Resources

PCR
- Real-Time Quantitative PCR
  - SYBR Green Detection
    - DNA
      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.
      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.
  - 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.
    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.
  - RNA
    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.
Next Generation Sequencing (NGS)
- DNA
  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.
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.
  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.
  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.

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
- Real-Time Quantitative PCR
  - SYBR Green Detection
    - DNA
      Oxidative Stress and X-ray Exposure Levels-Dependent Survival and Metabolic Changes in Murine HSPCs
      Melis Karabulutoglu - 2021
      Abstract
      Haematopoietic bone marrow cells are amongst the most sensitive to ionizing radiation (IR), initially resulting in cell death or genotoxicity that may later lead to leukaemia development, most frequently Acute Myeloid Leukaemia (AML). The target cells for radiation-induced Acute Myeloid Leukaemia (rAML) are believed to lie in the haematopoietic stem and progenitor cell (HSPC) compartment. Using the inbred strain CBA/Ca as a murine model of rAML, progress has been made in understanding the underlying mechanisms, characterisation of target cell population and responses to IR. Complex regulatory systems maintain haematopoietic homeostasis which may act to modulate the risk of rAML. However, little is currently known about the role of metabolic factors and diet in these regulatory systems and modification of the risk of AML development. This study characterises cellular proliferative and clonogenic potential as well as metabolic changes within murine HSPCs under oxidative stress and X-ray exposure. Ambient oxygen (normoxia; 20.8% O2) levels were found to increase irradiated HSPC-stress, stimulating proliferative activity compared to low oxygen (3% O2) levels. IR exposure has a negative influence on the proliferative capability of HSPCs in a dose-dependent manner (0–2 Gy) and this is more pronounced under a normoxic state. One Gy x-irradiated HSPCs cultured under normoxic conditions displayed a significant increase in oxygen consumption compared to those cultured under low O2 conditions and to unirradiated HSPCs. Furthermore, mitochondrial analyses revealed a significant increase in mitochondrial DNA (mtDNA) content, mitochondrial mass and membrane potential in a dose-dependent manner under normoxic conditions. Our results demonstrate that both IR and normoxia act as stressors for HSPCs, leading to significant metabolic deregulation and mitochondrial dysfunctionality which may affect long term risks such as leukaemia.
      The RNA binding protein IMP2 drives a stromal-Th17 cell circuit in autoimmune neuroinflammation
      Rami Bechara - 2021
      Abstract
      Stromal cells are emerging as key drivers of autoimmunity, in part by producing inflammatory chemokines that orchestrate inflammation. Chemokine expression is regulated transcriptionally but also through post-transcriptional mechanisms, the specific drivers of which are still incompletely defined. CCL2 (MCP1) is a multifunctional chemokine that drives myeloid cell recruitment. During experimental autoimmune encephalomyelitis (EAE), an IL-17-driven model of multiple sclerosis, CCL2 produced by lymph node (LN) stromal cells is essential for immunopathology. Here, we show that Ccl2 mRNA upregulation in human stromal fibroblasts in response to IL-17 requires the RNA binding protein (RBP) insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2, IMP2), which is expressed almost exclusively in non-hematopoietic cells. IMP2 binds directly to CCL2 mRNA, markedly extending its transcript half-life and thus required for efficient CCL2 secretion. Consistent with this, Imp2−/− mice showed reduced CCL2 production in LN during EAE, causing impairments in monocyte recruitment and Th17 cell polarization. Imp2-/- mice were fully protected from CNS inflammation. Moreover, deletion of IMP2 after EAE onset was sufficient to mitigate disease severity. These data show that posttranscriptional control of Ccl2 in stromal cells by IMP2 is required to permit IL-17-driven progression of EAE pathogenesis.
  - Probe-based Detection
    Point-of-Care Platform for Rapid Multiplexed Detection of SARS-CoV-2 Variants and Respiratory Pathogens
    Alexander Y. Trick - 2022
    Abstract
    The rise of highly transmissible SARS-CoV-2 variants brings new challenges and concerns with vaccine efficacy, diagnostic sensitivity, and public health responses to end the pandemic. Widespread detection of variants is critical to inform policy decisions to mitigate further spread, and postpandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. In this work, a portable, magnetofluidic cartridge platform for automated polymerase chain reaction testing in <30 min is developed. Cartridges are designed for multiplexed detection of SARS-CoV-2 with either identification of variant mutations or screening for Influenza A and B. Moreover, the platform can perform identification of B.1.1.7 and B.1.351 variants and the multiplexed SARS-CoV-2/Influenza assay using archived clinical nasopharyngeal swab eluates and saliva samples. This work illustrates a path toward affordable and immediate testing with potential to aid surveillance of viral variants and inform patient treatment.
    Specific inhibition of the NLRP3 inflammasome suppresses immune overactivation and alleviates COVID-19 like pathology in mice
    Jianxiong Zeng - 2022
    Abstract
    Background The Coronavirus Disease 2019 (COVID-19) pandemic has been a great threat to global public health since 2020. Although the advance on vaccine development has been largely achieved, a strategy to alleviate immune overactivation in severe COVID-19 patients is still needed. The NLRP3 inflammasome is activated upon SARS-CoV-2 infection and associated with COVID-19 severity. However, the processes by which the NLRP3 inflammasome is involved in COVID-19 disease remain unclear. Methods We infected THP-1 derived macrophages, NLRP3 knockout mice, and human ACE2 transgenic mice with live SARS-CoV-2 in Biosafety Level 3 (BSL-3) laboratory. We performed quantitative real-time PCR for targeted viral or host genes from SARS-CoV-2 infected mouse tissues, conducted histological or immunofluorescence analysis in SARS-CoV-2 infected mouse tissues. We also injected intranasally AAV-hACE2 or intraperitoneally NLRP3 inflammasome inhibitor MCC950 before SARS-CoV-2 infection in mice as indicated. Findings We have provided multiple lines of evidence that the NLRP3 inflammasome plays an important role in the host immune response to SARS-CoV-2 invasion of the lungs. Inhibition of the NLRP3 inflammasome attenuated the release of COVID-19 related pro-inflammatory cytokines in cell cultures and mice. The severe pathology induced by SARS-CoV-2 in lung tissues was reduced in Nlrp3−/− mice compared to wild-type C57BL/6 mice. Finally, specific inhibition of the NLRP3 inflammasome by MCC950 alleviated excessive lung inflammation and thus COVID-19 like pathology in human ACE2 transgenic mice. Interpretation Inflammatory activation induced by SARS-CoV-2 is an important stimulator of COVID-19 related immunopathology. Targeting the NLRP3 inflammasome is a promising immune intervention against severe COVID-19 disease.
    QUANTITATIVE ASSESSMENT OF METHODS FOR BACTERIAL AND VIRAL PURIFICATION AND CONCENTRATION
    Nina Sara Fraticelli-Guzmán - 2021
    Abstract
    The COVID-19 global pandemic has led to the exploration and implementation of rapid tests for viral load identification and thus, control of the spread. For this, there are varying approaches with the gold standard being quantitative Polymerase Chain Reaction [1]and a potential more rapid alternate approach being the use of an antibody coated biosensor such as a field effect transistor (FET). Analogously, bacterial pathogens such as anthrax need sensing and quantification as well. Therefore, methods for pathogen collection and detection for both viruses and bacteria are needed. Regardless of the analysis technique, it is ideal for samples to contain little to zero non-target particles or contaminants that might interfere with the detection approach. As such, traditional ways of purifying and concentrating samples prior to any type of analysis involve both chemical, physical, physiochemical, or biological approaches such as filtration, centrifugation, affinity chromatography, immunomagnetic separation, etc. Of these, we will focus on the physical approach, filtration, due to its simplicity, low cost, varying options, and ability to process large and small sample volumes. In this work, we investigate how well the method works for virus or bacteria in the presence of high interferent concentrations that could potentially be present in saliva samples or other hydrosol samples taken from an environment to be tested. The research presented here characterizes the applicability of syringe filters and a tangential flow filtration device for the purification and concentration of bacteria and virus samples, respectively. Furthermore, automation of such systems was explored. Varying syringe filter pore sizes yielded different recoveries of bacteria for purifying and concentrating the sample. Furthermore, varying volumes were analyzed for xviii optimal recovery and concentration of the target. We also developed a fully automated method for double filter filtration to enable hands-free purification and concentration in 5.5 minutes for 5 mL of input volume with a 42 ± 13-fold enrichment improvement (n = 3). Furthermore, the purification and concentration of virus using a manually operated tangential flow filtration device was also explored and yielded modest concentration increases of around 2× with an enrichment improvement of up to 1,916 ± 1,839-fold (n = 3) under one configuration. By characterizing and automating these readily available items, we can enhance the detection of samples by decreasing labor time and processing complexity required for the purification and concentration of the target pathogens.
    A Sensitive, Portable Microfluidic Device for SARS-CoV-2 Detection from Self-Collected Saliva
    Jianing Yang - 2021
    Abstract
    Since the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in December 2019, the spread of SARS-CoV2 infection has been escalating rapidly around the world. In order to provide more timely access to medical intervention, including diagnostic tests and medical treatment, the FDA authorized multiple test protocols for diagnostic tests from nasopharyngeal swab, saliva, urine, bronchoalveolar lavage and fecal samples. The traditional diagnostic tests for this novel coronavirus 2019 require standard processes of viral RNA isolation, reverse transcription of RNA to cDNA, then real-time quantitative PCR with the RNA templates extracted from the patient samples. Recently, many reports have demonstrated a direct detection of SARS-Co-V2 genomic material from saliva samples without any RNA isolation step. To make the rapid detection of SARS-Co-V2 infection more accessible, a point-of-care type device was developed for SARS-CoV-2 detection. Herein, we report a portable microfluidic-based integrated detectionanalysis system for SARS-CoV-2 nucleic acids detection directly from saliva samples. The saliva cartridge is self-contained and capable of microfluidic evaluation of saliva, from heating, mixing with the primers to multiplex real-time quantitative polymerase chain reaction, detecting SARSCoV- 2 with different primer sets and internal control. The approach has a detection sensitivity of 1000 copies/mL of SARS-CoV-2 RNA or virus, with consistency and automation, from saliva sample-in to result-out.
    Profiling Multiomic Biomarkers using Particle Detection Counters and Spectral-FLIM Microscopy
    Tam Vu - 2021
    Abstract
    Profiling multiomic biomarkers in bulk and in situ provides critical information which enables basic research and clinical applications. Unfortunately, most existing profiling methods are limited due to either low multiplexing, sensitivity, costs, or assay complexity. This thesis aims to develop two core technologies that address 1) bulk profiling issues with sensitivity and low throughput as well as 2) in situ profiling issues with low multiplexing capabilities, costs, and limited throughput. To address the first issue, this work introduces a novel liquid biopsy approach that utilizes a platform technology called Integrated Comprehensive Droplet Digital Detection (IC3D). This integrated approach combines microfluidic droplet partitioning technology, fluorescent multiplexed PCR chemistry, and our own unique and rapid particle counting technology to deliver ultrasensitive and ultrafast detection of colorectal cancer-specific genomic biomarkers from minimally processed blood samples. xv To address the second issue, this work introduces a new spatial multi-omics technology termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA) that integrates a) in situ labeling of molecular markers (e.g. mRNA, proteins) in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, and b) spectra and timeresolved fluorescence imaging and analysis to enable rapid, high-throughput, and cost-effective spatial profiling of multi-omics biomarkers. By utilizing both time and intensity domains for labeling and imaging, this technology seeks to discriminate a vast repertoire of lifetime and spectral components simultaneously within the same pixel or image of a sample to enable highly increased multiplexing capabilities with standard optical systems. Overall, these two technologies represent simple, versatile, and scalable tools which enable more rapid, sensitive, and/or multiplexed protein/transcriptomic analysis.
    RIPK1 or RIPK3 deletion prevents progressive neuronal cell death and improves memory function after traumatic brain injury
    Antonia Clarissa Wehn - 2021
    Abstract
    Traumatic brain injury (TBI) causes acute and subacute tissue damage, but is also associated with chronic inflammation and progressive loss of brain tissue months and years after the initial event. The trigger and the subsequent molecular mechanisms causing chronic brain injury after TBI are not well understood. The aim of the current study was therefore to investigate the hypothesis that necroptosis, a form a programmed cell death mediated by the interaction of Receptor Interacting Protein Kinases (RIPK) 1 and 3, is involved in this process. Neuron-specific RIPK1- or RIPK3-deficient mice and their wild-type littermates were subjected to experimental TBI by controlled cortical impact. Posttraumatic brain damage and functional outcome were assessed longitudinally by repetitive magnetic resonance imaging (MRI) and behavioral tests (beam walk, Barnes maze, and tail suspension), respectively, for up to three months after injury. Thereafter, brains were investigated by immunohistochemistry for the necroptotic marker phosphorylated mixed lineage kinase like protein(pMLKL) and activation of astrocytes and microglia. WT mice showed progressive chronic brain damage in cortex and hippocampus and increased levels of pMLKL after TBI. Chronic brain damage occurred almost exclusively in areas with iron deposits and was significantly reduced in RIPK1- or RIPK3-deficient mice by up to 80%. Neuroprotection was accompanied by a reduction of astrocyte and microglia activation and improved memory function. The data of the current study suggest that progressive chronic brain damage and cognitive decline after TBI depend on the expression of RIPK1/3 in neurons. Hence, inhibition of necroptosis signaling may represent a novel therapeutic target for the prevention of chronic post-traumatic brain damage.
    Extractionless Nucleic Acid Detection: A High Capacity Solution to COVID-19 Testing
    Shairaz Baksh - 2021
    Abstract
    We describe an extractionless real-time reverse transcriptase-PCR (rRT-PCR) protocol for SARS‐CoV‐2 nucleic acid detection using heat as an accurate cost-effective high-capacity solution to COVID‐19 testing. We present the effect of temperature, transport media, rRT-PCR mastermixes and gene assays on SARS‐CoV‐2 gene amplification and limits of detection. Utilizing our heated methodology, our limits of detection were 12.5 and 1 genome copy/reaction for singleplex E‐ and N1‐gene assays, respectively, and 1 genome copy/reaction by utilizing an E/N1 or Orf1ab/N1 multiplex assay combination. Using this approach, we detected up to 98% of COVID‐19 positive patient samples analyzed in our various cohorts including a significant percentage of weak positives. Importantly, this extractionless approach will allow for > 2‐fold increase in testing capacity with existing instruments, circumvent the additional need for expensive extraction devices, provide the sensitivity needed for COVID‐19 detection and significantly reduce the turn‐around time of reporting COVID‐19 test results.
    Autologous Transplantation of Skin-Derived Precursor Cells in a Porcine Model
    Anne-Laure Thomas - 2020
    Abstract
    Background Hirschprung's disease is characterized by aganglionic bowel and often requires surgical resection. Cell-based therapies have been investigated as potential alternatives to restore functioning neurons. Skin-derived precursor cells (SKPs) differentiate into neural and glial cells in vitro and generate ganglion-like structures in rodents. In this report, we aimed to translate this approach into a large animal model of aganglionosis using autologous transplantation of SKPs. Methods Juvenile pigs underwent skin procurement from the shoulder and simultaneous chemical denervation of an isolated colonic segment. Skin cells were cultured in neuroglial-selective medium and labeled with fluorescent dye for later identification. The cultured SKPs were then injected into the aganglionic segments of colon, and the specimens were retrieved within seven days after transplantation. SKPs in vitro and in vivo were assessed with histologic samples for various immunofluorescent markers of multipotency and differentiation. SKPs from the time of harvest were compared to those at the time of injection using PCR. Results Prior to transplantation, 72% of SKPs stained positive for nestin and S100b, markers of neural and glial precursor cells of neural crest origin, respectively. Markers of differentiated neurons and gliocytes, TUJ1 and GFAP, were detected in 47% of cultured SKPs. After transplantation, SKPs were identified in both myenteric and submucosal plexuses of the treated colon. Nestin co-expression was detected in the SKPs within the aganglionic colon in vivo. Injected SKPs appeared to migrate and express early neuroglial differentiation markers. Conclusions Autologous SKPs implanted into aganglionic bowel demonstrated immunophenotypes of neuroglial progenitors. Our results suggest that autologous SKPs may be potentially useful for cell-based therapy for patients with enteric nervous system disorders. Type of Study Basic science.
    Investigation of an outbreak caused by antibiotic‐susceptible Klebsiella oxytoca in a neonatal intensive care unit in Norway
    Torunn Gresdal Ronning - 2019
    Abstract
    Aim Klebsiella spp. have been stated to be the most frequent cause of neonatal intensive care unit (NICU) outbreaks. We report an outbreak of Klebsiella oxytoca in a NICU at a tertiary care hospital in Norway between April 2016 and April 2017. This study describes the outbreak, infection control measures undertaken and the molecular methods developed. Methods The outbreak prompted detailed epidemiological and microbial investigations, where whole‐genome sequencing (WGS) was particularly useful for both genotyping and development of two new K. oxytoca‐specific real‐time PCR assays. Routine screening of patients, as well as sampling from numerous environmental sites, was performed during the outbreak. A bundle of infection control measures was instigated to control the outbreak, among them strict cohort isolation. Results Five neonates had symptomatic infection, and 17 were found to be asymptomatically colonised. Infections varied in severity from conjunctivitis to a fatal case of pneumonia. A source of the outbreak could not be determined. Conclusion This report describes K. oxytoca as a significant pathogen in a NICU outbreak setting and highlights the importance of developing appropriate microbiological screening methods and implementing strict infection control measures to control the outbreak in a setting where the source could not be identified.
    Iron oxide nanoparticles enhance Toll-like receptor-induced cytokines in a particle size- and actin-dependent manner in human blood
    Susann Wolf-Grosse - 2018
    Abstract
    Aim: To assess the effects of different-sized iron oxide nanoparticles (IONPs) on inflammatory responses in human whole blood. Materials & methods: Human whole blood with and without 10 and 30 nm IONPs was incubated with Toll-like receptor (TLR) ligands. Cytokine levels, complement activation, reactive oxygen species and viability were determined. Results: The 10 nm IONPs enhanced the TLR2/6, TLR4 and partly TLR8-mediated cytokine production, whereas the 30 nm IONPs partly enhanced TLR2/6 and decreased TLR8-mediated cytokine production. Particle-mediated enhancement of TLR4-induced cytokines could not be explained by complement activation, but was dependent on TLR4/MD2 and CD14, as well as actin polymerization. Conclusion: The IONPs differentially affected the TLR ligand-induced cytokines, which has important implications for biomedical applications of IONPs.
  - RNA
    Point-of-Care Platform for Rapid Multiplexed Detection of SARS-CoV-2 Variants and Respiratory Pathogens
    Alexander Y. Trick - 2022
    Abstract
    The rise of highly transmissible SARS-CoV-2 variants brings new challenges and concerns with vaccine efficacy, diagnostic sensitivity, and public health responses to end the pandemic. Widespread detection of variants is critical to inform policy decisions to mitigate further spread, and postpandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. In this work, a portable, magnetofluidic cartridge platform for automated polymerase chain reaction testing in <30 min is developed. Cartridges are designed for multiplexed detection of SARS-CoV-2 with either identification of variant mutations or screening for Influenza A and B. Moreover, the platform can perform identification of B.1.1.7 and B.1.351 variants and the multiplexed SARS-CoV-2/Influenza assay using archived clinical nasopharyngeal swab eluates and saliva samples. This work illustrates a path toward affordable and immediate testing with potential to aid surveillance of viral variants and inform patient treatment.
    Specific inhibition of the NLRP3 inflammasome suppresses immune overactivation and alleviates COVID-19 like pathology in mice
    Jianxiong Zeng - 2022
    Abstract
    Background The Coronavirus Disease 2019 (COVID-19) pandemic has been a great threat to global public health since 2020. Although the advance on vaccine development has been largely achieved, a strategy to alleviate immune overactivation in severe COVID-19 patients is still needed. The NLRP3 inflammasome is activated upon SARS-CoV-2 infection and associated with COVID-19 severity. However, the processes by which the NLRP3 inflammasome is involved in COVID-19 disease remain unclear. Methods We infected THP-1 derived macrophages, NLRP3 knockout mice, and human ACE2 transgenic mice with live SARS-CoV-2 in Biosafety Level 3 (BSL-3) laboratory. We performed quantitative real-time PCR for targeted viral or host genes from SARS-CoV-2 infected mouse tissues, conducted histological or immunofluorescence analysis in SARS-CoV-2 infected mouse tissues. We also injected intranasally AAV-hACE2 or intraperitoneally NLRP3 inflammasome inhibitor MCC950 before SARS-CoV-2 infection in mice as indicated. Findings We have provided multiple lines of evidence that the NLRP3 inflammasome plays an important role in the host immune response to SARS-CoV-2 invasion of the lungs. Inhibition of the NLRP3 inflammasome attenuated the release of COVID-19 related pro-inflammatory cytokines in cell cultures and mice. The severe pathology induced by SARS-CoV-2 in lung tissues was reduced in Nlrp3−/− mice compared to wild-type C57BL/6 mice. Finally, specific inhibition of the NLRP3 inflammasome by MCC950 alleviated excessive lung inflammation and thus COVID-19 like pathology in human ACE2 transgenic mice. Interpretation Inflammatory activation induced by SARS-CoV-2 is an important stimulator of COVID-19 related immunopathology. Targeting the NLRP3 inflammasome is a promising immune intervention against severe COVID-19 disease.
    A Sensitive, Portable Microfluidic Device for SARS-CoV-2 Detection from Self-Collected Saliva
    Jianing Yang - 2021
    Abstract
    Since the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in December 2019, the spread of SARS-CoV2 infection has been escalating rapidly around the world. In order to provide more timely access to medical intervention, including diagnostic tests and medical treatment, the FDA authorized multiple test protocols for diagnostic tests from nasopharyngeal swab, saliva, urine, bronchoalveolar lavage and fecal samples. The traditional diagnostic tests for this novel coronavirus 2019 require standard processes of viral RNA isolation, reverse transcription of RNA to cDNA, then real-time quantitative PCR with the RNA templates extracted from the patient samples. Recently, many reports have demonstrated a direct detection of SARS-Co-V2 genomic material from saliva samples without any RNA isolation step. To make the rapid detection of SARS-Co-V2 infection more accessible, a point-of-care type device was developed for SARS-CoV-2 detection. Herein, we report a portable microfluidic-based integrated detectionanalysis system for SARS-CoV-2 nucleic acids detection directly from saliva samples. The saliva cartridge is self-contained and capable of microfluidic evaluation of saliva, from heating, mixing with the primers to multiplex real-time quantitative polymerase chain reaction, detecting SARSCoV- 2 with different primer sets and internal control. The approach has a detection sensitivity of 1000 copies/mL of SARS-CoV-2 RNA or virus, with consistency and automation, from saliva sample-in to result-out.
    RIPK1 or RIPK3 deletion prevents progressive neuronal cell death and improves memory function after traumatic brain injury
    Antonia Clarissa Wehn - 2021
    Abstract
    Traumatic brain injury (TBI) causes acute and subacute tissue damage, but is also associated with chronic inflammation and progressive loss of brain tissue months and years after the initial event. The trigger and the subsequent molecular mechanisms causing chronic brain injury after TBI are not well understood. The aim of the current study was therefore to investigate the hypothesis that necroptosis, a form a programmed cell death mediated by the interaction of Receptor Interacting Protein Kinases (RIPK) 1 and 3, is involved in this process. Neuron-specific RIPK1- or RIPK3-deficient mice and their wild-type littermates were subjected to experimental TBI by controlled cortical impact. Posttraumatic brain damage and functional outcome were assessed longitudinally by repetitive magnetic resonance imaging (MRI) and behavioral tests (beam walk, Barnes maze, and tail suspension), respectively, for up to three months after injury. Thereafter, brains were investigated by immunohistochemistry for the necroptotic marker phosphorylated mixed lineage kinase like protein(pMLKL) and activation of astrocytes and microglia. WT mice showed progressive chronic brain damage in cortex and hippocampus and increased levels of pMLKL after TBI. Chronic brain damage occurred almost exclusively in areas with iron deposits and was significantly reduced in RIPK1- or RIPK3-deficient mice by up to 80%. Neuroprotection was accompanied by a reduction of astrocyte and microglia activation and improved memory function. The data of the current study suggest that progressive chronic brain damage and cognitive decline after TBI depend on the expression of RIPK1/3 in neurons. Hence, inhibition of necroptosis signaling may represent a novel therapeutic target for the prevention of chronic post-traumatic brain damage.
    Extractionless Nucleic Acid Detection: A High Capacity Solution to COVID-19 Testing
    Shairaz Baksh - 2021
    Abstract
    We describe an extractionless real-time reverse transcriptase-PCR (rRT-PCR) protocol for SARS‐CoV‐2 nucleic acid detection using heat as an accurate cost-effective high-capacity solution to COVID‐19 testing. We present the effect of temperature, transport media, rRT-PCR mastermixes and gene assays on SARS‐CoV‐2 gene amplification and limits of detection. Utilizing our heated methodology, our limits of detection were 12.5 and 1 genome copy/reaction for singleplex E‐ and N1‐gene assays, respectively, and 1 genome copy/reaction by utilizing an E/N1 or Orf1ab/N1 multiplex assay combination. Using this approach, we detected up to 98% of COVID‐19 positive patient samples analyzed in our various cohorts including a significant percentage of weak positives. Importantly, this extractionless approach will allow for > 2‐fold increase in testing capacity with existing instruments, circumvent the additional need for expensive extraction devices, provide the sensitivity needed for COVID‐19 detection and significantly reduce the turn‐around time of reporting COVID‐19 test results.
    Autologous Transplantation of Skin-Derived Precursor Cells in a Porcine Model
    Anne-Laure Thomas - 2020
    Abstract
    Background Hirschprung's disease is characterized by aganglionic bowel and often requires surgical resection. Cell-based therapies have been investigated as potential alternatives to restore functioning neurons. Skin-derived precursor cells (SKPs) differentiate into neural and glial cells in vitro and generate ganglion-like structures in rodents. In this report, we aimed to translate this approach into a large animal model of aganglionosis using autologous transplantation of SKPs. Methods Juvenile pigs underwent skin procurement from the shoulder and simultaneous chemical denervation of an isolated colonic segment. Skin cells were cultured in neuroglial-selective medium and labeled with fluorescent dye for later identification. The cultured SKPs were then injected into the aganglionic segments of colon, and the specimens were retrieved within seven days after transplantation. SKPs in vitro and in vivo were assessed with histologic samples for various immunofluorescent markers of multipotency and differentiation. SKPs from the time of harvest were compared to those at the time of injection using PCR. Results Prior to transplantation, 72% of SKPs stained positive for nestin and S100b, markers of neural and glial precursor cells of neural crest origin, respectively. Markers of differentiated neurons and gliocytes, TUJ1 and GFAP, were detected in 47% of cultured SKPs. After transplantation, SKPs were identified in both myenteric and submucosal plexuses of the treated colon. Nestin co-expression was detected in the SKPs within the aganglionic colon in vivo. Injected SKPs appeared to migrate and express early neuroglial differentiation markers. Conclusions Autologous SKPs implanted into aganglionic bowel demonstrated immunophenotypes of neuroglial progenitors. Our results suggest that autologous SKPs may be potentially useful for cell-based therapy for patients with enteric nervous system disorders. Type of Study Basic science.
- Conventional PCR
  - DNA
    Performance of Conventional Urine Culture Compared to 16S rRNA Gene Amplicon Sequencing in Children with Suspected Urinary Tract Infection
    Christopher W. Marshall - 2021
    Abstract
    Because some organisms causing urinary tract infection (UTI) may be difficult to culture, examination of bacterial gene sequences in the urine may provide a more accurate view of bacteria present during a UTI. Our objective was to estimate how often access to 16S rRNA gene amplicon sequencing alters diagnosis and/or clinical management. The study was designed as a cross-sectional study of a convenience sample of children with suspected UTI. The setting was the emergency department or outpatient clinic at six pediatric centers. Participants included children 2 months to 10 years of age suspected of UTI. We categorized the results of urine culture as follows: “likely UTI” ($100,000 CFU/ml of a single uropathogen), “possible UTI” (10,000 to 99,000 CFU/ml of a uropathogen or $100,000 CFU/ ml of a single uropathogen plus other growth), and “unlikely UTI” (no growth or growth of nonuropathogens). Similarly, we categorized the results of 16S rRNA gene sequencing into the same three categories using the following criteria: likely UTI ($90% relative abundance of a uropathogen), possible UTI (50 to 89% relative abundance of a uropathogen), and unlikely UTI (remainder of samples). The main study outcome was concordance between conventional culture results and 16S rRNA gene sequencing. Concordance between the two methods was high in children with likely and unlikely UTI by conventional culture (95% and 87%, respectively). In children with possible UTI according to conventional culture, 71% had a single uropathogen at a relative abundance of $90% according to 16S rRNA gene sequencing data. Concordance between conventional culture and 16S rRNA gene amplicon sequencing appears to be high. In children with equivocal culture results, 16S rRNA gene results may provide information that may help clarify the diagnosis. IMPORTANCE Concordance between conventional culture and 16S rRNA gene amplicon sequencing appears to be high. In children with equivocal culture results, 16S rRNA gene results may provide information that may help clarify the diagnosis.
    Bacillus velezensis Identification and Recombinant Expression, Purification, and Characterization of Its Alpha-Amylase
    Xiaodong Zhang - 2021
    Abstract
    Amylases account for about 30% of the global market of industrial enzymes, and the current amylases cannot fully meet industrial needs. This study aimed to identify a high α-amylase producing bacterium WangLB, to clone its α-amylase coding gene, and to characterize the α-amylase. Results showed that WangLB belonged to Bacillus velezensis whose α-amylase gene was 1980 bp coding 659 amino acids designated as BvAmylase. BvAmylase was a hydrophilic stable protein with a signal peptide and a theoretical pI of 5.49. The relative molecular weight of BvAmylase was 72.35 kDa, and was verified by SDS-PAGE. Its modeled structure displayed that it was a monomer composed of three domains. Its optimum temperature and pH were 70 °C and pH 6.0, respectively. It also showed high activity in a wide range of temperatures (40–75 °C) and a relatively narrow pH (5.0–7.0). It was a Ca2+-independent enzyme, whose α-amylase activity was increased by Co2+, Tween 20, and Triton X-100, and severely decreased by SDS. The Km and the Vmax of BvAmylase were 3.43 ± 0.53 and 434.19 ± 28.57 U/mg. In conclusion, the α-amylase producing bacterium WangLB was identified, and one of its α-amylases was characterized, which will be a candidate enzyme for industrial applications.
    Microbiome Fingerprint as Biomarker for Geographical Origin and Heredity in Crocus sativus: A Feasibility Study
    Nancy Bhagat - 2021
    Abstract
    Host–microbiome interactions are specific and not random, making them defining entities for the host. The hypothesis proposed by various researchers earlier, that both plants and animals harbor specific inheritable core microbiome, is being augmented in the present study. Additionally, a case for using microbial fingerprint as a biomarker, not only for plant identification but also as a geographical indicator, has been investigated, taking Crocus sativus, saffron, as a study material. Crocus sativus, a monogenetic herb, on account of its male sterility and vegetative propagation, is reported to lack genome based molecular markers. Cormosphere microbiome (microbiome associated with corm) has been compared across three geographical locations, in two continents, to identify the core and unique microbiome, during the vegetative phase of its growth. Microbiome analysis done at phylum and genus level, using next generation sequencing technology, revealed that cormosphere at three locations harbored common phyla. At genus level, 24 genera were found common to all three geographical locations, indicating them to be part of the core microbiome of saffron. However, there were some bacterial genera unique to Kashmir, Kishtwar, and Morocco that can be used to develop microbial markers/geographical indicators for saffron grown in these regions. This is a preliminary study, indicating that the location specific bacterial community can be used to develop microbial barcodes but needs further augmentation with high coverage data from other saffron growing geographical regions.
    The effect of a mass distribution of insecticide-treated nets on insecticide resistance and entomological inoculation rates of Anopheles gambiae s.l. in Bandundu City, Democratic Repub`lic of Congo
    Emery Metelo-Matubi - 2021
    Abstract
    Introduction insecticide-treated nets (ITNs) remain the mainstay of malaria vector control in the Democratic Republic of Congo. However, insecticide resistance of malaria vectors threatens their effectiveness. Entomological inoculation rates and insecticide susceptibility in Anopheles gambiae s.l. were evaluated before and after mass distribution of ITNs in Bandundu City for possible occurrence of resistance. Methods a cross-sectional study was conducted from 15th July 2015 to 15th June 2016. Adult mosquitoes were collected using pyrethrum spray catches and human landing catches and identified to species level and tested for the presence of sporozoites. Bioassays were carried out before and after distribution of ITNs to assess the susceptibility of adult mosquitoes to insecticides. Synergist bioassays were also conducted and target site mutations assessed using Polymerase chain reaction (PCR). Results a total of 1754 female An. gambiae s.l. were collected before and after deployment of ITNs. Fewer mosquitoes were collected after the distribution of ITNs. However, there was no significant difference in sporozoite rates or the overall entomological inoculation rate before and after the distribution of ITNs. Test-mosquitoes were resistant to deltamethrin, permethrin, and Dichlorodiphenyltrichloroethane but susceptible to bendiocarb. Pre-exposure of mosquitoes to Piperonyl butoxide increased their mortality after exposure to permethrin and deltamethrin. The frequency of the Kinase insert domain receptor (kdr)-West gene increased from 92 to 99% before and after the distribution of nets, respectively. Conclusion seasonal impacts could be a limiting factor in the analysis of these data; however, the lack of decrease in transmission after the distribution of new nets could be explained by the high-level of resistance to pyrethroid.
    Genomic Characterization of the Fruity Aroma Gene, FaFAD1, Reveals a Gene Dosage Effect on γ-Decalactone Production in Strawberry (Fragaria × ananassa)
    Youngjae Oh - 2021
    Abstract
    Strawberries produce numerous volatile compounds that contribute to the unique flavors of fruits. Among the many volatiles, γ-decalactone (γ-D) has the greatest contribution to the characteristic fruity aroma in strawberry fruit. The presence or absence of γ-D is controlled by a single locus, FaFAD1. However, this locus has not yet been systematically characterized in the octoploid strawberry genome. It has also been reported that the volatile content greatly varies among the strawberry varieties possessing FaFAD1, suggesting that another genetic factor could be responsible for the different levels of γ-D in fruit. In this study, we explored the genomic structure of FaFAD1 and determined the allele dosage of FaFAD1 that regulates variations of γ-D production in cultivated octoploid strawberry. The genome-wide association studies confirmed the major locus FaFAD1 that regulates the γ-D production in cultivated strawberry. With the hybrid capture-based next-generation sequencing analysis, a major presence–absence variation of FaFAD1 was discovered among γ-D producers and non-producers. To explore the genomic structure of FaFAD1 in the octoploid strawberry, three bacterial artificial chromosome (BAC) libraries were developed. A deletion of 8,262 bp was consistently found in the FaFAD1 region of γ-D non-producing varieties. With the newly developed InDel-based codominant marker genotyping, along with γ-D metabolite profiling data, we revealed the impact of gene dosage effect for the production of γ-D in the octoploid strawberry varieties. Altogether, this study provides systematic information of the prominent role of FaFAD1 presence and absence polymorphism in producing γ-D and proposes that both alleles of FaFAD1 are required to produce the highest content of fruity aroma in strawberry fruit.
    Exploring the diversity of the deep sea—four new species of the amphipod genus Oedicerina described using morphological and molecular methods
    Anna M. Jazszewska - 2021
    Abstract
    Collections of the amphipod genus Oedicerina were obtained during six expeditions devoted to the study of deep-sea environments of the Pacific Ocean. The material revealed four species new to science. Two species (Oedicerina henrici sp. nov. and sp. nov.) were found at abyssal depths of the central eastern Pacific in the Clarion-Clipperton Zone; one species (sp. nov.) (Oedicerina claudei sp. nov.) was recovered in the Sea of Okhotsk (north-west Pacific), and one (Oedicerina lesci sp. nov.) in the abyss adjacent to the Kuril-Kamchatka Trench (KKT). The four new species differ from each other and known species by the shapes of the rostrum, coxae 1 and 4, basis of pereopod 7, armatures of pereonite 7, pleonites and urosomites. An identification key for all known species is provided. The study of the cytochrome c oxidase subunit I gene of the four new species and Oedicerina ingolfi collected in the North Atlantic confirmed their genetic distinction. However, small intraspecific variation within each of the studied species was observed. In the case of the new species occurring across the KKT, the same haplotype was found on both sides of the trench, providing evidence that the trench does not constitute an insurmountable barrier for population connectivity. None of the species have so far been found on both sides of the Pacific.
Next Generation Sequencing (NGS)
- DNA
  Microbiome Fingerprint as Biomarker for Geographical Origin and Heredity in Crocus sativus: A Feasibility Study
  Nancy Bhagat - 2021
  Abstract
  Host–microbiome interactions are specific and not random, making them defining entities for the host. The hypothesis proposed by various researchers earlier, that both plants and animals harbor specific inheritable core microbiome, is being augmented in the present study. Additionally, a case for using microbial fingerprint as a biomarker, not only for plant identification but also as a geographical indicator, has been investigated, taking Crocus sativus, saffron, as a study material. Crocus sativus, a monogenetic herb, on account of its male sterility and vegetative propagation, is reported to lack genome based molecular markers. Cormosphere microbiome (microbiome associated with corm) has been compared across three geographical locations, in two continents, to identify the core and unique microbiome, during the vegetative phase of its growth. Microbiome analysis done at phylum and genus level, using next generation sequencing technology, revealed that cormosphere at three locations harbored common phyla. At genus level, 24 genera were found common to all three geographical locations, indicating them to be part of the core microbiome of saffron. However, there were some bacterial genera unique to Kashmir, Kishtwar, and Morocco that can be used to develop microbial markers/geographical indicators for saffron grown in these regions. This is a preliminary study, indicating that the location specific bacterial community can be used to develop microbial barcodes but needs further augmentation with high coverage data from other saffron growing geographical regions.
  Ammonia-oxidizing bacterial communities are affected by nitrogen fertilization and grass species in native C4 grassland soils
  Jialin Hu - 2021
  Abstract
  Background Fertilizer addition can contribute to nitrogen (N) losses from soil by affecting microbial populations responsible for nitrification. However, the effects of N fertilization on ammonia oxidizing bacteria under C4 perennial grasses in nutrient-poor grasslands are not well studied. Methods In this study, a field experiment was used to assess the effects of N fertilization rate (0, 67, and 202 kg N ha−1) and grass species (switchgrass (Panicum virgatum) and big bluestem (Andropogon gerardii)) on ammonia-oxidizing bacterial (AOB) communities in C4 grassland soils using quantitative PCR, quantitative reverse transcription-PCR, and high-throughput amplicon sequencing of amoA genes. Results Nitrosospira were dominant AOB in the C4 grassland soil throughout the growing season. N fertilization rate had a stronger influence on AOB community composition than C4 grass species. Elevated N fertilizer application increased the abundance, activity, and alpha-diversity of AOB communities as well as nitrification potential, nitrous oxide (N2O) emission and soil acidity. The abundance and species richness of AOB were higher under switchgrass compared to big bluestem. Soil pH, nitrate, nitrification potential, and N2O emission were significantly related to the variability in AOB community structures (p < 0.05).
  Altered costimulatory signals and hypoxia support chromatin landscapes limiting the functional potential of exhausted T cells in cancer
  View ORCID ProfileB. Rhodes Ford - 2021
  Abstract
  Immunotherapy has changed cancer treatment with major clinical successes, but response rates remain low due in part to elevated prevalence of dysfunctional, terminally exhausted T cells. However, the mechanisms promoting progression to terminal exhaustion remain undefined. We profiled the histone modification landscape of tumor-infiltrating CD8 T cells throughout differentiation, finding terminally exhausted T cells possessed chromatin features limiting their transcriptional potential. Active enhancers enriched for bZIP/AP-1 transcription factor motifs lacked correlated gene expression, which were restored by immunotherapeutic costimulatory signaling. Epigenetic repression was also driven by an increase in histone bivalency, which we linked directly to hypoxia exposure. Our study is the first to profile the precise epigenetic changes during intratumoral differentiation to exhaustion, highlighting their altered function is driven by both improper costimulatory signals and environmental factors. These data suggest even terminally exhausted T cells remain poised for transcription in settings of increased costimulatory signaling and reduced hypoxia.
  Six de novo assemblies from pathogenic and non-pathogenic strains of Fusarium oxysporum f. sp. niveum
  James C. Fulton - 2021
  Abstract
  Fusarium wilt, caused by Fusarium oxysporum f. sp. niveum (Fon), is a soilborne disease which significantly limits yield in watermelon (Citrullus lanatus) and occasionally causes the loss of an entire year’s harvest. Reference-quality de novo genomic assemblies of pathogenic and non-pathogenic strains were generated using a combination of next-generation and third-generation sequencing technologies. Chromosomal-level genomes were produced with representatives from all Fon races facilitating comparative genomic analysis and the identification of chromosomal structural variation . Syntenic analysis between isolates allowed differentiation of the core and lineage-specific portions of their genomes. This research will support future efforts to refine the scientific understanding of the molecular and genetic factors underpinning the Fon host range, develop diagnostic assays for each of the four races, and decipher the evolutionary history of race 3.
Reverse Transcription
- Quantitative RT-qPCR
  Point-of-Care Platform for Rapid Multiplexed Detection of SARS-CoV-2 Variants and Respiratory Pathogens
  Alexander Y. Trick - 2022
  Abstract
  The rise of highly transmissible SARS-CoV-2 variants brings new challenges and concerns with vaccine efficacy, diagnostic sensitivity, and public health responses to end the pandemic. Widespread detection of variants is critical to inform policy decisions to mitigate further spread, and postpandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. In this work, a portable, magnetofluidic cartridge platform for automated polymerase chain reaction testing in <30 min is developed. Cartridges are designed for multiplexed detection of SARS-CoV-2 with either identification of variant mutations or screening for Influenza A and B. Moreover, the platform can perform identification of B.1.1.7 and B.1.351 variants and the multiplexed SARS-CoV-2/Influenza assay using archived clinical nasopharyngeal swab eluates and saliva samples. This work illustrates a path toward affordable and immediate testing with potential to aid surveillance of viral variants and inform patient treatment.
  Specific inhibition of the NLRP3 inflammasome suppresses immune overactivation and alleviates COVID-19 like pathology in mice
  Jianxiong Zeng - 2022
  Abstract
  Background The Coronavirus Disease 2019 (COVID-19) pandemic has been a great threat to global public health since 2020. Although the advance on vaccine development has been largely achieved, a strategy to alleviate immune overactivation in severe COVID-19 patients is still needed. The NLRP3 inflammasome is activated upon SARS-CoV-2 infection and associated with COVID-19 severity. However, the processes by which the NLRP3 inflammasome is involved in COVID-19 disease remain unclear. Methods We infected THP-1 derived macrophages, NLRP3 knockout mice, and human ACE2 transgenic mice with live SARS-CoV-2 in Biosafety Level 3 (BSL-3) laboratory. We performed quantitative real-time PCR for targeted viral or host genes from SARS-CoV-2 infected mouse tissues, conducted histological or immunofluorescence analysis in SARS-CoV-2 infected mouse tissues. We also injected intranasally AAV-hACE2 or intraperitoneally NLRP3 inflammasome inhibitor MCC950 before SARS-CoV-2 infection in mice as indicated. Findings We have provided multiple lines of evidence that the NLRP3 inflammasome plays an important role in the host immune response to SARS-CoV-2 invasion of the lungs. Inhibition of the NLRP3 inflammasome attenuated the release of COVID-19 related pro-inflammatory cytokines in cell cultures and mice. The severe pathology induced by SARS-CoV-2 in lung tissues was reduced in Nlrp3−/− mice compared to wild-type C57BL/6 mice. Finally, specific inhibition of the NLRP3 inflammasome by MCC950 alleviated excessive lung inflammation and thus COVID-19 like pathology in human ACE2 transgenic mice. Interpretation Inflammatory activation induced by SARS-CoV-2 is an important stimulator of COVID-19 related immunopathology. Targeting the NLRP3 inflammasome is a promising immune intervention against severe COVID-19 disease.
  Extractionless Nucleic Acid Detection: A High Capacity Solution to COVID-19 Testing
  Shairaz Baksh - 2021
  Abstract
  We describe an extractionless real-time reverse transcriptase-PCR (rRT-PCR) protocol for SARS‐CoV‐2 nucleic acid detection using heat as an accurate cost-effective high-capacity solution to COVID‐19 testing. We present the effect of temperature, transport media, rRT-PCR mastermixes and gene assays on SARS‐CoV‐2 gene amplification and limits of detection. Utilizing our heated methodology, our limits of detection were 12.5 and 1 genome copy/reaction for singleplex E‐ and N1‐gene assays, respectively, and 1 genome copy/reaction by utilizing an E/N1 or Orf1ab/N1 multiplex assay combination. Using this approach, we detected up to 98% of COVID‐19 positive patient samples analyzed in our various cohorts including a significant percentage of weak positives. Importantly, this extractionless approach will allow for > 2‐fold increase in testing capacity with existing instruments, circumvent the additional need for expensive extraction devices, provide the sensitivity needed for COVID‐19 detection and significantly reduce the turn‐around time of reporting COVID‐19 test results.
  Autologous Transplantation of Skin-Derived Precursor Cells in a Porcine Model
  Anne-Laure Thomas - 2020
  Abstract
  Background Hirschprung's disease is characterized by aganglionic bowel and often requires surgical resection. Cell-based therapies have been investigated as potential alternatives to restore functioning neurons. Skin-derived precursor cells (SKPs) differentiate into neural and glial cells in vitro and generate ganglion-like structures in rodents. In this report, we aimed to translate this approach into a large animal model of aganglionosis using autologous transplantation of SKPs. Methods Juvenile pigs underwent skin procurement from the shoulder and simultaneous chemical denervation of an isolated colonic segment. Skin cells were cultured in neuroglial-selective medium and labeled with fluorescent dye for later identification. The cultured SKPs were then injected into the aganglionic segments of colon, and the specimens were retrieved within seven days after transplantation. SKPs in vitro and in vivo were assessed with histologic samples for various immunofluorescent markers of multipotency and differentiation. SKPs from the time of harvest were compared to those at the time of injection using PCR. Results Prior to transplantation, 72% of SKPs stained positive for nestin and S100b, markers of neural and glial precursor cells of neural crest origin, respectively. Markers of differentiated neurons and gliocytes, TUJ1 and GFAP, were detected in 47% of cultured SKPs. After transplantation, SKPs were identified in both myenteric and submucosal plexuses of the treated colon. Nestin co-expression was detected in the SKPs within the aganglionic colon in vivo. Injected SKPs appeared to migrate and express early neuroglial differentiation markers. Conclusions Autologous SKPs implanted into aganglionic bowel demonstrated immunophenotypes of neuroglial progenitors. Our results suggest that autologous SKPs may be potentially useful for cell-based therapy for patients with enteric nervous system disorders. Type of Study Basic science.
- First-Strand cDNA Synthesis
  Bioreactor and Small Molecule Drug Applications in Hair Cell Differentiation
  Sylvia Yip - 2021
  Abstract
  Hair cells are mechanoreceptors of the inner ear that convert sounds into electrical signals to be perceived by the brain. The gradual loss of hair cells is common after a lifetime of chronic exposure to loud noises. Mammalian hair cells lack the ability to regenerate after embryonic development. Advancements in regenerative medicine strategies have been employed to restore function to damaged organs. The use of stem cells can regenerate and replace damaged hair cells. Understanding how stem cells differentiate into hair cells will provide insight into regeneration. To study hair cell regeneration, we employed an inner ear organoid system. Immortalized multipotent otic progenitor (iMOP) cells that differentiate into spiral ganglion neurons, supporting cells, and hair cells were used to generate organoids. This study tests whether a bioreactor facilitates sensory epithelial differentiation. The bioreactor agitates the medium to promote nutrient diffusion in the cultures. After ten days of differentiation, iMOP-derived organoids were collected, and relative changes in transcripts and protein markers that correspond to neurons, supporting cells, and hair cells were determined. The use of a bioreactor increased the percentage of cells expressing MYO6, a hair cell marker. The percentage of TUBB3 labeled neurons also increased while GFAP labeled supporting cells remained the same. Relative levels of Myo6 mRNA did not increase in any of the culture conditions while relative levels of Tubb3 mRNA significantly increased in all cultures compared to control samples. Although molecular mechanisms are unknown, the study shows that use of the bioreactor improves hair cell differentiation in otic progenitor-derived organoids.
  Muscle-specific functional deficits and lifelongfibrosisin response to paediatric radiotherapy and tumourelimination
  Jacob G. Kallenbach - 2022
  Abstract
  BackgroundAs paediatric cancer survivors are living into adulthood, they suffer from the age-related, accelerateddecline of functional skeletal muscle tissue, termed sarcopenia. With ionizing radiation (radiotherapy) at the core ofpaediatric cancer therapies, its direct and indirect effects can have lifelong negative impacts on paediatric growthand maintenance of skeletal muscle. Utilizing our recently developed preclinical rhabdomyosarcoma mouse model,we investigated the late effects of paediatric radiation treatment on skeletal muscles from late adolescent (8 weeksold) and middle-aged (16 months old) mice.MethodsPaediatric C57BL/6J male mice (3 weeks old) were injected with rhabdomyosarcoma cells into their righthindlimbs, and then fractionated irradiation (3 × 8.2 Gy) was administered to those limbs at 4 weeks old to eliminatethe tumours. Radiation-alone and tumour-irradiated mice were assessed at either 8 weeks (3 weeks post-irradiation) or16 months (14 months post-irradiation) of age for muscle physiology, myofibre characteristics, cell loss, histopathology,fibrosis, inflammatory gene expression, andfibrotic gene expression.ResultsMice that received only paediatric radiation demonstrated reduced muscle mass (17%,P<0.001), musclephysiological function (25%,P<0.01), muscle contractile kinetics (25%,P<0.05), satellite cell number (45%,P<0.05), myofibre cross-sectional area (30%,P<0.0001), and myonuclear number (17%,P<0.001). Paediatricradiation increased inflammatory gene expression, increasedfibrotic gene expression, and induced extracellular matrixprotein deposition (fibrosis) with tumour elimination exacerbating some phenotypes. Paediatric tumour-eliminatedmice demonstrated exacerbated deficits to function (20%,P<0.05) and myofibre size (17%,P<0.001) in somemuscles as well as further increases to inflammatory andfibrotic gene expression. Examining the age-related effectsof paediatric radiotherapy in middle-aged mice, we found persistent myofibre atrophy (20%,P<0.01), myonuclearloss (18%,P<0.001), up-regulated inflammatory andfibrotic signalling, and lifelongfibrosis.ConclusionsThe results from this paediatric radiotherapy model are consistent and recapitulate the clinical and mo-lecular features of accelerated sarcopenia, musculoskeletal frailty, and radiation-inducedfibrosis experienced by paedi-atric cancer survivors. We believe that this preclinical mouse model is well poised for future mechanistic insights andtherapeutic interventions that improve the quality of life for paediatric cancer survivors.
  The Betacoronavirus PHEV Replicates and Disrupts the Respiratory Epithelia and Upregulates Key Pattern Recognition Receptor Genes and Downstream Mediators, Including IL-8 and IFN-l
  Rahul Nelli - 2021
  Abstract
  The upper respiratory tract is the primary site of infection by porcine hemagglutinating encephalomyelitis virus (PHEV). In this study, primary porcine respiratory epithelial cells (PRECs) were cultured in an air-liquid interface (ALI) to differentiate into a pseudostratified columnar epithelium, proliferative basal cells, M cells, ciliated cells, and mucus-secreting goblet cells. ALI-PRECs recreates a cell culture environment morphologically and functionally more representative of the epithelial lining of the swine trachea than traditional culture systems. PHEV replicated actively in this environment, inducing cytopathic changes and progressive disruption of the mucociliary apparatus. The innate immunity against PHEV was comparatively evaluated in ALI-PREC cultures and tracheal tissue sections derived from the same cesarean-derived, colostrum-deprived (CDCD) neonatal donor pigs. Increased expression levels of TLR3 and/or TLR7, RIG1, and MyD88 genes were detected in response to infection, resulting in the transcriptional upregulation of IFN-l1 in both ALI-PREC cultures and tracheal epithelia. IFN-l1 triggered the upregulation of the transcription factor STAT1, which in turn induced the expression of the antiviral IFN-stimulated genes OAS1 and Mx1. No significant modulation of the major proinflammatory cytokines interleukin-1b (IL-1b), IL-6, and tumor necrosis factor alpha (TNF-a) was detected in response to PHEV infection. However, a significant upregulation of different chemokines was observed in ALI-PREC cultures (CCL2, CCL5, CXCL8, and CXCL10) and tracheal epithelium (CXCL8 and CXCL10). This study shed light on the molecular mechanisms driving the innate immune response to PHEV at the airway epithelium, underscoring the important role of respiratory epithelial cells in the maintenance of respiratory homeostasis and on the initiation, resolution, and outcome of the infectious process. IMPORTANCE The neurotropic betacoronavirus porcine hemagglutinating encephalomyelitis virus (PHEV) primarily infects and replicates in the swine upper respiratory tract, causing vomiting and wasting disease and/or encephalomyelitis in suckling pigs. This study investigated the modulation of key early innate immune genes at the respiratory epithelia in vivo, on tracheal tissue sections from experimentally infected pigs, and in vitro, on air-liquid interface porcine respiratory cell cultures. The results from the study underscore the important role of respiratory epithelial cells in maintaining respiratory homeostasis and on the initiation, resolution, and outcome of the PHEV infectious process
  Glucocorticoid-mediated induction of caveolin-1 disrupts cytoskeletal organization, inhibits cell migration and re-epithelialization of non-healing wounds
  Ivan Jozic - 2021
  Abstract
  Although impaired keratinocyte migration is a recognized hallmark of chronic wounds, the molecular mechanisms underpinning impaired cell movement are poorly understood. Here, we demonstrate that both diabetic foot ulcers (DFUs) and venous leg ulcers (VLUs) exhibit global deregulation of cytoskeletal organization in genomic comparison to normal skin and acute wounds. Interestingly, we found that DFUs and VLUs exhibited downregulation of ArhGAP35, which serves both as an inactivator of RhoA and as a glucocorticoid repressor. Since chronic wounds exhibit elevated levels of cortisol and caveolin-1 (Cav1), we posited that observed elevation of Cav1 expression may contribute to impaired actin-cytoskeletal signaling, manifesting in aberrant keratinocyte migration. We showed that Cav1 indeed antagonizes ArhGAP35, resulting in increased activation of RhoA and diminished activation of Cdc42, which can be rescued by Cav1 disruption. Furthermore, we demonstrate that both inducible keratinocyte specific Cav1 knockout mice, and MβCD treated diabetic mice, exhibit accelerated wound closure. Taken together, our findings provide a previously unreported mechanism by which Cav1-mediated cytoskeletal organization prevents wound closure in patients with chronic wounds.
Sample Preparation
- DNA
  Plaque-associated human microglia accumulate lipid droplets in a chimeric model of Alzheimer’s disease
  Christel Claes - 2021
  Abstract
  Background Disease-associated microglia (DAMs), that surround beta-amyloid plaques, represent a transcriptionally-distinct microglial profile in Alzheimer’s disease (AD). Activation of DAMs is dependent on triggering receptor expressed on myeloid cells 2 (TREM2) in mouse models and the AD TREM2-R47H risk variant reduces microglial activation and plaque association in human carriers. Interestingly, TREM2 has also been identified as a microglial lipid-sensor, and recent data indicates lipid droplet accumulation in aged microglia, that is in turn associated with a dysfunctional proinflammatory phenotype. However, whether lipid droplets (LDs) are present in human microglia in AD and how the R47H mutation affects this remains unknown. Methods To determine the impact of the TREM2 R47H mutation on human microglial function in vivo, we transplanted wild-type and isogenic TREM2-R47H iPSC-derived microglial progenitors into our recently developed chimeric Alzheimer mouse model. At 7 months of age scRNA-seq and histological analyses were performed. Results Here we report that the transcriptome of human wild-type TREM2 and isogenic TREM2-R47H DAM xenografted microglia (xMGs), isolated from chimeric AD mice, closely resembles that of human atherosclerotic foam cells. In addition, much like foam cells, plaque-bound xMGs are highly enriched in lipid droplets. Somewhat surprisingly and in contrast to a recent in vitro study, TREM2-R47H mutant xMGs exhibit an overall reduction in the accumulation of lipid droplets in vivo. Notably, TREM2-R47H xMGs also show overall reduced reactivity to plaques, including diminished plaque-proximity, reduced CD9 expression, and lower secretion of plaque-associated APOE. Conclusions Altogether, these results indicate lipid droplet accumulation occurs in human DAM xMGs in AD, but is reduced in TREM2-R47H DAM xMGs, as it occurs secondary to TREM2-mediated changes in plaque proximity and reactivity.