sex determination a primary signal starts the genetic sex determination cascade that, in most insect orders, is subsequently transduced down the cascade by a transformer (tra) ortholog. Only a female-specifically spliced tra mRNA yields a functional TRA-protein that forms a complex with TRA2, encoded by a transformer-2 (tra2) ortholog, to act as a sex specific splicing regulator of the downstream transcription factors doublesex (dsx) and fruitless (fru). Here, we identify the tra2 ortholog of the haplodiploid parasitoid wasp N. vitripennis (Nv-tra2) and confirm its function in N. vitripennis sex determination. Knock down of Nv-tra2 by parental RNA interference (pRNAi) results in complete sex reversal of diploid offspring from female to male, indicating the requirement of Nv-tra2 for female sex determination. As Nv-tra2 pRNAi leads to frequent lethality in early developmental stages, maternal provision of Nv-tra2 transcripts is apparently also required for another, non-sex determining function during embryogenesis. In addition, lethality following Nv-tra2 pRNAi appears more pronounced in diploid than in haploid offspring. This diploid lethal effect was also observed following Nv-tra pRNAi, which served as a positive control in our experiments. As diploid embryos from fertilized eggs have a paternal chromosome set in addition to the maternal one, this suggests that either the presence of this paternal chromosome set or the dosage effect resulting from the diploid state is incompatible with the induced male development in N. vitripennis caused by either Nv-tra2 or Nv-tra pRNAi. The role of Nv-tra2 in activating the female sex determination pathway yields more insight into the sex determination mechanism of Nasonia.
    Cooperative regulation of Gja1 expression by members of the AP-1 family cJun and cFos in TM3 Leydig and TM4 Sertoli cells
    Firas Ghouili, Gene - 2017
    Abstract
    Within the testis, connexin43 encoded by Gja1 plays an important role in cell-to-cell communication between Leydig cells as well as between Sertoli cells and spermatogonia. In the adult male, Leydig cells are the principal producers of testosterone sustaining spermatogenesis, while Sertoli cells nourish, protect and support the differentiating germ cells. It has been shown previously that members of the AP-1 family regulate Gja1 expression in myometrial cells, suggesting that such regulatory mechanism may also be relevant within the testis. Thus, we performed cotransfections of AP-1 expression plasmids with different mouse Gja1 promoter/luciferase reporter constructs within TM3 Leydig and TM4 Sertoli cells. We showed that a functional cooperation between cJun and cFos activates Gja1 expression and requires an AP-1 DNA regulatory element located between − 132 and − 26 bp. In addition, such synergy relies on the recruitment of cFos to this region of the mouse Gja1 promoter. Hence, our data indicate that AP-1 members are important for optimal expression of Gja1 within Sertoli and Leydig cells from the testis.
    Identification of novel biomarkers for MLL translocated acute myeloid leukemia
    Karine Lagacé, Experimental Hematology - 2017
    Abstract
    Acute myeloid leukemias (AML) with translocations of the Mixed Lineage Leukemia (MLL/KMT2A) gene are common in young patients and are generally associated with poor clinical outcomes. The molecular biology of MLL fusion genes remains incompletely characterized and is complicated by the fact that over 100 different partner genes have been identified in fusions with MLL. The continually growing list of MLL fusions also represents a clinical challenge with respect to identification of novel fusions and tracking the fusions to monitor progression of the disease after treatment. We have recently developed a novel single donor model leukemia system that permits the development of human AML from normal cord blood cells. Gene expression analysis of this model and MLL-AML patient samples has identified a number of candidate biomarker genes with highly biased expression on leukemic cells. Here we present the data demonstrating the potential clinical utility of several of these candidate genes for identifying known and novel MLL fusions.
    An LC-MS Approach to Quantitative Measurement of Ammonia Isotopologues
    Jessica B. Spinelli, Scientific Reports - 2017
    Abstract
    Ammonia is a fundamental aspect of metabolism spanning all of phylogeny. Metabolomics, including metabolic tracing studies, are an integral part of elucidating the role of ammonia in these systems. However, current methods for measurement of ammonia are spectrophotometric, and cannot distinguish isotopologues of ammonia, significantly limiting metabolic tracing studies. Here, we describe a novel LC-MS-based method that quantitatively assesses both 14N-and 15N-isotopologues of ammonia in polar metabolite extracts. This assay (1) quantitatively measures the concentration of ammonia in polar metabolite isolates used for metabolomic studies, and (2) accurately determines the percent isotope abundance of 15N-ammonia in a cell lysate for 15N-isotope tracing studies. We apply this assay to quantitatively measure glutamine-derived ammonia in lung cancer cell lines with differential expression of glutaminase.
    Acute blood loss stimulates fibroblast growth factor 23 production
    Seham Rabadi, American Journal of Physiology - Renal Physiology - 2017
    Abstract
    Fibroblast growth factor 23 (FGF23) production is upregulated by iron deficiency and hypoxia. However, the influence of acute blood loss, and the resulting increases in circulating erythropoietin, on FGF23 production is unknown. Using wild-type C57BL/6 mice, we show that acute loss of 10% total blood volume leads to an increase in plasma C-terminal FGF23 (cFGF23) levels within six hours, while plasma levels of intact FGF23, phosphate, calcium, parathyroid hormone, iron, and ferritin remain similar to control mice without acute blood loss. Volume resuscitation with PBS did not significantly alter these findings. The increase in plasma cFGF23 levels in bled animals was accompanied by increased plasma erythropoietin levels at 6 hours. Administration of erythropoietin led to an acute increase in plasma cFGF23 levels similar to that observed in acute blood loss. Fgf23 mRNA expression was increased 20-fold in bone marrow, but not in bone, of bled versus control mice, suggesting bone marrow as a key source of elevated plasma FGF23 levels following acute blood loss. To extend these findings to humans, we measured plasma cFGF23 levels in 131 critically ill patients admitted to the intensive care unit. In univariate and multivariate models, we found a positive association between number of red blood cell transfusions, an indirect indicator of acute blood loss, and plasma cFGF23 levels. We conclude that FGF23 production is rapidly increased after acute blood loss, and that erythropoietin may be the mediator of this increase. Thus, erythropoietin may represent a novel physiologic regulator of FGF23 production.
    Expression of the chemokine receptors CCR1 and CCR2B is up-regulated in peripheral blood B cells upon EBV infection and in established lymphoblastoid cell lines
    Irina Kholodnyuk, Virology - 2017
    Abstract
    In immunocompetent individuals, EBV establishes in B cells an asymptomatic lifelong latent infection controlled by the immune system. Chemokine receptors regulate immune system function. CCR1 and CCR2 share protein sequence similarity and exert responses to multiple chemokines. The role of these receptors in B cells is largely unknown. We show that the mRNA and functional protein expression of CCR1 and CCR2 is induced in ex vivo B cells upon EBV infection and in established lymphoblastoid cell lines (LCLs). The CCR1 and CCR2B ORF transcripts were determined in LCLs. In contrast, in both the EBV-negative and EBV-positive Burkitt lymphoma cell lines, neither the CCR1, CCR2A, and CCR2B ORF transcripts nor their corresponding proteins were detected. Our data suggest that CCR1/CCR2B could be involved in clearing EBV-infected latency III B cells in immunocompetent individuals via directing the migration of these cells and attracting the chemokines-expressing immune cells.
    Dietary broccoli impacts microbial community structure and attenuates chemically induced colitis in mice in an Ah receptor dependent manner
    Troy D. Hubbard, Journal of Functional Foods - 2017
    Abstract
    Consumption of broccoli mediates numerous chemo-protective benefits through the intake of phytochemicals, some of which modulate aryl hydrocarbon receptor (AHR) activity. Whether AHR activation is a critical aspect of the therapeutic potential of dietary broccoli is not known. Here we administered isocaloric diets, with or without supplementation of whole broccoli (15% w/w), to congenic mice expressing the high-affinity Ahrb/b or low-affinity Ahrd/d alleles, for 24 days and examined the effects on AHR activity, intestinal microbial community structure, inflammatory status, and response to chemically induced colitis. Cecal microbial community structure and metabolic potential were segregated according to host dietary and AHR status. Dietary broccoli associated with heightened intestinal AHR activity, decreased microbial abundance of the family Erysipelotrichaceae, and attenuation of colitis. In summary, broccoli consumption elicited an enhanced response in ligand-sensitive Ahrb/b mice, demonstrating that in part the beneficial aspects of dietary broccoli upon intestinal health are associated with heightened AHR activity.
    Reptin regulates insulin-stimulated Akt phosphorylation in hepatocellular carcinoma via the regulation of SHP-1/PTPN6
    Anne-Aurélie Raymond, Cell Biochemistry and Function - 2017
    Abstract
    Hepatocellular carcinoma (HCC) is the main primary cancer of the liver. Many studies have shown that insulin resistance is a risk factor for HCC. We previously discovered the overexpression and oncogenic role of the Reptin/RUVBL2 ATPase in HCC. Here, we found that Reptin silencing enhanced insulin sensitivity in 2 HCC cell lines, as shown by a large potentiation of insulin-induced AKT phosphorylation on Ser473 and Thr308, and of downstream signalling. Reptin silencing did not affect the tyrosine phosphorylation of the insulin receptor nor of IRS1, but it enhanced the tyrosine phosphorylation of the p85 subunit of PI3K. The expression of the SHP-1/PTPN6 phosphatase, which dephosphorylates p85, was reduced after Reptin depletion. Forced expression of SHP-1 restored a normal AKT phosphorylation after insulin treatment in cells where Reptin was silenced, demonstrating that the downregulation of SHP1 is mechanistically linked to increased Akt phosphorylation. In conclusion, we have uncovered a new function for Reptin in regulating insulin signalling in HCC cells via the regulation of SHP-1 expression. We suggest that the regulation of insulin sensitivity by Reptin contributes to its oncogenic action in the liver.
    Long-term correction of diabetic hyperglycemia through glucose-responsive hepatic insulin production using lentivirus
    Handorf Am, Journal of Diabetology and Endocrinolog - 2017
    Abstract
    Type 1 diabetes mellitus (T1DM) is caused by the autoimmune destruction of the insulin-producing β cells of the pancreas. Insulin gene therapy is a promising strategy capable of overcoming the limitations of current treatments, but to become a viable option, it must provide long-term, glucose-responsive control of insulin production. We have previously achieved glucose-responsivity by incorporating glucose-inducible response elements (GIREs) upstream of a liver-specific insulin expression cassette (3xGIRE.ALB.Ins1-2xfur). In this study, 3xGIRE.ALB.Ins1-2xfur was delivered into streptozotocin-induced diabetic rats using lentivirus, resulting in remission of diabetic hyperglycemia for at least 482 days while restoring rate of weight gain in a dose-dependent fashion. Insulin immunostaining showed abundant insulin production in the liver, and qPCR showed 13-20 lentiviral integrations per cell in the liver of rats treated with high dose lentivirus. Negligible integration was found in the pancreas, kidney, spleen and muscle of LV-treated rats, confirming liver specificity. In vitro, LV.3xGIRE.ALB.Ins1-2xfur produced a 4.5-fold increase in insulin production in high glucose conditions, and in vivo, a 1.7-fold increase in insulin levels was found during an intraperitoneal glucose tolerance test. Unfortunately, limitations in large-scale lentivirus production and use of a tissue-specific promoter prevented treatment of more than one rat per batch of lentivirus. Thus, two of the LV-treated diabetic rats were undertreated, while another two rats were over treated, becoming hypoglycemic in the fed state. Nonetheless, we have established the framework for a long-term, glucose-responsive treatment for T1DM from which further improvements can be made.
    Sakacin G is the main responsible bacteriocin for the anti-listerial activity of meat-borne Lactobacillus curvatus ACU-1
    Mónica Adriana Mechoud, Annals of Microbiology - 2017
    Abstract
    The present study was conducted to quantify the expression of the sakacins produced by Lactobacillus curvatus ACU-1, a strain isolated from artisanal dry fermented sausages of Argentina. Polymerase chain reaction (PCR) screening indicated the presence of sakacin G, P, and Q genes in L. curvatus ACU-1. Purification and activity assays determined that anti-Listeria activity was mainly associated to sakacin G, as mass spectrometry analysis revealed a single peak of 3832.60 Da. Further characterization by quantitative PCR demonstrated that L. curvatus ACU-1 transcription of the sakacin G structural gene was three orders of magnitude higher than the others. Interestingly, L. curvatus ACU-1 had skgA1/skgA2 as well as sppQ genes encoded in a plasmid, while the sppA gene that encodes for sakacin P was present in the bacterial chromosome. These results point out that sakacin G is the main peptide responsible for the anti-listerial activity of L. curvatus ACU-1, with little or no contribution of sakacin P and sakacin Q. The high level of expression of sakacin G demonstrated in the present work would facilitate its potential use in food preservation, improving the food quality, safety, and shelf life. In addition, the sakacin G promoter may serve as an interesting tool for the expression of other bacteriocins at higher levels.
    PMK-1 p38 MAPK promotes cadmium stress resistance, the expression of SKN-1/Nrf and DAF-16 target genes, and protein biosynthesis in Caenorhabditis elegans
    Alex Keshet, Molecular Genetics and Genomics - 2017
    Abstract
    The mechanisms of cadmium (Cd) resistance are complex and not sufficiently understood. The present study, therefore, aimed at assessing the roles of important components of stress-signaling pathways and of ABC transporters under severe Cd stress in Caenorhabditis elegans. Survival assays on mutant and control animals revealed a significant promotion of Cd resistance by the PMK-1 p38 MAP kinase, the transcription factor DAF-16/FoxO, and the ABC transporter MRP-1. Transcriptome profiling by RNA-Seq on wild type and a pmk-1 mutant under control and Cd stress conditions revealed, inter alia, a PMK-1-dependent promotion of gene expression for the translational machinery. PMK-1 also promoted the expression of target genes of the transcription factors SKN-1/Nrf and DAF-16 in Cd-stressed animals, which included genes for molecular chaperones or immune proteins. Gene expression studies by qRT-PCR confirmed the positive effects of PMK-1 on DAF-16 activity under Cd stress and revealed negative effects of DAF-16 on the expression of genes for MRP-1 and DAF-15/raptor. Additional studies on pmk-1 RNAi-treated wild type and mutant strains provided further information on the effects of PMK-1 on SKN-1 and DAF-16, which resulted in a model of these relationships. The results of this study demonstrate a central role of PMK-1 for the processing of cellular responses to abiotic and biotic stressors, with the promoting effects of PMK-1 on Cd resistance mostly mediated by the transcription factors SKN-1 and DAF-16.
    Blood and Nasal Epigenetics Correlate to Allergic Rhinitis Symptom Development in the Environmental Exposure Unit
    Michelle L. North, Allergy - 2017
    Abstract
    Background Epigenetic alterations may represent new therapeutic targets and/or biomarkers of allergic rhinitis (AR). Our aim was to examine genome-wide epigenetic changes induced by controlled pollen exposure in the Environmental Exposure Unit (EEU). Methods 38 AR-sufferers and 8 non-allergic controls were exposed to grass pollen for 3h on two consecutive days. We interrogated DNA methylation at baseline and 3h in peripheral blood mononuclear cells (PBMCs) using the Infinium Methylation 450K array. We corrected for demographics, cell composition, and multiple testing (Benjamini-Hochberg), and verified hits using bisulfite PCR-pyrosequencing and qPCR. To extend these findings to a clinically relevant tissue, we investigated DNA methylation and gene expression of mucin 4 (MUC4), in nasal brushings from a separate validation cohort exposed to birch pollen. Results In PBMCs of allergic rhinitis participants, 42 sites showed significant DNA methylation changes of 2% or greater. DNA methylation changes in tryptase gamma 1 (TPSG1), schlafen 12 (SLFN12) and MUC4 in response to exposure were validated by pyrosequencing. SLFN12 DNA methylation significantly correlated with symptoms (p<0.05), and baseline DNA methylation pattern was found to be predictive of symptom severity upon grass allergen exposure (p<0.05). Changes in MUC4 DNA methylation in nasal brushings in the validation cohort correlated with drop in peak nasal inspiratory flow (Spearman r = 0.314, p = 0.034), and MUC4 gene expression was significantly increased (p<0.0001). Conclusion This study revealed novel and rapid epigenetic changes upon exposure in a controlled allergen challenge facility, identified baseline epigenetic status as a predictor of symptom severity. This article is protected by copyright. All rights reserved.
    Selective Inhibition of Janus Kinase 3 Has No Impact on Infarct Size or Neurobehavioral Outcomes in Permanent Ischemic Stroke in Mice
    Kelly M. DeMars, Frontiers in Neurology - 2017
    Abstract
    Janus kinase 3 (JAK3) is associated with the common gamma chain of several interleukin receptors essential to inflammatory signaling. To study the potential role of JAK3 in stroke-induced neuroinflammation, we subjected mice to permanent middle cerebral artery occlusion, and investigated the effects of JAK3 inhibition with decernotinib (VX-509) on infarct size, behavior, and levels of several inflammatory mediators. Results from our double immunofluorescence staining showed JAK3 expression on neurons, endothelial cells, and microglia/macrophages in the ischemic mouse brain (n=3). We found for the first time that total as well as phosphorylated/activated JAK3 are dramatically increased after stroke in the ipsilateral hemisphere (**P<0.01; n=5-13/group) in addition to increased IL-21 expression after stroke (**P<0.01; n=5-7/group). However, inhibition of JAK3 confirmed by reduced phosphorylation of its activation loop at tyrosine residues 980/981 does not reduce infarct volume measured at 48 h after stroke (n=6-10/group) nor does it alter behavioral outcomes sensitive to neurological deficits or stroke-induced neuroinflammatory response (n=9-10/group). These results do not support a detrimental role for JAK3 in acute neuroinflammation following permanent focal cerebral ischemia. The functional role of increased JAK3 activation after stroke remains to be further investigated.
    Treatment with antioxidants ameliorates oxidative damage in a mouse model of propionic acidemia
    Ana Rivera-Barahona, Molecular Genetics and Metabolism - 2017
    Abstract
    Oxidative stress contributes to the pathogenesis of propionic acidemia (PA), a life threatening disease caused by the deficiency of propionyl CoA-carboxylase, in the catabolic pathway of branched-chain amino acids, odd-number chain fatty acids and cholesterol. Patients develop multisystemic complications including seizures, extrapyramidal symptoms, basal ganglia deterioration, pancreatitis and cardiomyopathy. The accumulation of toxic metabolites results in mitochondrial dysfunction, increased reactive oxygen species and oxidative damage, all of which have been documented in patients' samples and in a hypomorphic mouse model. Here we set out to investigate whether treatment with a mitochondria-targeted antioxidant, MitoQ, or with the natural polyphenol resveratrol, which is reported to have antioxidant and mitochondrial activation properties, could ameliorate the altered redox status and its functional consequences in the PA mouse model. The results show that oral treatment with MitoQ or resveratrol decreases lipid peroxidation and the expression levels of DNA repair enzyme OGG1 in PA mouse liver, as well as inducing tissue-specific changes in the expression of antioxidant enzymes. Notably, treatment decreased the cardiac hypertrophy marker BNP that is found upregulated in the PA mouse heart. Overall, the results provide in vivo evidence to justify more in depth investigations of antioxidants as adjuvant therapy in PA.
    Loss of Calreticulin Uncovers a Critical Role for Calcium in Regulating Cellular Lipid Homeostasis
    Wen-An Wang, Scientific Reports - 2017
    Abstract
    A direct link between Ca2+ and lipid homeostasis has not been definitively demonstrated. In this study, we show that manipulation of ER Ca2+ causes the re-distribution of a portion of the intracellular unesterified cholesterol to a pool that is not available to the SCAP-SREBP complex. The SREBP processing pathway in ER Ca2+ depleted cells remained fully functional and responsive to changes in cellular cholesterol status but differed unexpectedly in basal activity. These findings establish the role of Ca2+ in determining the reference set-point for controlling cellular lipid homeostasis. We propose that ER Ca2+ status is an important determinant of the basal sensitivity of the sterol sensing mechanism inherent to the SREBP processing pathway.
    BITC and S-Carvone Restrain High-Fat Diet-Induced Obesity and Ameliorate Hepatic Steatosis and Insulin Resistance
    Sary Alsanea, Pharmaceutical Research - 2017
    Abstract
    PurposeTo investigate the preventative activity of benzyl isothiocyante and S-carvone against high-fat diet-induced obesity and metabolic complications.MethodsTen-week-old C57BL/6 male mice were fed a high-fat diet and injected intraperitoneally twice per week with benzyl isothiocyante, S-carvone, or vehicle for 8 weeks. The body weight, food intake, and body composition were monitored, and glucose tolerance and insulin tolerance tests were performed at the end of the experiment. Serum and tissue samples were studied using serum biochemistry, histological, and gene expression analysis to define the effects of benzyl isothiocyante and S-carvone treatments on lipid and glucose metabolism and inflammatory responses.ResultsBenzyl isothiocyante and S-carvone blocked high-fat diet-induced weight gain, fat accumulation in the liver, and insulin resistance. The beneficial effects were found to be associated with an improvement of expression of macrophage marker genes in white adipose tissue, including F4/80, Cd11b, Cd11c, Cd206, and Tnf-α, and reduced expression of genes (Pparγ2, Scd1, Cd36) responsible for lipid synthesis and transport in the liver.ConclusionBenzyl isothiocyante and S-carvone block high-fat diet-induced obesity and metabolism disorders and can be considered for management of the obesity epidemic that affects approximately 36% of adults and 17% of children in the USA.
    Impact of environmental microbiota on human microbiota of workers in academic mouse research facilities: An observational study
    Peggy S. Lai, PLOS ONE - 2017
    Abstract
    Objectives To characterize the microbial environment of workers in academic mouse research facilities using endotoxin, 16S qPCR, and 16S amplicon sequencing. To determine whether the work microbiome contributes to the human microbiome of workers. Methods We performed area air sampling from the animal rooms, dirty, middle, and setup cage wash locations in four academic mouse research facilities. 10 workers in the dirty cage wash area underwent personal air sampling as well as repeated collection of nasal, oral, and skin samples before and after the work shift. Environmental samples underwent measurement of endotoxin, mouse allergen, bacteria copy number via 16S qPCR, and microbial identification via 16S rDNA sequencing. 16S rDNA sequencing was also performed on human samples before and after the work shift. SourceTracker was used to identify the contribution of the work microbiome to the human microbiome. Results Median endotoxin levels ranged from undetectable to 1.0 EU/m3. Significant differences in mouse allergen levels, bacterial copy number, microbial richness, and microbial community structure were identified between animal, dirty, middle, and setup cage wash locations. Endotoxin levels had only a moderate correlation with microbial composition. Location within a facility was a stronger predictor of microbial community composition (R2 = 0.41, p = 0.002) than facility. The contribution of the work microbiome to the pre-shift human microbiome of workers was estimated to be 0.1 ± 0.1% for the oral microbiome; 3.1 ± 1.9% for the nasal microbiome; and 3.0 ± 1.5% for the skin microbiome. Conclusions The microbial environment of academic animal care facilities varies significantly by location rather than facility. Endotoxin is not a proxy for assessment of environmental microbial exposures using 16S qPCR or 16S rDNA sequencing. The work microbiome contributes to the composition of the nasal and skin microbiome of workers; the clinical implications of this observation should be further studied.
    Numerical Relationships Between Archaeal and Bacterial amoA Genes Vary by Icelandic Andosol Classes
    Hendrikus J. Laanbroek, Microbial Ecology - 2017
    Abstract
    Bacterial amoA genes had not been detectable by qPCR in freshly sampled Icelandic Andosols thus far. Hence, a new primer set yielding shorter gene fragments has been designed to verify the absence of ammonia-oxidizing bacteria in different Icelandic Andosol classes. At the same time, a new primer set was also constructed for archaeal amoA genes that should improve the quality of PCR products. Although a large part of the soil samples were found to be amoA-negative, bacterial amoA genes were detectable with new as well as old primer sets. The same results were obtained for the archaeal amoA genes. The relative distribution of archaeal and bacterial amoA genes varied between Andosol classes. Archaeal amoA genes were significantly more abundant in Brown than in Histic Andosols, while the opposite was observed for bacterial amoA genes. The numbers of archaeal and bacterial amoA genes in Gleyic Andosols were not significantly different from those in Histic and Brown Andosols. The numbers of bacterial amoA genes, but not the numbers of archaeal amoA genes, correlated significantly and positively with potential ammonia oxidation activities. The presence of the bacterial nitrification inhibitor allylthiourea inhibited the potential ammonia oxidation activities during the first 12 h of incubation. Hence, it was concluded that ammonia-oxidizing bacteria profited most from the conditions during the measurements of potential ammonia oxidation activities.
    Bioaccessibility, bioavailability and anti-inflammatory effects of anthocyanins from purple root vegetables using mono- and co-culture cell models
    Hua Zhang, Molecular Nutrition & Food Research - 2017
    Abstract
    Scope Immune-inflammatory, signalling and metabolic effects are the main pillars for bioactivity of anthocyanins derived from highly pigmented root vegetables. This study aims to assess the bioaccessibility and bioavailability of purple carrot and potato derived anthocyanins and the molecular mechanisms of their ability to ameliorate cellular inflammation in a mono- and co-culture cell models. Methods and Results An in vitro gastrointestinal model was used and demonstrated bioaccessibility of 44.62% and 71.8% for anthocyanins of purple carrot and potato, respectively. These accessible anthocyanins significantly inhibited cellular inflammation in Caco-2 cells. Intact cyanidin glycoside or petunidin glycoside (respectively from carrots and potatoes) were transported across a transmembrane cell model and detected by LC-MS/MS. Computational docking and glucose uptake analyses suggested uptake of anthocyanins was mediated by hexose transporters. Subsequent experiment using an inflamed Caco-2 BBe1/THP-1 co-culture cell model showed these transported anthocyanins inhibited IL-8 and TNF-α secretion, and expression of pro-inflammatory cytokines by blocking NF-κB, and MAPK mediated inflammatory cellular signalling cascades, but with varying degrees due to structural features. Conclusion Anthocyanins from purple carrots and potatoes possess a promising anti-inflammatory effect in model gut system. They can be absorbed and act differently but are in general beneficial for inflammation-mediated diseases. This article is protected by copyright. All rights reserved
    Use of a real-time PCR to explore the intensity of Plasmodium spp. infections in native, endemic and introduced New Zealand birds
    D. C. Sijbranda, Cambridge Core - 2017
    Abstract
    SUMMARY Avian malaria, caused by Plasmodium spp., is an emerging disease in New Zealand (NZ). To detect Plasmodium spp. infection and quantify parasite load in NZ birds, a real-time polymerase chain reaction (PCR) (qPCR) protocol was used and compared with a nested PCR (nPCR) assay. A total of 202 blood samples from 14 bird species with known nPCR results were tested. The qPCR prevalences for introduced, native and endemic species groups were 70, 11 and 21%, respectively, with a sensitivity and specificity of 96·7 and 98%, respectively, for the qPCR, while a sensitivity and specificity of 80·9 and 85·4% were determined for the nPCR. The qPCR appeared to be more sensitive in detecting lower levels of parasitaemia. The mean parasite load was significantly higher in introduced bird species (2245 parasites per 10 000 erythrocytes) compared with endemic species (31·5 parasites per 10 000 erythrocytes). In NZ robins (Petroica longipes), a significantly lower packed cell volume was found in birds that were positive for Plasmodium spp. compared with birds that were negative. Our data suggest that introduced bird species, such as blackbirds (Turdus merula), have a higher tolerance for circulating parasite stages of Plasmodium spp., indicating that introduced species are an important reservoir of avian malaria due to a high infection prevalence and parasite load.
    Pigment epithelium derived factor play a positive role in bone mineralization of osteoblasts derived from diabetic patients
    Na Song, Gene - 2017
    Abstract
    Pigment epithelium-derived factor (PEDF) is a multifunctional secreted protein which plays important role in anti-angiogenic, anti-tumorigenic, as well as involves in the metabolism and regeneration of bone. In this study, our aim is to investigate the role of PEDF in regulating mineralization of osteoblasts from diabetic patients (DP). We isolated and cultured osteoblasts derived from DP and non-diabetic patients (NDP), in order to analyze the variable differences via gene expression and calcification assay in vitro. Gene expression analysis and alizarin red S staining revealed that osteoblasts from DP exhibited defective mineralization. PEDF and vascular endothelial growth factor (VEGF) levels were lower in osteoblasts from DP than those from NDP. Interestingly, exogenous PEDF could upregulate the gene expression levels of VEGF and osteoblast-related genes, further to restore mineralization ability in osteoblasts from DP. Our results demonstrated that PEDF played a positive role in maintaining bone development in diabetic osteoblasts, therefore, we confidently believe that PEDF may be a promising cytokine to consider in development of treatments for diabetic bone diseases.
    Simple and fast quantification of DNA damage by real-time PCR, and its application to nuclear and mitochondrial DNA from multiple tissues of aging zebrafish
    Shusen Zhu, BMC Research Notes - 2017
    Abstract
    We describe a real-time (rt) PCR-based method of quantifying DNA damage, adapted from the long-run rtPCR method of DNA damage quantification (LORD-Q) developed by Lehle et al. (Nucleic Acids Res 42(6):e41, 2014). We show that semi-long run rtPCR, which generates amplicons half the length of those generated in LORD-Q, provides equivalent sensitivity for detecting low lesion frequencies, and better sensitivity for detecting high frequencies. The smaller amplicon size greatly facilitates PCR optimization and allows greater flexibility in the use of detection dyes, and a modified data analysis method simplifies the calculation of lesion frequency. The method was used to measure DNA damage in the nuclear and mitochondrial genomes of different tissues in zebrafish of different ages. We find that nuclear DNA damage generally increases with age, and that the amount of mitochondrial DNA damage varies substantially between tissues, increasing with age in liver and brain but not in heart or skeletal muscle, the latter having the highest levels of damage irrespective of age.
    Curcumin Protects Skin against UVB-Induced Cytotoxicity via the
    Maya Ben Yehuda Greenwald, Hindawi Oxidative Medicine and Cellular Longevity - 2017
    Abstract
    Curcumin was found to be beneficial in treating several skin pathologies and diseases, providing antioxidant protection due to its reducing properties and its electrophilic properties (the ability to activate the Nrf2 pathway and induce phase II cytoprotective enzymes). Nevertheless, clinical applications of curcumin are being hampered by its insufficient solubility, chemical instability,and poor absorption, leading to low efficacy in preventing skin pathologies. These limitations can be overcome by using a nanotechnology-based delivery system. Here, we elucidated the possibility of using curcumin encapsulated in a microemulsion preserving its unique chemical structure. We also examined whether curcumin microemulsion would reduce UVB-induced toxicity in skin. A significant curcumin concentration was found in the human skin dermis following topical application of a curcumin microemulsion. Moreover, curcumin microemulsion enhanced the reduction of UV-induced cytotoxicity in epidermal cells, paving the way for other incorporated electrophiles in encapsulated form protecting skin against stress-related diseases.
    Increased incidence of non-alcoholic fatty liver disease in male rat offspring exposed to fluoxetine during fetal and neonatal life involves the NLRP3 inflammasome and augmented de novo hepatic lipogenesis
    Nicole E.De Long, Journal of Applied Toxicology - 2017
    Abstract
    Up to 10% of women take selective serotonin reuptake inhibitors (SSRI) during pregnancy. Children exposed to SSRIs in utero have an increased risk of being overweight suggesting that fetal exposure to SSRIs can cause permanent metabolic changes. We have previously shown in rats that fetal and neonatal exposure to the SSRI antidepressant fluoxetine results in metabolic perturbations including increased hepatic triglyceride content; a hallmark of non-alcoholic fatty liver disease (NAFLD). Therefore, the aim of this study was to identify the mechanism(s) underlying the fluoxetine-induced increase in intrahepatic triglyceride content. Female nulliparous Wistar rats were given vehicle or fluoxetine (10 mg/kg/day) orally for 2 weeks prior to mating until weaning. At 6 months of age, we assessed whether SSRI exposure altered components of the hepatic triglyceride biosynthesis pathway in the offspring and examined the molecular mechanisms underlying these changes. Male SSRI-exposed offspring had a significant increase in the steady-state mRNA levels of Elovl6 and Dgat1 and core components of the NLRP3 inflammasome (apoptosis-associated speck-like protein containing a caspase activation recruitment domain [ASC] and caspase-1). Augmented expression of Asc in the SSRI-exposed offspring coincided with increased histone acetylation in the proximal promoter region. Given that we have previously demonstrated that antenatal exposure to SSRIs can lead to fatty liver in the offspring, this raises concerns regarding the long-term metabolic sequelae of fetal SSRI exposure. Moreover, this study suggests that elevated hepatic triglyceride levels observed in the SSRI-exposed offspring may be due, in part, to activation of the NLRP3 inflammasome and augmentation of de novo lipogenesis.
    YY1 Is Required for Posttranscriptional Stability of SOX2 and OCT4 Proteins
    Mary C.Wallingford, Cellular Reprogramming - 2017
    Abstract
    Yinyang1 (YY1) participates in protein-DNA, protein-RNA, and protein–protein interactions and regulates developmental processes and disease mechanisms. YY1 interactions regulate a range of important biological functions, including oocyte maturation, epithelial to mesenchymal transition, and vascular endothelial growth factor (VEGF) signaling. We tested the hypothesis that YY1 is required for inner cell mass (ICM) lineage commitment during preimplantation development. In this study, we document gene expression patterns and protein localization of key transcription factors in Yy1 global, tissue-specific, and dsRNA-mediated knockout/down embryos. YY1 protein was found in cells of preimplantation and peri-implantation embryos, and adult tissues where two isoforms are observed. In the absence of YY1, OCT4 and SOX2 protein were lost in the ICM during preimplantation and naive neuroectoderm during gastrulation stages, yet no difference in Oct4 or Sox2 mRNA levels was observed. The loss of OCT4 and SOX2 protein occurred specifically in cells that normally express both OCT4 and SOX2 protein. These observations support a role for YY1 meditating and/or regulating the interaction of OCT4 and SOX2 at a posttranscriptional level. Our results suggest that distinct mechanisms of YY1-mediated molecular regulation are present in the early embryo, and may offer insight to promote lineage commitment in in vitro cell lines.
    Increasing corn distillers solubles alters the liquid fraction of the ruminal microbiome
    J. C.McCann, Journal of Animal Science - 2017
    Abstract
    Five ruminally fistulated steers were used in a 5 × 5 Latin square design to determine the effects of increasing dietary fat and sulfur from condensed distiller’s solubles (CDS) on the ruminal microbiome. Treatments included a corn-based control (CON) and 4 levels of CDS (0, 10, 19, and 27%) in a coproduct-based (corn gluten feed and soybean hulls) diet. Fat concentrations were 1.79, 4.43, 6.80, and 8.91% for diets containing 0, 10, 19, and 27% CDS, respectively. Steers were fed for ad libitum intake once daily. After feeding each diet for 18 d, ruminal samples were collected 3 h after feeding on d 19. Samples were separated into solid and liquid fractions. Microbial DNA was extracted for bacterial analysis using paired-end sequencing of the V3 through V4 region of the 16S rRNA gene on the MiSeq Illumina platform and quantitative PCR of selected species. Orthogonal contrasts were used to determine linear and quadratic effects of CDS inclusion. Increasing CDS inclusion decreased (linear, P < 0.05) α-diversity and species richness in the liquid fraction. Analysis of Bray–Curtis similarity indicated a treatment effect (P = 0.01) in the liquid fraction. At the phyla level, relative abundance of Bacteroidetes decreased in steers fed increasing dietary inclusion of CDS as Firmicutes increased to 82% of sequences for the 27% CDS treatment. Family Ruminococcaceae increased (linear, P < 0.01) 2-fold in the liquid fraction when feeding CDS increased from 0 to 27% CDS, yet genera Ruminococcus tended (P = 0.09) to decrease in steers fed greater CDS. The most abundant family of sulfate-reducing bacteria, Desulfovibrionaceae, increased (P < 0.03) in the solid and liquid fraction in steers fed additional dietary CDS and sulfur. Relative abundance of family Veillonellaceae and Selenomonas ruminantium were increased (linear, P ≤ 0.02) in the solid fraction as steers were fed increasing CDS. There were no effects (P > 0.10) of feeding increasing dietary fat from CDS on fibroylytic genus Fibrobacter in either fraction. Results demonstrate increasing fat and sulfur from CDS in a coproduct-based diet markedly alters the liquid fraction ruminal microbiome but does not elicit negative effects on relative abundance of identified fiber-fermenting bacteria.
    Pancreatic Beta Cells Express the Fetal Islet Hormone Gastrin in Rodent and Human Diabetes
    Tehila Dahan, Diabetes - 2016
    Abstract
    Beta-cell failure in type 2 diabetes (T2D) was recently proposed to involve dedifferentiation of beta-cells and ectopic expression of other islet hormones, including somatostatin and glucagon. Here we show that gastrin, a stomach hormone typically expressed in the pancreas only during embryogenesis, is expressed in islets of diabetic rodents and humans with T2D. While in mice gastrin is expressed insulin+ cells, in humans with T2D gastrin expression occurs in both insulin+ and somatostatin+ cells. Genetic lineage tracing in mice indicates that gastrin expression is turned on in a subset of differentiated beta-cells following exposure to severe hyperglycemia. Gastrin expression in adult beta-cells does not involve the endocrine progenitor cell regulator NeuroG3 but requires membrane depolarization, calcium influx and calcineurin signaling. In vivo and in vitro experiments show that gastrin expression is rapidly eliminated upon exposure of beta cells to normal glucose levels. These results reveal the fetal hormone gastrin as a novel marker for reversible human beta-cell reprogramming in diabetes.
    Contribution of asparagine catabolism to Salmonella virulence
    Patrick A. McLaughlin, Infection and Immunity - 2016
    Abstract
    Salmonellae are pathogenic bacteria that cause significant morbidity and mortality in humans worldwide. Salmonellae establish infection and avoid clearance by the immune system by mechanisms that are not well understood. We previously showed that L-Asparaginase II produced by Salmonella enterica serovar Typhimurium (S. Typhimurium) inhibits T cell responses and mediates virulence. In addition, we previously showed that asparagine deprivation such as that mediated by L-Asparaginase II of S. Typhimurium causes suppression of activation-induced T cell metabolic reprogramming. Here, we report that STM3997, which encodes a homolog of disulfide bond protein A (dsbA) of Escherichia coli, is required for L-Asparaginase II stability and function. Furthermore, we report that L-Asparaginase II localizes primarily to the periplasm and acts together with L-Asparaginase I to provide S. Typhimurium the ability to catabolize asparagine and assimilate nitrogen. Importantly, we determined that, in a murine model of infection, S. Typhimurium lacking both L-Asparaginase I and II genes compete poorly with wild-type S. Typhimurium for colonization of target tissues. Collectively, these results indicate that asparagine catabolism contributes to S. Typhimurium virulence, providing new insights into the competition for nutrients at the host pathogen interface.
    Physiological characterization of drought stress response and expression of two transcription factors and two LEA genes in three Prunus genotypes
    Beatriz Bielsa, Scientia Horticulturae - 2016
    Abstract
    Global warming has led to a progressive decrease in rainfall, which is reflected by a reduction of water resources in the soil and a negative effect on crop production in Mediterranean areas. Under drought stress, many plants react by inducing a different series of responses at both physiological and molecular levels, allowing them to survive for a variable period of time. Therefore, in order to understand the response of roots to drought conditions, the genotypes peach × almond ‘Garnem’ [P. amygdalus Batsch × P. persica (L.) Batsch] and their progeny, the hybrid ‘P.2175’ × ‘Garnem’-3 and OP-‘P.2175’ (P. cerasifera Ehrh.) were subjected to a period of water deficit. Drought conditions with a subsequent re-watering period were tested for potted plants for one month. Stomatal conductance and leaf water potential were measured to monitor the plant physiological responses. Significant differences among the drought stress and drought stress recovery treatments and among the genotypes were observed. In addition, four genes related to the ABA biosynthesis pathway were studied for their expression by RT-qPCR: an AN20/AN1 zinc finger protein (ppa012373m); a bZIP transcription factor (ppa013046m); a dehydrin (ppa005514m) and a LEA protein (ppa008651m). Their expression profiles correlated with our physiological results of drought response, being higher in roots than in phloem tissue. In general, the expression of the four studied genes was higher after 15 days under drought conditions. Under drought and recovery conditions, the zinc finger and bZIP transcription factors showed significant differences in their relative expression levels from LEA and dehydrin. These results suggest the role of LEA and dehydrin in the regulatory response to drought stress in Prunus genotypes. Therefore, the dehydrin and the protein LEA might be potential biomarkers to select rootstocks for tolerance to drought conditions.
    Evolution of subcutaneous adipose tissue fibrosis after bariatric surgery
    M.S Chabot, Diabetes & Metabolism - 2016
    Abstract
    Obesity is associated with the development of metabolic complications such as insulin resistance (IR). The mechanisms leading to IR remain unclear. This study aimed to investigate the relationship between adipose tissue fibrosis and IR in obese patients before and after bariatric surgery.
    Ribosome biogenesis is dynamically regulated during osteoblast differentiation
    Cynthia L. Nebena,, Science Direct - 2016
    Abstract
    Changes in ribosome biogenesis are tightly linked to cell growth, proliferation, and differentiation. The rate of ribosome biogenesis is established by RNA Pol I-mediated transcription of ribosomal RNA (rRNA). Thus, rRNA gene transcription is a key determinant of cell behavior. Here, we show that ribosome biogenesis is dynamically regulated during osteoblast differentiation. Upon osteoinduction, osteoprogenitor cells transiently silence a subset of rRNA genes through a reversible mechanism that is initiated through biphasic nucleolar depletion of UBF1 and then RNA Pol I. Nucleolar depletion of UBF1 is coincident with an increase in the number of silent but transcriptionally permissible rRNA genes. This increase in the number of silent rRNA genes reduces levels of ribosome biogenesis and subsequently, protein synthesis. Together these findings demonstrate that fluctuations in rRNA gene transcription are determined by nucleolar occupancy of UBF1 and closely coordinated with the early events necessary for acquisition of the osteoblast cell fate.
    Sphingomyelinase-like phosphodiesterase 3b mediates radiation-induced damage of renal podocytes
    Anis Ahmad, The FASEB Journal - 2016
    Abstract
    The molecular mechanisms responsible for the development of proteinuria and glomerulosclerosis in radiation nephropathy remain largely unknown. Podocytes are increasingly recognized as key players in the pathogenesis of proteinuria in primary and secondary glomerular disorders. The lipid-modulating enzyme sphingomyelin phosphodiesterase acid-like 3B (SMPDL3b) is a key determinant of podocyte injury and a known off target of the anti-CD20 antibody rituximab (RTX). The current study investigates the role of sphingolipids in radiation-induced podocytopathy. After a single dose of radiation (8 Gy), several ceramide species were significantly elevated. In particular, C16:00, C24:00, and C24:1 ceramides were the most abundant ceramide species detected. These changes were paralleled by a time-dependent drop in SMPDL3b protein, sphingosine, and sphingosine-1-phosphate levels. Interestingly, SMPDL3b overexpressing podocytes had higher basal levels of sphingosine-1-phosphate and maintained basal ceramide levels after irradiation. Morphologically, irradiated podocytes demonstrated loss of filopodia and remodeling of cortical actin. Furthermore, the actin binding protein ezrin relocated from the plasma membrane to the cytosol as early as 2 h after radiation. In contrast, SMPDL3b overexpressing podocytes were protected from radiation-induced cytoskeletal remodeling. Treatment with RTX before radiation exposure partially protected podocytes from SMPDL3b loss, cytoskeletal remodeling, and caspase 3 cleavage. Our results demonstrate that radiation injury induces early cytoskeletal remodeling, down-regulation of SMPDL3b, and elevation of cellular ceramide levels. Overexpression of SMPDL3b and pretreatment with RTX confer a radioprotective effect in cultured podocytes. These findings indicate a potential role for SMPDL3b and RTX in radiation-induced podocytopathy.—Ahmad, A., Mitrofanova, A., Bielawski, J., Yang, Y., Marples, B., Fornoni, A., Zeidan, Y. H. Sphingomyelinase-like phosphodiesterase 3b mediates radiation-induced damage of renal podocytes.
    Comparison of miRNA signature versus conventional biomarkers before and after off-pump coronary artery bypass graft
    Fatemeh Pourrajab, Journal of Pharmaceutical and Biomedical Analysis - 2016
    Abstract
    Circulating levels of microRNAs (miRNAs) and their expression patterns are supposed to serve as signatures for diagnosis or prognosis of cardiovascular events. The present study aimed at determining if there is any correlation between the release pattern of 2 miRNAs and the plasma levels of conventional biomarkers cardiac troponin I (cTnI), creatine kinase (CK) and uric acid (UA) in patients undergoing their first off-pump coronary artery bypass graft (OCABG). Seventy OCABG patients (69% men, aged 59.2 ± 8.2 years) were enrolled. Emergencies, re-operations, abnormal preoperative serum cTnI and combined procedures were excluded from this study. Pre-operative mean ejection fraction was 45.8 ± 8.6%, the average number of grafts was 3 ± 0.87/patient, and the internal mammary artery was used for all. Beside conventional clinical assays, we performed real-time quantitative PCR to analyze the circulating levels of miR-155, miR-126 and miR-499 at 1 day before surgery as well as 4 days after surgery. Importantly, there was no report of myocardial infarction in our patients, pre- or post-operatively. In contrast to conventional biomarkers cTnI and CK, circulating levels of miRNAs decreased significantly (P < 0.01) after revascularization surgery. A significant positive correlation was seen between the cTnI and miR-499 (r ∼ 0.53, P < 0.01) and between miR-126 and UA (r ∼ 0.5, P < 0.01). Time course study of circulating miR-499, miR-126 and miR-155 in cardiac surgery clarified their advantage and correlations to the traditional biomarkers cTnI, total CK, CK-MB and UA. Our results suggest that this signature is a novel, early biomarker which indicates myocardial ischemia in cardiac surgery. It could be postulated that the application of these miRNAs may be considered for monitoring of response to pharmacological interventions aimed at reducing cardiac ischemia, especially in OCABG candidates.
    Secretoglobin Superfamily Protein SCGB3A2 Alleviates House Dust Mite-Induced Allergic Airway Inflammation in Mice
    M. Yoneda, International Archives of Allergy and Immunology - 2016
    Abstract
    Background: Secretoglobin (SCGB) 3A2, a novel, lung-enriched, cytokine-like, secreted protein of small molecular weight, was demonstrated to exhibit various biological functions includin
    Functional Roles of Acetylated Histone Marks at Mouse Meiotic Recombination Hotspots
    Irina V.Getun, Molecular and Cellular Biology - 2016
    Abstract
    Meiotic recombination initiates following the formation of DNA double strand breaks (DSBs) by the Spo11 endonuclease early in prophase I at discrete regions in the genome coined hotspots. In mammals, meiotic DSB site selection is directed in part by sequence specific binding of PRDM9, a polymorphic histone H3 (H3K4Me3) methyltransferase. However, other chromatin features needed for meiotic hotspot specification are largely unknown. Here, we show that the recombinogenic cores of active hotspots in mice harbor several histone H3 and H4 acetylation and methylation marks that are typical of open, active chromatin. Further, deposition of these open chromatin-associated histone marks is dynamic and is manifest at spermatogonia and/or pre-leptotene meiotic stage cells, which would facilitate PRDM9 binding and access for Spo11 to direct the formation of DSBs, which are initiated at leptotene. Importantly, manipulating histone acetylase and deacetylase activity established that histone acetylation marks are necessary for both hotspot activity and crossover resolution. We conclude there are functional roles for histone acetylation marks at mammalian meiotic recombination hotspots.
    The Kallikrein-Kinin System: A Novel Mediator of IL-17-Driven Anti-Candida Immunity in the Kidney
    Kritika Ramani, PLOS Pathogens - 2016
    Abstract
    Author Summary Candida albicans is the causative agent of oropharyngeal candidiasis (OPC, thrush), dermal and vaginal candidiasis. However, the most severe C. albicans-induced disease is disseminated candidiasis, a frequent nosocomial infection associated with a high mortality rate. During disseminated candidiasis, C. albicans form invasive hyphae that damage target organs, particularly kidney and liver. Previous studies have identified an essential role of interleukin-17 (IL-17) in controlling systemic infection through regulation of neutrophils. We show here for the first time that IL-17 also regulates the renal protective Kallikrein-kinin system (KKS). Our discovery of a connection between IL-17 and the KKS suggests a new, previously unanticipated avenue for the treatment of renal damage in disseminated candidiasis. These findings have potential translational significance, as agonists of the KKS are in routine clinical use. Therefore, these results not only identify downstream mediators that could serve as novel drug targets, but could possibly be used to guide decisions on whether targeting these mediators could be a useful therapeutic option in conjunction with current antifungal therapies.
    Pharmacological treatment and BBB-targeted genetic therapy for MCT8-dependent hypomyelination in zebrafish
    David Zada, Disease Models & Mechanisms - 2016
    Abstract
    Skip to Next Section Hypomyelination is a key symptom of Allan-Herndon-Dudley syndrome (AHDS), a psychomotor retardation associated with mutations in the thyroid-hormone (TH) transporter MCT8 (monocarboxylate transporter 8). AHDS is characterized by severe intellectual deficiency, neuromuscular impairment and brain hypothyroidism. In order to understand the mechanism for TH-dependent hypomyelination, we developed an mct8 mutant (mct8−/−) zebrafish model. The quantification of genetic markers for oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes revealed reduced differentiation of OPCs into oligodendrocytes in mct8−/− larvae and adults. Live imaging of single glial cells showed that the number of oligodendrocytes and the length of their extensions are reduced, and the number of peripheral Schwann cells is increased, in mct8−/− larvae compared with wild type. Pharmacological analysis showed that TH analogs and clemastine partially rescued the hypomyelination in the CNS of mct8−/− larvae. Intriguingly, triiodothyronine (T3) treatment rescued hypomyelination in mct8−/− embryos before the maturation of the blood–brain barrier (BBB), but did not affect hypomyelination in older larvae. Thus, we expressed Mct8-tagRFP in the endothelial cells of the vascular system and showed that even relatively weak mosaic expression completely rescued hypomyelination in mct8−/− larvae. These results suggest potential pharmacological treatments and BBB-targeted gene therapy that can enhance myelination in AHDS and possibly in other TH-dependent brain disorders.
    The neuropeptide galanin modulates natural killer cell function
    Andreas Koller, Neuropeptides - 2016
    Abstract
    Natural killer (NK) cells are part of the innate immune system and combat pathogens and tumors by secreting pro-inflammatory cytokines like interferon gamma (IFN-γ) and by their cytotoxic action. Galanin is a neuropeptide also expressed in peripheral tissue where it impacts several physiological functions, including inflammation. The effects of galanin are mediated via three receptors, GAL1–3. Since other neuropeptides have been shown to regulate NK cell activity, we investigated the potential of galanin to modulate human NK cell function. NK cells were isolated from human peripheral blood mononuclear cells. mRNA expression was analyzed by qRT-PCR. The dynamic mass redistribution of NK cells upon regulatory peptide stimulation was determined by label-free biochip technology. IFN-γ producing NK cells were identified by flow cytometry analysis and IFN-γ secretion was measured by ELISA. NK cell cytotoxicity was analyzed by flow cytometry via CD107a mobilization. NK cells were found to express the receptor GAL2 but not GAL1, GAL3 or galanin. Galanin per se did not affect the dynamic mass redistribution of NK cells, but significantly enhanced the response of NK cells to IL-18. Galanin significantly modulated the IFN-γ production of the CD56bright NK cell population upon IL-12 and IL-18 stimulation. Furthermore, galanin significantly modulated the IL-12 and IL-18 stimulated IFN-γ secretion. NK cell cytotoxicity was not modulated by galanin treatment. Galanin can be classified as an immunomodulatory peptide as it is able to sensitize NK cells toward specific cytokines.
    Effects of soybean meal on digestive enzymes activity, expression of inflammation-related genes, and chromatin modifications in marine fish (Sparus aurata L.) larvae
    Erick Perera, Fish Physiology and Biochemistry - 2016
    Abstract
    The effects of soybean meal (SBM) in early diet of Sparus aurata larvae at two developmental windows were assessed. Prolonged (beyond 14 days post-hatch, dph) feeding with SBM decreased the activity of pancreatic enzymes of larvae. In the absence of SBM these larvae later resumed enzyme activities, but exhibited a significant delay in development. Larvae response to SBM involved up-regulation of extracellular matrix remodeling enzymes and pro-inflammatory cytokines, coupled with a drop in putative intestinal enzymes. Larvae receiving SBM at first feeding appear later to have lower expression of inflammation-related genes, especially those fed SBM until 14 dph. Multivariate analysis confirmed that the duration of the SBM early feeding period drives the physiology of larvae in different directions. Feeding larvae with SBM increased global histone H3 acetylation, whereas upon removal of SBM the process was reverted. A more in deep analysis revealed a dynamic interplay among several reversible histone modifications such as H3K14ac and H3K27m3. Finally, we showed that SBM feeding of larvae results in global hypomethylation that persist after SBM removal. This study is the first demonstrating an effect of diet on marine fish epigenetics. It is concluded that there are limitations for extending SBM feeding of S. aurata larvae beyond 14 dph even under co-feeding with live feed, affecting key physiological processes and normal growth. However, up to 14 dph, SBM does not affect normal development, and produces apparently lasting effects on some key enzymes, genes, and chromatin modifications.
    ADAM17 in tumor associated leukocytes regulates inflammatory mediators and promotes mammary tumor formation
    Laura R. Bohrer1,*, Genes and Cancer - 2016
    Abstract
    The presence of inflammatory cells within the tumor microenvironment has been tightly linked to mammary tumor formation and progression. Specifically, interactions between tumor cells and infiltrating macrophages can contribute to the generation of a pro-tumorigenic microenvironment. Understanding the complex mechanisms that drive tumor cell-macrophage cross-talk will ultimately lead to the development of approaches to prevent or treat early stage breast cancers. As described here, we demonstrate that the cell surface protease a disintegrin and metalloproteinase 17 (ADAM17) is expressed by macrophages in mammary tumors and contributes to regulating the expression of pro-inflammatory mediators, including inflammatory cytokines and the inflammatory mediator cyclooxygenase-2 (Cox-2). Furthermore, we demonstrate that ADAM17 is expressed on leukocytes, including macrophages, within polyoma middle T (PyMT)-derived mammary tumors. Genetic deletion of ADAM17 in leukocytes resulted in decreased onset of mammary tumor growth, which was associated with reduced expression of the Cox-2 within the tumor. These findings demonstrate that ADAM17 regulates key inflammatory mediators in macrophages and that leukocyte-specific ADAM17 is an important promoter of mammary tumor initiation. Understanding the mechanisms associated with early stage tumorigenesis has implications for the development of preventive and/or treatment strategies for early stage breast cancers.
    PHACTR1 Is a Genetic Susceptibility Locus for Fibromuscular Dysplasia Supporting Its Complex Genetic Pattern of Inheritance
    Soto Romuald, PLOS Genet - 2016
    Abstract
    Author Summary Fibromuscular Dysplasia (FMD) is a vascular disease characterized by a succession of occlusions and dilatation of medium-sized arteries (e.g renal, carotid or coronary arteries) with important health consequences, mainly resistant hypertension and stroke. FMD is an atypical vascular disease because it is not associated with overweight or dyslipidemia and 80% of patients are early middle aged women. Our genetic study conducted in >1100 patients and >3800 controls demonstrate that a common variant rs9349379 located on chromosome 6 in the phosphatase and actin regulator 1 gene ( PHACTR1 ) increases by ~40% the risk of FMD. This is the first time a genetic risk factor is reported for FMD because it has been longtime considered rare and potentially under a Mendelian mode of inheritance. We also show that rs9349379 correlates with the expression of PHACTR1 in fibroblasts from FMD patients and controls. Interestingly, the same allele that increases the risk of FMD is at risk for cervical artery dissection and migraine, often reported in FMD patients but protective from myocardial infarction and coronary disease, where atherosclerosis is more common. The clear role of PHACTR1 in maintaining vascular well integrity is not fully elucidated. Using a specific antibody we detected PHACTR1 both on endothelial and smooth muscle cells of human FMD and control carotids, which suggests that PHACTR1 may have multiple functions depending on the cell type and the degree of atherosclerosis of the arteries.
    Gli transcription factors mediate the oncogenic transformation of prostate basal cells induced by a Kras-androgen receptor axis
    Meng Wu, Journal of Biological Chemistry - 2016
    Abstract
    Although the differentiation of oncogenically transformed basal progenitor cells is one of the key steps in prostate tumorigenesis, the mechanisms mediating this cellular process are still largely unknown. Here we demonstrated that an expanded p63+ and CK5+ basal/progenitor cell population, induced by the concomitant activation of oncogenic Kras(G12D) and androgen receptor (AR) signaling, underwent cell differentiation in vivo. The differentiation process led to suppression of the p63 expressing cells with a decreased number of CK5+ basal cells, but an increase of CK8+ luminal tumorigenic cells, and revealed a hierarchal lineage pattern consisting of p63+/CK5+ progenitor, CK5+/CK8+ transitional progenitor, and CK8+ differentiated luminal cells. Further analysis of the phenotype showed that the Kras-AR axis induced tumorigenesis was mediated by Gli transcription factors. Combined blocking of the activators of this family of proteins (Gli1 and Gli2) inhibited the proliferation of p63+ and CK5+ basal/progenitor cells and development of tumors. Finally, we identified that Gli1 and Gli2 exhibited different functions in regulation of p63 expression or proliferation of p63+ cells in Kras-AR driven tumors. Gli2, but not Gli1, transcriptionally regulated the expression levels of p63 and prostate sphere formation. Our study provides evidence of a novel mechanism mediating pathological dysregulation of basal/progenitor cells through the differential activation of the Gli transcription factors. Also, these findings define Gli proteins as new downstream mediators of the Kras-AR axis in prostate carcinogenesis and open a potential therapeutic avenue of targeting prostate cancer progression by inhibiting Gli signaling.
    CRH peptide evolution occurred in three phases: Evidence from characterizing sea lamprey CRH system members
    Matthew J. Endsin, General and Comparative Endocrinology - 2016
    Abstract
    The corticotropin releasing hormone (CRH) system, which includes the CRH family of peptides, their receptors (CRHRs) and a binding protein (CRHBP), has been strongly conserved throughout vertebrate evolution. The identification of invertebrate homologues suggests this system evolved over 500 million years ago. However, the early vertebrate evolution of the CRH system is not understood. Current theory indicates that agnathans (hagfishes and lampreys) are monophyletic with a conservative evolution over the past 500 million years and occupy a position at the root of vertebrate phylogeny. We isolated the cDNAs for three CRH family members, two CRHRs and a CRHBP from the sea lamprey, Petromyzon marinus. Two of the CRH peptides are related to the CRH/urotensin-1 (UI) lineage, whereas the other is a urocortin (Ucn) 3 orthologue. The predicted amino acid identity of CRH and UI is 61% but they possess distinct motifs indicative of each peptide, suggesting an early divergence of the two genes. Based on our findings we propose the CRH peptides evolved in at least 3 distinct phases. The first occurring prior to the agnathans gave rise to the CRH/UI-like and Ucn2/3-like paralogous lineages. The second was a partial sub-genomic duplication of the ancestral CRH/UI-like gene, but not the Ucn2/3-like gene, giving rise to the CRH and UI (Ucn) lineages. The third event which resulted in the appearance of Ucn2 and Ucn3 must have occurred after the evolution of the cartilaginous fishes. Interestingly, unlike other vertebrate CRHRs, we were unable to classify our two P. marinus receptors (designated CRHRα and CRHRβ) as either type 1 or type 2, indicating that this split evolved later in vertebrate evolution. A single CRHBP gene was found suggesting that either this gene has not been affected by the vertebrate genome duplications or there have been a series of paralogous gene deletions. This study suggests that P. marinus possess a functional CRH system that differs from that of the gnathostomes and may represent a model for the earliest functioning CRH system in vertebrates.
    Frontiers | The Arabidopsis Transcription Factor ANAC032 Represses Anthocyanin Biosynthesis in Response to High Sucrose and Oxidative and Abiotic Stresses | Plant Physiology
    Kashif Mahmood, Frontiers in Plant Science - 2016
    Abstract
    Production of anthocyanins is one of the adaptive responses employed by plants during stress conditions. During stress, anthocyanin biosynthesis is mainly regulated at the transcriptional level via a complex interplay between activators and repressors of anthocyanin biosynthesis genes. In this study, we investigated the role of a NAC transcription factor, ANAC032, in the regulation of anthocyanin biosynthesis during stress conditions. ANAC032 expression was found to be induced by exogenous sucrose as well as high light (HL) stress. Using biochemical, molecular and transgenic approaches, we show that ANAC032 represses anthocyanin biosynthesis in response to sucrose treatment, HL and oxidative stress. ANAC032 was found to negatively affect anthocyanin accumulation and the expression of anthocyanin biosynthesis (DFR, ANS/LDOX) and positive regulatory (TT8) genes as demonstrated in overexpression line (35S:ANAC032) compared to wild-type under HL stress. The chimeric repressor line (35S:ANAC032-SRDX) exhibited the opposite expression patterns for these genes. The negative impact of ANAC032 on the expression of DFR, ANS/LDOX and TT8 was found to be correlated with the altered expression of negative regulators of anthocyanin biosynthesis, AtMYBL2 and SPL9. In addition to this, ANAC032 also repressed the MeJA- and ABA-induced anthocyanin biosynthesis. As a result, transgenic lines overexpressing ANAC032 (35S:ANAC032) produced drastically reduced levels of anthocyanin pigment compared to wild-type when challenged with salinity stress. However, transgenic chimeric repressor lines (35S:ANAC032-SRDX) exhibited the opposite phenotype. Our results suggest that ANAC032 functions as a negative regulator of anthocyanin biosynthesis in Arabidopsis thaliana during stress conditions
    Acute and long‐term effects of blood flow restricted training on heat shock proteins and endogenous antioxidant systems
    K. T. Cumming, Scandinavian Journal of Medicine & Science in Sports - 2016
    Abstract
    Blood flow restricted exercise (BFRE) with low loads has been demonstrated to induce considerable stress to exercising muscles. Muscle cells have developed a series of defensive systems against exercise-induced stress. However, little is known about acute and long-term effects of BFRE training on these systems. Nine previously untrained females trained low-load BFRE and heavy load strength training (HLS) on separate legs and on separate days to investigate acute and long-term effects on heat shock proteins (HSP) and endogenous antioxidant systems in skeletal muscles. BFRE and HLS increased muscle strength similarly by 12 ± 7% and 12 ± 6%, respectively, after 12 weeks of training. Acutely after the first BFRE and HLS exercise session, αB-crystallin and HSP27 content increased in cytoskeletal structures, accompanied by increased expression of several HSP genes. After 12 weeks of training, this acute HSP response was absent. Basal levels of αB-crystallin, HSP27, HSP70, mnSOD, or GPx1 remained unchanged after 12 weeks of training, but HSP27 levels increased in the cytoskeleton. Marked translocation of HSP to cytoskeletal structures at the commencement of training indicates that these structures are highly stressed from BFRE and HLS. However, as the muscle gets used to this type of exercise, this response is abolished.
    Mutations in TSPEAR , Encoding a Regulator of Notch Signaling, Affect Tooth and Hair Follicle Morphogenesis
    Alon Peled, PLOS Genet - 2016
    Abstract
    Author Summary Ectodermal dysplasias refer to a large group of inherited disorders characterized by developmental defects in tissues of ectodermal origin. The study of these conditions has been instrumental in the discovery of biological pathways involved in the regulation of epithelial tissue morphogenesis. In this report, through the delineation of the molecular basis of a novel form of autosomal recessive ectodermal dysplasia, we identified a new key player in ectodermal development. We detected a number of mutations in TSPEAR co-segregating with abnormal hair and tooth development in three families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR was found to be strongly expressed in murine hair and tooth. Using a reporter assay, we showed that it regulates Notch activity. Accordingly, NOTCH1 expression was altered in patient skin, and NOTCH1, as well as many of its known targets, was down-regulated in TSPEAR deficient keratinocytes. Moreover, Tspear silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch pathway. As such, these new data are likely to lead to further investigations aimed at characterizing the role of Notch signaling pathway in other forms of ectodermal dysplasias as well as acquired hair and tooth pathologies.
    Extensive cryptic splicing upon loss of RBM17 and TDP43 in neurodegeneration models
    Qiumin Tan, Human Molecular Genetics - 2016
    Abstract
    Splicing regulation is an important step of post-transcriptional gene regulation. It is a highly dynamic process orchestrated by RNA-binding proteins (RBPs). RBP dysfunction and global splicing dysregulation have been implicated in many human diseases, but the in vivo functions of most RBPs and the splicing outcome upon their loss remain largely unexplored. Here we report that constitutive deletion of Rbm17, which encodes an RBP with a putative role in splicing, causes early embryonic lethality in mice and that its loss in Purkinje neurons leads to rapid degeneration. Transcriptome profiling of Rbm17-deficient and control neurons and subsequent splicing analyses using CrypSplice, a new computational method that we developed, revealed that more than half of RBM17-dependent splicing changes are cryptic. Importantly, RBM17 represses cryptic splicing of genes that likely contribute to motor coordination and cell survival. This finding prompted us to re-analyze published datasets from a recent report on TDP-43, an RBP implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as it was demonstrated that TDP-43 represses cryptic exon splicing to promote cell survival. We uncovered a large number of TDP-43-dependent splicing defects that were not previously discovered, revealing that TDP-43 extensively regulates cryptic splicing. Moreover, we found a significant overlap in genes that undergo both RBM17- and TDP-43-dependent cryptic splicing repression, many of which are associated with survival. We propose that repression of cryptic splicing by RBPs is critical for neuronal health and survival.
    TGF-β1 promotes linear invadosome formation in hepatocellular carcinoma cells, through DDR1 up-regulation and collagen I cross-linking
    Zakaria Ezzoukhry, European Journal of Cell Biology - 2016
    Abstract
    Transforming growth factor-β1 (TGF-β1) is an important player in chronic liver diseases inducing fibrogenesis and hepatocellular carcinoma (HCC) development. TGF-β1 promotes pleiotropic modifications at the cellular and matrix microenvironment levels. TGF-β1 was described to enhance production of type I collagen and its associated cross-linking enzyme, the lysyl oxidase-like2 (LOXL2). In addition, TGF-β1 and type I collagen are potent inducers of invadosomes. Indeed, type I collagen fibers induce the formation of active linear invadosomes through the discoidin domain receptor 1 (DDR1). The goal of our study was to address the role of TGF-β1 in collagen cross-linking and its impact on the formation of linear invadosomes in liver cancer cells. We first report a significant correlation between expressions of TGF-β1, and type I collagen, LOXL2, DDR1 and MT1-MMP in human HCCs. We demonstrate that TGF-β1 promotes a Smad4-dependent up-regulation of DDR1, together with LOXL2, in cultured HCC cells. Moreover, we show that LOXL2-induced collagen cross-linking enhances linear invadosome formation. Altogether, our data demonstrate that TGF-β1 favors linear invadosome formation through the expressions of both the inducers, such as collagen and LOXL2, and the components such as DDR1 and MT1-MMP of linear invadosomes in cancer cells. Meanwhile, our data uncover a new TGF-β1-dependent regulation of DDR1 expression.
    Expression of T helper cell–associated inflammatory mediator mRNAs in cells of bronchoalveolar lavage fluid samples and oxygen concentration in arterial blood samples from healthy horses exposed to hyperbaric oxygen
    Maty G. P. Looijen, American Journal of Veterinary Research - 2016
    Abstract
    OBJECTIVE To evaluate the mRNA expression of T helper (Th)1, Th2, and Th17 cell–associated inflammatory mediators in cells of bronchoalveolar lavage fluid samples collected from healthy horses exposed to hyperbaric oxygen (HBO) and to monitor blood oxygen concentration during and following HBO therapy. ANIMALS 8 healthy horses. PROCEDURES In a randomized controlled crossover design study, each horse was exposed (beginning day 1) to 100% oxygen at a maximum of 3 atmospheres absolute (304 kPa) daily for 10 days or ambient air at atmospheric pressure in the HBO chamber for an equivalent amount of time (control). Bronchoalveolar lavage fluid samples were collected on days 0 and 10. After validation of candidate reference genes, relative mRNA expressions of various innate inflammatory, Th1 cell–derived, Th2 cell–derived (including eotaxin-2), Th17 cell–derived, and regulatory cytokines were measured by quantitative PCR assays. For 3 horses, arterial blood samples were collected for blood gas analysis during a separate HBO session. RESULTS The optimal combination of reference genes was glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine ribosyltransferase, and ribosomal protein L32. Compared with day 0 findings, expression of eotaxin-2 mRNA was significantly lower (0.12-fold reduction) and the percentage of neutrophils in bronchoalveolar lavage fluid samples was significantly lower on day 10 when horses received HBO therapy. Values of Pao2 rapidly increased (> 800 mm Hg) but immediately decreased to pretreatment values when HBO sessions ended. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that HBO therapy does not increase mRNA expression of inflammatory cytokines, but reduces eotaxin-2 mRNA transcription. The Pao2 increase was transient with no cumulative effects of HBO.
    Influence of Donor Age and Stimulation Intensity on Osteogenic Differentiation of Rat Mesenchymal Stromal Cells in Response to Focused Low-Intensity Pulsed Ultrasound
    Regina Puts, Ultrasound in Medicine & Biology - 2016
    Abstract
    A focused low-intensity pulsed ultrasound (FLIPUS) was used to investigate the effects of stimulation period, acoustic intensity and donor age on the osteogenic differentiation potential of rat mesenchymal stromal cells (rMSCs). rMSCs from 3- and 12-mo-old female Sprague Drawly rats were isolated from bone marrow and stimulated 20 min/d with either 11.7 or 44.5 mW/cm2 (spatial average temporal average intensity) for 7 or 14 d. Osteogenic differentiation markers, i.e., Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and degree of matrix calcification were analyzed. On day 7 of stimulation, OCN gene expression was enhanced 1.9-fold in cells from young rats when stimulated with low intensity. The low intensity also led to a 40% decrease in RUNX2 expression on day 7 in aged cells, whereas high intensity enhanced expression of RUNX2 on day 14. FLIPUS treatment with low intensity resulted in a 15% increase in extracellular matrix mineralization in young but not old rMSCs. These differences suggest the necessity of a donor-age related optimization of stimulation parameters.
    Targeted Gene Activation Using RNA-Guided Nucleases
    Ulf Andersson Ørom, Methods in Molecular Biology - 2016
    Abstract
    The discovery of the prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) system and its adaptation for targeted manipulation of DNA in diverse species has revolutionized the field of genome engineering. In particular, the fusion of catalytically inactive Cas9 to any number of transcriptional activator domains has resulted in an array of easily customizable synthetic transcription factors that are capable of achieving robust, specific, and tunable activation of target gene expression within a wide variety of tissues and cells. This chapter describes key experimental design considerations, methods for plasmid construction, gene delivery protocols, and procedures for analysis of targeted gene activation in mammalian cell lines using CRISPR-Cas transcription factors.
    Expression of the aryl hydrocarbon receptor contributes to the establishment of intestinal microbial community structure in mice
    Iain A. Murray, Scientific Reports - 2016
    Abstract
    Environmental and genetic factors represent key components in the establishment/maintenance of the intestinal microbiota. The aryl hydrocarbon receptor (AHR) is emerging as a pleiotropic factor, modulating pathways beyond its established role as a xenobiotic sensor.
    Effect of rearing temperature on the hypoxia response of embryonic zebrafish
    Kelly Levesque, The University of Guelph - 2016
    Abstract
    Environmental stressors, such as warm temperatures and hypoxia, can interact and pose a threat to aquatic species. Cross-talk between the hypoxia and heat stress cellular pathways can lead to enhanced cross-tolerance between these environmental stressors. In this study, I asked whether elevated temperatures(from 27°C to 32°C)during rearing would enhance the hypoxia-inducible transcription factor-1(HIF-1) mediated transcriptional response to hypoxia in early stages of zebrafish development and whether these differences would be associated with enhanced larval tolerance and survivalto hypoxia. I found that embryos reared at 32°C had an enhanced cellular HIF-1 response and that acute hypoxia activated the heat-shock response. Rearing at 32°C and/ or embryonichypoxia exposure had no effect on thehypoxia tolerance(Pcrit) of four day-old larvae and did not protect larvae against the lethal effects of a second acute hypoxia exposure.Overall, cross-talk at the gene expression level did not predict whole animal responses when larvae were reexposed to hypoxic conditions .
    Potential vectors of Xylella fastidiosa: a study of leafhoppers and treehoppers in citrus agroecosystems affected by Citrus Variegated Chlorosis
    G. Dellapé, Entomologia Experimentalis et Applicata - 2016
    Abstract
    This study investigated the predominant leafhopper and treehopper (Hemiptera, Auchenorrhyncha) species in Citrus Variegated Chlorosis (CVC)-affected citrus agroecosystems in Argentina, their seasonal fluctuation, and their potential role as vectors of Xylella fastidiosa Wells et al., using molecular methods for detection. More than 6 000 Auchenorrhyncha were collected from three citrus agroecosystems over a period of 3 years using yellow sticky traps and entomological nets. Cicadellidae and Membracidae were the most abundant families. Of the 43 species identified, five were predominant in citrus orchards, and three were predominant in weeds surrounding citrus plants. All predominant species and another four non-predominant species tested positive for X. fastidiosa in PCR and real-time PCR assays. In a transmission assay, Dechacona missionum (Berg), Tapajosa rubromarginata (Signoret), and Cyphonia clavigera (Fabricius) transmitted X. fastidiosa successfully. Scaphytopius bolivianus Oman and Frequenamia spiniventris (Linnavuori) populations increased once (during the summer), possibly due to favorable weather conditions, and Bucephalogonia xanthophis (Berg), Molomea lineiceps Young, and T. rubromarginata populations increased twice a year: once in summer and once in winter, coinciding with the increase in early citrus shoots (flush). Among the X. fastidiosa-positive species, those with the higher population densities during the sprouting period, where trees are highly susceptible to infection, must be considered as most relevant vectors of CVC in the citrus-growing areas in Argentina.
    Biased Signalling is an Essential Feature of TLR4 in Glioma Cells
    Marie-Theres Zeuner, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research - 2016
    Abstract
    A distinct feature of the Toll-like receptor 4 (TLR4) is its ability to trigger both MyD88-dependent and MyD88-independent signalling, culminating in activation of pro-inflammatory NF-κB and/or the antiviral IRF3. Although TLR4 agonists (lipopolysaccharides; LPSs) derived from different bacterial species have different endotoxic activity, the impact of LPS chemotype on the downstream signalling is not fully understood. Notably, different TLR4 agonists exhibit anti-tumoural activity in animal models of glioma, but the underlying molecular mechanisms are largely unknown. Thus, we investigated the impact of LPS chemotype on the signalling events in the human glioma cell line U251. We found that LPS of Escherichia coli origin (LPSEC) leads to NF-κB-biased downstream signalling compared to Salmonella minnesota-derived LPS (LPSSM). Exposure of U251 cells to LPSEC resulted in faster nuclear translocation of the NF-κB subunit p65, higher NF-κB-activity and expression of its targets genes, and higher amount of secreted IL-6 compared to LPSSM. Using super-resolution microscopy we showed that the biased agonism of TLR4 in glioma cells is neither a result of differential regulation of receptor density nor of formation of higher order oligomers. Consistent with previous reports, LPSEC-mediated NF-κB activation led to significantly increased U251 proliferation, whereas LPSSM-induced IRF3 activity negatively influenced their invasiveness. Finally, treatment with methyl-β-cyclodextrin (MCD) selectively increased LPSSM-induced nuclear translocation of p65 and NF-κB activity without affecting IRF3. Our data may explain how TLR4 agonists differently affect glioma cell proliferation and migration.
    Recovery of antigen-specific T cell responses from dogs infected with Leishmania (L.) infantum by use of vaccine associated TLR-agonist adjuvant
    Robert G. Schaut, Vaccine - 2016
    Abstract
    Visceral leishmaniasis (VL), caused by infection with the obligate intracellular protozoan parasite Leishmania infantum, is a fatal disease of dogs and humans. Protection against VL requires a T helper 1 (Th1) skewed CD4+ T response, but despite this knowledge, there are currently no approved-to-market vaccines for humans and only three veterinary-use vaccines globally. As VL progresses from asymptomatic to symptomatic, L. infantum–specific interferon gamma (IFNγ) driven-Th1 responses become dampened and a state of immune exhaustion established. T cell exhaustion and other immunoregulatory processes, starting during asymptomatic disease, are likely to hinder vaccine-induced responses if vaccine is administered to infected, but asymptomatic and seronegative, individuals. In this study we evaluated how immune exhaustion, shown previously by our group to worsen in concert with VL progression, effected the capacity of vaccine candidate antigen/toll-like receptor (TLR) agonist combinations to promote protective CD4+ T cell responses during progressive VL. In conjunction with Th1 responses, we also evaluated concomitant stimulation of immune-balanced IL-10 regulatory cytokine production by these vaccine products in progressive VL canine T cells. Vaccine antigen L111f in combination with TLR agonists significantly recovered CD4+ T cell IFNγ intracellular production in T cells from asymptomatic VL dogs. Vaccine antigen NS with TLR agonists significantly recovered CD4+ T cell production in both endemic control and VL dogs. Combinations of TLR agonists and vaccine antigens overcame L. infantum induced cellular exhaustion, allowing robust Th1 CD4+ T cell responses from symptomatic dogs that previously had dampened responses to antigen alone. Antigen-agonist adjuvants can be utilized to promote more robust vaccine responses from infected hosts in endemic areas where vaccination of asymptomatic, L. infantum-infected animals is likely.
    Characterization of heme oxygenase and biliverdin reductase gene expression in zebrafish (Danio rerio): Basal expression and response to pro-oxidant exposures
    Andrew Holowiecki, Toxicology and Applied Pharmacology - 2016
    Abstract
    While heme is an important cofactor for numerous proteins, it is highly toxic in its unbound form and can perpetuate the formation of reactive oxygen species. Heme oxygenase enzymes (HMOX1 and HMOX2) degrade heme into biliverdin and carbon monoxide, with biliverdin subsequently being converted to bilirubin by biliverdin reductase (BVRa or BVRb). As a result of the teleost-specific genome duplication event, zebrafish have paralogs of hmox1 (hmox1a and hmox1b) and hmox2 (hmox2a and hmox2b). Expression of all four hmox paralogs and two bvr isoforms were measured in adult tissues (gill, brain and liver) and sexually dimorphic differences were observed, most notably in the basal expression of hmox1a, hmox2a, hmox2b and bvrb in liver samples. hmox1a, hmox2a and hmox2b were significantly induced in male liver tissues in response to 96 h cadmium exposure (20 μM). hmox2a and hmox2b were significantly induced in male brain samples, but only hmox2a was significantly reduced in male gill samples in response to the 96 h cadmium exposure. hmox paralogs displayed significantly different levels of basal expression in most adult tissues, as well as during zebrafish development (24 to 120 hpf). Furthermore, hmox1a, hmox1b and bvrb were significantly induced in zebrafish eleutheroembryos in response to multiple pro-oxidants (cadmium, hemin and tert-butylhydroquinone). Knockdown of Nrf2a, a transcriptional regulator of hmox1a, was demonstrated to inhibit the Cd-mediated induction of hmox1b and bvrb. These results demonstrate distinct mechanisms of hmox and bvr transcriptional regulation in zebrafish, providing initial evidence of the partitioning of function of the hmox paralogs.
    Toll-like receptor 4 mutation suppresses hyperhomocysteinemia-mediated hypertension. - viewcontent.cgi
    Anastasia Familtseva, University of Louisville - 2016
    Abstract
    Background: Hyperhomocysteinemia (HHcy) has been observed to promote hypertension, but the mechanisms are unclear. Toll-like receptor 4 (TLR-4) is a cellular membrane protein that is ubiquitously expressed in all cell types of the vasculature. TLR4 activation has been shown to promote inflammation that has been associated with pathogenesis of hypertension. In this study, we hypothesize that HHcy induces hypertension by TLR-4 activation that promotes inflammatory cytokine up-regulation (IL1β, IL 6, TNF-α) and initiation of mitochondrial dysfunction leading to cell death and chronic vascular inflammation.
    Downregulation of NEDD9 by apigenin suppresses migration, invasion, and metastasis of colorectal cancer cells
    Jin Dai, Toxicology and Applied Pharmacology - 2016
    Abstract
    Apigenin is a natural flavonoid which possesses multiple anti-cancer properties such as anti-proliferation, anti-inflammation, and anti-metastasis in many types of cancers including colorectal cancer. Neural precursor cell expressed developmentally downregulated 9 (NEDD9) is a multi-domain scaffolding protein of the Cas family which has been shown to correlate with cancer metastasis and progression. The present study investigates the role of NEDD9 in apigenin-inhibited cell migration, invasion, and metastasis of colorectal adenocarcinoma DLD1 and SW480 cells. The results show that knockdown of NEDD9 inhibited cell migration, invasion, and metastasis and that overexpression of NEDD9 promoted cell migration and invasion of DLD1 cells and SW4890 cells. Apigenin treatment attenuated NEDD9 expression at protein level, resulting in reduced phosphorylations of FAK, Src, and Akt, leading to inhibition on cell migration, invasion, and metastasis of both DLD1 and SW480 cells. The present study has demonstrated that apigenin inhibits cell migration, invasion, and metastasis through NEDD9/Src/Akt cascade in colorectal cancer cells. NEDD9 may function as a biomarker for evaluation of cancer aggressiveness and for selection of drug target for therapeutic potential against cancer progression.
    Agkihpin, a novel SVAE may inhibit the migration and invasion of liver cancer cells associated with the inversion of EMT induced by Wnt/β-catenin signaling inhibition
    Miao Huang, Biochemical and Biophysical Research Communications - 2016
    Abstract
    In our previous work, agkihpin, a snake venom arginine esterase (SVAE), was isolated from the Gloydius halys Pallas, which could attenuate the migration of liver cancer cells. However, the mechanism of the effect of agkihpin on attenuating migration of liver cancer cell is unknown yet. Here, to learn more about agkihpin and explore the possibility of agkihpin as an anti-metastatic drug in the future, a series of experiments about the migration and invasion of liver cancer cells with agkihpin, HepG 2 and SMMC-7721, was conducted. Epithelial-mesenchymal transition (EMT) is an initial step and a major phenotype of cancer metastasis and invasion, while a number of EMT opposite phenomenons were observed, for example, epithelial marker E-cadherin was up-regulated, mesenchymal markers N-cadherin and Vimentin, and transcription regulators Snail and twist were down-regulated after treating with agkihpin in liver cancer cells; canonical Wnt/β-catenin pathway, one of the signals initiated EMT, was inhibited by decreased expressions of FZD7 and β-catenin, phosphorylation of GSK3β (Ser9), and nuclear β-catenin accumulation in agkihpin treated cancer cells. By using bioinformatics analysis and protease activity analysis in vitro we also found that agkihpin might bind and degrade FZD7. As a result, we hypothesized that agkihpin could inhibit the Wnt/β-catenin signaling pathway by cleaving FZD7, leading to the inactivation of the TCF/LEF transcription factor, which contributed to the inversion of EMT, and finally attenuated the migration and invasion of liver cancer cells. Therefore, our findings provided novel mechanistic insights into the role of SVAEs in liver cancer controlling, and raised the possibility that agkihpin may be used therapeutically in liver cancer.
    SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics
    Dana Chen, Scientific Reports - 2016
    Abstract
    Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities.
    The Matrix Protein of Nipah Virus Targets the E3-Ubiquitin Ligase TRIM6 to Inhibit the IKKε Kinase-Mediated Type-I IFN Antiviral Response
    Preeti Bharaj, PLOS Pathogens - 2016
    Abstract
    For efficient replication, viruses have developed mechanisms to evade innate immune responses, including the antiviral type-I interferon (IFN-I) system. Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae family (genus Henipavirus), is known to encode for four P gene-derived viral proteins (P/C/W/V) with IFN-I antagonist functions. Here we report that NiV matrix protein (NiV-M), which is important for virus assembly and budding, can also inhibit IFN-I responses. IFN-I production requires activation of multiple signaling components including the IκB kinase epsilon (IKKε). We previously showed that the E3-ubiquitin ligase TRIM6 catalyzes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, and activate IKKε for induction of IFN-I mediated antiviral responses. Using co-immunoprecipitation assays and confocal microscopy we show here that the NiV-M protein interacts with TRIM6 and promotes TRIM6 degradation. Consequently, NiV-M expression results in reduced levels of unanchored K48-linked polyubiquitin chains associated with IKKε leading to impaired IKKε oligomerization, IKKε autophosphorylation and reduced IFN-mediated responses. This IFN antagonist function of NiV-M requires a conserved lysine residue (K258) in the bipartite nuclear localization signal that is found in divergent henipaviruses. Consistent with this, the matrix proteins of Ghana, Hendra and Cedar viruses were also able to inhibit IFNβ induction. Live NiV infection, but not a recombinant NiV lacking the M protein, reduced the levels of endogenous TRIM6 protein expression. To our knowledge, matrix proteins of paramyxoviruses have never been reported to be involved in innate immune antagonism. We report here a novel mechanism of viral innate immune evasion by targeting TRIM6, IKKε and unanchored polyubiquitin chains. These findings expand the universe of viral IFN antagonism strategies and provide a new potential target for development of therapeutic interventions against NiV infections.
    NLRC4 suppresses melanoma tumor progression independently of inflammasome activation
    Ann Janowski, The Journal of Clinical Investigation - 2016
    Abstract