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PerfeCTa SYBR® Green FastMix

Super. Fast. PerfeCTa.
Features & Benefits
  • 2x concentrated reagents minimize pipetting steps, simplify reaction assembly and improve accuracy
  • Superior assay sensitivity and specificity with ultrapure AccuStart enzyme technology – maximum-yielding Taq DNA polymerase mutant controlled by stringent, multi-epitope antibody hot start
  • Supports efficient vortex mixing with proprietary anti-foaming formulation
  • FastMix formulation supports both fast and standard thermal cycling conditions

 

PerfeCTa SYBR Green FastMix is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

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PerfeCTa SYBR Green FastMix for IQ
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Kit Size:  5000 x 20 μL rxns (1 x 50 mL)
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PerfeCTa SYBR Green FastMix
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Price:  $258.00
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Kit Size:  1250 x 20 μL rxns (10 x 1.25 mL)
Part Number:  95072-012
Price:  $831.00
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Kit Size:  5000 x 20 μL rxns (1 x 50 mL)
Part Number:  95072-05K
Price:  $2,849.00
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PerfeCTa SYBR Green FastMix ROX
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Kit Size:  1250 x 20 μL rxns (10 x 1.25 mL)
Part Number:  95073-012
Price:  $831.00
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Kit Size:  5000 x 20 μL rxns (1 x 50 mL)
Part Number:  95073-05K
Price:  $2,849.00
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PerfeCTa SYBR Green FastMix Low ROX
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Kit Size:  250 x 20 μL rxns (2 x 1.25 mL)
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Kit Size:  1250 x 20 μL rxns (10 x 1.25 mL)
Part Number:  95074-012
Price:  $831.00
Add to Cart
Kit Size:  5000 x 20 μL rxns (1 x 50 mL)
Part Number:  95074-05K
Price:  $2,849.00
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Description

PerfeCTa SYBR® Green FastMix is a 2X concentrated, ready-to-use reaction cocktail that contains all components, except primers and DNA template. This rigorously optimized master mix contains of proprietary buffer technology, stabilizers and AccuFast Taq DNA polymerase to deliver maximum assay precision, sensitivity, and PCR efficiency for accelerated or conventional thermal cycling conditions for SYBR Green detection. Dye-based detection methods are critically dependent on highly specific amplification because dsDNA dyes will bind to any amplicon, including off-target primer elongation and primer dimerization. AccuFast hot start Taq DNA polymerase contains a proprietary mixture of ultra-pure monoclonal antibodies that stringently suppress primer elongation prior to the initial PCR denaturation step and allows for setup and multi-day storage at ambient room temperature prior to thermal cycling. AccuFast provides rapid release of fully active enzyme to support accelerated thermal cycling conditions.

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Details

Details

Contents

Single-tube, 2X concentrated reagent containing:

  • Reaction buffer with optimized concentrations of molecular-grade MgCl2, dATP, dCTP, dGTP, and dTTP.
  • AccuStart II Taq DNA Polymerase
  • SYBR Green I dye
  • Proprietary enzyme stabilizers and performance-enhancing additives.
  • Titrated reference dye (if applicable)
Instrument Capability

Instrument Capability

ROX

  • Applied Biosystems 5700
  • Applied Biosystems 7000
  • Applied Biosystems 7300
  • Applied Biosystems 7700
  • Applied Biosystems 7900
  • Applied Biosystems 7900HT
  • Applied Biosystems 7900 HT Fast
  • Applied Biosystems StepOne™
  • Applied Biosystems StepOnePlus™

Low ROX

  • Applied Biosystems 7500
  • Applied Biosystems 7500 Fast
  • Stratagene Mx3000P®
  • Stratagene Mx3005P™
  • Stratagene Mx4000™
  • Applied Biosystems ViiA 7
  • Applied Biosystems QuantStudio™ (all models)
  • Douglas Scientific IntelliQube®

No ROX

  • Quantabio Q
  • BioRad CFX
  • Roche LightCycler 480
  • QIAGEN Rotor-Gene Q
  • Agilent AriaMx
  • Azure Cielo™
  • Other

Bio-Rad iCycler iQ systems

  • BioRad iCycler iQ™
  • BioRad MyiQ™
  • BioRad iQ™5
Customer Testimonials

Customer Testimonials

PerfeCTa SYBR Green FastMix

"With the PerfeCTa SYBR Green FastMix we had less variability in our triplicates and more reproducible results between repeat experiments."

With the PerfeCTa SYBR Green FastMix we had less variability in our triplicates and more reproducible results between repeat experiments. | Professor, UCSF

Details

Contents

Single-tube, 2X concentrated reagent containing:

  • Reaction buffer with optimized concentrations of molecular-grade MgCl2, dATP, dCTP, dGTP, and dTTP.
  • AccuStart II Taq DNA Polymerase
  • SYBR Green I dye
  • Proprietary enzyme stabilizers and performance-enhancing additives.
  • Titrated reference dye (if applicable)

Instrument Capability

ROX

  • Applied Biosystems 5700
  • Applied Biosystems 7000
  • Applied Biosystems 7300
  • Applied Biosystems 7700
  • Applied Biosystems 7900
  • Applied Biosystems 7900HT
  • Applied Biosystems 7900 HT Fast
  • Applied Biosystems StepOne™
  • Applied Biosystems StepOnePlus™

Low ROX

  • Applied Biosystems 7500
  • Applied Biosystems 7500 Fast
  • Stratagene Mx3000P®
  • Stratagene Mx3005P™
  • Stratagene Mx4000™
  • Applied Biosystems ViiA 7
  • Applied Biosystems QuantStudio™ (all models)
  • Douglas Scientific IntelliQube®

No ROX

  • Quantabio Q
  • BioRad CFX
  • Roche LightCycler 480
  • QIAGEN Rotor-Gene Q
  • Agilent AriaMx
  • Azure Cielo™
  • Other

Bio-Rad iCycler iQ systems

  • BioRad iCycler iQ™
  • BioRad MyiQ™
  • BioRad iQ™5

Performance Data

Resources

Customer Profile Stories

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Product Manuals

Application Notes

CofA (PSFs)

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SDSs

FAQs

  • We use Perfecta Sybr Green Fastmix. We are starting to use uMELT software to predict qPCR melt curves. Could you please provide me with this info so that I can accurately use uMELT? [Mono+] : Concentration of monovalent cations in solution (mM).
    The PerfeCta SYBR Green Fastmix has 50 mM salt and 2.2mM Free Mg at 1X. However, the co-solvents and the concentration of SYBR green I dye in the mastermix also affect amplicon Tm, so you may still not be able to accurately use uMELT. Our R&D has found uMELT mostly useful for predicting multiple melting domains in an amplicon.
  • Can I use PerfeCTa® SYBR® Green FastMix® instead of SYBR Green SuperMix?
    You can use FastMix instead of SuperMix. We have observed similar results with both PCR mixes, however, slightly lower background was observed using SuperMix when using 10 ng of cDNA or more in the qPCR. The qScript microRNA Quantification system has been validated using SYBR Green SuperMix.
  • Publications

    Arc1: a regulator of triglyceride homeostasis in male Drosophila
    Samuel D. Swope - 2023
    Abstract
    Achieving metabolic homeostasis is necessary for survival, and many genes are required to control organismal metabolism. A genetic screen in Drosophila larvae identified putative fat storage genes including Arc1. Arc1 has been shown to act in neurons to regulate larval lipid storage; however, whether Arc1 functions to regulate adult metabolism is unknown. Arc1 esm18 males store more fat than controls while both groups eat similar amounts. Arc1 esm18 flies express more brummer lipase and less of the glycolytic enzyme triose phosphate isomerase, which may contribute to excess fat observed in these mutants. These results suggest that Arc1 regulates adult Drosophila lipid homeostasis.
    Differences in both expression and protein activity contribute to the distinct functions of AINTEGUMENTA compared with AINTEGUMENTA-LIKE 5 and AINTEGUMENTA-LIKE 7
    Beth A. Krizek - 2023
    Abstract
    Three members of the Arabidopsis AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) transcription factor family, AIL5/PLT5, AIL6/PLT3, and AIL7/PLT7, exhibit partially overlapping roles with AINTEGUMENTA (ANT) during flower development. Loss of ANT function alone results in smaller floral organs and female sterility indicating that some ANT functions cannot be provided by these related transcription factors. Previously, we showed that expression of AIL6 at the same levels and spatial pattern as ANT could largely rescue the defects of ant mutants. This suggested that the functional differences between ANT and AIL6 were primarily a consequence of expression differences. Here, we investigated the functional differences between ANT and both AIL5 and AIL7 by expressing these two AILs under the control of the ANT promoter. We found that only ANT:gAIL5 lines with much higher amounts of AIL5 mRNA as compared with ANT could compensate for loss of ANT function. ANT:gAIL7 lines with AIL7 mRNA levels similar to those of ANT were able to rescue some but not all aspects of the ant mutant phenotype. Thus, expression differences alone cannot explain the functional differences between ANT and these two related proteins. Studies in yeast show that AIL5 and AIL7 have lower transcriptional activation activities as compared with ANT and AIL6 when bound to the consensus ANT DNA binding site. Our results suggest that differences in both expression and protein activity contribute to the functional specificity of ANT compared with AIL5 and AIL7.
    DPPA3-HIF1α axis controls colorectal cancer chemoresistance by imposing a slow cell-cycle phenotype
    Estefania Cuesta-Borràs - 2023
    Abstract
    Tumor relapse is linked to rapid chemoresistance and represents a bottleneck for cancer therapy success. Engagement of a reduced proliferation state is a non-mutational mechanism exploited by cancer cells to bypass therapy-induced cell death. Through combining functional pulse-chase experiments in engineered cells and transcriptomic analyses, we identify DPPA3 as a master regulator of slow-cycling and chemoresistant phenotype in colorectal cancer (CRC). We find a vicious DPPA3-HIF1α feedback loop that downregulates FOXM1 expression via DNA methylation, thereby delaying cell-cycle progression. Moreover, downregulation of HIF1α partially restores a chemosensitive proliferative phenotype in DPPA3-overexpressing cancer cells. In cohorts of CRC patient samples, DPPA3 overexpression acts as a predictive biomarker of chemotherapeutic resistance that subsequently requires reduction in its expression to allow metastatic outgrowth. Our work demonstrates that slow-cycling cancer cells exploit a DPPA3/HIF1α axis to support tumor persistence under therapeutic stress and provides insights on the molecular regulation of disease progression.
    THE ALPHAVIRAL CAPSID PROTEIN INHIBITS IRAK1-DEPENDENT TLR SIGNALING TO PROMOTE PATHOGENESIS
    V “Trey” Douglas Landers - 2023
    Abstract
    Alphaviruses are positive-sense RNA viruses spread by mosquitos. They can cause a severe multi-joint febrile arthritis or encephalitis resulting in death or life-long cognitive impairments. To date there are no approved antiviral therapeutics or vaccine strategies for the treatment of alphaviruses creating a critical need to better understand the host pathogen interactions of alphaviruses that enable pathogenesis. It is known that alphaviruses evade innate immune responses by shutting down host transcription and translation, but these methods are dependent on viral gene expression and leave a critical time frame during early infection before viral gene expression has begun that the virus can be identified and responded to by host cells. In order to identify potential viral products that could serve to mask the virus during early infection Sindbis virus, a model alphavirus, was tested for the expression of proteins that could interfere with intracellular immune defenses. vi Through these efforts we discovered that the alphavirus capsid protein (CP) interacts with host Interleukin 1 Receptor Associated Kinase 1 (IRAK1) in order to block Toll-Like Receptor Signaling (TLR). IRAK1 is a key signaling kinase for several pro-inflammatory immune pathways and CP interacting with it provides a possible mechanism of inhibiting detection by host cells. Pursuing this interaction, we discovered that CP from several members of the Alphavirus family are capable of binding IRAK1 and inhibiting IRAK1-dependent TLR signaling. We were able to map the necessary interaction determinates on CP which, when mutated, ablated IRAK1 binding and restored IRAK1-depdent signaling. Host cells infected with this mutant virus increased their expression of IFN-β in vitro, relative to cells infected with wild-type Sindbis virus. The mutant virus was also impaired in an in vivo model of infection where it failed to cause symptoms of alphavirus infection. Collectively these data show a novel interaction between alphavirus CP and host IRAK1 that allows the virus to evade the immune detection during early infection a crucial time when the virus has yet not been able to shut down host transcription and translation. This interaction was also found to be crucial for viral pathogenesis and the absence of it leaves the virus attenuated. The data presented in this dissertation offer insight into a newly observed method Alphavirus uses to evade detection by the innate immune system which could be a potential target for therapeutic intervention as well as a mutant virus that could be a candidate for a live-attenuated vaccine.
    Distinct Requirements for Adaptor Proteins NCK1 and NCK2 in Mammary Gland Development
    Adam P. Golding - 2023
    Abstract
    The adaptor proteins NCK1 and NCK2 are well-established signalling nodes that regulate diverse biological processes including cell proliferation and actin dynamics in many tissue types. Here we have investigated the distribution and function of Nck1 and Nck2 in the developing mouse mammary gland. Using publicly available single-cell RNA sequencing data, we uncovered distinct expression profiles between the two paralogs. Nck1 showed widespread expression in luminal, basal, stromal and endothelial cells, while Nck2 was restricted to luminal and basal cells, with prominent enrichment in hormone-sensing luminal subtypes. Next, using mice with global knockout of Nck1 or Nck2, we assessed mammary gland development during and after puberty (5, 8 and 12 weeks of age). Mice lacking Nck1 or Nck2 displayed significant defects in ductal outgrowth and branching at 5 weeks compared to controls, and the defects persisted in Nck2 knockout mice at 8 weeks before normalizing at 12 weeks. These defects were accompanied by an increase in epithelial cell proliferation at 5 weeks and a decrease at 8 weeks in both Nck1 and Nck2 knockout mice. We also profiled expression of several key genes associated with mammary gland development at these timepoints and detected temporal changes in transcript levels of hormone receptors as well as effectors of cell proliferation and migration in Nck1 and Nck2 knockout mice, in line with the distinct phenotypes observed at 5 and 8 weeks. Together these studies reveal a requirement for NCK proteins in mammary gland morphogenesis, and suggest that deregulation of Nck expression could drive breast cancer progression and metastasis.
    Microbial and Immune Control of Intestinal Stem Cells
    Meghan Patricia Ferguson - 2023
    Abstract
    Intestinal epithelial damage and homeostatic cell shedding sends evolutionarily conserved growth signals to activate division programs in stem cells, which renews the epithelium to maintain barrier integrity[1]. In addition to conserved growth and stress signaling, microbes and innate immune pathways regulate stem cell function during homeostasis and disease. For instance, the microbiome promotes stem cell proliferation and differentiation while overactive innate immune signaling causes stem cell hyperproliferation and exacerbates tumorigenesis[2]. It is clear bacteria and immune pathways are important regulators of stem cell function, however the complexity of the microbiome has made it difficult to elucidate the specific effects individual bacterial species have on stem cells. In addition, the intestine is a heterologous tissue composed of multiple different specialized cell types, including intestinal stem cells, absorptive enterocytes and secretory enteroendocrine cells. Given the heterogeneity of cell types in the intestine and the limitations of vertebrate genetics, cell-type specific contributions of immune pathway activation on intestinal growth have been hard to determine, especially the impact of immune activity in stem cells. To determine how individual bacterial species and immune pathways impact stem cell function I used Drosophila melanogaster as a model system. Drosophila provides a number of advantages for this work. For example, Drosophila can be easily reared without a microbiome and associated with single bacterial species to determine their effects. In addition, Drosophila genetics allow for the perturbation of immune signaling in distinct cell types of the intestine, including stem cells. Using Drosophila, I identified the commensal bacteria Lactobacillus brevis as a potent stimulator of stem cell divisions and tumorigenesis. L. brevis altered the expression and intracellular localization of integrins in stem cells, leading to symmetric stem cell expansion. Next, I asked what iii the consequences of immune signaling in stem cells are by activating or inhibiting the immune deficiency (IMD) pathway. Activation of IMD in progenitor cells resulted in intestinal hyperplasia and exacerbation of tumorigenesis. Furthermore, inhibition of IMD reduced homeostatic proliferation and disrupted differentiation. Finally, I asked if the NF-kB family transcription factor Relish acts in stem cells to modulate damage response. Damage of the intestine results in elevated stem cell divisions to repair the injury. However, stem cell specific Relish depletion caused stem cells and their progeny to undergo apoptosis, rendering intestines incapable of effective epithelial repair, leading to host lethality. Thus, bacterial species can have profound effects on stem cell physiology and that immune pathways act directly in stem cells to modify proliferation, tumorigenesis, differentiation and epithelial repair responses.
    Functional expression of the thermally activated transient receptor potential channels TRPA1 and TRPM8 in human myotubes
    Christine Skagen - 2023
    Abstract
    Transient potential (TRP) ion channels expressed in primary sensory neurons act as the initial detectors of environmental cold and heat, information which controls muscle energy expenditure. We hypothesize that non-neuronal TRPs have direct cellular responses to thermal exposure, also affecting cellular metabolism. In the present study we show expression of TRPA1, TRPM8 and TRPV1 in rat skeletal muscle and human primary myotubes by qPCR. Effects of TRP activity on metabolism in human myotubes were studied using radiolabeled glucose. FURA-2 was used for Ca2+ imaging. TRPA1, TRPM8 and TRPV1 were expressed at low levels in primary human myotubes and in m. gastrocnemius, m. soleus, and m. trapezius from rat. Activation of TRPA1 by ligustilide resulted in an increased glucose uptake and oxidation in human myotubes, whereas activation of TRPM8 by menthol and icilin significantly decreased glucose uptake and oxidation. Activation of heat sensing TRPV1 by capsaicin had no effect on glucose metabolism. Agonist-induced increases in intracellular Ca2+ levels by ligustilide and icilin in human myotubes confirmed a direct activation of TRPA1 and TRPM8, respectively. The mRNA expression of some genes involved in thermogenesis, i.e. peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), uncoupling protein (UCP) 1 and UCP3, were downregulated in human myotubes following TRPA1 activation, while the mRNA expression of TRPM8 and TRPA1 were downregulated following TRPM8 activation by menthol and icilin, respectively. Cold exposure (18 °C) of cultured myotubes followed by a short recovery period had no effect on glucose uptake and oxidation in the basal situation, however when TRPA1 and TRPM8 channels were chemically inhibited a temperature-induced difference in glucose metabolism was found. In conclusion, mRNA of TRPA1, TRPM8 and TRPV1 was expressed in rat skeletal muscle and human skeletal muscle cells. Modulation of TRPA1 and TRPM8 by chemical agents induced changes in Ca2+ levels and glucose metabolism in human skeletal muscle cells, indicating functional receptors.
    miR-33a Expression Attenuates ABCA1-Dependent Cholesterol Efflux and Promotes Macrophage-Like Cell Transdifferentiation in Cultured Vascular Smooth Muscle Cells
    Ikechukwu C. Esobi - 2023
    Abstract
    Recent evidence suggests that the majority of cholesterol-laden cells found in atherosclerotic lesions are vascular smooth muscle cells (VSMC) that have transdifferentiated into macrophage-like cells (MLC). Furthermore, cholesterol-laden MLC of VSMC origin have demonstrated impaired ABCA1-dependent cholesterol efflux, but it is poorly understood why this occurs. A possible mechanism which may at least partially be attributed to cholesterol-laden MLC demonstrating attenuated ABCA1-dependent cholesterol efflux is a miR-33a expression, as a primary function of this microRNA is to silence ABCA1 expression, but this has yet to be rigorously investigated. Therefore, the VSMC line MOVAS cells were used to generate miR-33a knockout (KO) MOVAS cells, and we used KO and wild-type (WT) MOVAS cells to delineate any possible proatherogenic role of miR-33a expression in VSMC. When WT and KO MOVAS cells were cholesterol-loaded to convert into MLC, this resulted in the WT MOVAS cells to exhibit impaired ABCA1-dependent cholesterol efflux. In the cholesterol-loaded WT MOVAS MLC, we also observed a delayed restoration of the VSMC phenotype when these cells were exposed to the ABCA1 cholesterol acceptor, apoAI. These results imply that miR-33a expression in VSMC drives atherosclerosis by triggering MLC transdifferentiation via attenuated ABCA1-dependent cholesterol efflux.
    Chronodisruption and Loss of Melatonin Rhythm, Associated with Alterations in Daily Motor Activity and Mitochondrial Dynamics in Parkinsonian Zebrafish, Are Corrected by Melatonin Treatment
    Paula Aranda-Martínez - 2023
    Abstract
    Beyond sleep/wake, clock genes regulate the daily rhythms of melatonin production, motor activity, innate immunity, and mitochondrial dynamics, among others. All these rhythms are affected in Parkinson’s disease (PD), suggesting that chronodisruption may be an early stage of the disease. The aim of this study was to evaluate the connection between clock genes and these rhythms in PD, and whether melatonin administration reestablished the normal clock function. Parkinsonism was induced with 600 µM MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in 24–120 h post fertilization (hpf) zebrafish embryos and melatonin was administered at a dose of 1 µM. Day–night melatonin rhythm disappeared in MPTP-treated embryos, which showed an advance in the activity phase in parallel with changes in the rhythm of clock genes. An alteration in the fission-to-fusion mitochondrial dynamics was also detected in parkinsonian embryos, increasing the former and leading to apoptosis. Melatonin administration to MPTP-treated embryos fully restored the circadian system, including the rhythms of clock genes, motor activity, melatonin rhythm, and mitochondrial dynamics, and decreasing apoptosis. Because clock-controlled rhythms such as sleep/wake alterations are early events in PD, the data here reported may point to chronodisruption as one initial pathophysiological event of the disease.
    Lifetime Evaluation of Left Ventricular Structure and Function in Male C57BL/6J Mice after Gamma and Space-Type Radiation Exposure
    Agnieszka Brojakowska - 2023
    Abstract
    The lifetime effects of space irradiation (IR) on left ventricular (LV) function are unknown. The cardiac effects induced by space-type IR, specifically 5-ion simplified galactic cosmic ray simulation (simGCRsim), are yet to be discovered. Three-month-old, age-matched, male C57BL/6J mice were irradiated with 137Cs gamma (γ; 100, 200 cGy) and simGCRsim (50 and 100 cGy). LV function was assessed via transthoracic echocardiography at 14 and 28 days (early), and at 365, 440, and 660 (late) days post IR. We measured the endothelial function marker brain natriuretic peptide in plasma at three late timepoints. We assessed the mRNA expression of the genes involved in cardiac remodeling, fibrosis, inflammation, and calcium handling in LVs harvested at 660 days post IR. All IR groups had impaired global LV systolic function at 14, 28, and 365 days. At 660 days, 50 cGy simGCRsim-IR mice exhibited preserved LV systolic function with altered LV size and mass. At this timepoint, the simGCRsim-IR mice had elevated levels of cardiac fibrosis, inflammation, and hypertrophy markers Tgfβ1, Mcp1, Mmp9, and βmhc, suggesting that space-type IR may induce the cardiac remodeling processes that are commonly associated with diastolic dysfunction. IR groups showing statistical significance were modeled to calculate the Relative Biological Effectiveness (RBE) and Radiation Effects Ratio (RER). The observed dose-response shape did not indicate a lower threshold at these IR doses. A single full-body IR at doses of 100–200 cGy for γ-IR, and 50–100 cGy for simGCRsim-IR decreases the global LV systolic function in WT mice as early as 14 and 28 days after exposure, and at 660 days post IR. Interestingly, there is an intermediate time point (365 days) where the impairment in LV function is observed. These findings do not exclude the possibility of increased acute or degenerative cardiovascular disease risks at lower doses of space-type IR, and/or when combined with other space travel-associated stressors such as microgravity.
    Comparative Phenotypic, Genomic, and Transcriptomic Analyses of Two Contrasting Strains of the Plant Beneficial Fungus Trichoderma virens
    Shikha Pachauri - 2023
    Abstract
    Trichoderma virens is a beneficial fungus that helps plants fight pathogens and abiotic stresses and thereby enhances crop yields. Unlike other Trichoderma spp., there are two well-defined strains (P and Q) of T. virens, classified by secondary metabolites profiling, primarily the biosynthesis of the nonribosomal, strong antimicrobial agents gliotoxin (Q) and gliovirin (P). We have studied the phenotypic and biocontrol properties of two well-studied representative isolates (T. virens Gv29-8 and T. virens GvW/IMI304061) that represent a Q strain and a P strain of T. virens, respectively. We refined the genome assembly of the P strain using nanopore technology, and we compared it with the Q strain. The differences between the genomes include gene expansion in the Q strain. T. virens Gv29-8 is weaker than GvW as a mycoparasite on the broad host-range plant pathogen Sclerotium rolfsii, and it is ineffective as a biocontrol agent when applied to pathogen-infested soil. T. virens Gv29-8 proved to be phytotoxic to Arabidopsis seedlings, whereas the effect of T. virens GvW was not major. Both strains colonized the surface and outer cortex layer of tomato roots, with about 40% higher colonization by T. virens Gv29- 8. T. virens Gv29-8 induced the expression of a larger set of tomato genes than did T. virens GvW, although some tomato genes were uniquely induced in response to T. virens GvW. We studied the comparative transcriptome response of T. virens Gv29-8 and T. virens GvW to S. rolfsii. A larger set of genes was regulated in T. virens GvW than in T. virens Gv29-8 in the presence of the plant pathogen. IMPORTANCE Trichoderma virens populations that were earlier classified into two strains (P and Q) based on secondary metabolites profiling are also phenotypically and genetically distinct, with the latter being ineffective in controlling the devastating, broad host range plant pathogen Sclerotium rolfsii. The two strains also provoke distinct as well as overlapping transcriptional responses to the presence of the plant and the pathogen. This study enriches our knowledge of Trichoderma-plant-pathogen interactions and identifies novel candidate genes for further research and deployment in agriculture.
    Characterization of Hyaluronan Localization in the Developing Mammary Gland and Mammary Tumors
    Patrice M. Witschen - 2023
    Abstract
    The extracellular matrix (ECM) is biochemically and biomechanically important for the structure and function of the mammary gland, which undergoes vast structural changes throughout pubertal and reproductive development. Although hyaluronan (HA) is a ubiquitous glycosaminoglycan (GAG) of the mammary gland ECM, extensive characterization of HA deposition in the mammary gland is lacking. Understanding physiologic HA metabolism is critical as this tightly controlled system is often hijacked in cancer. In the current studies, we characterize HA regulation throughout mammary gland development to better understand subsequent dysregulation of HA in mammary tumors. Using immunofluorescence (IF) imaging, we demonstrate that organized HA-rich septa exist in the mammary gland stroma throughout puberty, pregnancy, and involution. Furthermore, we find heterogeneous HA deposition within two murine models of breast cancer. Using cell specific isolation techniques, we characterize expression of genes associated with HA binding, synthesis, and degradation within EpCAM + epithelial cells, CD90.2 + fibroblasts, and F4/80 + macrophages isolated from mammary glands and tumors. Most notably, we identify elevated levels of the hyaluronidases Hyal1 and Hyal2 in tumor-association macrophages (TAMs), suggesting a role for TAM-mediated turnover of HA in the tumor microenvironment (TME). Gene expression is supported functionally by in vitro experiments in which macrophages treated with tumor-cell conditioned media exhibit increased hyaluronidase activity. These findings link TAMs to the direct degradation of HA within the TME of mammary tumors, which has negative implications for patient survival.
    Variable effects of Wolbachia on alphavirus infection in Aedes aegypti
    Brittany L. Dodson - 2023
    Abstract
    Wolbachia pipientis (=Wolbachia) has promise as a tool to suppress virus transmission by Aedes aegypti mosquitoes. However, Wolbachia can have variable effects on mosquitoborne viruses. This variation remains poorly characterized, yet the multimodal effects of Wolbachia on diverse pathogens could have important implications for public health. Here, we examine the effects of somatic infection with two strains of Wolbachia (wAlbB and wMel) on the alphaviruses Sindbis virus (SINV), O’nyong-nyong virus (ONNV), and Mayaro virus (MAYV) in Ae. aegypti. We found variable effects of Wolbachia including enhancement and suppression of viral infections, with some effects depending on Wolbachia strain. Both wAlbB- and wMel-infected mosquitoes showed enhancement of SINV infection rates one week post-infection, with wAlbB-infected mosquitoes also having higher viral titers than controls. Infection rates with ONNV were low across all treatments and no significant effects of Wolbachia were observed. The effects of Wolbachia on MAYV infections were strikingly strain-specific; wMel strongly blocked MAYV infections and suppressed viral titers, while wAlbB did not influence MAYV infection. The variable effects of Wolbachia on vector competence underscore the importance of further research into how this bacterium impacts the virome of wild mosquitoes including the emergent human pathogens they transmit.
    Distinct effect of exercise modes on mood-related behavior in mice
    Ki Hoon Yuk - 2023
    Abstract
    Exercise can afford several benefits to combat mood disorders in both rodents and humans. Engagement in various physical activities upregulates levels of neurotrophic factors in several brain regions and improves mental health. However, the type of exercise that regulates mood and the underlying mechanisms in the brain remain elusive. Herein, we performed two distinct types of exercise and RNA sequencing analyses to investigate the effect of exercise on mood-related behaviors and explain the distinct patterns of gene expression. Specifically, resistance exercise exhibited reduced immobility time in the forced swim test when compared with both no exercise and treadmill exercise (in the aerobic training [AT] group). Interestingly, anxiety-like behaviors in the open field and nest-building tests were ameliorated in the AT group when compared with those in the control group; however, this was not observed in the RT group. To elucidate the mechanism underlying these different behavioral changes caused by distinct exercise types, we examined the shift in the gene expression pattern in the hippocampus, a brain region that plays a critical role in regulating mood. We discovered that 38 and 40 genes were altered in the AT and RT groups, respectively, compared with the control group. Both exercises regulated 16 common genes. Compared with the control group, mitogen-activated protein kinase (MAPK) was enriched in the AT group and phosphatidylinositol-3-kinase (PI3K)/AKT and neurotrophin signaling pathways were enriched in the RT group, as determined by bioinformatics pathway analysis. PCR results revealed that Cebpβ expression was increased in AT group, and Dcx expression was upregulated in both groups. Our findings indicate that different exercise types may exert substantially distinct effects on mood-like behaviors. Accordingly, appropriate types of exercise can be undertaken based on the mood disorder to be regulated.
    CCR1 Mediates Müller Cell Activation and Photoreceptor Cell Death in Macular and Retinal Degeneration
    Sarah Elbaz-Hayoun - 2023
    Abstract
    Mononuclear cells are involved in the pathogenesis of retinal diseases, including age- related macular degeneration (AMD). Here, we examined the mechanisms that underlie macrophage-driven retinal cell death. Monocytes were extracted from patients with AMD and differentiated into macrophages (hMdɸs), which were characterized based on proteomics, gene expression, and ex vivo and in vivo properties. Using bioinformatics, we identified the signaling pathway involved in macrophage-driven retinal cell death, and we assessed the therapeutic potential of targeting this pathway. We found that M2a hMdɸs were associated with retinal cell death in retinal explants and following adoptive transfer in a photic injury model. Moreover, M2a hMdɸs express several CCRI (C-C chemokine receptor type 1) ligands. Importantly, CCR1 was upregulated in Müller cells in models of retinal injury and aging, and CCR1 expression was correlated with retinal damage. Lastly, inhibiting CCR1 reduced photic-induced retinal damage, photoreceptor cell apoptosis, and retinal inflammation. These data suggest that hMdɸs, CCR1, and Müller cells work together to drive retinal and macular degeneration, suggesting that CCR1 may serve as a target for treating these sight-threatening conditions.
    Central Role of Sibling Small RNAs NgncR_162 and NgncR_163 in Main Metabolic Pathways of Neisseria gonorrhoeae
    Thomas Steiner - 2023
    Abstract
    Small bacterial regulatory RNAs (sRNAs) have been implicated in the regulation of numerous metabolic pathways. In most of these studies, sRNA-dependent regulation of mRNAs or proteins of enzymes in metabolic pathways has been predicted to affect the metabolism of these bacteria. However, only in a very few cases has the role in metabolism been demonstrated. Here, we performed a combined transcriptome and metabolome analysis to define the regulon of the sibling sRNAs NgncR_162 and NgncR_163 (NgncR_162/163) and their impact on the metabolism of Neisseria gonorrhoeae. These sRNAs have been reported to control genes of the citric acid and methylcitric acid cycles by posttranscriptional negative regulation. By transcriptome analysis, we now expand the NgncR_162/163 regulon by several new members and provide evidence that the sibling sRNAs act as both negative and positive regulators of target gene expression. Newly identified NgncR_162/163 targets are mostly involved in transport processes, especially in the uptake of glycine, phenylalanine, and branched-chain amino acids. NgncR_162/163 also play key roles in the control of serine-glycine metabolism and, hence, probably affect biosyntheses of nucleotides, vitamins, and other amino acids via the supply of one-carbon (C1) units. Indeed, these roles were confirmed by metabolomics and metabolic flux analysis, which revealed a bipartite metabolic network with glucose degradation for the supply of anabolic pathways and the usage of amino acids via the citric acid cycle for energy metabolism. Thus, by combined deep RNA sequencing (RNA-seq) and metabolomics, we significantly extended the regulon of NgncR_162/163 and demonstrated the role of NgncR_162/163 in the regulation of central metabolic pathways of the gonococcus.
    Chromosome-level assembly of the Rangifer tarandus genome and validation of cervid and bovid evolution insights
    William Poisson - 2022
    Abstract
    Background:Genome assembly into chromosomes facilitates several analyses including cytogenetics, genomics and phylogenetics. Despite rapid development in bioinformatics, however, assembly beyond scaffolds remains challenging, especially in species without closely related well-assembled and available reference genomes. So far, four draft genomes of Rangifer tarandus (caribou or reindeer, a circumpolar distributed cervid species) have been published, but none with chromosome-level assembly. This emblematic northern species is of high interest in ecological studies and conservation since most populations are declining. Results:We have designed specific probes based on Oligopaint FISH technology to upgrade the latest published reindeer and caribou chromosome-level genomes. Using this oligonucleotide-based method, we found six mis-assembled scaffolds and physically mapped 68 of the largest scaffolds representing 78% of the most recent R. tarandus genome assembly. Combining physical mapping and comparative genomics, it was possible to document chromosomal evolution among Cervidaeand closely related bovids. Conclusions:Our results provide validation for the current chromosome-level genome assembly as well as resources to use chromosome banding in studies of Rangifer tarandus.
    Steroid treatment suppresses the CD4+ T-cell response to the third dose of mRNA COVID-19 vaccine in systemic autoimmune rheumatic disease patients
    Avishai Maliah, - 2022
    Abstract
    Prolonged steroid treatment has a suppressive effect on the immune system, however, its effect on the cellular response to mRNA vaccine is unknown. Here we assessed the impact of prolonged steroid treatment on the T-cell and humoral response to the SARS-CoV-2 spike (S) peptide following the third dose of the BNT162b2 vaccine in systemic autoimmune rheumatic disease patients. We found that CD4 T-cell response to the S peptide in patients on high-dose long-term steroid treatment showed significantly less S-peptide specific response, compare to low-dose or untreated patients. Remarkably, these results were not reflected in their humoral response, since almost all patients in the cohort had sufficient antibody levels. Moreover, S-peptide activation failed to induce significant mRNA levels of IFNγ and TNFα in patients receiving high-dose steroids. RNA-sequencing datasets analysis implies that steroid treatments' inhibitory effect of nuclear factor kappa-B signaling may interfere with the activation of S-specific CD4 T-cells. This reveals that high-dose steroid treatment inhibits T-cell response to the mRNA vaccine, despite having sufficient antibody levels. Since T-cell immunity is a crucial factor in the immune response to viruses, our findings highlight the need for enhancing the efficiency of vaccines in immune-suppressive patients, by modulation of the T-cell response.
    Characterization of an RNA binding protein interactome reveals a context-specific post-transcriptional landscape of MYC-amplified medulloblastoma
    Michelle M. Kameda-Smith, - 2022
    Abstract
    Pediatric medulloblastoma (MB) is the most common solid malignant brain neoplasm, with Group 3 (G3) MB representing the most aggressive subgroup. MYC amplification is an independent poor prognostic factor in G3 MB, however, therapeutic targeting of the MYC pathway remains limited and alternative therapies for G3 MB are urgently needed. Here we show that the RNA-binding protein, Musashi-1 (MSI1) is an essential mediator of G3 MB in both MYC-overexpressing mouse models and patient-derived xenografts. MSI1 inhibition abrogates tumor initiation and significantly prolongs survival in both models. We identify binding targets of MSI1 in normal neural and G3 MB stem cells and then cross referenced these data with unbiased large-scale screens at the transcriptomic, translatomic and proteomic levels to systematically dissect its functional role. Comparative integrative multi-omic analyses of these large datasets reveal cancer-selective MSI1-bound targets sharing multiple MYC associated pathways, providing a valuable resource for context-specific therapeutic targeting of G3 MB.
    The Biomimetics of Mg2+-Concentration-Resolved Microenvironment for Bone and Cartilage Repairing Materials Design
    Zhengqiang Li - 2022
    Abstract
    With the increase in population aging, the tendency of osteochondral injury will be accelerated, and repairing materials are increasingly needed for the optimization of the regenerative processes in bone and cartilage recovery. The local environment of the injury sites and the deficiency of Mg2+ retards the repairing period via inhibiting the progenitor osteogenesis and chondrogenesis cells’ recruitment, proliferation, and differentiation, which results in the sluggish progress in the osteochondral repairing materials design. In this article, we elucidate the Mg2+-concentration specified effect on the cell proliferation, osteochondral gene expression, and differentiation of modeling chondrocytes (extracted from New Zealand white rabbit) and osteoblasts (MC3T3-E1). The concentration of Mg2+ in the culture medium affects the proliferation, chondrogenesis, and osteogenesis: (i) Appropriate concentrations of Mg2+ promote the proliferation of chondrocytes (1.25–10.0 mM) and MC3T3-E1 cells (2.5–30.0 mM); (ii) the optimal concentration of Mg2+ that promotes the gene expression of noncalcified cartilage is 15 mM, calcified cartilage 10 mM, and subchondral bone 5 mM, respectively; (iii) overdosed Mg2+ leads to the inhibition of cell activity for either chondrocytes (>20 mM) or osteoblasts (>30 mM). The biomimetic elucidation for orchestrating the allocation of gradient concentration of Mg2+ in accordance of the physiological condition is crucial for designing the accurate microenvironment in osteochondral injury defects for optimization of bone and cartilage repairing materials in the future.
    Specific inhibition of NADPH oxidase 2 modifies chronic epilepsy
    Prince Kumar Singh - 2022
    Abstract
    Recent work by us and others has implicated NADPH oxidase (NOX) enzymes as main producers of reactive oxygen species (ROS) following a brain insult such as status epilepticus, contributing to neuronal damage and development of epilepsy. Although several NOX isoforms have been examined in the context of epilepsy, most attention has focused on NOX2. In this present study, we demonstrate the effect of gp91ds-tat, a specific competitive inhibitor of NOX2, in in vitro epileptiform activity model as well as in temporal lobe epilepsy (TLE) model in rats. We showed that in in vitro seizure model, gp91ds-tat modulated Ca2+ oscillation, prevented epileptiform activity-induced ROS generation, mitochondrial depolarization, and neuronal death. Administration of gp91ds-tat 1 h after kainic acid-induced status epilepticus significantly decreased the expression of NOX2, as well as the overall NOX activity in the cortex and the hippocampus. Finally, we showed that upon continuous intracerebroventricular administration to epileptic rats, gp91ds-tat significantly reduced the seizure frequency and the total number of seizures post-treatment compared to the scrambled peptide-treated animals. The results of the study suggest that NOX2 may have an important effect on modulation of epileptiform activity and has a critical role in mediating seizure-induced NOX activation, ROS generation and oxidative stress in the brain, and thus significantly contributes to development of epilepsy following a brain insult.
    The μ2 and λ1 Proteins of Mammalian Reovirus Modulate Early Events Leading to Induction of the Interferon Signaling Network
    Guillaume David Després - 2022
    Abstract
    It has been previously shown that amino acid polymorphisms in reovirus proteins μ2 and λ1 are associated with differing levels of interferon induction. In the present study, viruses carrying these polymorphisms in either or both proteins, were further studied. The two viral determinants exert a synergistic effect on the control of β-interferon induction at the protein and mRNA level, with a concomitant increase in RIG-I. In contrast, levels of phospho-Stat1 and interferon-stimulated genes are increased in singly substituted viruses but with no further increase when both substitutions were present. This suggests that the viral determinants are acting during initial events of viral recognition. Accordingly, difference between viruses was reduced when infection was performed with partially uncoated virions (ISVPs) and transfection of RNA recovered from early-infected cells recapitulates the differences between viruses harboring the different polymorphisms. Altogether, the data are consistent with a redundant or complementary role of μ2 and λ1, affecting either early disassembly or the nature of the viral RNA in the incoming viral particle. Proteins involved in viral RNA synthesis are thus involved in this likely critical aspect of the ability of different reovirus variants to infect various cell types, and to discriminate between parental and transformed/cancer cells.
    Silencing of a mannitol transport gene in Phelipanche aegyptiaca by the tobacco rattle virus system reduces the parasite germination on the host root
    Vinay Kumar Bari - 2022
    Abstract
    Root parasitic weed Phelipanche aegyptiaca is an obligate plant parasite that causes severe damage to host crops. Agriculture crops mainly belong to the Brassicaceae, Leguminosae, Cruciferae, and Solanaceae plant families affected by this parasitic weed, leading to the devastating loss of crop yield and economic growth. This root-specific parasitic plant is not able to complete its life cycle without a suitable host and is dependent on the host plant for nutrient uptake and germination. Therefore, selected parasitic genes of P. aegyptiaca which were known to be upregulated upon interaction with the host were chosen. These genes are essential for parasitism, and reduced activity of these genes could affect host-parasitic interaction and provide resistance to the host against these parasitic weeds. To check and examine the role of these parasitic genes which can affect the development of host resistance, we silenced selected genes in the P. aegyptiaca using the tobacco rattle virus (TRV) based virus-induced gene silencing (VIGS) method. Our results demonstrated that the total number of P. aegyptiaca parasite tubercles attached to the root of the host plant Nicotiana benthamiana was substantially decreased in all the silenced plants. However, silencing of the P. aegyptiaca MNT1 gene which encodes the mannitol transporter showed a significantly reduced number of germinated shoots and tubercles. Thus, our study indicates that the mannitol transport gene of P. aegyptiaca plays a crucial role in parasitic germination, and silencing of the PaMNT1 gene abolishes the germination of parasites on the host roots.
    Mutations in FBXL4 Cause Mitochondrial Encephalopathy and a Disorder of Mitochondrial DNA Maintenance
    Mohammad Nasim Saleh - 2013
    Abstract
    Nuclear genetic disorders causing mitochondrial DNA (mtDNA) depletion are clinically and genetically heterogeneous, and the molecular etiology remains undiagnosed in the majority of cases. Through whole-exome sequencing, we identified recessive nonsense and splicing mutations in FBXL4 segregating in three unrelated consanguineous kindreds in which affected children present with a fatal encephalopathy, lactic acidosis, and severe mtDNA depletion in muscle. We show that FBXL4 is an F-box protein that colocalizes with mitochondria and that loss-of-function and splice mutations in this protein result in a severe respiratory chain deficiency, loss of mitochondrial membrane potential, and a disturbance of the dynamic mitochondrial network and nucleoid distribution in fibroblasts from affected individuals. Expression of the wild-type FBXL4 transcript in cell lines from two subjects fully rescued the levels of mtDNA copy number, leading to a correction of the mitochondrial biochemical deficit. Together our data demonstrate that mutations in FBXL4 are disease causing and establish FBXL4 as a mitochondrial protein with a possible role in maintaining mtDNA integrity and stability
    Infection by Salmonella enterica Serovar Typhimurium DT104 Modulates Immune Responses, the Metabolome, and the Function of the Enteric Microbiota in Neonatal Broiler Chickens
    Danisa M. Bescucci - 2022
    Abstract
    Salmonella enterica serovar Typhimurium incites salmonellosis in many different species including chickens and human beings. Acute salmonellosis was studied in neonatal broiler chicks by orally inoculating 2-day-old chicks with S. Typhimurium DT104. The temporal impact of disease (1, 2, and 4 days post-inoculation) on the structure and function of the enteric microbiota, on the bird’s immune response in the ileum, cecum, and colon, and on the metabolome of digesta, breast muscle, liver, serum, and hippocampus were examined. Substantive histopathologic changes were observed in the small and large intestine, including the colon of chicks inoculated with S. Typhimurium, and increased in magnitude over the experimental time period. A variety of inflammatory genes (IFNγ, IL8, IL10, INOS, MIP1β, TGFβ2, TLR4, and TLR15) were temporally regulated. In addition, the metabolome of ileal digesta, breast muscle, liver, serum, and hippocampus was temporally altered in infected chicks. Although the structure of bacterial communities in digesta was not affected by S. Typhimurium infection, metabolomic analysis indicated that the function of the microbiota was changed. Collectively, the study findings demonstrate that infection of neonatal chicks by S. Typhimurium imparts a temporal and systemic impact on the host, affecting the immune system, the metabolome, and the function of the enteric microbiota.
    The Role of Chromatin Remodeler BAF in Neuronal Activity-Induced Transcription
    Brenda Gutierrez Ruiz - 2022
    Abstract
    The BAF complex is an ATP dependent chromatin remodeling complex that is known to facilitate gene transcription. However, underlying mechanisms are not fully understood. We expected neuronal activity-induced gene transcription of delayed IEGs to require BAF complex-dependent chromatin remodeling and to be impaired when the latter functions sub-optimally, while rapid IEGs were expected to not need BAF as they already have accessible chromatin. Pharmacological inhibition or degradation of cBAF, but not the PBAF, and RNAi knockdown (KD) of nBAF subunit 170 and 53b significantly attenuated transcription of neuronal immediate early genes including rapid IEGs. BAF subunit KD didn’t alter phosphorylation of MAPK-ERK, a necessary signaling cascade for transcription. Also, pulse-chase with the nBAF inhibitor showed that the BAF complex is required for IEG transcription on a continuous basis. Taken together, using multiple experimental approaches, this is the first study to demonstrate a critical role of nBAF in neuronal activity-induced transcription.
    Spatio-temporal and cell type-specific expression of Nrf2 following an acute epileptic seizure in rats
    Sereen Sandouka - 2022
    Abstract
    The modulation of Nrf2 activity has been reported to be implicated in the pathology of various neurological disorders, including epilepsy. Previous studies have demonstrated that Nrf2 is activated in the post-status epilepticus rat model, however, the spatio-temporal, as well as cell type-specific expression of Nrf2 following brief epileptic seizures remains unclear. Here, we evaluated how an acute epileptic seizure affected the expression of Nrf2 and its downstream genes in the cortex and the hippocampus up to 1-week following the induced seizure. We found that after a pentylenetetrazol-induced seizure, Nrf2 significantly increased at 24 h at the mRNA level and 3 to 6 h at the protein level in the cortex. In the hippocampus, the Nrf2 mRNA level peaked at 3 h after the seizure, and no significant changes were observed in the protein level. Interestingly, the mRNA level of Nrf2 downstream genes peaked at 3-6 h after seizure in both the cortex and the hippocampus. A significant increase in the expression of Nrf2 was observed in the neuronal population of CA1 and CA3 regions of the hippocampus, as well as in the cortex. Moreover, we observed no change in the co-localization of Nrf2 with astrocytes neither in the cortex nor in CA1 and CA3. Our results revealed that following a brief acute epileptic seizure, the expression of Nrf2 and its downstream genes is transiently increased and peaked at early timepoints after seizure predominantly in the hippocampus, and this expression is restricted to the neuronal population.
    Kisspeptin Is Upregulated at the Maternal-Fetal Interface of the Preeclamptic-like BPH/5 Mouse and Normalized after Synchronization of Sex Steroid Hormones
    Viviane C. L. Gomes - 2022
    Abstract
    Insufficient invasion of conceptus-derived trophoblast cells in the maternal decidua is a key event in the development of early-onset preeclampsia (PE), a subtype of PE associated with high maternal and fetal morbidity and mortality. Kisspeptins, a family of peptides previously shown to inhibit trophoblast cell invasion, have been implicated in the pathogenesis of early-onset PE. However, a role of kisspeptin signaling during the genesis of this syndrome has not been elucidated. Herein, we used the preeclamptic-like BPH/5 mouse model to investigate kisspeptin expression and potential upstream regulatory mechanisms in a PE-like syndrome. Expression of the kisspeptin encoding gene, Kiss1, and the 10-amino-acid kisspeptide (Kp-10), are upregulated in the non-pregnant uterus of BPH/5 females during diestrus and in the maternal-fetal interface during embryonic implantation and decidualization. Correspondingly, the dysregulation of molecular pathways downstream to kisspeptins also occurs in this mouse model. BPH/5 females have abnormal sex steroid hormone profiles during early gestation. In this study, the normalization of circulating concentrations of 17β-estradiol (E2) and progesterone (P4) in pregnant BPH/5 females not only mitigated Kiss1 upregulation, but also rescued the expression of multiple molecules downstream to kisspeptin and ameliorated adverse fetoplacental outcomes. Those findings suggest that uterine Kiss1 upregulation occurs pre-pregnancy and persists during early gestation in a PE-like mouse model. Moreover, this study highlights the role of sex steroid hormones in uteroplacental Kiss1 dysregulation and the improvement of placentation by normalization of E2, P4 and Kiss1.
    THE ROLE OF ACTIVATOR E2FS IN ADULT NEURAL STEM CELL QUIESCENCE AND ACTIVATION
    Daniel O'Neil - 2022
    Abstract
    Within the adult mammalian brain, Neural Stem Cell (NSC)s are maintained in distinct neurogenic niches in a mostly quiescent state. Activation of quiescent NSCs first requires re-entry into the cell cycle in order for the pool to proliferate and eventually commit to a neural fate, giving rise to newborn neurons. The canonical Retinoblastoma (Rb)-E2 Promoter Binding Factor (E2f) pathway is not only key in overcoming the Gap 1 Phase (G1)/S-phase restriction, but novelly appears to be involved in adult neurogenesis and NSC activation. I hypothesized that activator transcription factors E2 Promoter Binding Factor 1 (E2f1) and E2 Promoter Binding Factor 3 (E2f3) are crucial for exit from a quiescent state in adult NSCs. The contribution of the activator E2fs in this transition was studied using a Nestin-driven Cre Recombinase-Estrogen Receptor Tamoxifen-2 Ligand Binding Domain (Cre-ERT2) system to induce targeted deletion of E2f1/3 within NSCs in adult mice. We show that loss of E2f1/3 causes significant neurogenic defects, including pro-neural activation and decreased pools of adult NSCs, that preferentially adopt a quiescent profile in the subventricular zone. We employed this model to further isolate subventricular zone-derived NSCs using a Rosa26:Yellow Fluorescent Protein (YFP) reporter and subsequently analysed transcriptional profiles by RNA sequencing. Loss of E2f1/3 shifts NSC transcriptomes towards one overlapping with quiescent neural stem cell signatures (Codega et al., 2014; Basak et al., 2018), further highlighting the requirement of these E2fs for initial activation. A significant portion of these differentially expressed genes are putative E2f targets. Transcriptionally, major pathways involving cell metabolism, cellular signaling, and neural development are perturbed without activator E2f expression. In effect, this combined approach based on in vivo data and bioinformatics analyses offers a method of prospective identification of novel regulators of adult neurogenesis that require the activator E2fs. Preliminary data suggests that AT-Hook Transcription Factor (Akna) is one such target worth pursuing. Cumulatively, this project describes a unique role for E2f1 and E2f3 during NSC exit from quiescence and subsequent activation towards differentiation. As ongoing maintenance of quiescent NSCs is a necessary prerequisite for lifelong neurogenesis, conclusions from this study could determine the therapeutic potential of targeting activator E2fs to combat the niche exhaustion associated with aging, injury, and neurodegenerative diseases.
    A diet rich in fermentable fiber promotes robust changes in the intestinal microbiota, mitigates intestinal permeability, and attenuates autoimmune uveitis
    Yukiko K. Nakamura - 2022
    Abstract
    Therapeutic approaches for noninfectious uveitis have expanded greatly over the past 10 years, but are limited by potential side effects and limited efficacy. Thus, therapeutic approaches that include less toxic, potentially preventative strategies to manage noninfectious uveitis are essential areas of study. Diets rich in fermentable fiber are potentially preventative in various conditions such as metabolic syndrome and type 1 diabetes. We studied the effects of various fermentable dietary fibers in an inducible model of experimental autoimmune uveitis (EAU) and found that they differentially modulated uveitis severity. A high pectin diet was the most protective, reducing clinical disease severity through the induction of regulatory T lymphocytes and the suppression of Th1 and Th17 lymphocytes at peak ocular inflammation in either intestinal or extra-intestinal lymphoid tissues. The high pectin diet also promoted intestinal homeostasis as shown by changes in intestinal morphology and gene expression, as well as intestinal permeability. Pectin-induced modulation of intestinal bacteria appeared to be associated with protective changes in immunophenotype in the intestinal tract, and correlated with reduced uveitis severity. In summary, our current findings support the potential for dietary intervention as a strategy to mitigate noninfectious uveitis severity.
    Establishment and Genetic Profiling of Platinum/Taxane Doublet-Resistant Cells Generated by Hybridizing Single Resistant Cells
    Seiji Isonishi - 2022
    Abstract
    Background: Current standard chemotherapy for gynecologic malignancies consists of platinum agent and taxane though, many patients experience the relapse of disease with drug resistance making the following therapy unsuccessful. It’s a compelling question whether the mechanisms of doubly resistance is a simple combination of single agent resistance or whether the core novel mechanism common to platinum and taxane resistance stands out as a result of combination therapy. The purpose of this study is to establish the doublet drug resistant cell line and to find its genetic characteristics. Methods: Platinum/taxane doublet resistant cell lines (F3 and F4) were generated by hybridizing two independent, platinum or taxane resistant subline (C13 or PX24) stemmed from grand parental ME180 cells. The resistant cells were selected through repeated exposure to cisplatin and paclitaxel. For the assessment of drug sensitivity, colony forming assay was used. For the gene expression analysis, genome-wide expression profiling was done using the Human Genome U133A Array. Protein-protein interaction network (PPI) scaffold networks were retrieved from the Search Tool for the Retrieval of Interacting Genes database and, for the enrichment of pathway analysis, WebGestalt was used. Results: Colony forming assay showed C13 was 5.8-fold cisplatin resistant while PX24 was 5.3-fold paclitaxel resistant compared with parental ME180 cells. F3 and F4 acquired resistance to cisplatin and paclitaxel by 8.3/4.9- and 3.7/3.3-fold (F3/4) respectively. Microarray analysis demonstrated, out of 22284 genes, 103 genes were > 4-fold up-regulated in F3/4 and 33 (32%) were identified as simultaneously upregulated genes (SUG) in C13, PX24 and F3/4. The Protein-protein interaction analysis of 33 SUG displayed a scaffold network pivoting aldo-keto reductase 1C1 (AKR1C1), aldo-keto reductase1C2 (AKR1C2) and aldo-keto reductase1C3 (AKR1C3). The enrichment pathway analysis demonstrated AKR1C gene family anchored to molecular function of oxidoreductase and aldo-keto reductase activity and biological process of daunorubicin and doxorubicin metabolism. Conclusions: We report here the establishment of doubly drug-resistant hybridoma to platinum and taxane. Analysis of SUG indicated the AKR1C gene family plays a key role for doubly resistant mechanism that would be possible targets for therapeutic strategies.
    Highly tailorable gellan gum nanoparticles as a platform for the development of T cell activator systems
    Daniel Rodrigues - 2022
    Abstract
    Background T cell priming has been shown to be a powerful immunotherapeutic approach for cancer treatment in terms of efficacy and relatively weak side effects. Systems that optimize the stimulation of T cells to improve therapeutic efficacy are therefore in constant demand. A way to achieve this is through artificial antigen presenting cells that are complexes between vehicles and key molecules that target relevant T cell subpopulations, eliciting antigen-specific T cell priming. In such T cell activator systems, the vehicles chosen to deliver and present the key molecules to the targeted cell populations are of extreme importance. In this work, a new platform for the creation of T cell activator systems based on highly tailorable nanoparticles made from the natural polymer gellan gum (GG) was developed and validated. Methods GG nanoparticles were produced by a water in oil emulsion procedure, and characterized by dynamic light scattering, high resolution scanning electronic microscopy and water uptake. Their biocompatibility with cultured cells was assessed by a metabolic activity assay. Surface functionalization was performed with anti-CD3/CD28 antibodies via EDC/NHS or NeutrAvidin/Biotin linkage. Functionalized particles were tested for their capacity to stimulate CD4+ T cells and trigger T cell cytotoxic responses. Results Nanoparticles were approximately 150 nm in size, with a stable structure and no detectable cytotoxicity. Water uptake originated a weight gain of up to 3200%. The functional antibodies did efficiently bind to the nanoparticles, as confirmed by SDS-PAGE, which then targeted the desired CD4+ populations, as confirmed by confocal microscopy. The developed system presented a more sustained T cell activation over time when compared to commercial alternatives. Concurrently, the expression of higher levels of key cytotoxic pathway molecules granzyme B/perforin was induced, suggesting a greater cytotoxic potential for future application in adoptive cancer therapy. Conclusions Our results show that GG nanoparticles were successfully used as a highly tailorable T cell activator system platform capable of T cell expansion and re-education.
    Integrin-specific hydrogels for growth factor-free vasculogenesis
    Helena R. Moreira - 2022
    Abstract
    Integrin-binding biomaterials have been extensively evaluated for their capacity to enable de novo formation of capillary-like structures/vessels, ultimately supporting neovascularization in vivo. Yet, the role of integrins as vascular initiators in engineered materials is still not well understood. Here, we show that αvβ3 integrin-specific 3D matrices were able to retain PECAM1+ cells from the stromal vascular fraction (SVF) of adipose tissue, triggering vasculogenesis in vitro in the absence of extrinsic growth factors. Our results suggest that αvβ3-RGD-driven signaling in the formation of capillary-like structures prevents the activation of the caspase 8 pathway and activates the FAK/paxillin pathway, both responsible for endothelial cells (ECs) survival and migration. We also show that prevascularized αvβ3 integrin-specific constructs inosculate with the host vascular system fostering in vivo neovascularization. Overall, this work demonstrates the ability of the biomaterial to trigger vasculogenesis in an integrin-specific manner, by activating essential pathways for EC survival and migration within a self-regulatory growth factor microenvironment. This strategy represents an improvement to current vascularization routes for Tissue Engineering constructs, potentially enhancing their clinical applicability.
    Neddylation in Schwann cell myelination
    Miguel Tamayo Caro - 2022
    Abstract
    Schwann cells (SCs) are the main glial cells in the peripheral nervous system. The main function of these glial cells is to produce the myelin sheets that isolate neurons and improve the nerve conduction velocity. This activity is vital for the correct functioning of the nervous system. Schwann cells also provide trophic and metabolic support to the neurons. Over the last few years, there has been an explosion in studies aimed at identifying the signals and molecules that drive the Schwann cell phenotype during development and in disease situations. Thus, Schwann cell myelination has been shown to be regulated by several extrinsic and intrinsic signals, including neuregulin (NRG) type III, laminin, and mTOR amongst others. In addition, a complex transcriptional and epigenetic regulatory program has been uncovered, with key roles described for master transcription factors such Egr2, Sox10 and Zeb2 in positively regulating the myelinating phenotype. This process is opposed by negative regulators of myelination, including Notch, Sox2, mTOR and c-Jun signaling pathways that are downregulated during myelinogenesis. Post-translational mechanisms, on the other hand, have barely been studied, even though these modifications can fine-tune the interactions, trafficking, stability, localization and activity of proteins, which could have a key role in the dynamic processes of myelin formation and breakdown. Neddylation, a ubiquitylation-like pathway that conjugates a ubiquitin-like protein NEDD8 to target proteins, has emerged as a critical regulatory process controlling ubiquitination, protein transcription and signaling transduction. Dysregulation of neddylation has been linked to a broad spectrum of pathological conditions ranging from tumorigenesis to neurodegeneration. So far, its role in Schwann cell development and function has not been examined. In this thesis, we have found that neddylation is a critical regulator of Schwann cell myelination. We found that genetic ablation of Nae 1 (Nae1 cKO), a key enzyme in the neddylation pathway, in Schwann cells, led to striking defects in peripheral nerves that had all the hallmarks of a severe neuropathy. The conditional knockout mice developed gait abnormalities, muscle weakness, and hindlimb clasping, a typical presentation of neuromuscular dysfunction very early after birth, and most mice did not survive past three weeks of age. In neurophysiological tests, we recorded a severe reduction in nerve conduction velocity (NCV) in Nae1 cKO compared to control mice. Strikingly, electron microscopy (EM) revealed that Nae1-6 deficient mice lacked peripheral myelination and exhibited active myelin breakdown of the few formed myelin sheaths. This lack of myelin was accompanied by an absence of the myelin structural proteins, and the master myelination transcriptional regulator Egr2. We found widespread changes in the transcriptomic and proteomic profile of Nae1 cKO nerves. Notably, we found an upregulation of the negative regulator of myelination c-Jun, and that neddylation via regulation of the activity of Cullin Ring Ligases (CRL), E3 ubiquitin ligases that control the stability of numerous proteins, was responsible for regulating protein expression of c-Jun during myelination. On the other hand, using a tamoxifen-inducible Nae1 knockout model, we did not find any robust role of neddylation for the maintenance of mature myelin sheaths, and in pilot studies, on the regeneration of nerves after nerve injury. In summary, we found that neddylation is a critical regulator of the Schwann cell myelination and that one of the key mechanisms behind its biological function is the regulation of expression of negative regulators of myelination.
    MOVAS Cells: A Versatile Cell Line for Studying Vascular Smooth Muscle Cell Cholesterol Metabolism
    Ikechukwu Collins Esobi - 2022
    Abstract
    Cholesterol metabolism is paramount to cells. Aberrations to cholesterol metabolism affects cholesterol homeostasis, which may impact the risk of several diseases. Recent evidence has suggested that vascular smooth muscle cell (VSMC) cholesterol metabolism may play a role in atherosclerosis. However, there is scant in vitro mechanistic data involving primary VSMC that directly tests how VSMC cholesterol metabolism may impact atherosclerosis. One reason for this lack of data is due to the impracticality of gene manipulation studies in primary VSMC, as cultured primary VSMC become senescent and lose their morphology rapidly. However, there are no immortalized VSMC lines known to be suitable for studying VSMC cholesterol metabolism. The purpose of this study was to determine whether MOVAS cells, a commercially available VSMC line, are suitable to use for studying VSMC cholesterol metabolism. Using immunoblotting and immunofluorescence, we showed that MOVAS cells express ABCA1, ABCG1, and SREBP-2. We also determined that MOVAS cells efflux cholesterol to apoAI and HDL, which indicates functionality of ABCA1/ABCG1. In serum-starved MOVAS cells, SREBP-2 target gene expression was increased, confirming SREBP-2 functionality. We detected miR-33a expression in MOVAS cells and determined this microRNA can silence ABCA1 and ABCG1 via identifying conserved miR-33a binding sites within ABCA1/ABCG1 3’UTRs in MOVAS cells. We showed that cholesterol-loading MOVAS cells results in this cell line to transdifferentiate into a macrophage-like cell, which also occurs when VSMC accumulate cholesterol. Our characterization of MOVAS cells sufficiently demonstrates that they are suitable to use for studying VSMC cholesterol metabolism in the context of atherosclerosis.
    Efficiency-corrected PCR quantification for identification of prevalence and load of respiratory disease-causing agents in feedlot cattle
    RJ Barnewall - 2022
    Abstract
    Bovine respiratory disease (BRD) is the most prevalent disease in feedlot cattle worldwide with Bovine alphaherpesvirus 1 (BoAHV1), Histophilus somni, Mannheimia haemolytica, Mycoplasma bovis, Pasteurella multocida and Trueperella pyogenes accepted to be common etiological agents associated with BRD. Although these agents are common in the upper and lower airways in clinical BRD cases, some also exist as normal flora suggesting their presence in the upper airways alone is not necessarily informative with respect to disease status or risk. To determine the relationship between presence, load and disease status, we investigated the relationship between load in the upper airways at induction and active BRD cases in feedlot cattle using efficiency-corrected PCR quantification. By this approach, we were able to accurately determine the prevalence and load of the key BRD agents in the upper respiratory tract showing that cattle in the hospital pen had a higher prevalence, and load, of these agents both singly and in combination compared to cattle sampled at feedlot induction. A combination of agents was the most accurate indicator of BRD risk with cattle with four or more agents detected in the upper airway more likely to be undergoing treatment for BRD than non-BRD ailments. In addition, M. bovis was rarely detected at feedlot induction but was identified at high prevalence in cattle in the hospital pen. These findings present a potential new technological approach for the investigation, analysis and identification of BRD-associated viral and bacterial agents for Australian feedlot systems as well as for BRD disease management and treatment
    Quantifying the effect of human population mobility on malaria risk in the Peruvian Amazon
    Gabriel Carrasco-Escobar - 2022
    Abstract
    The impact of human population movement (HPM) on the epidemiology of vector-borne diseases, such as malaria, has been described. However, there are limited data on the use of new technologies for the study of HPM in endemic areas with difficult access such as the Amazon. In this study conducted in rural Peruvian Amazon, we used self-reported travel surveys and GPS trackers coupled with a Bayesian spatial model to quantify the role of HPM on malaria risk. By using a densely sampled population cohort, this study highlighted the elevated malaria transmission in a riverine community of the Peruvian Amazon. We also found that the high connectivity between Amazon communities for reasons such as work, trading or family plausibly sustains such transmission levels. Finally, by using multiple human mobility metrics including GPS trackers, and adapted causal inference methods we identified for the first time the effect of human mobility patterns on malaria risk in rural Peruvian Amazon. This study provides evidence of the causal effect of HPM on malaria that may help to adapt current malaria control programmes in the Amazon.
    Ubiquitination and deubiquitination of 4E-T regulate neural progenitor cell maintenance and neurogenesis by controlling P-body formation
    Shreeya Kedia - 2022
    Abstract
    During embryogenesis, neural stem/progenitor cells (NPCs) proliferate and differentiate to form brain tissues. Here, we show that in the developing murine cerebral cortex, the balance between the NPC maintenance and differentiation is coordinated by ubiquitin signals that control the formation of processing bodies (P-bodies), cytoplasmic membraneless organelles critical for cell state regulation. We find that the deubiquitinase Otud4 and the E3 ligase Trim56 counter-regulate the ubiquitination status of a core P-body protein 4E-T to orches- trate the assembly of P-bodies in NPCs. Aberrant induction of 4E-T ubiquitination promotes P-body assem- bly in NPCs and causes a delay in their cell cycle progression and differentiation. In contrast, loss of 4E-T ubiquitination abrogates P-bodies and results in premature neurogenesis. Thus, our results reveal a critical role of ubiquitin-dependent regulation of P-body formation in NPC maintenance and neurogenesis during brain development.
    Comprehensive landscape of tRNA-derived fragments in lung cancer
    Zitong Gao - 2022
    Abstract
    tRNA-derived fragment (tRDF) is novel small non-coding RNA that presences in different types of cancer. The comprehensive understanding of tRDFs in non-small cell lung cancer remains largely unknown. In this study, 1550 patient samples of NSCLC were included, and 52 tRDFs with four subtypes were identified. Six tRDFs were picked as diagnostic signatures based on the tRDFs expression patterns, and under the curve (AUC) in independent validations is up to 0.90. Two signatures were validated successfully in plasma samples, and six signatures were confirmed the consistency of distinguished expression in NSCLC cell lines. Ten tRDFs along with independent risk scores can be used to predict survival outcomes by stages, 5a_tRF-Ile-AAT/GAT can be prognosis biomarker for early stage. Association analysis of tRDFs signatures correlated mRNAs and miRNA were targeted to the cell cycle and oocyte meiosis signaling pathways. Five tRDFs were assessed to associate with PD-L1 immune checkpoint and correlated with the genes that targets in PD-L1 checkpoint signaling pathway. Our study is the first to provide a comprehensive analysis of tRDFs in lung cancer including four subtypes of tRDFs, investigating the diagnostic and prognostic values, and demonstrated their biological function and transcriptional role as well as potential immune therapeutic value.
    Differences between the global transcriptomes of Salmonella enterica serovars Dublin and Cerro infecting bovine epithelial cells
    Serajus Salaheen - 2022
    Abstract
    Background The impact of S. enterica colonization in cattle is highly variable and often serovar-dependent. The aim of this study was to compare the global transcriptomes of highly pathogenic bovine-adapted S. enterica serovar Dublin and the less pathogenic, bovine-adapted, serovar Cerro during interactions with bovine epithelial cells, to identify genes that impact serovar-related outcomes of S. enterica infections in dairy animals. Result Bovine epithelial cells were infected with S. enterica strains from serovars Dublin and Cerro, and the bacterial RNA was extracted and sequenced. The total number of paired-end reads uniquely mapped to non-rRNA and non-tRNA genes in the reference genomes ranged between 12.1 M (Million) and 23.4 M (median: 15.7 M). In total, 360 differentially expressed genes (DEGs) were identified with at least two-fold differences in the transcript abundances between S. Dublin and S. Cerro (false discovery rate ≤ 5%). The highest number of DEGs (17.5%, 63 of 360 genes) between the two serovars were located on the genomic regions potentially associated with Salmonella Pathogenicity Islands (SPIs). DEGs potentially located in the SPI-regions that were upregulated (≥ 2-fold) in the S. Dublin compared with S. Cerro included: 37 SPI-1 genes encoding mostly Type 3 Secretion System (T3SS) apparatus and effectors; all of the six SPI-4 genes encoding type I secretion apparatus (siiABCDEF); T3SS effectors and chaperone (sopB, pipB, and sigE) located in SPI-5; type VI secretion system associated protein coding genes (sciJKNOR) located in SPI-6; and T3SS effector sopF in SPI-11. Additional major functional categories of DEGs included transcription regulators (n = 25), amino acid transport and metabolism (n = 20), carbohydrate transport and metabolism (n = 20), energy production and metabolism (n = 19), cell membrane biogenesis (n = 18), and coenzyme transport and metabolism (n = 15). DEGs were further mapped to the metabolic pathways listed in the KEGG database; most genes of the fatty acid β-oxidation pathway were upregulated/uniquely present in the S. Dublin strains compared with the S. Cerro strains. Conclusions This study identified S. enterica genes that may be responsible for symptomatic or asymptomatic infection and colonization of two bovine-adapted serovars in cattle.
    RIP140 regulates POLK gene expression and the response to alkylating drugs in colon cancer cells
    Pascale Palassin - 2022
    Abstract
    Aim: The transcription factor RIP140 (receptor interacting protein of 140 kDa) is involved in intestinal tumorigenesis. It plays a role in the control of microsatellite instability (MSI), through the regulation of MSH2 and MSH6 gene expression. The aim of this study was to explore its effect on the expression of POLK, the gene encoding the specialized translesion synthesis (TLS) DNA polymerase κ known to perform accurate DNA synthesis at microsatellites. Methods: Different mouse models and engineered human colorectal cancer (CRC) cell lines were used to analyze by RT-qPCR, while Western blotting and luciferase assays were used to elucidate the role of RIP140 on POLK gene expression. Published DNA microarray datasets were reanalyzed. The in vitro sensitivity of CRC cells to methyl methane sulfonate and cisplatin was determined. Results: RIP140 positively regulates, at the transcriptional level, the expression of the POLK gene, and this effect involves, at least partly, the p53 tumor suppressor. In different cohorts of CRC biopsies (with or without MSI), a strong positive correlation was observed between RIP140 and POLK gene expression. In connection with its effect on POLK levels and the TLS function of this polymerase, the cellular response to methyl methane sulfonate was increased in cells lacking the Rip140 gene. Finally, the association of RIP140 expression with better overall survival of CRC patients was observed only when the corresponding tumors exhibited low levels of POLK, thus strengthening the functional link between the two genes in human CRC. Conclusion: The regulation of POLK gene expression by RIP140 could thus contribute to the maintenance of microsatellite stability, and more generally to the control of genome integrity.
    A STUDY OF THE EFFEC Y OF THE EFFECT OF PH T OF PHYSIOLOGICAL STRESSORS ON OGICAL STRESSORS ON HYPOTHALAMIC REGUL AMIC REGULATION OF REPRODUC TION OF REPRODUCTION USING AN IN TION USING AN IN VITRO SYSTEM
    Lisa L. Amelse - 2022
    Abstract
    Stressors have a negative impact on reproductive efficiency in humans and other animals, which has an economic cost due to infertility treatments for humans and reduced income for food producers. We wished to determine the molecular pathways by which metabolic disturbances and low-level inflammation impact the hypothalamic pituitary gonadal (HPG) axis using an in vitro model. To this end, we used the GT1-7 cell line, an immortalized line expressing the Kiss1R receptor that responds to kisspeptin stimulation with the release of GnRH to assess the impact of stressors on the GnRH-releasing cells. Additionally, we used the KTaV-3 and KTaR-1 cell lines, immortalize lines derived from the rodent anteroventral periventricular and arcuate nuclei, respectively, to assess the impact of stressors on kisspeptin-producing cells. We modeled metabolic disease and negative energy balance by exposing cells to betahydroxybutyric acid (BHB) and inflammation by exposure to tumor necrosis factor alpha (TNFα [alpha]). We determined that exposure to BHB significantly increased the intrinsic production of GnRH by GT1-7 cells, but that there were no additional changes in the expression of GnRH mRNA or release of GnRH protein. Additionally, there were no significant changes in calcium signaling or in the phosphorylation of ERK1/2, suggesting that the direct effect of stressors does not impact the GnRH-producing cells of the hypothalamus. Exposure to stressors in culture did not change the expression of kiss mRNA in KTaV-3 cells upon exposure to estradiol. However, KTaV-1 cells exhibited a significantly decreased expression of kiss1 mRNA and kiss1 protein release upon exposure to both BHB and tumor necrosis factor alpha. There was also a reduction in the expression of neurokinin B (nkb) mRNA under these conditions, suggesting that the impact of stressors on the HPG axis may be acting through the suppression of signaling in the arcuate nucleus. Disruption of neurokinin B and kisspeptin expression in the arcuate nucleus could impact the production of tonic levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH), resulting in a deficiency in follicle production and oocyte maturation.
    A dual-function phage regulator controls the response of cohabiting phage elements via regulation of the bacterial SOS response
    Gil Azulay - 2022
    Abstract
    Listeria monocytogenes strain 10403S harbors two phage elements in its chromosome; one produces infective virions and the other tailocins. It was previously demonstrated that induction of the two elements is coordinated, as they are regulated by the same anti-repressor. In this study, we identified AriS as another phage regulator that controls the two elements, bearing the capacity to inhibit their lytic induction under SOS conditions. AriS is a two-domain protein that possesses two distinct activities, one regulating the genes of its encoding phage and the other downregulating the bacterial SOS response. While the first activity associates with the AriS N-terminal AntA/AntB domain, the second associates with its C-terminal ANT/KilAC domain. The ANT/KilAC domain is conserved in many AriS-like proteins of listerial and non-listerial prophages, suggesting that temperate phages acquired such dual-function regulators to align their response with the other phage elements that cohabit the genome.
    Cerebrospinal fluid of progressive multiple sclerosis patients reduces differentiation and immune functions of oligodendrocyte progenitor cells
    Omri Zveik - 2022
    Abstract
    Oligodendrocyte progenitor cells (OPCs) are responsible for remyelination in the central nervous system (CNS) in health and disease. For patients with multiple sclerosis (MS), remyelination is not always successful, and the mechanisms differentiating successful from failed remyelination are not well-known. Growing evidence suggests an immune role for OPCs, in addition to their regenerative role; however, it is not clear if this helps or hinders the regenerative process. We studied the effect of cerebrospinal fluid (CSF) from relapsing MS (rMS) and progressive MS (pMS) patients on primary OPC differentiation and immune gene expression and function. We observed that CSF from either rMS or pMS patients has a differential effect on the ability of mice OPCs to differentiate into mature oligodendrocytes and to express immune functions. CSF of pMS patients impaired differentiation into mature oligodendrocytes. In addition, it led to decreased major histocompatibility complex class (MHC)-II expression, tumor necrosis factor (TNF)-α secretion, nuclear factor kappa-B (NFκB) activation, and less activation and proliferation of T cells. Our findings suggest that OPCs are not only responsible for remyelination, but they may also play an active role as innate immune cells in the CNS.
    Molecular and Functional Signatures Associated with CAR T Cell Exhaustion and Impaired Clinical Response in Patients with B Cell Malignancies
    Katia Beider - 2022
    Abstract
    Despite the high rates of complete remission following chimeric antigen receptor (CAR) T cell therapy, its full capacity is currently limited by the generation of dysfunctional CAR T cells. Senescent or exhausted CAR T cells possess poor targeting and effector functions, as well as impaired cell proliferation and persistence in vivo. Strategies to detect, prevent or reverse T cell exhaustion are therefore required in order to enhance the effectiveness of CAR T immunotherapy. Here we report that CD19 CAR T cells from non-responding patients with B cell malignancies show enrichment of CD8+ cells with exhausted/senescent phenotype and display a distinct transcriptional signature with dysregulation of genes associated with terminal exhaustion. Furthermore, CAR T cells from non-responding patients exhibit reduced proliferative capacity and decreased IL-2 production in vitro, indicating functional impairment. Overall, our work reveals potential mediators of resistance, paving the way to studies that will enhance the efficacy and durability of CAR T therapy in B cell malignancies.
    The dynamic nature of the coronavirus receptor, angiotensin-converting enzyme 2 (ACE2) in differentiating airway epithelia
    Vincent J. Manna - 2022
    Abstract
    Once inhaled, SARS-CoV-2 particles enter respiratory ciliated cells by interacting with angiotensin converting enzyme 2 (ACE2). Understanding the nature of ACE2 within airway tissue has become a recent focus particularly in light of the COVID-19 pandemic. Airway mucociliary tissue was generated in-vitro using primary human nasal epithelial cells and the air-liquid interface (ALI) model of differentiation. Using ALI tissue, three distinct transcript variants of ACE2 were identified. One transcript encodes the documented full-length ACE2 protein. The other two transcripts are unique truncated isoforms, that until recently had only been predicted to exist via sequence analysis software. Quantitative PCR revealed that all three transcript variants are expressed throughout differentiation of airway mucociliary epithelia. Immunofluorescence analysis of individual ACE2 protein isoforms exogenously expressed in cell-lines revealed similar abilities to localize in the plasma membrane and interact with the SARS CoV 2 spike receptor binding domain. Immunohistochemistry on differentiated ALI tissue using antibodies to either the N-term or C-term of ACE2 revealed both overlapping and distinct signals in cells, most notably only the ACE2 C-term antibody displayed plasma-membrane localization. We also demonstrate that ACE2 protein shedding is different in ALI Tissue compared to ACE2-transfected cell lines, and that ACE2 is released from both the apical and basal surfaces of ALI tissue. Together, our data highlights various facets of ACE2 transcripts and protein in airway mucociliary tissue that may represent variables which impact an individual's susceptibility to SARS-CoV-2 infection, or the severity of Covid-19.
    Members of the AP-1 Family of Transcription Factors Regulate the Expression of Gja1 in Mouse GC-1 Spermatogonial Cells
    Mustapha Najih - 2021
    Abstract
    Gap junctions, mainly formed by Gja1 (Connexin43), play an essential role in the regulation of proliferation and differentiation of spermatogonia in the testis. Regulation of the abundance of Gja1 in spermatogonia involves various processes, including gene transcription, mRNA maturation, protein synthesis, post-translational modifications, plasma membrane integration and protein degradation. However, gene expression of Gja1 is abnormally decreased in most testicular germ cell tumors. Hence, a better understanding of the mechanisms of transcriptional regulation of Gja1 in spermatogonia is essential to understand how the loss of its expression occurs during the development of testicular cancer. As in other cell types, activator protein-1 (AP-1) transcription factors may be involved in such regulatory process. Thus, AP-1 members were overexpressed in GC-1 cells to assess their impact on Gja1 expression. We showed that Jun and Fosl2 cooperate to activate the Gja1 promoter in GC-1 cells. Furthermore, the recruitment of Jun to the proximal region (−153 to +46 bp) of the Gja1 promoter has been confirmed via chromatin immunoprecipitation. Protein kinase A and calcium-calmodulin protein kinase I also contribute to the activation of Gja1 expression by improving the cooperation between AP-1 factors. Therefore, the reduction in Gja1 expression in testicular germ cell tumors may involve a loss of cooperation between AP-1 factors.
    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.
    Anti-psoriatic and anti-inflammatory effects of Kaempferia parviflora in keratinocytes and macrophage cells
    Mingkwan Na Takuathung - 2021
    Abstract
    Kaempferia parviflora (KP) has been used as folk medicine for curing various conditions, including anti-inflammatory diseases. However, anti-psoriatic effects in an aspect of suppression of NF-κB activation have not been explored. Therefore, our current study aimed to elucidate the anti-inflammation of KP in lipopolysaccharide (LPS)-induced RAW264.7 cells and anti-psoriatic effects of KP in cytokine-induced human keratinocytes, HaCaT cells. We discovered that KP extract significantly suppressed LPS-induced inflammation at both gene expression and protein production. Specifically, dramatic reduction of nitric oxide (NO) was explored by using Griess method. Consistently, data from RT-qPCR, ELISA, and western blot analysis confirmed that crucial inflammatory and psoriatic markers including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, IL-17, IL-22, and IL-23 were significantly decreased by the action of KP. These events were associated with the results from immunofluorescence study and western blot analysis where the activation of NF-κB upon LPS stimulation was clearly inhibited by KP through its ability to suppress IκB-α degradation resulting in inhibition of NF-κB nuclear translocation. Furthermore, KP extract significantly inhibited LPS-stimulated phosphorylation of ERK1/2, JNK, and p38 in a dose-dependent manner, along with inhibition of ERK1/2 activation in both TNF-α- and EGF-induced HaCaT cells. Interestingly, HaCaT cells exposed to 15 μg/mL of KP also exhibited significant decrease of cell migration and proliferation. Our results revealed that KP extract has a potential to be developed as a promising agent for treating inflammation and psoriasis, in part through targeting the proliferation and the NF-κB pathways.
    Influenza A virus infection in turkeys induces respiratory and enteric bacterial dysbiosis correlating with cytokine gene expression
    Ngunjiri JM - 2021
    Abstract
    Turkey respiratory and gut microbiota play important roles in promoting health and production performance. Loss of microbiota homeostasis due to pathogen infection can worsen the disease or predispose the bird to infection by other pathogens. While turkeys are highly susceptible to influenza viruses of different origins, the impact of influenza virus infection on turkey gut and respiratory microbiota has not been demonstrated. In this study, we investigated the relationships between low pathogenicity avian influenza (LPAI) virus replication, cytokine gene expression, and respiratory and gut microbiota disruption in specific-pathogen-free turkeys. Differential replication of two LPAI H5N2 viruses paralleled the levels of clinical signs and cytokine gene expression. During active virus shedding, there was significant increase of ileal and nasal bacterial contents, which inversely corresponded with bacterial species diversity. Spearman’s correlation tests between bacterial abundance and local viral titers revealed that LPAI virus-induced dysbiosis was strongest in the nasal cavity followed by trachea, and weakest in the gut. Significant correlations were also observed between cytokine gene expression levels and relative abundances of several bacteria in tracheas of infected turkeys. For example, interferon γ/λ and interleukin-6 gene expression levels were correlated positively with Staphylococcus and Pseudomonas abundances, and negatively with Lactobacillus abundance. Overall, our data suggest a potential relationship where bacterial community diversity and enrichment or depletion of several bacterial genera in the gut and respiratory tract are dependent on the level of LPAI virus replication. Further work is needed to establish whether respiratory and enteric dysbiosis in LPAI virus-infected turkeys is a result of host immunological responses or other causes such as changes in nutritional uptake.
    SIRT4 is an early regulatorof branched-chain amino acid catabolismthat promotes adipogenesis
    Elma Zaganjor - 2021
    Abstract
    Upon nutrient stimulation, pre-adipocytes undergo differentiation to transform into mature adipocytescapable of storing nutrients as fat. We profiled cellular metabolite consumption to identify early metabolicdrivers of adipocyte differentiation. We find that adipocyte differentiation raises the uptake and consumptionof numerous amino acids. In particular, branched-chain amino acid (BCAA) catabolism precedes and pro-motes peroxisome proliferator-activated receptor gamma (PPARg), a key regulator of adipogenesis. In earlyadipogenesis, the mitochondrial sirtuin SIRT4 elevates BCAA catabolism through the activation of methylcro-tonyl-coenzyme A (CoA) carboxylase (MCCC). MCCC supports leucine oxidation by catalyzing the carboxyl-ation of 3-methylcrotonyl-CoA to 3-methylglutaconyl-CoA. Sirtuin 4 (SIRT4) expression is decreased in adi-pose tissue of numerous diabetic mouse models, and its expression is most correlated with BCAA enzymes,suggesting a potential role for SIRT4 in adipose pathology through the alteration of BCAA metabolism. Insummary, this work provides a temporal analysis of adipocyte differentiation and uncovers early metabolicevents that stimulate transcriptional reprogramming.
    Novel Role of Tieg1 in Muscle Metabolism and Mitochondrial Oxidative Capacities
    Malek Kammoun - 2021
    Abstract
    Aim: Tieg1 is involved in multiple signaling pathways, human diseases, and is highly expressed in muscle where its functions are poorly understood. Methods: We have utilized Tieg1 KO mice to identify novel and important roles for this transcription factor in regulating muscle ultrastructure, metabolism and mitochondrial functions in the soleus and EDL muscles. RNA sequencing, immunoblotting, TEM, MRI, NMR, histochemical and mitochondrial function assays were performed. Results: Loss of Tieg1 expression resulted in altered sarcomere organization and a significant decrease in mitochondrial number. Histochemical analyses demonstrated an absence of SDH staining and a decrease in COX enzyme activity in KO soleus with similar, but diminished, effects in the EDL. Decreased complex I, COX and citrate synthase activities were detected in the soleus muscle of KO mice indicating altered mitochondrial function. Complex I activity was also diminished in KO EDL. Significant decreases in citrate synthase and respiratory chain complex activities were identified in KO soleus. 1H-NMR spectra revealed no significant metabolic difference between WT and KO muscles. However, 31P spectra revealed a significant decrease in phosphocreatine and ATP. Altered expression of 279 genes, many of which play roles in mitochondrial and muscle function, were identified in KO soleus muscle. Ultimately, all of these changes resulted in an exercise intolerance phenotype in Tieg1 KO mice. Conclusion: Our findings have implicated novel roles for Tieg1 in muscle including regulation of gene expression, metabolic activity and organization of tissue ultrastructure. This muscle phenotype resembles diseases associated with exercise intolerance and myopathies of unknown consequence.
    Cardiomyocytes Derived from Induced Pluripotent Stem Cells as a Disease Model for Propionic Acidemia
    Esmeralda Alonso-Barroso - 2021
    Abstract
    Propionic acidemia (PA), one of the most frequent life-threatening organic acidemias, is caused by mutations in either the PCCA or PCCB genes encoding both subunits of the mitochondrial propionyl-CoA carboxylase (PCC) enzyme. Cardiac alterations (hypertrophy, dilated cardiomyopathy, long QT) are one of the major causes of mortality in patients surviving the neonatal period. To overcome limitations of current cellular models of PA, we generated induced pluripotent stem cells (iPSCs) from a PA patient with defects in the PCCA gene, and successfully differentiated them into cardiomyocytes. PCCA iPSC-derived cardiomyocytes exhibited reduced oxygen consumption, an accumulation of residual bodies and lipid droplets, and increased ribosomal biogenesis. Furthermore, we found increased protein levels of HERP, GRP78, GRP75, SIG-1R and MFN2, suggesting endoplasmic reticulum stress and calcium perturbations in these cells. We also analyzed a series of heart-enriched miRNAs previously found deregulated in the heart tissue of a PA murine model and confirmed their altered expression. Our novel results show that PA iPSC-cardiomyocytes represent a promising model for investigating the pathological mechanisms underlying PA cardiomyopathies, also serving as an ex vivo platform for therapeutic evaluation.
    An ionising radiation-induced specic transcriptional signature of inammationassociated genes in human whole blood: a pilot study
    Lourdes Cruz-Garcia - 2021
    Abstract
    This communication reports the identication of a new panel of transcriptional changes in inammationassociated genes observed in response to ionising radiation received by radiotherapy patients. Peripheral blood samples were taken with ethical approval and informed consent from a total of 20 patients undergoing external beam radiotherapy for breast, lung, gastrointestinal or genitourinary tumours. Nanostring nCounter analysis of transcriptional changes was carried out in samples prior and 24 hours post-delivery of the 1st radiotherapy fraction, just prior to the 5th or 6th fraction, and just before the last fraction. Statistical analysis with BRB Array Tools, GLM MANOVA and nSolver, revealed a radiation responsive panel of genes which varied by patient group (type of cancer) and with time since exposure (as an analogue for dose received), which may be useful as a biomarker of radiation response. Further validation in a wider group of patients is ongoing, together with work towards a full understanding of patient specic responses in support of personalised approaches to radiation medicine.
    SIRT7 deficiency suppresses inflammation, induces EndoMT, and increases vascular permeability in primary pulmonary endothelial cells
    Anne E. Wyman - 2020
    Abstract
    Acute lung injury (ALI), a common condition in critically ill patients, has limited treatments and high mortality. Aging is a risk factor for ALI. Sirtuins (SIRTs), central regulators of the aging process, decrease during normal aging and in aging-related diseases. We recently showed decreased SIRT7 expression in lung tissues and fibroblasts from patients with pulmonary fibrosis compared to controls. To gain insight into aging-related mechanisms in ALI, we investigated the effects of SIRT7 depletion on lipopolysaccharide (LPS)-induced inflammatory responses and endothelial barrier permeability in human primary pulmonary endothelial cells. Silencing SIRT7 in pulmonary artery or microvascular endothelial cells attenuated LPS-induced increases in ICAM1, VCAM1, IL8, and IL6 and induced endomesenchymal transition (EndoMT) with decreases in VE-Cadherin and PECAM1 and increases in collagen, alpha-smooth muscle actin, TGFβ receptor 1, and the transcription factor Snail. Loss of endothelial adhesion molecules was accompanied by increased F-actin stress fibers and increased endothelial barrier permeability. Together, these results show that an aging phenotype induced by SIRT7 deficiency promotes EndoMT with impaired inflammatory responses and dysfunction of the lung vascular barrier.
    CTCF loss mediates unique DNA hypermethylation landscapes in human cancers
    Nathan A. Damaschke - 2020
    Abstract
    Background The chromatin insulator CCCTC-binding factor (CTCF) displays tissue-specific DNA binding sites that regulate transcription and chromatin organization. Despite evidence linking CTCF to the protection of epigenetic states through barrier insulation, the impact of CTCF loss on genome-wide DNA methylation sites in human cancer remains undefined. Results Here, we demonstrate that prostate and breast cancers within The Cancer Genome Atlas (TCGA) exhibit frequent copy number loss of CTCF and that this loss is associated with increased DNA methylation events that occur preferentially at CTCF binding sites. CTCF sites differ among tumor types and result in tissue-specific methylation patterns with little overlap between breast and prostate cancers. DNA methylation and transcriptome profiling in vitro establish that forced downregulation of CTCF leads to spatially distinct DNA hypermethylation surrounding CTCF binding sites, loss of CTCF binding, and decreased gene expression that is also seen in human tumors. DNA methylation inhibition reverses loss of expression at these CTCF-regulated genes. Conclusion These findings establish CTCF loss as a major mediator in directing localized DNA hypermethylation events in a tissue-specific fashion and further support its role as a driver of the cancer phenotype.
    Microbial community similarity and dissimilarity inside and across full-scale activated sludge processes for simultaneous nitrification and denitrification
    Jianfeng Wen - 2020
    Abstract
    Simultaneous nitrification and denitrification under low dissolved oxygen conditions is an energy-saving modification of the activated sludge process to achieve efficient nitrogen removal. Geographically distinct full-scale treatment plants are excellent platforms to address the links of microbial community with operating parameters. Mixed liquor samples were collected from a sequencing batch reactor plant, oxidation ditch plant, and step-feed activated sludge plant. Next-Generation Sequencing of the samples showed that the microbial communities were similar at the phylum level among the plants, being dominated by Proteobacteria. Microbial composition of functional groups was similar between the react fill and react phases of the sequencing batch reactors, among four sequencing batch reactors, and among four oxidation ditches. Nitrospira was the only identified genus of autotropic nitrifying bacteria with a relative abundance of 2.2–2.5% in the oxidation ditches and 0.4–0.7% at the other plants. Heterotrophic nitrifying–aerobic denitrifying bacteria were dominated by Dechloromonas with a relative abundance of 0.4–1.0%. Microbial community composition and nitrogen removal mechanisms were related to overall level and local zonation of dissolved oxygen, mixed liquor suspended solids concentration, nitrogen and organic loadings, and solids retention time. Low dissolved oxygen and low organic and nitrogen loadings favored growth of Nitrospira.
    Inhibition of PIM2 in liver cancer decreases tumor cell proliferation in vitro and in vivo primarily through the modulation of cell cycle progression
    Pia Kronschnabl - 2020
    Abstract
    Liver cancer is the fourth leading cause of cancer‑related mortality worldwide with limited therapeutic options. Thus, novel treatment strategies are urgently required. While the oncogenic kinase, proviral integration site for Moloney murine leukemia virus 2 (PIM2), has been shown to be overexpressed in liver cancer, little is known about the role of PIM2 in this tumor entity. In this study, we explored the functional relevance and therapeutic potential of PIM2 in liver cancer. Using PIM2‑specific siRNAs, we examined the effects of PIM2 knockdown on proliferation (WST‑1 assays and spheroid assays), 3D‑colony formation and colony spread, apoptosis (flow cytometry and caspase 3/caspase 7 activity), as well as cell cycle progression (flow cytometry, RT‑qPCR and western blot analysis) in the two liver cancer cell lines, HepG2 and Huh‑7. In subcutaneous liver cancer xenografts, we assessed the effects of PIM2 knockdown on tumor growth via the systemic delivery of polyethylenimine (PEI)‑complexed siRNA. The knockdown of PIM2 resulted in potent anti‑proliferative effects in cells grown on plastic dishes, as well as in spheroids. This was due to G0/G1 cell cycle blockade and the subsequent downregulation of genes related to the S phase as well as the G2/M phase of the cell cycle, whereas the apoptotic rates remained unaltered. Furthermore, colony formation and colony spread were markedly inhibited by PIM2 knockdown. Notably, we found that HepG2 cells were more sensitive to PIM2 knockdown than the Huh‑7 cells. In vivo, the therapeutic nanoparticle‑mediated delivery of PIM2 siRNA led to profound anti‑tumor effects in a liver cancer xenograft mouse model. On the whole, the findings of this study underscore the oncogenic role of PIM2 and emphasize the potential of targeted therapies based on the specific inhibition of PIM2 in liver cancer.
    Assessment of the ptxD gene as a growth and selective marker in Trichoderma atroviride using Pccg6, a novel constitutive promoter
    Nohemí Carreras-Villaseñor - 2020
    Abstract
    Background Trichoderma species are among the most effective cell factories to produce recombinant proteins, whose productivity relies on the molecular toolkit and promoters available for the expression of the target protein. Although inducible promoter systems have been developed for producing recombinant proteins in Trichoderma, constitutive promoters are often a desirable alternative. Constitutive promoters are simple to use, do not require external stimuli or chemical inducers to be activated, and lead to purer enzyme preparations. Moreover, most of the promoters for homologous and heterologous expression reported in Trichoderma have been commonly evaluated by directly assessing production of industrial enzymes, requiring optimization of laborious protocols. Results Here we report the identification of Pccg6, a novel Trichoderma atroviride constitutive promoter, that has similar transcriptional strength as that of the commonly used pki1 promoter. Pccg6 displayed conserved arrangements of transcription factor binding sites between promoter sequences of Trichoderma ccg6 orthologues genes, potentially involved in their regulatory properties. The predicted ccg6-encoded protein potentially belongs to the SPE1/SPI1 protein family and shares high identity with CCG6 orthologue sequences from other fungal species including Trichoderma reesei, Trichoderma virens, Trichoderma asperellum, and to a lesser extent to that of Neurospora crassa. We also report the use of the Pccg6 promoter to drive the expression of PTXD, a phosphite oxidoreductase of bacterial origin, which allowed T. atroviride to utilize phosphite as a sole source of phosphorus. We propose ptxD as a growth reporter gene that allows real-time comparison of the functionality of different promoters by monitoring growth of Trichoderma transgenic lines and enzymatic activity of PTXD. Finally, we show that constitutive expression of ptxD provided T. atroviride a competitive advantage to outgrow bacterial contaminants when supplied with phosphite as a sole source of phosphorus. Conclusions A new constitutive promoter, ccg6, for expression of homologous and heterologous proteins has been identified and tested in T. atroviride to express PTXD, which resulted in an effective and visible phenotype to evaluate transcriptional activity of sequence promoters. Use of PTXD as a growth marker holds great potential for assessing activity of other promoters and for biotechnological applications as a contamination control system.
    The Cutaneous Inflammatory Response to Thermal Burn Injury in a Murine Model
    Zabeen Lateef - 2019
    Abstract
    Many burn interventions aim to target the inflammatory response as a means of enhancing healing or limiting hypertrophic scarring. Murine models of human burns have been developed, but the inflammatory response to injury in these models has not been well defined. The aim of this study was to profile inflammatory cell populations and gene expression relative to healing and scarring in a murine model of thermal burns. Cutaneous injuries were created on the dorsal region of C57Bl/6 mice using a heated metal rod. Animals were euthanized at selected time points over ten weeks, with the lesions evaluated using macroscopic measurements, histology, immunofluorescent histochemistry and quantitative PCR. The burn method generated a reproducible, partial-thickness injury that healed within two weeks through both contraction and re-epithelialization, in a manner similar to human burns. The injury caused an immediate increase in pro-inflammatory cytokine and chemokine expression, coinciding with an influx of neutrophils, and the disappearance of Langerhans cells and mast cells. This preceded an influx of dendritic cells and macrophages, a quarter of which displayed an inflammatory (M1) phenotype, with both populations peaking at closure. As with human burns, the residual scar increased in size, epidermal and dermal thickness, and mast cell numbers over 10 weeks, but abnormal collagen I-collagen III ratios, fibre organization and macrophage populations resolved 3–4 weeks after closure. Characterisation of the inflammatory response in this promising murine burn model will assist future studies of burn complications and aid in the preclinical testing of new anti-inflammatory and anti-scarring therapies.
    Fructose-1,6-bisphosphate prevents pulmonary fibrosis by regulating extracellular matrix deposition and inducing phenotype reversal of lung myofibroblasts
    Henrique Bregolin Dias - 2019
    Abstract
    Pulmonary fibrosis (PF) is the result of chronic injury where fibroblasts become activated and secrete large amounts of extracellular matrix (ECM), leading to impaired fibroblasts degradation followed by stiffness and loss of lung function. Fructose-1,6-bisphosphate (FBP), an intermediate of glycolytic pathway, decreases PF development, but the underlying mechanism is unknown. To address this issue, PF was induced in vivo using a mouse model, and pulmonary fibroblasts were isolated from healthy and fibrotic animals. In PF model mice, lung function was improved by FBP as revealed by reduced collagen deposition and downregulation of ECM gene expression such as collagens and fibronectin. Fibrotic lung fibroblasts (FLF) treated with FBP for 3 days in vitro showed decreased proliferation, contraction, and migration, which are characteristic of myofibroblast to fibroblast phenotype reversal. ECM-related genes and proteins such as collagens, fibronectin and α-smooth muscle actin, were also downregulated in FBP-treated FLF. Moreover, matrix metalloproteinase (MMP) 1, responsible for ECM degradation, was produced only in fibroblasts obtained from healthy lungs (HLF) and FBP did not alter its expression. On the other hand, tissue inhibitor of metalloproteinase (TIMP)-1, a MMP1 inhibitor, and MMP2, related to fibroblast tissue-invasion, were predominantly produced by FLF and FBP was able to downregulate its expression. These results demonstrate that FBP may prevent bleomycin-induced PF development through reduced expression of collagen and other ECM components mediated by a reduced TIMP-1 and MMP2 expression.
    Glucocorticoid receptor modulation decreases ER-positive breast cancer cell proliferation and suppresses wild-type and mutant ER chromatin association
    Eva Tonsing-Carter - 2019
    Abstract
    Background: Non-ER nuclear receptor activity can alter estrogen receptor (ER) chromatin association and resultant ER-mediated transcription. Consistent with GR modulation of ER activity, high tumor glucocorticoid receptor (GR) expression correlates with improved relapse-free survival in ER+ breast cancer (BC) patients. Methods: In vitro cell proliferation assays were used to assess ER-mediated BC cell proliferation following GR modulation. ER chromatin association following ER/GR co-liganding was measured using global ChIP sequencing and directed ChIP analysis of proliferative gene enhancers. Results: We found that GR liganding with either a pure agonist or a selective GR modulator (SGRM) slowed estradiol (E2)-mediated proliferation in ER+ BC models. SGRMs that antagonized transcription of GR-unique genes both promoted GR chromatin association and inhibited ER chromatin localization at common DNA enhancer sites. Gene expression analysis revealed that ER and GR co-activation decreased proliferative gene activation (compared to ER activation alone), specifically reducing CCND1, CDK2, and CDK6 gene expression. We also found that ligand-dependent GR occupancy of common ER-bound enhancer regions suppressed both wild-type and mutant ER chromatin association and decreased corresponding gene expression. In vivo, treatment with structurally diverse SGRMs also reduced MCF-7 Y537S ER-expressing BC xenograft growth. Conclusion: These studies demonstrate that liganded GR can suppress ER chromatin occupancy at shared ER-regulated enhancers, including CCND1 (Cyclin D1), regardless of whether the ligand is a classic GR agonist or antagonist. Resulting GR-mediated suppression of ER+ BC proliferative gene expression and cell division suggests that SGRMs could decrease ER-driven gene expression.
    The TLR4 adaptor TRAM controls the phagocytosis of Gram-negative bacteria by interacting with the Rab11-family interacting protein 2
    Astrid Skjesol - 2019
    Abstract
    Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria.
    Influence of the concentration of dietary digestible calcium on growth performance, bone mineralization, plasma calcium, and abundance of genes involved in intestinal absorption of calcium in pigs from 11 to 22 kg fed diets with different concentrations o
    L. Vanessa Lagos - 2019
    Abstract
    A 21-day experiment was conducted to test the hypothesis that Ca requirements to maximize growth performance expressed as the standardized total tract digestible (STTD) Ca to STTD P ratio is less than 1.40:1. The second hypothesis was that increasing dietary Ca increases plasma Ca concentration and downregulates abundance of genes related to Ca absorption (TRPV6, S100G, and ATP2B1) in the duodenum, and tight junction proteins (OCLN, CLDN1, and ZO1) in the duodenum and ileum. Methods Twenty corn-soybean meal diets were formulated using a 4 × 5 factorial design with diets containing 0.16%, 0.33%, 0.42%, or 0.50% STTD P, and 0.14%, 0.29%, 0.44%, 0.59%, or 0.74% STTD Ca. Six hundred and forty pigs (initial weight: 11.1 ± 1.4 kg) were allotted to 20 diets and 5 blocks in a randomized complete block design. On day 21, weights of pigs and feed left in feeders were recorded and blood, duodenal tissue, ileal mucosa, and the right femur were collected from 1 pig per pen. Abundance of mRNA was determined in duodenal and ileal tissue via quantitative RT-PCR. Data were analyzed using a response surface model. Results The predicted maximum ADG (614 g), G:F (0.65), and bone ash (11.68 g) was obtained at STTD Ca:STTD P ratios of 1.39:1, 1.25:1, and 1.66:1, respectively, when STTD P was provided at the requirement (0.33%). If dietary STTD P was below the requirement, increasing dietary Ca resulted in reduced (P < 0.05) ADG and G:F. However, if dietary STTD P was above the requirement, negative effects (P < 0.05) on ADG and G:F of increasing STTD Ca were observed only if dietary STTD Ca exceeded 0.6%. Plasma Ca concentration was positively affected by STTD Ca over the range studied (quadratic, P < 0.01) and negatively affected by increasing STTD P (linear, P < 0.01). There was a linear negative effect (P < 0.05) of STTD Ca on the abundance of S100G, TRPV6, OCLN, and ZO1 in duodenum, and CLDN and ZO1 in ileum. Conclusions The STTD Ca:STTD P ratio needed to maximize growth performance of 11- to 25-kg pigs is less than 1.40:1, if P is at the estimated requirement. Increasing dietary Ca reduces transcellular absorption of Ca and increases paracellular absorption of Ca.
    Brf1 loss and not overexpression disrupts tissues homeostasis in the intestine, liver and pancreas
    Dritan Liko - 2019
    Abstract
    RNA polymerase III (Pol-III) transcribes tRNAs and other small RNAs essential for protein synthesis and cell growth. Pol-III is deregulated during carcinogenesis; however, its role in vivo has not been studied. To address this issue, we manipulated levels of Brf1, a Pol-III transcription factor that is essential for recruitment of Pol-III holoenzyme at tRNA genes in vivo. Knockout of Brf1 led to embryonic lethality at blastocyst stage. In contrast, heterozygous Brf1 mice were viable, fertile and of a normal size. Conditional deletion of Brf1 in gastrointestinal epithelial tissues, intestine, liver and pancreas, was incompatible with organ homeostasis. Deletion of Brf1 in adult intestine and liver induced apoptosis. However, Brf1 heterozygosity neither had gross effects in these epithelia nor did it modify tumorigenesis in the intestine or pancreas. Overexpression of BRF1 rescued the phenotypes of Brf1 deletion in intestine and liver but was unable to initiate tumorigenesis. Thus, Brf1 and Pol-III activity are absolutely essential for normal homeostasis during development and in adult epithelia. However, Brf1 overexpression or heterozygosity are unable to modify tumorigenesis, suggesting a permissive, but not driving role for Brf1 in the development of epithelial cancers of the pancreas and gut.
    No evidence that gut microbiota impose a net cost on their butterfly host
    Alison Ravenscraft - 2019
    Abstract
    Gut microbes are believed to play a critical role in most animal life, yet fitness effects and cost–benefit trade‐offs incurred by the host are poorly understood. Unlike most hosts studied to date, butterflies largely acquire their nutrients from larval feeding, leaving relatively little opportunity for nutritive contributions by the adult's microbiota. This provides an opportunity to measure whether hosting gut microbiota comes at a net nutritional price. Because host and bacteria may compete for sugars, we hypothesized that gut flora would be nutritionally neutral to adult butterflies with plentiful food, but detrimental to semistarved hosts, especially when at high density. We held field‐caught adult Speyeria mormonia under abundant or restricted food conditions. Because antibiotic treatments did not generate consistent variation in their gut microbiota, we used interindividual variability in bacterial loads and operational taxonomic unit abundances to examine correlations between host fitness and the abdominal microbiota present upon natural death. We detected strikingly few relationships between microbial flora and host fitness. Neither total bacterial load nor the abundances of dominant bacterial taxa were related to butterfly fecundity, egg mass or egg chemical content. Increased abundance of a Commensalibacter species did correlate with longer host life span, while increased abundance of a Rhodococcus species correlated with shorter life span. Contrary to our expectations, these relationships were unchanged by food availability to the host and were unrelated to reproductive output. Our results suggest the butterfly microbiota comprises parasitic, commensal and beneficial taxa that together do not impose a net reproductive cost, even under caloric stress.
    Altered Gene Response to Aflatoxin B1 in the Spleens of Susceptible and Resistant Turkeys
    Kent M. Reed - 2019
    Abstract
    Susceptibility and/or resistance to aflatoxin B1 (AFB1) is a threshold trait governed principally by glutathione S transferase (GST)-mediated detoxification. In poultry, domesticated turkeys are highly sensitive to AFB1, most likely due to dysfunction in hepatic GSTs. In contrast, wild turkeys are comparatively resistant to aflatoxicosis due to the presence of functional hepatic GSTAs and other possible physiological and immunological interactions. The underlying genetic basis for the disparate GST function in turkeys is unknown as are the broader molecular interactions that control the systemic response. This study quantifies the effects of dietary AFB1 on gene expression in the turkey spleen, specifically contrasting genetically distinct domesticated (DT, susceptible) and Eastern wild (EW, resistant) birds. Male turkey poults were subjected to a short-term AFB1 treatment protocol with feed supplemented with 320 ppb AFB1 beginning on day 15 of age and continuing for 14 days. Spleen tissues were harvested and subjected to deep RNA sequencing and transcriptome analysis. Analysis of differential gene expression found the effects of AFB1 treatment on the spleen transcriptomes considerably more prominent in the DT birds compared to EW. However, expression of the differentially expressed genes (DEGs) was directionally biased, with the majority showing higher expression in EW (i.e., down-regulation in DT). Significantly altered pathways included FXR/RXR and LXR/RXR activation, coagulation system, prothrombin activation, acute phase response, and atherosclerosis signaling. Differential extra-hepatic expression of acute phase protein genes was confirmed by quantitative real time PCR (qRT-PCR) in the original experiment and additional turkey lines. Results demonstrate that wild turkeys possess a capacity to more effectively respond to AFB1 exposure
    Plant growth-promoting rhizobacteria induce changes in Arabidopsis thaliana gene expression of nitrate and ammonium uptake genes
    Pamela Calvo - 2019
    Abstract
    Plant growth-promoting rhizobacteria (PGPR) enhance plant growth under the influence of multigenic processes, including nitrate (NO−3) and ammonium (NH+4) uptake genes, which could potentially explain the improvement in plant nutrition and plant growth promotion. Studies on the effects of PGPR inoculation on regulation of NO−3 and NH+4 plant uptake genes and nutrient accumulation using soil or soil-like substrates are limited. Here, we tested the hypothesis that the application of PGPR Bacillus mixtures increases overall plant growth, nutrient uptake and the transcript levels of nitrate and ammonium uptake genes in Arabidopsis thaliana. All three PGPR mixtures tested in this study significantly increased plant shoot fresh weight, root fresh, chlorophyll content, nutrient uptake and plant diameter. The transcript levels of five nitrate and four ammonium uptake genes were significantly higher in PGPR-treated plants compared to untreated plants. These results demonstrate that plant growth promotion and enhanced nutrient uptake by select PGPR mixtures.
    The effect of maxillary sinus antrostomy size on the sinus microbiome
    Alexander S. Kim BSE - 2019
    Abstract
    Background The optimal maxillary antrostomy size to surgically treat sinusitis is not well known. In this study, we examined clinical metrics of disease severity and symptom scores, measured secreted inflammatory markers, and characterized the sinus microbiome to determine if there were significant differences in outcome between different maxillary ostial sizes. Methods Prospective randomized, single‐blinded clinical trial enrolling 12 individuals diagnosed with recurrent acute or chronic rhinosinusitis. Each patient was blinded and randomized to receive minimal maxillary ostial dilation via balloon sinuplasty on 1 side vs a mega‐antrostomy on the contralateral side. Data collected included symptom scores (20‐item Sino‐Nasal Outcome Test [SNOT‐20]), endoscopy, and radiologic Lund‐Mackay scores. During surgery and at their postoperative visit swabs were obtained from each maxillary sinus, and 16S DNA and inflammatory cytokine levels analyzed. The use of each patient as their own control allowed us to minimize confounding variables. Results There was statistically significant improvement in SNOT‐20 symptom scores postoperatively in all patients. There were no significant differences between maxillary ostial size in postoperative endoscopy scores, cytokine profile, or bacterial burden. There were statistically significant differences in relative postoperative abundance of Staphylococcus, Lactococcus, and Cyanobacteria between the mega‐antrostomy and mini‐antrostomy. Conclusions The method used in surgical maxillary antrostomies had no effect on endoscopy scores or cytokine profiles. Microbiome analysis determined significant differences between the different antrostomy sizes in postoperative Staphylococcus, Lactococcus, and Cyanobacteria abundance. The clinical significance of these changes in the sinus microbiome are not known but may be a result of increased access to postoperative sinonasal irrigations.
    An Fc-Optimized CD133 Antibody for Induction of Natural Killer Cell Reactivity Against Colorectal Cancer
    Bastian J. Schmied - 2019
    Abstract
    The introduction of monoclonal antibodies (mAbs) has largely improved treatment options for cancer patients. The ability of antitumor mAbs to elicit antibody-dependent cellular cytotoxicity (ADCC) contributes to a large extent to their therapeutic efficacy. Many efforts accordingly aim to improve this important function by engineering mAbs with Fc parts that display enhanced affinity to the Fc receptor CD16 expressed, e.g., on natural killer (NK) cells. Here we characterized the CD133 mAb 293C3-SDIE that contains an engineered Fc part modified by the amino acid exchanges S239D/I332E—that reportedly increase the affinity to CD16—with regard to its ability to induce NK reactivity against colorectal cancer (CRC). 293C3-SDIE was found to be a stable protein with favorable binding characteristics achieving saturating binding to CRC cells at concentrations of approximately 1 µg/mL. While not directly affecting CRC cell growth and viability, 293C3-SDIE potently induced NK cell activation, degranulation, secretion of Interferon-γ, as well as ADCC resulting in potent lysis of CRC cell lines. Based on the preclinical characterization presented in this study and the available data indicating that CD133 is broadly expressed in CRC and represents a negative prognostic marker, we conclude that 293C3-SDIE constitutes a promising therapeutic agent for the treatment of CRC and thus warrants clinical evaluation.
    Epigenetic Changes at the Birc5 Promoter Induced by YM155 in Synovial Sarcoma
    Aleksander Mika - 2019
    Abstract
    YM155 is an anti-cancer therapy that has advanced into 11 different human clinical trials to treat various cancers. This apoptosis-inducing therapy indirectly affects the protein levels of survivin (gene: Birc5), but the molecular underpinnings of the mechanism remain largely unknown. Synovial sarcoma is a rare soft-tissue malignancy with high protein expression of survivin. We investigated whether YM155 would be a viable therapeutic option to treat synovial sarcoma. YM155 therapy was applied to human synovial sarcoma cell lines and to a genetically engineered mouse model of synovial sarcoma. We discovered that YM155 exhibited nanomolar potency against human synovial sarcoma cell lines and the treated mice with synovial sarcoma demonstrated a 50% reduction in tumor volume compared to control treated mice. We further investigated the mechanism of action of YM155 by looking at the change of lysine modifications of the histone tails that were within 250 base pairs of the Birc5 promoter. Using chromatin immunoprecipitation (ChIP)-qPCR, we discovered that the histone epigenetic marks of H3K27 for the Birc5 promoter changed upon YM155 treatment. H3K27me3 and H3K27ac increased, but the net result was decreased Birc5/survivin expression. Furthermore, the combination of molecular events resulted in caspase 3/7/8 upregulation and death of the sarcoma cells.
    Chemokine Receptor Redundancy and Specificity Are Context Dependent
    Douglas P. Dyer - 2019
    Abstract
    Currently, we lack an understanding of the individual and combinatorial roles for chemokine receptors in the inflammatory process. We report studies on mice with a compound deletion of Ccr1, Ccr2, Ccr3, and Ccr5, which together control monocytic and eosinophilic recruitment to resting and inflamed sites. Analysis of resting tissues from these mice, and mice deficient in each individual receptor, provides clear evidence for redundant use of these receptors in establishing tissue-resident monocytic cell populations. In contrast, analysis of cellular recruitment to inflamed sites provides evidence of specificity of receptor use for distinct leukocyte subtypes and no indication of comprehensive redundancy. We find no evidence of involvement of any of these receptors in the recruitment of neutrophils or lymphocytes to resting or acutely inflamed tissues. Our data shed important light on combinatorial inflammatory chemokine receptor function and highlight Ccr2 as the primary driver of myelomonocytic cell recruitment in acutely inflamed contexts.
    Localization of the 1,25-dihydroxyvitamin d-mediated response in the intestines of mice
    Carmen J. Reynolds - 2019
    Abstract
    1,25-Dihydroxyvitamin D3 (1,25(OH)2D) elicits a transcriptional response in the intestines. Assessments of this response are often derived from crude tissue homogenates and eliminate the ability to discriminate among different cell types. Here, we used an RNA in situ hybridization assay, RNAScope (Advanced Cell Diagnostics, Newark, CA), to identify the cells in the intestine that respond to 1,25(OH)2D with expression of cytochrome P450 family 24 subfamily A member 1 (Cyp24a1) mRNA. Mice were gavaged with a single bolus dose of 1,25(OH)2D to target the duodenum or a glucuronic acid conjugate of 1,25(OH)2D, β-G-1,25(OH)2D, to target the colon. QRT-PCR analysis of Cyp24a1 mRNA verified that the 1,25(OH)2D-induced responses were present. RNAScope revealed that the mRNA response present after six hours is limited to mature enterocytes exposed to the intestinal lumen in both the duodenum and colon. No detectable expression was observed in goblet cells, lamina propria, muscularis mucosa muscle, submucosa and submucosal lymphoid follicles, or tunica muscularis. Our findings have identified epithelial enterocytes to be the intestinal targets for 1,25(OH)2D in both the duodenum and colon.
    Characterization of myofibroblasts isolated from the intestine of patients with inflammatory bowel disease [version 1; peer review: 1 approved, 1 approved with reservations]
    Serge Dionne - 2019
    Abstract
    Background: Intestinal fibrosis represents a serious complication of inflammatory bowel diseases (IBD), often necessitating surgical resections. Myofibroblasts are primarily responsible for interstitial matrix accumulation in fibrotic diseases. However intestinal myofibroblasts (IMF) remain inadequately characterized. The aim was to examine fibroblast markers and fibrosis-associated gene expression in IMF isolated from resected intestine from IBD and control patients. As well as determining the effect of the fibrogenic cytokine TGFβ. Methods: Intestinal resections were obtained (n =35) from consenting patients undergoing elective surgery (2014-16). Primary cultures of IMF were isolated using DTT and EDTA and cultured. Viability and phenotypic characterization of IMF was carried out by flow cytometry and fluorescence microscopy. IMF (passages 3-8) were treated for 24 hours. Cytokines were quantified in IMF by real time PCR and in supernatants using the human pro-inflammatory cytokine panel Results: All markers and most fibrosis mediators studied were preferentially expressed by IMF compared to mucosal tissue. Metalloproteinases (MMP) 2 and 3, as well as their inhibitor TIMP1, are highly expressed by IMF. They also highly expressed inflammatory mediators, including IL-6, IL-8, CCL2 and PTGS2. Whereas mucosal expression of pro-inflammatory cytokines such as TNFα and IL-17 is increased in IBD, that of fibrosis mediators was not different. Fibrosis-related gene expression in IMF from IBD patients and controls was similar, but IMF from IBD expressed higher levels of several inflammatory genes. IMF from CD and UC had mostly similar expression profiles. TGFβ induced expression of fibrogenic genes αSMA, COL1A1, CTGF, FN1 and LOX. TGFβ-stimulated IMF released increased levels of IL-6, whereas IL-6, IL-8, as well as small amounts of IFN-γ and IL12p70 were produced following stimulation with IL-1β+IL-23. Conclusions: This study extends knowledge about the pathogenesis of fibrosis in IBD. Further research in the identification of mechanisms involved in IMF activation and fibrogenesis are required.
    Hairless regulates p53 target genes to exert tumor suppressive functions in glioblastoma
    Lemlem Brook - 2019
    Abstract
    Glioblastoma (GBM) is the most common malignant brain tumor and is associated with a poor prognosis, with most patients living less than a year after diagnosis. Given that GBM nearly always recurs after conventional treatments, there is an urgent need to identify novel molecular targets. Hairless (HR) is a nuclear factor enriched in the skin and has been previously implicated in hair cycling. HR is also highly expressed in the brain, but its significance is unknown. We found that human hairless gene (HR) expression is significantly decreased in all GBM subtypes compared with normal brain tissue and is predictive of prognosis, which suggests that loss of HR expression can contribute to GBM pathogenesis. HR was recently discovered to bind to and regulate p53 responsive elements, and thus we hypothesized that HR may have a tumor suppressive function in GBM by modulating p53 target gene expression. We found that HR indeed regulates p53 target genes, including those implicated in cell cycle progression and apoptosis in the GBM‐derived U87 cell line, and restoring HR expression triggered G2/M arrest and apoptosis. An analysis of sequenced genomes from patients with GBM revealed 10 HR somatic mutations in patients with glioma, two of which are located in the histone demethylase domain of HR. These two mutations, P996S and K1004N, were reconstructed and found to have impaired p53 transactivating properties. Collectively, the results of our study suggest that HR has tumor suppressive functions in GBM, which may be clinically relevant and a potential avenue for therapeutic intervention.
    Neuroendocrine Whiplash: Slamming the Breaks on Anabolic-Androgenic Steroids Following Repetitive Mild Traumatic Brain Injury in Rats May Worsen Outcomes
    Jason Tabor - 2019
    Abstract
    Sport-related concussion is an increasingly common injury among adolescents, with repetitive mild traumatic brain injuries (RmTBI) being a significant risk factor for long-term neurobiological and psychological consequences. It is not uncommon for younger professional athletes to consume anabolic-androgenic steroids (AAS) in an attempt to enhance their performance, subjecting their hormonally sensitive brains to potential impairment during neurodevelopment. Furthermore, RmTBI produces acute neuroendocrine dysfunction, specifically in the anterior pituitary, disrupting the hypothalamic-pituitary adrenal axis, lowering cortisol secretion that is needed to appropriately respond to injury. Some AAS users exhibit worse symptoms post-RmTBI if they quit their steroid regime. We sought to examine the pathophysiological outcomes associated with the abrupt cessation of the commonly abused AAS, Metandienone (Met) on RmTBI outcomes in rats. Prior to injury, adolescent male rats received either Met or placebo, and exercise. Rats were then administered RmTBIs or sham injuries, followed by steroid and exercise cessation (SEC) or continued treatment. A behavioral battery was conducted to measure outcomes consistent with clinical representations of post-concussion syndrome and chronic AAS exposure, followed by analysis of serum hormone levels, and qRT-PCR for mRNA expression and telomere length. RmTBI increased loss of consciousness and anxiety-like behavior, while also impairing balance and short-term working memory. SEC induced hyperactivity while Met treatment alone increased depressive-like behavior. There were cumulative effects whereby RmTBI and SEC exacerbated anxiety and short-term memory outcomes. mRNA expression in the prefrontal cortex, amygdala, hippocampus, and pituitary were modified in response to Met and SEC. Analysis of telomere length revealed the negative impact of SEC while Met and SEC produced changes in serum levels of testosterone and corticosterone. We identified robust changes in mRNA to serotonergic circuitry, neuroinflammation, and an enhanced stress response. Interestingly, Met treatment promoted glucocorticoid secretion after injury, suggesting that maintained AAS may be more beneficial than abstaining after mTBI.
    Fine-scale spatial and temporal dynamics of kdr haplotypes in Aedes aegypti from Mexico
    Marissa K. Grossman - 2019
    Abstract
    Background As resistance to insecticides increases in disease vectors, it has become exceedingly important to monitor populations for susceptibility. Most studies of field populations of Aedes aegypti have largely characterized resistance patterns at the spatial scale of the city or country, which may not be completely informative given that insecticide application occurs at the scale of the house or city block. Phenotypic resistance to pyrethroids dominates in Ae. aegypti, and it has been partially explained by mutations in the voltage-gated sodium channel gene. Here, we assess community-level patterns of four knockdown resistance (kdr) haplotypes (C1534/I1016, F1534/I1016, C1534/V1016 and F1534/V1016) in Ae. aegypti in 24 randomly chosen city blocks from a city in Yucatán State, Mexico, during both the dry and wet season and over two years. Results Three of the four haplotypes, C1534/I1016, C1534/V1016 and F1534/V1016 were heterogeneous between city blocks at all four sampling time points, and the double mutant haplotype, C1534/I1016, showed a significant increase following the wet season. The F1534/I1016 haplotype was rarely detected, similar to other studies. However, when haplotype frequencies were aggregated to a coarser spatial scale, the differences in space and time were obscured. Conclusions Our results provide empirical evidence that the selection of kdr alleles is occurring at fine spatial scales, indicating that future studies should include this scale to better understand evolutionary processes of resistance in natural populations.
    Loss-of-function mutations in caspase recruitment domain-containing protein 14 (CARD14) are associated with a severe variant of atopic dermatitis
    Alon Peled BMedSci - 2019
    Abstract
    Background Atopic dermatitis (AD) is a highly prevalent chronic inflammatory skin disease that is known to be, at least in part, genetically determined. Mutations in caspase recruitment domain-containing protein 14 (CARD14) have been shown to result in various forms of psoriasis and related disorders. Objective We aimed to identify rare DNA variants conferring a significant risk for AD through genetic and functional studies in a cohort of patients affected with severe AD. Methods Whole-exome and direct gene sequencing, immunohistochemistry, real-time PCR, ELISA, and functional assays in human keratinocytes were used. Results In a cohort of patients referred with severe AD, DNA sequencing revealed in 4 patients 2 rare heterozygous missense mutations in the gene encoding CARD14, a major regulator of nuclear factor κB (NF-κB). A dual luciferase reporter assay demonstrated that both mutations exert a dominant loss-of-function effect and result in decreased NF-κB signaling. Accordingly, immunohistochemistry staining showed decreased expression of CARD14 in patients' skin, as well as decreased levels of activated p65, a surrogate marker for NF-κB activity. CARD14-deficient or mutant-expressing keratinocytes displayed abnormal secretion of key mediators of innate immunity. Conclusions Although dominant gain-of-function mutations in CARD14 are associated with psoriasis and related diseases, loss-of-function mutations in the same gene are associated with a severe variant of AD.
    Superresolution microscopy reveals linkages between ribosomal DNA on heterologous chromosomes
    Tamara A. Potapova - 2019
    Abstract
    The spatial organization of the genome is enigmatic. Direct evidence of physical contacts between chromosomes and their visualization at nanoscale resolution has been limited. We used superresolution microscopy to demonstrate that ribosomal DNA (rDNA) can form linkages between chromosomes. We observed rDNA linkages in many different human cell types and demonstrated their resolution in anaphase. rDNA linkages are coated by the transcription factor UBF and their formation depends on UBF, indicating that they regularly occur between transcriptionally active loci. Overexpression of c-Myc increases rDNA transcription and the frequency of rDNA linkages, further suggesting that their formation depends on active transcription. Linkages persist in the absence of cohesion, but inhibition of topoisomerase II prevents their resolution in anaphase. We propose that linkages are topological intertwines occurring between transcriptionally active rDNA loci spatially colocated in the same nucleolar compartment. Our findings suggest that active DNA loci engage in physical interchromosomal connections that are an integral and pervasive feature of genome organization
    Serum FHR1 binding to necrotic-type cells activates monocytic inflammasome and marks necrotic sites in vasculopathies
    Sarah Irmscher - 2019
    Abstract
    Persistent inflammation is a hallmark of many human diseases, including anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) and atherosclerosis. Here, we describe a dominant trigger of inflammation: human serum factor H-related protein FHR1. In vitro, this protein selectively binds to necrotic cells via its N-terminus; in addition, it binds near necrotic glomerular sites of AAV patients and necrotic areas in atherosclerotic plaques. FHR1, but not factor H, FHR2 or FHR3 strongly induces inflammasome NLRP3 in blood-derived human monocytes, which subsequently secrete IL-1β, TNFα, IL-18 and IL-6. FHR1 triggers the phospholipase C-pathway via the G-protein coupled receptor EMR2 independent of complement. Moreover, FHR1 concentrations of AAV patients negatively correlate with glomerular filtration rates and associate with the levels of inflammation and progressive disease. These data highlight an unexpected role for FHR1 during sterile inflammation, may explain why FHR1-deficiency protects against certain diseases, and identifies potential targets for treatment of auto-inflammatory diseases.
    RAL GTPases Drive Intestinal Stem Cell Function and Regeneration through Internalization of WNT Signalosomes
    Joel Johansson - 2019
    Abstract
    Ral GTPases are RAS effector molecules and by implication a potential therapeutic target for RAS mutant cancer. However, very little is known about their roles in stem cells and tissue homeostasis. Using Drosophila, we identified expression of RalA in intestinal stem cells (ISCs) and progenitor cells of the fly midgut. RalA was required within ISCs for efficient regeneration downstream of Wnt signaling. Within the murine intestine, genetic deletion of either mammalian ortholog, Rala or Ralb, reduced ISC function and Lgr5 positivity, drove hypersensitivity to Wnt inhibition, and impaired tissue regeneration following damage. Ablation of both genes resulted in rapid crypt death. Mechanistically, RALA and RALB were required for efficient internalization of the Wnt receptor Frizzled-7. Together, we identify a conserved role for RAL GTPases in the promotion of optimal Wnt signaling, which defines ISC number and regenerative potential.
    A Neuronal Relay Mediates a Nutrient Responsive Gut/Fat Body Axis Regulating Energy Homeostasis in Adult Drosophila
    Alessandro Scopelliti - 2019
    Abstract
    The control of systemic metabolic homeostasis involves complex inter-tissue programs that coordinate energy production, storage, and consumption, to maintain organismal fitness upon environmental challenges. The mechanisms driving such programs are largely unknown. Here, we show that enteroendocrine cells in the adult Drosophila intestine respond to nutrients by secreting the hormone Bursicon α, which signals via its neuronal receptor DLgr2. Bursicon α/DLgr2 regulate energy metabolism through a neuronal relay leading to the restriction of glucagon-like, adipokinetic hormone (AKH) production by the corpora cardiaca and subsequent modulation of AKH receptor signaling within the adipose tissue. Impaired Bursicon α/DLgr2 signaling leads to exacerbated glucose oxidation and depletion of energy stores with consequent reduced organismal resistance to nutrient restrictive conditions. Altogether, our work reveals an intestinal/neuronal/adipose tissue inter-organ communication network that is essential to restrict the use of energy and that may provide insights into the physiopathology of endocrine-regulated metabolic homeostasis.
    A Role for FACT in RNA Polymerase II Promoter-Proximal Pausing
    Theophilus T. Tettey - 2019
    Abstract
    FACT (facilitates chromatin transcription) is an evolutionarily conserved histone chaperone that was initially identified as an activity capable of promoting RNA polymerase II (Pol II) transcription through nucleosomes in vitro. In this report, we describe a global analysis of FACT function in Pol II transcription in Drosophila. We present evidence that loss of FACT has a dramatic impact on Pol II elongation-coupled processes including histone H3 lysine 4 (H3K4) and H3K36 methylation, consistent with a role for FACT in coordinating histone modification and chromatin architecture during Pol II transcription. Importantly, we identify a role for FACT in the maintenance of promoter-proximal Pol II pausing, a key step in transcription activation in higher eukaryotes. These findings bring to light a broader role for FACT in the regulation of Pol II transcription.
    Suppression of UV-B stress induced flavonoids by biotic stress: Is there reciprocal crosstalk?
    Dirk Schenke - 2019
    Abstract
    Plants respond to abiotic UV-B stress with enhanced expression of genes for flavonoid production, especially the key-enzyme chalcone synthase (CHS). Some flavonoids are antioxidative, antimicrobial and/or UV-B protective secondary metabolites. However, when plants are challenged with concomitant biotic stress (simulated e.g. by the bacterial peptide flg22, which induces MAMP triggered immunity, MTI), the production of flavonoids is strongly suppressed in both Arabidopsis thaliana cell cultures and plants. On the other hand, flg22 induces the production of defense related compounds, such as the phytoalexin scopoletin, as well as lignin, a structural barrier thought to restrict pathogen spread within the host tissue. Since all these metabolites require the precursor phenylalanine for their production, suppression of the flavonoid production appears to allow the plant to focus its secondary metabolism on the production of pathogen defense related compounds during MTI. Interestingly, several flavonoids have been reported to display anti-microbial activities. For example, the plant flavonoid phloretin targets the Pseudomonas syringae virulence factors flagella and type 3 secretion system. That is, suppression of flavonoid synthesis during MTI might have also negative side-effects on the pathogen defense. To clarify this issue, we deployed an Arabidopsis flavonoid mutant and obtained genetic evidence that flavonoids indeed contribute to ward off the virulent bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Finally, we show that UV-B attenuates expression of the flg22 receptor FLS2, indicating that there is negative and reciprocal interaction between this abiotic stress and the plant-pathogen defense responses.
    Diet and diet‐associated bacteria shape early microbiome development in Yellowtail Kingfish (Seriola lalandi)
    Jackson Wilkes Walburn - 2019
    Abstract
    The supply of quality juveniles via land‐based larviculture represents a major bottleneck to the growing finfish aquaculture industry. As the microbiome plays a key role in animal health, this study aimed to assess the microbial community associated with early larval development of commercially raised Yellowtail Kingfish (Seriola lalandi). We used qPCR and 16S rRNA gene amplicon sequencing to monitor changes in the microbiome associated with the development of S. lalandi from larvae to juveniles. We observed an increase in the bacterial load during larval development, which consisted of a small but abundant core microbiota including taxa belonging to the families Rhodobacteraceae, Lactobacillaceae and Vibrionaceae. The greatest change in the microbiome occurred as larvae moved from a diet of live feeds to formulated pellets, characterized by a transition from Proteobacteria to Firmicutes as the dominant phylum. A prediction of bacterial gene functions found lipid metabolism and secondary metabolite production were abundant in the early larval stages, with carbohydrate and thiamine metabolism functions increasing in abundance as the larvae age and are fed formulated diets. Together, these results suggest that diet is a major contributor to the early microbiome development of commercially raised S. lalandi.
    Therapeutic Targeting of Stat3 Using Lipopolyplex Nanoparticle-Formulated siRNA in a Syngeneic Orthotopic Mouse Glioma Model
    Benedikt Linder - 2019
    Abstract
    Glioblastoma (GBM), WHO grade IV, is the most aggressive primary brain tumor in adults. The median survival time using standard therapy is only 12–15 months with a 5-year survival rate of around 5%. Thus, new and effective treatment modalities are of significant importance. Signal transducer and activator of transcription 3 (Stat3) is a key signaling protein driving major hallmarks of cancer and represents a promising target for the development of targeted glioblastoma therapies. Here we present data showing that the therapeutic application of siRNAs, formulated in nanoscale lipopolyplexes (LPP) based on polyethylenimine (PEI) and the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), represents a promising new approach to target Stat3 in glioma. We demonstrate that the LPP-mediated delivery of siRNA mediates efficient knockdown of Stat3, suppresses Stat3 activity and limits cell growth in murine (Tu2449) and human (U87, Mz18) glioma cells in vitro. In a therapeutic setting, intracranial application of the siRNA-containing LPP leads to knockdown of STAT3 target gene expression, decreased tumor growth and significantly prolonged survival in Tu2449 glioma-bearing mice compared to negative control-treated animals. This is a proof-of-concept study introducing PEI-based lipopolyplexes as an efficient strategy for therapeutically targeting oncoproteins with otherwise limited druggability
    Infection with Toxoplasma gondii (Eucoccidiorida: Sarcocystidae) in bats of Campeche and Yucatán, Mexico
    Marco Torres-Castro - 2019
    Abstract
    Toxoplasma gondii is a protozoan parasite recognized as the causative agent of toxoplasmosis, a zoonotic disease that affects humans and domestic or wild animals. In Mexico, it represents a public and animal health problem, especially in regions with tropical and subtropical climates. Bats have been reported as accidental hosts in the transmission cycle; however, there is no preceding information in Mexico. Therefore, the aim of the present study is to report the T. gondii infection in bats captured in sites of Campeche and Yucatan states, Mexico. Bats were captured in two sites in Yucatan (X’matkuil and Panaba) and one in Campeche (Hampolol), located in the Yucatan Peninsula. Kidneys, spleen, and liver were collected and used in the total DNA extraction. Toxoplasma gondii infection was detected through the amplification of a B1 gene fragment, using nested PCR. The positive PCR products were purified and sent to sequencing for a posterior sequence identity analysis. Additionally, a phylogenetic tree was made. A total of 69 bats belonging to eight different species were processed: 41 (59.4 %, 41/69) Artibeus jamaicensis; six (8.7 %, 6/69) Pteronotus parnellii; six (8.7 %, 6/69) Noctilio leporinus; six (8.7 %, 6/69) Chiroderma villosum; four (5.8 %, 4/69) Glossophaga soricina; two (2.9 %, 2/69) Carollia sowelli; two (2.89 %, 2/69) Artibeus lituratus; and two (2.9 %, 2/69) Rhogeessa aeneus. The nested PCR identified eight (11.6 %, 8/69) infected bats: six (75 %, 6/8) A. jamaicensis, captured in X'matkuil and Panaba, one (12.5 %, 1/8) G. soricina, and one (12.5 %, 1/8) C. villosum, both captured in Panaba. The alignment analysis yielded 99-100 % for cover and 97-99 % for identity to T. gondii sequences. Our results contribute to the understanding of the T. gondii transmission cycle in the region; however, future research is needed to determine circulating genotypes, as previous studies have demonstrated that these animals might be infected with identified genotypes in other domestic or wild animals and even in humans.
    Overlapping Activities of Two Neuronal Splicing Factors Switch the GABA Effect from Excitatory to Inhibitory by Regulating REST
    Yoko Nakano - 2019
    Abstract
    A truncating mutation in the mouse Srrm4 gene, which encodes a neuronal splicing factor, causes alternative splicing defects selectively in the ear. The mechanism by which splicing is preserved in the brain of these mice is not known. Here, we show that SRRM3 limits the Srrm4 mutation-associated defects to the ear and that, in cortical neurons, overlapping SRRM3-SRRM4 activity regulates the development of interneuronal inhibition. In vitro, SRRM3 and SRRM4 regulate the same splicing events, but a mutation in mouse Srrm3 causes tremors and mild defects in neuronal alternative splicing, demonstrating unique SRRM3 roles in vivo. Mice harboring mutations in both Srrm3 and Srrm4 die neonatally and exhibit severe splicing defects. In these mice, splicing alterations prevent inactivation of the gene repressor REST, which maintains immature excitatory GABAergic neurotransmission by repressing K-Cl cotransporter 2. Thus, our data reveal that SRRM3 and SRRM4 act redundantly to regulate GABAergic neurotransmission by inactivating REST.
    The chemokine receptor CXCR2 contributes to murine adipocyte development
    Douglas P. Dyer - 2019
    Abstract
    Chemokines are members of a large family of chemotactic cytokines that signal through their receptors to mediate leukocyte recruitment during inflammation and homeostasis. The chemokine receptor CXCR2 has largely been associated with neutrophil recruitment. However, there is emerging evidence of roles for chemokines and their receptors in processes other than leukocyte migration. We have previously demonstrated that CXCR2 knockout (KO) mice have thinner skin compared to wild‐type mice. Herein we demonstrate that this is due to a thinner subcutaneous adipose layer, as a result of fewer and smaller individual adipocytes. We observe a similar phenotype in other fat depots and present data that suggests this may be due to reduced expression of adipogenesis related genes associated with adipocyte specific CXCR2 signaling. Interestingly, this phenotype is evident in female, but not male, CXCR2 KO mice. These findings expand our understanding of nonleukocyte related chemokine receptor functions and help to explain some previously observed adipose‐related phenotypes in CXCR2 KO mice.
    Effects of 50 Hz magnetic fields on circadian rhythm control in mice
    Louise Lundberg - 2019
    Abstract
    Artificial light and power frequency magnetic fields are ubiquitous in the built environment. Light is a potent zeitgeber but it is unclear whether power frequency magnetic fields can influence circadian rhythm control. To study this possibility, 8–12‐week‐old male C57BL/6J mice were exposed for 30 min starting at zeitgeber time 14 (ZT14, 2 h into the dark period of the day) to 50 Hz magnetic fields at 580 μT using a pair of Helmholtz coils and/or a blue LED light at 700 lux or neither. Our experiments revealed an acute adrenal response to blue light, in terms of increased adrenal per1 gene expression, increased serum corticosterone levels, increased time spent sleeping, and decreased locomotor activity (in all cases, P < 0.0001) compared to an unexposed control group. There appeared to be no modulating effect of the magnetic fields on the response to light, and there was also no effect of the magnetic fields alone (in both cases, P > 0.05) except for a decrease in locomotor activity (P < 0.03). Gene expression of the cryptochromes cry1 and cry2 in the adrenals, liver, and hippocampus was also not affected by exposures (in all cases, P > 0.05). In conclusion, these results suggest that 50 Hz magnetic fields do not significantly affect the acute light response to a degree that can be detected in the adrenal response. Bioelectromagnetics. 2019;9999:XX–XX. © 2019 Bioelectromagnetics Society.
    Transcriptome Response of Female Culicoides sonorensis Biting Midges (Diptera: Ceratopogonidae) to Early Infection with Epizootic Hemorrhagic Disease Virus (EHDV-2)
    Dana Nayduch - 2019
    Abstract
    Female Culicoides sonorensis biting midges are vectors of epizootic hemorrhagic disease virus (EHDV), which causes morbidity and mortality in wild and domesticated ruminants. The aims in this study were to identify key changes in female midge transcriptome profiles occurring during early infection with EHDV-2. Midges were fed either negative control bloodmeals or bloodmeals containing EHDV-2 and transcriptomes were acquired at 36 h through deep sequencing. Reads were de novo assembled into a transcriptome comprised of 18,754 unigenes. Overall, there were 2401 differentially expressed unigenes and ~60% were downregulated in response to the virus (953 up; 1448 down). Downstream Gene Ontology enrichment, KEGG pathway mapping, and manual analyses were used to identify the effect of virus ingestion at both the gene and pathway levels. Downregulated unigenes were predominantly assigned to pathways related to cell/tissue structure and integrity (actin cytoskeleton, adherens junction, focal adhesion, hippo signaling), calcium signaling, eye morphogenesis and axon guidance. Unigenes attributed to sensory functions (especially vision), behavior, learning and memory were largely downregulated. Upregulated unigenes included those coding for innate immune processes, olfaction and photoreceptor pigments. Our results suggest that midges respond to virus infection as soon as 36 h post-ingestion, and that EHDV-2 may have a significant phenotypic effect on sensory and neural tissues.
    S-adenosyl methionine prevents ASD like behaviors triggered by earlypostnatal valproic acid exposure in very young mice
    asher Ornoy - 2019
    Abstract
    Introduction:A common animal model of ASD is the one induced by valproic acid (VPA), inducing epigeneticchanges and oxidative stress. We studied the possible preventive effect of the methyl donor for epigenetic en-zymatic reactions, S-adenosine methionine (SAM), on ASD like behavioral changes and on redox potential in thebrain and liver in this model.Methods:ICR albino mice were injected on postnatal day 4 with one dose of 300 mg/kg of VPA, with normalsaline (controls) or with VPA and SAM that was given orally for 3 days at the dose of 30 mg/kg body weight.From day 50, we carried out neurobehavioral tests and assessment of the antioxidant status of the prefrontalcerebral cortex, liver assessing SOD and CAT activity, lipid peroxidation and the expression of antioxidant genes.Results:Mice injected with VPA exhibited neurobehavioral deficits typical of ASD that were more prominent inmales. Changes in the activity of SOD and CAT increased lipid peroxidation and changes in the expression ofantioxidant genes were observed in the prefrontal cortex of VPA treated mice, more prominent in females, whileASD like behavior was more prominent in males. There were no changes in the redox potential of the liver. Theco-administration of VPA and SAM alleviated most ASD like neurobehavioral symptoms and normalized theredox potential in the prefrontal cortex.Conclusions:Early postnatal VPA administration induces ASD like behavior that is more severe in males, whilethe redox status changes are more severe in females; SAM corrects both. VPA-induced ASD seems to result fromepigenetic changes, while the redox status changes may be secondary.
    Human-based fibrillar nanocomposite hydrogels as bioinstructive matrices to tune stem cell behavior
    Bárbara B. Mendes - 2018
    Abstract
    The extracellular matrix (ECM)-biomimetic fibrillar structure of platelet lysate (PL) gels along with their enriched milieu of biomolecules has drawn significant interest in regenerative medicine applications. However, PL-based gels have poor structural stability, which severely limits their performance as a bioinstructive biomaterial. Here, rod-shaped cellulose nanocrystals (CNC) are used as a novel approach to modulate the physical and biochemical microenvironment of PL gels enabling their effective use as injectable human-based cell scaffolds with a level of biomimicry that is difficult to recreate with synthetic biomaterials. The incorporation of CNC (0 to 0.61 wt%) into the PL fibrillar network during the coagulation cascade leads to decreased fiber branching, increased interfiber porosity (from 66 to 83%) and modulates fiber (from 1.4 ± 0.7 to 27 ± 12 kPa) and bulk hydrogel (from 18 ± 4 to 1256 ± 82 Pa) mechanical properties. As a result of these physicochemical alterations, nanocomposite PL hydrogels resist the typical extensive clot retraction (from 76 ± 1 to 24 ± 3 at day 7) and show favored retention of PL bioactive molecules. The feedback of these cues on the fate of human adipose-derived stem cells is evaluated, showing how it can be explored to modulate the commitment of encapsulated stem cells toward different genetic phenotypes without the need for additional external biological stimuli. These fibrillar nanocomposite hydrogels allow therefore the exploration of the outstanding biological properties of human-based PL as an efficient engineered ECM which can be tailored to trigger specific regenerative pathways in minimal invasive strategies.
    Maternal malnourishment induced upregulation of fetuin-B blunts nephrogenesis in the low birth weight neonate
    May M. Rabadi - 2018
    Abstract
    Maternal undernutrition during pregnancy (MUN) often leads to low birth weight (LBW) neonates that have a reduced total nephron endowment, leaving these neonates susceptible to kidney disease throughout their lives. For reasons unknown, these LBW neonates have impaired kidney development due to a severe reduction in renal SIX2+ stem cells during nephrogenesis. Using a mouse model of MUN, we investigated SIX2+ stem cell reduction in the LBW neonate. Significant upregulation of the protein fetuin-B (measured by PCR and immunoblotting) in the MUN mother's placenta, organs and circulation yielded a 3-fold increase of this protein in the embryonic kidney. Recombinant fetuin-B, administered to healthy pregnant mothers at the concentration equivalent to that in the MUN mother, crossed the placenta and reduced both SIX2+ stem cells by 50% and nephron formation by 66% in embryonic kidneys (measured by immunofluorescence and the physical dissector/fractionator stereological method). Administration of fetuin-B to kidney explants yielded similar reductions in renal SIX2+ stem cells and nephron formation. Fetuin-B treatment of isolated embryonic renal SIX2+ stem cell primary cultures 1) increased NF-kB activity and apoptosis, 2) reduced cell proliferation due to upregulated p21 nuclear activity and subsequent cell cycle arrest, and 3) enhanced generation of reactive oxygen species (measured by fluorescence microscopy). In conclusion, MUN increases fetuin-B in the developing embryonic kidney. The increase in fetuin-B blunts nephrogenesis by reducing SIX2+ stem cells by promoting their apoptosis (via NF-kB upregulation), blunting their proliferative renewal (via p21 upregulation) and enhancing oxidative stress.
    Polyunsaturated Fatty Acids Induce ROS Synthesis in Microvascular Endothelial Cells
    Simon Trommer - 2018
    Abstract
    In sepsis, endothelial dysfunction is a crucial driver known to limit the survival rate of affected patients. For this, ROS-mediated signaling plays an important role in endothelial communication and functionality. In the management of sepsis, polyunsaturated fatty acids (PUFA) have received increasing attention regarding their anti-inflammatory potential neglecting the oxidative properties of these substances. Therefore, in the present study we examined the capacity of PUFA to interfere with the expression of major ROS-producing enzymes, as well as endothelial ROS production itself. The human microvascular endothelial cells TIME (ATCC number: CRL-4025) were used. Cells were cultured in medium enriched with LNA (C18:3n3), EPA (C20:5n3), DHA (C22:6n3), LA (C18:2n6), or AA (C20:4n6) in concentrations of 15 μM totaling 144 h. Stimulation of cells was performed in the last 24 h of fatty acid supplementation by addition of the cytokines TNF-α + IL-1β + IFN-γ (5 ng/ml each). Gene expression of eNOS, COX-2, and NOX-4 was evaluated by qPCR. ROS synthesis was analyzed by means of a flow cytometry-based rhodamine 123 assay. Cytokine stimulation was found to differentially affect gene expression of major ROS synthesizing enzymes: eNOS was decreased whereas COX-2 and NOX-4 were increased. As a consequence, cytokine stimulation had no effect on rhodamine accumulation in endothelial cells. PUFA supplementation alone did not affect the gene expression of eNOS, COX-2, and NOX-4. Nevertheless, an increasing action of PUFA on the stimulation-induced reduction in eNOS expression was found. More importantly, the number of rhodamine positive endothelial cells almost doubled following enrichment with the PUFA EPA, DHA or AA. This effect was independent of the stimulation status of the cells but seemed to be related to the number of double bonds of a supplemented fatty acid. Our data warrant further studies to ensure that increased endothelial cell oxidative stress is not boosted by PUFA in septic patients.
    Detection of coliphages and human adenoviruses in a subtropical estuarine lake
    Emily M. Cooksey - 2018
    Abstract
    Fecal indicator bacteria (FIB) have been used to assess fecal contamination in recreational water. However, enteric viruses have been shown to be more persistent in the environment and resistant to wastewater treatment than bacteria. Recently, U.S Environmental Protection Agency has proposed the use of coliphages as viral indicators to better protect against viral waterborne outbreaks. This study aimed to detect and determine correlation between coliphages (F-specific and somatic), fecal indicator bacteria (enterococci and fecal coliforms), and human enteric viruses (human adenovirus) in a subtropical brackish estuarine lake. Water samples were collected from 9 estuarine recreation sites on Lake Pontchartrain in southeast Louisiana. Water samples (n = 222, collected weekly) were analyzed for coliphages and fecal indicator bacteria using culture-based methods and large volume water samples (n = 54, collected monthly) were analyzed for human adenovirus using quantitative PCR. Somatic coliphage and F-specific coliphage were found in 93.7 and 65.2% of samples with geometric mean concentrations of 30 and 3 plaque forming units (PFU) per 100 mL, respectively. Enterococci, fecal coliforms, and adenovirus were found in all samples with geometric mean concentrations of 27 most probable number (MPN), 77 MPN, and 3.0 × 104 gene copies per 100 mL, respectively. Watersheds in suburban areas exhibited significantly higher concentrations of coliphages and fecal indicator bacteria, indicating potential fecal contamination from septic systems. There was no significant correlation (p > 0.05) observed between the presence of adenoviruses and fecal indicator bacteria and coliphages. The presence of human adenovirus in Lake Pontchartrain poses a significant public health problem for both recreational use and seafood harvesting as it increases exposure risks. This study demonstrated the lack of relationship between fecal indicators and human viral pathogen in Lake Pontchartrain supporting an alternative microbial surveillance system such as direct pathogen detection.
    Restoring Endothelial Function by Targeting Desert Hedgehog Downstream of Klf2 Improves Critical Limb Ischemia in Adults
    Caroline Caradu - 2018
    Abstract
    Rationale: Klf2 is critical to establish and maintain endothelial integrity. Objective:Therefore, determining upstream and downstream mediators of Klf2 would lead to alternative therapeutic targets in cardiovascular disease management. Methods and Results: Here we identify Desert Hedgehog (Dhh) as a downstream effector of Klf2, whose expression in endothelial cells (ECs) is upregulated by shear stress and decreased by inflammatory cytokines. Consequently, we show that Dhh knock down in ECs promotes endothelial permeability and EC activation and that Dhh agonist prevents TNFα or glucose-induced EC dysfunction. Moreover, we demonstrate that human critical limb ischemia (CLI), a pathological condition linked to diabetes and inflammation, is associated to major EC dysfunction. By recreating a complex model of CLI in diabetic mice, we found that Dhh-signaling agonist significantly improved EC function without promoting angiogenesis, which subsequently improved muscle perfusion. Conclusions: Restoring EC functi...
    AAV8-mediated overexpression of mPCSK9 in liver differs between male and female mice
    Lemlem Brook - 2018
    Abstract
    Glioblastoma (GBM) is the most common malignant brain tumor and is associated with a poor prognosis, with most patients living less than a year after diagnosis. Given that GBM nearly always recurs after conventional treatments, there is an urgent need to identify novel molecular targets. Hairless (HR) is a nuclear factor enriched in the skin and has been previously implicated in hair cycling. HR is also highly expressed in the brain, but its significance is unknown. We found that human hairless gene (HR) expression is significantly decreased in all GBM subtypes compared with normal brain tissue and is predictive of prognosis, which suggests that loss of HR expression can contribute to GBM pathogenesis. HR was recently discovered to bind to and regulate p53 responsive elements, and thus we hypothesized that HR may have a tumor suppressive function in GBM by modulating p53 target gene expression. We found that HR indeed regulates p53 target genes, including those implicated in cell cycle progression and apoptosis in the GBM-derived U87 cell line, and restoring HR expression triggered G2/M arrest and apoptosis. An analysis of sequenced genomes from patients with GBM revealed 10 HR somatic mutations in patients with glioma, two of which are located in the histone demethylase domain of HR. These two mutations, P996S and K1004N, were reconstructed and found to have impaired p53 transactivating properties. Collectively, the results of our study suggest that HR has tumor suppressive functions in GBM, which may be clinically relevant and a potential avenue for therapeutic intervention.
    AAV8-mediated overexpression of mPCSK9 in liver differs between male and female mice
    Aimee E. Vozenilek - 2018
    Abstract
    Abstrat Background and aims The recombinant adeno-associated viral vector serotype 8 expressing the gain-of-function mutation of mouse proprotein convertase subtilisin/kexin type 9 (AAV8- PCSK9) is a new model for the induction of hypercholesterolemia. AAV8 preferentially infects hepatocytes and the incorporated liver-specific promoter should ensure expression of PCSK9 in the liver. Since tissue distribution of AAVs can differ between male and female mice, we investigated the differences in PCSK9 expression and hypercholesterolemia development between male and female mice using the AAV8-PCSK9 model. Methods Male and female C57BL/6 mice were injected with either a low-dose or high-dose of AAV8-PCSK9 and fed a high-fat diet. Plasma lipid levels were evaluated as a measure of the induction of hypercholesterolemia. Results Injection of mice with low dose AAV8-PCSK9 dramatically elevated both serum PCSK9 and cholesterol levels in male but not female mice. Increasing the dose of AAV8-PCSK9 threefold in female mice rescued the hypercholesterolemia phenotype but did not result in full restoration of AAV8-PCSK9 transduction of livers in female mice compared to the low-dose male mice. Our data demonstrate female mice respond differently to AAV8-PCSK9 injection compared to male mice. Conclusions These differences do not hinder the use of female mice when AAV8-PCSK9 doses are taken into consideration. However, localization to and production of AAV8-PCSK9 in organs besides the liver in mice may introduce confounding factors into studies and should be considered during experimental design.
    Genetic diversity of Hepatitis C Virus in Pakistan using Next Generation Sequencing
    Sana Saleem - 2018
    Abstract
    Background In Pakistan, HCV disease is considered a major public health issue with about 10–17 million people suffering with this infection and rate is increasing every day without any hindrance. The currently available Pyrosequencing approach used to analyze complex viral genomes as it can determine minor variants. It is crucial to understand viral evolution and quasispecies diversity in complex viral strains. Objectives To assess genetic diversity in patients with HCV using Next Generation Sequencing (NGS) and compare nucleotide diversity of genotype 3a with respect to other genotypes. Study design Intra-host viral diversity of HCV was determined using NGS from 13 chronically HCV infected individuals. NGS of three different regions (E2 (HVR1), NS3 and NS5B) of HCV-3a allowed for a comprehensive analysis of the viral population. Result Phylogenetic analysis of different HCV genes revealed great variability within the Pakistani population. The average nucleotide diversity for HVR1, NS3 and NS5B was 0.029, 0.011 and 0.010 respectively. Conclusion Our findings clearly indicate that patient-2 greater quasispecies heterogeneity than other patients of same genotype-3a using phylogenetic and one step network analyses. Initially phylogenetic analysis of these three genes showed that genotype 3a samples have greater genetic diversity. However, no significant difference was determined when nucleotide variability of genotype 3a compared with other genotypes (1a, 1b, 2a & 4a).
    Sperm capacitation is associated with phosphorylation of the testis-specific radial spoke protein Rsph6a
    Bidur Paudel - 2018
    Abstract
    A sequence corresponding to the N-terminal domain of the radial spoke protein Rsph6 was found phosphorylated in capacitated sperm. Rsph6 expression is a testis-
    Localization of the 1,25-dihydroxyvitamin D-mediated response in the intestines of mice
    Carmen J. Reynolds - 2018
    Abstract
    1,25-Dihydroxyvitamin D3 (1,25(OH)2D) elicits a transcriptional response in the intestines. Assessments of this response are often derived from crude tissue homogenates and eliminate the ability to discriminate among different cell types. Here, we used an RNA in situ hybridization assay, RNAScope (Advanced Cell Diagnostics, Newark, CA), to identify the cells in the intestine that respond to 1,25(OH)2D with expression of cytochrome P450 family 24 subfamily A member 1 (Cyp24a1) mRNA. Mice were gavaged with a single bolus dose of 1,25(OH)2D to target the duodenum or a glucuronic acid conjugate of 1,25(OH)2D, β-G-1,25(OH)2D, to target the colon. QRT-PCR analysis of Cyp24a1 mRNA verified that the 1,25(OH)2D-induced responses were present. RNAScope revealed that the mRNA response present after six hours is limited to mature enterocytes exposed to the intestinal lumen in both the duodenum and colon. No detectable expression was observed in goblet cells, lamina propria, muscularis mucosa muscle, submucosa and submucosal lymphoid follicles, or tunica muscularis. Our findings have identified epithelial enterocytes to be the intestinal targets for 1,25(OH)2D in both the duodenum and colon.
    Loss-of-function mutations in CARD14 are associated with a severe variant of atopic dermatitis
    Alon Peled - 2018
    Abstract
    Background Atopic dermatitis (AD) is a highly prevalent chronic inflammatory skin disease which is known to be, at least in part, genetically determined. Mutations in CARD14 have been shown to result in various forms of psoriasis and related disorders. Objective We aimed to identify rare DNA variants conferring a significant risk for AD through genetic and functional studies in a cohort of patients affected with severe atopic dermatitis. Methods Whole exome and direct gene sequencing, immunohistochemistry, real-time PCR, ELISA and functional assays in human keratinocytes were used. Results In a cohort of individuals referred with severe atopic dermatitis, DNA sequencing revealed in 4 patients two rare heterozygous missense mutations in CARD14 encoding the Caspase Recruitment Domain-Containing Protein 14, a major regulator of NF-κB. A dual luciferase reporter assay demonstrated that both mutations exert a dominant loss-of-function effect and result in decreased NF-κB signaling. Accordingly, immunohistochemistry staining showed decreased expression of CARD14 in patient skin as well as decreased levels of activated p65, a surrogate marker for NF-κB activity. CARD14-deficient or mutant-expressing keratinocytes displayed abnormal secretion of key mediators of innate immunity. Conclusions While dominant gain-of-function mutations in CARD14 are associated with psoriasis and related diseases, loss-of-function mutations in the same gene are associated with a severe variant of atopic dermatitis.
    Tissue-specific gene regulation corresponds with seasonal plasticity in female testosterone
    Alexandra B. Bentz - 2018
    Abstract
    Testosterone (T) is a sex steroid hormone that often varies seasonally and mediates trade-offs between territorial aggression and parental care. Prior work has provided key insights into the ‘top-down’ hypothalamic control of this seasonal plasticity in T, yet mechanisms acting outside of the brain may also influence circulating T levels. We hypothesized that peripheral mechanisms may be especially critical for females, because peripheral regulation may mitigate the costs of systemically elevated T. Here, we begin to test this hypothesis using a seasonal comparative approach, measuring gene expression in peripheral tissues in tree swallows (Tachycineta bicolor), a songbird with intense female-female competition and T-mediated aggression. We focused on the gonad and liver for their role in T production and metabolism, respectively, and we contrasted females captured during territory establishment versus incubation. During territory establishment, when T levels are highest, we found elevated gene expression of the hepatic steroid metabolizing enzyme CYP2C19 along with several ovarian steroidogenic enzymes, including the androgenic 5α-reductase. Despite these seasonal changes in gene expression along the steroidogenic pathway, we did not observe seasonal changes in sensitivity to upstream signals, measured as ovarian mRNA abundance of luteinizing hormone receptor. Together, these data suggest that differential regulation of steroidogenic gene expression in the ovary is a potentially major contributor to seasonal changes in T levels in females. Furthermore, these data provide a unique and organismal glimpse into tissue-specific gene regulation and its potential role in hormonal plasticity in females.
    Potent in vivo lung cancer Wnt signaling inhibition via cyclodextrin-LGK974 inclusion complexes
    Pedro P. G. Guimaraes - 2018
    Abstract
    Activation of the Wnt signaling pathway promotes lung cancer progression and contributes to poor patient prognosis. The porcupine inhibitor LGK974, a novel orally bioavailable cancer therapeutic in Phase I clinical trials, induces potent Wnt inhibition leading to suppressed growth and progression of multiple types of cancers. The clinical use of LGK974, however, is limited in part due to its low solubility and high toxicity in tissues that rely on Wnt signaling for normal homeostasis. Here, we report the use of host-guest chemistry to enhance solubility and bioavailability of LGK974 in mice through complexation with cyclodextrins (CD). We assessed the effects of these complexes to inhibit Wnt signaling in lung adenocarcinomas that are typically driven by overactive Wnt signaling. 2D H1 NMR confirmed host-guest complexation of CDs with LGK974. CD:LGK974 complexes significantly decreased the expression of Wnt target genes both in vitro and in vivo. Further, CD:LGK974 complexes increased the bioavailability upon oral administration in mice compared to free LGK974. In a mouse lung cancer allograft model, CD:LGK974 complexes induced potent Wnt signaling inhibition with reduced intestinal toxicity compared to administration of free drug. Collectively, the development of these complexes enables safer and repeated oral or parenteral administration of porcupine inhibitors, which hold promise for the treatment of multiple types of malignancies.
    Alterations of EDEM1 functions enhance ATF6 pro-survival signaling
    Alexandra Papaioannou - 2018
    Abstract
    Activating transcription factor 6 alpha (referred to as ATF6 hereafter) is an endoplasmic reticulum (ER)-resident glycoprotein and one of the 3 sensors of the unfolded protein response (UPR). Upon ER stress, ATF6 is exported to the Golgi complex where it is cleaved by the S1P and S2P proteases thus releasing ATF6 cytosolic fragment and leading to the transcription of ATF6 target genes. In this study, we performed a phenotypic small interfering RNA (siRNA) screening to better characterize the ER mechanisms involved in ATF6 activation upon ER stress. This revealed that silencing of ER-degradation enhancing alpha-mannosidase-like protein-1 (EDEM1) increased the bioavailability of ER stress-induced ATF6 export to the Golgi complex through the stabilization of the natively unstable ATF6 protein. Moreover, we characterized a somatic variant of EDEM1 (N198I) found in hepatocellular carcinoma that alters ATF6 signaling and might provide a selective advantage to the transforming cells. Hence, our work confirms the natively unstable nature of ATF6 and links this property to potentially associated pro-oncogenic functions. This article is protected by copyright. All rights reserved.
    Expression of a hyperthermophilic endoglucanase in hybrid poplar modifies the plant cell wall and enhances digestibility
    Yao Xiao - 2018
    Abstract
    Expression of glycosyl hydrolases in lignocellulosic biomass has been proposed as an alternative to improve efficiency of cellulosic ethanol production. In planta production of hyperthermophilic hydrolytic enzymes could prevent the detrimental effects often seen resulting from the expression of recombinant mesophilic enzymes to plant hosts. Utilizing lignocellulosic feedstocks to produce hyperthermophilic hydrolases provides additional benefits for ethanol production in the way of transgenic feedstocks serving as both enzyme providers and cellulosic substrates.
    Endocrine-immune signaling as a predictor of survival: A prospective study in developing songbird chicks
    Emily E. Virgin - 2018
    Abstract
    Immune function varies with an animal’s endocrine physiology and energy reserves, as well as its abiotic and biotic environment. This context-dependency is thought to relate to adaptive trade-off resolution that varies from one context to the next; however, it is less clear how state- and environmentally-dependent differences in endocrine-immune signaling relate to survival in natural populations. We begin to address this question in a prospective study on a free-living passerine bird, the tree swallow (Tachycineta bicolor), by capitalizing upon naturally-occurring variation in ectoparasitism in 12-day old chicks. We measured body mass, hematological gene expression of the pro-inflammatory cytokine interleukin-6 (IL-6) as well as corticosterone (CORT) secretion at baseline and in response to 30 min of handling. We found that chicks with ectoparasites had smaller body mass and higher levels of IL-6 gene expression at this critical stage of post-natal growth and development. Mass and IL-6 were positively correlated, but only among parasitized chicks, suggesting that larger chicks mount stronger immune responses when necessary, i.e. in the presence of ectoparasites that are known to induce inflammation. IL-6 mRNA expression was negatively correlated with stress-induced CORT levels, suggesting that this proxy of inflammation may be co-regulated with or coordinated by glucocorticoids. More importantly, these endocrine-immune parameters predicted survival to fledging, which was positively associated with IL-6 mRNA abundance and, to a lesser degree, CORT reactivity. These results suggest a link between endocrine-immune interactions and performance in nature, and as a consequence, they shed light on the potentially adaptive, context-dependent interplay between body mass, immunity, and endocrine physiology during development.
    time-ChIP: A Method to Determine Long-Term Locus-Specific Nucleosome Inheritance
    Wojciech Siwek - 2018
    Abstract
    Understanding chromatin dynamics is essential to define the contribution of chromatin to heritable gene silencing and the long-term maintenance of gene expression. Here we present a detailed protocol for time-ChIP, a novel method to measure histone turnover at high resolution across long timescales. This method is based on the SNAP-tag, a self-labeling enzyme that can be pulse labeled with small molecules in cells. Upon pulse biotinylation of a cohort of SNAP-tagged histones we can determine their abundance and fate across a chase period using a biotin-specific chromatin pulldown followed by DNA sequencing or quantitative PCR. This method is unique in its ability to trace the long-term fate of a chromatin bound histone pool, genome wide. In addition to a step by step protocol, we outline advantages and limitations of the method in relation to other existing techniques. time-ChIP can define regions of high and low histone turnover and identify the location of pools of long lived histones.
    Exploiting the impact of the secretome of MSCs isolated from different tissue sources on neuronal differentiation and axonal growth
    Rita C. Assunção-Silva - 2018
    Abstract
    Cell transplantation free-based therapies using Mesenchymal stem cell (MSC) secretome have recently been presented as a possible for CNS related disorders. MSC secretome is rich in several bio-factors that act synergically towards the repair of damaged tissues, thus making it an ideal candidate for regenerative applications. Great effort is currently being made to map the molecules that compose the MSC secretome. Previous proteomic characterization of the secretome (in the form of conditioned media - CM) of MSCs derived from adipose tissue (ASC), bone-marrow (BMSC) and umbilical cord (HUCPVC) was performed by our group, where proteins relevant for neuroprotection, neurogenic, neurodifferentiation, axon guidance and growth functions were identified. Moreover, we have found significant differences among the expression of several molecules, which may indicate that their therapeutic outcome might be distinct. Having this in mind, in the present study, the neuroregulatory potential of ASC, BMSC and HUCPVC CM in promoting neurodifferentiation and axonal outgrowth was tested in vitro, using human telencephalon neuroprogenitor cells and dorsal root ganglion explants, respectively. The CM from the three MSC populations induced neuronal differentiation from human neural progenitor cells, as well as neurite outgrowth from dorsal root ganglion explants. Moreover, all the MSC populations promoted the same extent of neurodifferentiation, while ASC CM demonstrated higher potential in promoting axonal growth.
    Bifidobacterium pseudolongum in the Ceca of Rats Fed Hi-Maize Starch Has Characteristics of a Keystone Species in Bifidobacterial Blooms
    Manuela Centanni - 2018
    Abstract
    Starches resistant to mammalian digestion are present in foods and pass to the large bowel, where they may be degraded and fermented by the microbiota. Increases in relative abundances of bifidobacteria (blooms) have been reported in rats whose diet was supplemented with Hi-Maize resistant starch. We determined that the bifidobacterial species present in the rat cecum under these circumstances mostly belonged to Bifidobacterium animalis. However, cultures of B. animalis isolated from the rats failed to degrade Hi-Maize starch to any extent. In contrast, Bifidobacterium pseudolongum also detected in the rat microbiota had high starch-degrading ability. Transcriptional comparisons showed increased expression of a type 1 pullulanase, alpha-amylase, and glycogen debranching enzyme by B. pseudolongum when cultured in medium containing Hi-Maize starch. Maltose was released into the culture medium, and B. animalis cultures had shorter doubling times in maltose medium than did B. pseudolongum. Thus, B. pseudolongum, which was present at a consistently low abundance in the microbiota, but which has extensive enzymatic capacity to degrade resistant starch, showed the attributes of a keystone species associated with the bifidobacterial bloom. IMPORTANCE This study addresses the microbiology and function of a natural ecosystem (the rat gut) using DNA-based observations and in vitro experimentation. The microbial community of the large bowel of animals, including humans, has been studied extensively through the use of high-throughput DNA sequencing methods and advanced bioinformatics analysis. These studies reveal the compositions and genetic capacities of microbiotas but not the intricacies of how microbial communities function. Our work, combining DNA sequence analysis and laboratory experiments with cultured strains of bacteria, revealed that the increased abundance of bifidobacteria in the rat gut, induced by feeding indigestible starch, involved a species that cannot itself degrade the starch (Bifidobacterium animalis) but cohabits with a species that can (Bifidobacterium pseudolongum). B. pseudolongum has the characteristics of a keystone species in the community because it had low abundance but high ability to perform a critical function, the hydrolysis of resistant starch.
    Increased Alternative Splicing as a Host Response to Edwardsiella ictaluri Infection in Catfish
    Suxu Tan - 2018
    Abstract
    Alternative splicing is the process of generating multiple transcripts from a single pre-mRNA used by eukaryotes to regulate gene expression and increase proteomic complexity. Although alternative splicing profiles have been well studied in mammalian species, they have not been well studied in aquatic species, especially after biotic stresses. In the present study, genomic information and RNA-Seq datasets were utilized to characterize alternative splicing profiles and their induced changes after bacterial infection with Edwardsiella ictaluri in channel catfish (Ictalurus punctatus). A total of 27,476 alternative splicing events, derived from 9694 genes, were identified in channel catfish. Exon skipping was the most abundant while mutually exclusive exon was the least abundant type of alternative splicing. Alternative splicing was greatly induced by E. ictaluri infection with 21.9% increase in alternative splicing events. Interestingly, genes involved in RNA binding and RNA splicing themselves were significantly enriched in differentially alternatively spliced genes after infection. Sequence analyses of splice variants of a representative alternatively spliced gene, splicing factor srsf2, revealed that certain spliced transcripts may undergo nonsense-mediated decay (NMD), suggesting functional significance of the induced alternative splicing. Although statistical analysis was not possible with such large datasets, results from quantitative real-time PCR from representative differential alternative splicing events provided general validation of the bacterial infection-induced alternative splicing. This is the first comprehensive study of alternative splicing and its changes in response to bacterial infection in fish species, providing insights into the molecular mechanisms of host responses to biotic stresses.
    Segregation of dopamine and glutamate release sites in dopamine neuron axons: regulation by striatal target cells
    Guillaume M. Fortin - 2018
    Abstract
    Dopamine (DA) is a key regulator of circuits controlling movement and motivation. A subset of midbrain DA neurons has been shown to express the vesicular glutamate transporter (VGLUT)2, underlying their capacity for glutamate release. Glutamate release is found mainly by DA neurons of the ventral tegmental area (VTA) and can be detected at terminals contacting ventral, but not dorsal, striatal neurons, suggesting the possibility that target-derived signals regulate the neurotransmitter phenotype of DA neurons. Whether glutamate can be released from the same terminals that release DA or from a special subset of axon terminals is unclear. Here, we provide in vitro and in vivo data supporting the hypothesis that DA and glutamate-releasing terminals in mice are mostly segregated and that striatal neurons regulate the cophenotype of midbrain DA neurons and the segregation of release sites. Our work unveils a fundamental feature of dual neurotransmission and plasticity of the DA system.—Fortin, G. M., Ducrot, C., Giguère, N., Kouwenhoven, W. M., Bourque, M.-J., Pacelli, C., Varaschin, R. K., Brill, M., Singh, S., Wiseman, P. W., Trudeau, L.-E. Segregation of dopamine and glutamate release sites in dopamine neuron axons: regulation by striatal target cells.
    Differentiation of the granulosa layer from hen prehierarchal follicles associated with follicle stimulating hormone receptor signaling
    Dongwon Kim - 2018
    Abstract
    Recruitment of a single follicle into the preovulatory hierarchy of the domestic hen ovary occurs from a small cohort of prehierarchal follicles measuring 6-8 mm in diameter. We have previously reported that granulosa cells (GC) collected from prehierarchal follicles express highest levels of membrane-localized follicle-stimulating hormone receptor (FSHR) during follicle development, yet fail to initiate signaling via cAMP following short-term incubation with FSH. Consequently, GC from prehierarchal follicles remain in an undifferentiated state and lack the capacity for steroidogenesis due to a deficiency of cAMP-dependent STAR protein and CYP11A1 gene expression. The present studies investigate FSH responsiveness in GC before and after the transition from undifferentiated to a differentiated state at follicle recruitment. Prior to recruitment focus is directed towards the inhibition of FSHR signaling by β-ARRESTIN (βARR). Specifically, knockdown of βARR mRNA in cultured, undifferentiated GC using small interfering RNA (siRNA) facilitated FSH-induced cAMP formation, STAR expression and progesterone production. Furthermore, over-expression of bovine βARR1 and G PROTEIN-COUPLED RECEPTOR KINASE2 in actively differentiating GC significantly decreased cAMP accumulation and progesterone production following a challenge with FSH. We propose that a βARR-mediated mechanism maintains FSHR unresponsiveness in undifferentiated GC from prehierarchal follicles, and as a result prevents GC differentiation until the time of follicle recruitment. This article is protected by copyright. All rights reserved.
    Thymoquinone inhibits cell proliferation, migration, and invasion by regulating the elongation factor 2 kinase (eEF-2K) signaling axis in triple-negative breast cancer
    Nashwa Kabil - 2018
    Abstract
    Background/purposeTriple-negative breast cancer (TNBC) is the most aggressive and chemoresistant subtype of breast cancer. Therefore, new molecular targets and treatments need to be developed to improve poor patient prognosis and survival. We have previously shown that eukaryotic elongation factor 2 kinase (eEF-2K) is highly expressed in TNBC cells, is associated with poor patient survival and prognosis, and promotes cell proliferation, migration, and invasion. In vivo targeting of eEF-2K significantly reduces the tumor growth of orthotopic TNBC xenograft mouse models, suggesting that eEF-2K may serve as a potential novel therapeutic target.Methods/resultsIn the current study, we identified thymoquinone (TQ), an active ingredient of Nigella sativa, as a potential safe and effective eEF-2K inhibitor in TNBC. We demonstrated for the first time that TQ inhibits the protein and mRNA expression of eEF-2K, as well as the clinically relevant downstream targets, including Src/FAK and Akt, and induces the tumor suppressor miR-603, in response to NF-kB inhibition. This effect was associated with a significant decrease in the proliferation, colony formation, migration, and invasion of TNBC cells. Furthermore, systemic in vivo injection of TQ (20 and 100 mg/kg) significantly reduced the growth of MDA-MB-231 tumors and inhibited the eEF-2K expression in an orthotopic tumor model in mice.ConclusionOur study provides first evidence that TQ treatment inhibits cell proliferation, migration/invasion, and tumor growth, in part through the inhibition of eEF-2K signaling in TNBC. Thus, our findings suggest that systemic TQ treatment may be used as a targeted therapeutic strategy for the inhibition of eEF-2K in TNBC tumor growth and progression.
    Exercise Preconditioning Diminishes Skeletal Muscle Atrophy after Hindlimb Suspension in Mice*
    Nicholas T. Theilen - 2018
    Abstract
    The aim of the present study was to investigate if short-term, concurrent exercise training prior to hindlimb suspension (HLS) prevents or diminishes both soleus and gastrocnemius atrophy and to analyze if changes in mitochondrial molecular markers were associated. Male C57BL/6 (WT) mice (12-14 weeks old) were assigned to control, 7-day HLS (HLS), 2-weeks exercise training prior to 7-day HLS (Ex+HLS), and 2-weeks exercise training (Ex) groups. HLS resulted in a 27.1% and 21.5% decrease in soleus and gastrocnemius muscle weight to bodyweight ratio, respectively, in WT mice. Exercise training prior to HLS resulted in a 5.6% and 8.1% decrease in soleus and gastrocnemius weight to bodyweight ratio, respectively. Exercise increased mitochondrial biogenesis and function associated markers, slow myosin heavy chain (SMHC) expression, and reduced the fiber-type transitioning marker myosin heavy chain 4 (Myh4). Ex+HLS revealed decreased reactive oxygen species (ROS) and oxidative stress compared to HLS. Our data indicated the time prior to an atrophic setting, particularly caused by muscle unloading, may be a useful period to intervene short-term, progressive exercise training to prevent skeletal muscle atrophy and is associated with mitochondrial biogenesis, function, and redox balance.
    In Silico Analyses of Rice Thionin genes and the Antimicrobial Activity of OsTHION15 against Phytopathogens
    Krissana Boonpa - 2018
    Abstract
    Thionins are a family of antimicrobial peptides. We performed in silico expression analyses of the 44 rice (Oryza sativa L.) thionins (OsTHIONs). Modulated expression levels of OsTHIONs under different treatments suggest their involvement in many processes, including biotic, abiotic and nutritional stress responses, and in hormone signaling. OsTHION15 (LOC_Os06g32600) was selected for further characterization based on several in silico analyses. OsTHION15 in O. sativa L. ssp. indica ‘KDML 105’ was expressed in all of the tissues/organs examined, including germinating seeds, leaves and roots of seedlings and mature plants, and inflorescences. To investigate the antimicrobial activity of OsTHION15, we produced a recombinant peptide in Escherichia coli Rosetta-gami (DE3). The recombinant OsTHION15 exhibited inhibitory activities toward rice pathogenic bacteria, such as Xanthomonas oryzae pv. oryzae and Pectobacterium carotovorum pv. atroseptica, with minimum inhibitory concentrations of 112.6 and 14.1 µg ml-1, respectively. A significant hyphal growth inhibition was also observed towards Fusarium oxysporum ssp. cubense and Helminthosporium oryzae. In addition, we demonstrated the in planta antibacterial activity of this peptide in Nicotiana benthamiana against Xanthomonas campestris pv. glycines. These activities suggest the possible application of OsTHION15 in plant disease control.
    Pax-5 Inhibits NF-κB Activity in Breast Cancer Cells Through IKKε and miRNA-155 Effectors
    Jason Harquail - 2018
    Abstract
    Pax-5, an essential transcription factor in B cell development, is aberrantly expressed in various B cell cancer lesions and solid tumors such as breast carcinoma. We have recently shown that Pax-5 regulates NF-κB activity which lead to the modulation of breast cancer phenotypic features (EMT-MET). NF-κB is known as a central mediator in inflammation, stress response as well as being a gatekeeper of pro-tumorigenic activity. However, little is known as to how Pax-5 affects this modulation. We thus turned our attention to microRNAs as potential regulatory effectors. In this study, we set out to elucidate the regulatory network between differential Pax-5 expression and NF-κB activity which dictate breast cancer malignancy. Through next-generation sequencing (NGS) of breast cancer cells conditionally expressing Pax-5, we profile significantly upregulated microRNAs; including microRNA-155, a known regulator of pathological processes and suppressor of malignant growth. Through the conditional expression of microRNA-155 in breast cancer models, we identify and validate IKKε (IKBKE) as a downstream target and an essential effector of Pax-5-mediated suppression of NF-κB signaling. Using rescue experiments, we also confirm that Pax-5 modulates NF-κB activity via IKKε downregulation. Interestingly, we also show that microRNA-155, in turn, supresses Pax-5 expression, indicative of an auto-regulatory feedback loop. Altogether, we demonstrate that Pax-5 inhibits NF-κB signalling through the regulation of microRNA-155 and its downstream target IKKε. The elucidation of this signaling network is relevant as Pax-5 and NF-κB are potent transcriptional regulators of breast cancer aggressivity. In addition, IKKε is relevant oncogene aberrantly expressed in 30% of breast carcinomas. Further insight into the regulatory pathways of breast cancer progression will eventually identify strategic therapeutic and prognostic targets to improve cancer patient outcome.
    The effects of electronic cigarette vapor on placental trophoblast cell function: Short title : E-cigarette and trophoblast function
    Sergio Raez-Villanueva - 2018
    Abstract
    Despite evidence that maternal smoking is associated with numerous adverse outcomes, 10-35% of women still smoke during pregnancy. Recently, many smokers have turned to electronic cigarettes (e-cigarettes) as a smoking cessation tool. However, there is considerable uncertainty regarding their safety for use during pregnancy. The goal of this study was to examine the effects of e-cigarette vapor on placental trophoblast function. HTR-8/SVneo cells were exposed to unflavored e-cigarette vapor-conditioned media with and without nicotine to assess cell viability, proliferation, migration (wound healing assay), invasion (transwell extracellular matrix invasion assay), and tube formation, a surrogate for angiogenesis. While there was no effect on cell viability, proliferation or migration (all p > 0.05), e-cigarette conditioned media significantly reduced trophoblast invasion and tube formation; these effects could not be solely attributed to the presence of nicotine. These results suggest that an evaluation of the safety of e-cigarette use during pregnancy is urgently required.
    Post-exposure effects of the piscicide 3-trifluoromethyl-4-nitrophenol (TFM) on the stress response and liver metabolic capacity in rainbow trout (Oncorhynchus mykiss)
    Oana Birceanu - 2018
    Abstract
    The piscicide 3-trifluoromethyl-4-nitrophenol (TFM) has been used to control invasive sea lamprey (Petromyzon marinus) populations in the Great Lakes for almost 60 years. Applied to rivers and streams containing larval lampreys, TFM seldom harms non-target fishes, but the effects of sub-lethal treatments on fish physiology are not well understood. We examined the effects of 9 h exposure to TFM on the stress axis and liver metabolic capacity of rainbow trout (Oncorhynchus mykiss) using in vivo and in vitro approaches. The fish that had been acutely exposed to TFM in vivo had increased plasma cortisol levels at 12 h post-treatment, but TFM exposure did not interfere with in vitro cortisol production in head kidney preparations. Subjecting trout to an acute handling stressor 12 h post-TFM exposure resulted in a relative attenuation of the plasma cortisol and glucose response compared to pre-stress levels. We conclude that routine TFM treatments can lead to elevations of plasma cortisol following exposure, plus a relative dampening of the stress response in rainbow trout, with high cortisol levels lasting at least 12 h post-treatment. Since the ability of the fish to produce cortisol and the liver metabolic capacity were not compromised following TFM exposure, it is likely that their ability to cope with other stressors is not altered in the long-term.
    Antimicrobial peptide expression in swine granulosa cells in response to lipopolysaccharide
    Xiaofeng Sun - 2018
    Abstract
    Antimicrobial peptides (AMP) are host defense peptides present in all species examined. The objective of the current study was to characterize the expression of a group of antimicrobial peptides in ovarian cells, and to investigate their expression response to pathogen ligands. It was found that while PG1 transcript was not detected in the ovary, the expression of BD2 is the highest in small follicle derived granulosa cells (SGC), and its expression decreases during follicular development to large follicle stage (LGC; p < 0.05). The expression of BD2 in cumulus cells also decreased from GV to MII stage of oocyte maturation. ANG4 expression increased in granulosa cells during follicular development from SGC to LGC stage (p < 0.05), although no significant difference was observed in cumulus cells from different stages of oocyte maturation. We further examined AMP expression in follicle cells treated with different toll-like receptor (TLR) ligands which mimic pathogen exposure in the ovary. Of the four TLR ligands examined, lipopolysaccharide (LPS) exposure resulted in a 11.5 fold increase of BD2 expression, and a significant decrease of LYZ in LGC. A similar response pattern in BD2 and LYZ expression was also observed in SGC. These responses of AMP expression to LPS are associated with increased TLR4 signaling pathway component in mRNA and protein level, such as MyD88 and NFkB, and pro-inflammatory cytokines/chemokines, such as IL-6, TNFα and IL-8 (p < 0.05). Our data suggest that AMPs may play a role in innate defense as well as other physiological functions during ovarian follicular development and oocyte maturation.
    Effect of Tim23 Knockdown in vivo on Mitochondrial Protein Import and Retrograde Signaling to the UPRmt in Muscle
    Ashley N Oliveira - 2018
    Abstract
    The mitochondrial unfolded protein response (UPRmt) is a protein quality control mechanism that strives to achieve proteostasis in the face of misfolded proteins. Due to the reliance of mitochondria on both the nuclear and mitochondrial genomes, a perturbation of the coordination of these genomes results in a mito-nuclear imbalance in which holoenzymes are unable to assume mature stoichiometry and thereby activates the UPRmt. Thus, we sought to perturb this genomic coordination by using a systemic anti-sense oligonucleotide (in-vivo Morpholino) targeted to Tim23, the major channel of the inner membrane. This resulted in a 40% reduction in Tim23 protein content, a 32% decrease in matrix-destined protein import, and a trend to elevate ROS emission under maximal respiration conditions. This import defect activated the CHOP-branch of the UPRmt, as evident from increases in ClpP and cpn10, but not the ATF5 arm. Thus, in the face of proteotoxic stress, CHOP and ATF5 could be activated independently to regain proteostasis. Our second aim was to investigate the role of proteolytically-derived peptides in mediating retrograde signaling. Peptides released from the mitochondrion following basal proteolysis were isolated and incubated with import reactions. Dose- and time-dependent effect of peptides on protein import was observed. Our data suggest that mitochondrial proteolytic byproducts exert an inhibitory effect on protein import, possibly to reduce excessive protein import as a potential negative feedback mechanism. The inhibition of import into the organelle also serves a retrograde function, possibly via ROS emission, to modify nuclear gene expression and ultimately improve folding capacity.
    HISTONE DEACETYLASE 19 and the flowering time gene FD maintain reproductive meristem identity in an age-dependent manner
    Sasha R. Gorham - 2018
    Abstract
    The shoot apical meristem (SAM) undergoes developmental transitions that include a shift from vegetative to reproductive growth. This transition is triggered by flowering time genes, which up-regulate floral meristem (FM) identity genes that, in turn, control flower development by activating floral organ identity genes. This cascade of transcriptional activation is refined by repression mechanisms that temporally and spatially restrict gene expression to ensure proper development. Here, we demonstrate that HISTONE DEACETYLASE 19 (HDA19) maintains the identity of the reproductive SAM, or inflorescence meristem (IM), late in Arabidopsis thaliana development. At late stages of growth, hda19 IMs display a striking patterning defect characterized by ectopic expression of floral organ identity genes and the replacement of flowers with individual stamenoid organs. We further show that the flowering time gene FD has a specific function in this regulatory process, as fd hastens the emergence of these patterning defects in hda19 growth. Our work therefore identifies a new role for FD in reproductive patterning, as FD regulates IM function together with HDA19 in an age-dependent fashion. To effect these abnormalities, hda19 and fd may accentuate the weakening of transcriptional repression that occurs naturally with reproductive meristem proliferation.
    Rnd3/RhoE expression is regulated by G-actin through MKL1-SRF signaling pathway
    Léo Piquet - 2018
    Abstract
    Rnd3/RhoE is an atypical member of the Rho family of small GTPases, devoid of intrinsic GTP hydrolytic activity and a general modulator of important cellular processes such as migration and proliferation. Here, we show that Rnd3 is a target of the transcription factor SRF and its co-activator MKL1. The MKL1-SRF pathway assures the translation of physical forces into a transcriptional response. Rho GTPases can modulate the activity of this mechanotransduction pathway through actin cytoskeleton regulation, and many MKL1-SRF targets are involved in the regulation of actin. We found that Rnd3 expression is altered by G-actin signaling and sensitive to actin-targeting drugs and MKL1 mutants. We further characterized a consensus SRF binding site in the Rnd3 promoter. We found that MKL1-SRF modulation regulates Rnd3 promoter activity and Rnd3 expression can affect MKL1-SRF pathway activity in return. We demonstrated that this novel MKL1-SRF target is required in mechanosensitive mechanisms such as cell spreading and spheroid formation. Thus, Rnd3 is a MKL1-SRF target that plays a key role in the feedback loop described between the MKL1-SRF pathway and the organization of the actin cytoskeleton.
    Improved fatty acid profiles in seeds of Camelina sativa by artificial microRNA mediated FATB gene suppression
    Mehmet E. Ozseyhan - 2018
    Abstract
    The fatty acid profile of plant oils determines their quality and uses. Saturated fatty acids are often not desirable from the standpoints of nutrition and some industrial applications. Camelina sativa is a re-emerged oilseed crop, however its oil needs to be improved to meet different application requirements. In this study, saturated fatty acids were greatly reduced by down-regulating genes encoding the fatty acyl-ACP thioesterases (FATB). An artificial microRNA (amiFATB) was created by replacing a microRNA sequence in the camelina Csa-miR159a gene with a FATB gene specific sequence. Seed-specific expression of amiFATB caused a 45% reduction of palmitic acid (16:0) and a 38% reduction of stearic acid (18:0) compared to wildtype seeds. The total saturated fatty acid content was decreased by 35% from 14.6% to 9.4% of total fatty acids. When amiFATB was expressed in a high-oleic acid transgenic line, it caused further increased oleic acid content. This work demonstrates that the FATB genes in camelina can be effectively knocked down by an artificial microRNA targeting gene-specific sequences, thus provides an additional tool to improve seed oils for desired properties.
    Mechanical load increase–induced changes in cytoskeletal structure and cellular barrier function in human cerebral endothelial cells
    Dongjoo Kim - 2018
    Abstract
    Globally, approximately a billion patients are estimated to suffer from neurological disorders. Although there are many therapeutic candidates for the central nervous system, treatment of brain disorders is restricted by the blood–brain barrier (BBB), which is a highly selective membrane that protects the brain from exogenous substances. This study was undertaken to develop a novel strategy to overcome the BBB and improve the efficiency of drug delivery to the brain by mechanical load increase using hypergravity. Human cerebral microvascular endothelial cells were exposed three times to 20 min hypergravity (10g), with a 20-min rest period between each exposure. The applied hypergravity reversibly decreased the cellular metabolic activity and increased the permeation rate of fluorescein sodium salt, fluorescein isothiocyanate–labeled dextran (FD-4), and fluorescein-labeled jacalin. Following the exposure to hypergravity, we also observed structural changes of the cytoskeleton and tight junctions, and an alteration in the expression levels of related genes. These results indicate that increased mechanical load due to the applied hypergravity affects the cytoskeletal arrangement and tight junctions, thereby weakening the cell barrier function and enhancing the permeability of the paracellular pathway. Thus, the mechanical load increase by hypergravity has the potential of being used as a novel strategy to overcome the BBB for brain drug delivery.
    The ATP-stimulated translocation promoter (ASTP) activity of glycerol kinase plays central role in adipogenesis
    Lilly S. Parr - 2018
    Abstract
    Glycerol kinase (GK) is a multifunctional enzyme located at the interface of carbohydrate and fat metabolism. It contributes to both central carbon metabolism and adipogenesis; specifically, through its role as the ATP-stimulated translocation promoter (ASTP). GK overexpression leads to increased ASTP activity and increased fat storage in H4IIE cells. We performed metabolic flux analysis in human GK-overexpressing H4IIE cells and found that overexpressing cells had significantly altered fluxes through central carbon and lipid metabolism including increased flux through the pentose phosphate pathway and increased production of lipids. We also observed an equal contribution of glycerol to carbohydrate metabolism in all cell lines, suggesting that GK's alternate functions rather than its enzymatic function are important for these processes. To further elucidate the contributions of the enzymatic (phosphorylation) and alternative (ASTP) functions of GK in adipogenesis, we performed experiments on mammalian GK and E. coli GK. We determined that the ASTP function of GK (which is absent in E. coli GK) plays a greater role than the enzymatic activity in these processes. These studies further emphasize GK's diverse functionality and provides fundamental insights into the multiple protein functions of glycerol kinase.
    IL-36 and IL-1/IL-17 Drive Immunity to Oral Candidiasis via Parallel Mechanisms
    Akash H. Verma - 2018
    Abstract
    Protection against microbial infection by the induction of inflammation is a key function of the IL-1 superfamily, including both classical IL-1 and the new IL-36 cytokine families. Candida albicans is a frequent human fungal pathogen causing mucosal infections. Although the initiators and effectors important in protective host responses to C. albicans are well described, the key players in driving these responses remain poorly defined. Recent work has identified a central role played by IL-1 in inducing innate Type-17 immune responses to clear C. albicans infections. Despite this, lack of IL-1 signaling does not result in complete loss of immunity, indicating that there are other factors involved in mediating protection to this fungus. In this study, we identify IL-36 cytokines as a new player in these responses. We show that C. albicans infection of the oral mucosa induces the production of IL-36. As with IL-1α/β, induction of epithelial IL-36 depends on the hypha-associated peptide toxin Candidalysin. Epithelial IL-36 gene expression requires p38-MAPK/c-Fos, NF-κB, and PI3K signaling and is regulated by the MAPK phosphatase MKP1. Oral candidiasis in IL-36R−/− mice shows increased fungal burdens and reduced IL-23 gene expression, indicating a key role played by IL-36 and IL-23 in innate protective responses to this fungus. Strikingly, we observed no impact on gene expression of IL-17 or IL-17–dependent genes, indicating that this protection occurs via an alternative pathway to IL-1–driven immunity. Thus, IL-1 and IL-36 represent parallel epithelial cell–driven protective pathways in immunity to oral C. albicans infection.
    Molecular cloning and characterization of a sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) from Y-organs of the blue crab (Callinectes sapidus)
    Megan E. Roegner - 2018
    Abstract
    Existing data indicate that a Ca2+ signal stimulates ecdysteroid hormone production by crustacean molting glands (Y-organs). Ca2+ signaling is dependent on a tightly regulated Ca2+ gradient, with intracellular free Ca2+ maintained at a low basal level (typically sub-micromolar). This is achieved through the action of proteins intrinsic to the plasma membrane and the membranes of organelles. One such protein, the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), pumps Ca2+ from cytosol to the lumen of the endoplasmic reticulum. As a step toward understanding Ca2+-mediated regulation of ecdysteroidogenesis, we have begun investigating Ca2+ transport proteins in Y-organs. In studies reported here, we used a PCR-based strategy to clone from Y-organs of the blue crab (Callinectes sapidus) a cDNA encoding a putative SERCA protein. The cloned Cas-SERCA cDNA (3806 bp) includes a 3057-bp open reading frame that encodes a 1019-residue protein (Cas-SERCA). The conceptually translated protein has a predicted molecular mass of 111.42 × 103 and contains all signature domains of an authentic SERCA, including ten transmembrane domains and a phosphorylation site at aspartate 351. A homology model of Cas-SERCA closely resembles models of related SERCA proteins. Phylogenetic analysis shows Cas-SERCA clusters with SERCA proteins from other arthropods. An assessment of tissue distribution indicates the Cas-SERCA transcript is widely distributed across tissues. Studies using quantitative PCR showed Cas-SERCA transcript abundance increased significantly in Y-organs activated by eyestalk ablation, a pattern consistent with the hypothesis that Cas-SERCA functions to maintain Ca2+ homeostasis in Y-organs.
    Accelerated neural differentiation of mouse embryonic stem cells on aligned GYIGSR-functionalized nanofibers
    Elena A. Silantyeva - 2018
    Abstract
    Substrates for embryonic stem cell culture are typified by poorly defined xenogenic, whole proteins or cellular components that are difficult and expensive to generate, characterize, and recapitulate. Herein, the generation of well-defined scaffolds of Gly-Tyr-Ile-Gly-Ser-Arg (GYIGSR) peptide-functionalized poly(ε-caprolactone) (PCL) aligned nanofibers are used to accelerate the neural lineage commitment and differentiation of D3 mouse embryonic stem cells (mESCs). Gene expression trends and immunocytochemistry analysis were similar to laminin-coated glass, and indicated an earlier differentiation progression than D3 mESCs on laminin. Further, GYIGSR-functionalized nanofiber substrates yielded an increased gene expression of Sox1, a neural progenitor cell marker, and Tubb3, Cdh2, Syp, neuronal cell markers, at early time points. In addition, guidance of neurites was found to parallel the fiber direction. We demonstrate the fabrication of a well-defined, xeno-free functional nanofiber scaffold and demonstrates its use as a surrogate for xenogenic and complex matrixes currently used for the neural differentiation of stem cells ex vivo. Statement of Significance In this paper, we report the use of GYIGSR-functionalized poly(ε-caprolactone) aligned nanofibers as a tool to accelerate the neural lineage commitment and differentiation of D3 mouse embryonic stem cells. The results indicate that functional nanofiber substrates promote faster differentiation than laminin coated substrates. The data suggest that aligned nanofibers and post-electrospinning surface modification with bioactive species can be combined to produce translationally relevant xeno-free substrates for stem cell therapy. Future development efforts are focused on additional bioactive species that are able to function as surrogates for other xenogenic factors found in differentiation media.
    Leaderless mRNAs are circularized in Chlamydomonas reinhardtii mitochondria
    A. Bruce Cahoon - 2018
    Abstract
    The mitochondrial genome of Chlamydomonas reinhardtii encodes eight protein coding genes transcribed on two polycistronic primary transcripts. The mRNAs are endonucleolytically cleaved from these transcripts directly upstream of their AUG start codons, creating leaderless mRNAs with 3′ untranslated regions (UTR) comprised of most or all of their downstream intergenic regions. In this report, we provide evidence that these processed linear mRNAs are circularized, which places the 3′ UTR upstream of the 5′ start codon, creating a leader sequence ex post facto. The circular mRNAs were found to be ribosome associate by polysome profiling experiments suggesting they are translated. Sequencing of the 3′–5′ junctions of the circularized mRNAs found the intra-molecular ligations occurred between fully processed 5′ ends (the start AUG) and a variable 3′ terminus. For five genes (cob, cox, nd2, nd4, and nd6), some of the 3′ ends maintained an oligonucleotide addition during ligation, and for two of them, cob and nd6, these 3′ termini were the most commonly recovered sequence. Previous reports have shown that after cleavage, three untemplated oligonucleotide additions may occur on the 3′ termini of these mRNAs—adenylation, uridylylation, or cytidylation. These results suggest oligo(U) and oligo(C) additions may be part of the maturation process since they are maintained in the circular mRNAs. Circular RNAs occur in organisms across the biological spectrum, but their purpose in some systems, such as organelles (mitochondria and chloroplasts) is unclear. We hypothesize, that in C. reinhardtii mitochondria it may create a leader sequence to facilitate translation initiation, which may negate the need for an alternative translation initiation mechanism in this system, as previously speculated. In addition, circularization may play a protective role against exonucleases, and/or increase translational productivity.
    The Yeast Three-Hybrid System for Screening RNA-Binding Proteins in Plants
    Sung Ki Cho - 2018
    Abstract
    Yeast-hybrid methods have been successfully applied for screening interacting partners of DNAs or proteins. A yeast-based method, the yeast three-hybrid system, using a chimeric protein of a DNA-binding domain (LexA or GAL4BD) with a protein (MS2 coat protein or HIV Rev. M10) having a hybrid RNA at the 3′ end of a target RNA sequence, has been developed for screening RNA-binding proteins. When the target RNA interacts with RNA-binding proteins fused with an activation domain (AD), yeast cells having all the interacting components can survive on selection media, and interacting reporters, HIS3 and LacZ, are activated. Based on this selection, interaction can be easily monitored and detected by simple biochemical assays. The in vivo screening strategy has been widely applied for characterizing and evaluating specific interactions between target RNAs and RNA-binding proteins. Here, we describe a library screening strategy for isolating RNA-binding proteins of select target RNAs using the yeast three-hybrid method. We also describe strategies to verify binding specificity using both a yeast-dependent reporter system and a yeast-independent method, in vivo RNA immunoprecipitation (RIP).
    PAK1 regulates ATXN1 levels providing an opportunity to modify its toxicity in spinocerebellar ataxia type 1
    Vitaliy V. Bondar - 2018
    Abstract
    Spinocerebellar ataxia type 1 (SCA1) is caused by the expansion of a trinucleotide repeat that encodes a polyglutamine tract in ataxin-1 (ATXN1). The expanded polyglutamine in ATXN1 increases the protein’s stability and results in its accumulation and toxicity. Previous studies have demonstrated that decreasing ATXN1 levels ameliorates SCA1 phenotypes and pathology in mouse models. We rationalized that reducing ATXN1 levels through pharmacological inhibition of its modulators could provide a therapeutic avenue for SCA1. Here, through a forward genetic screen in Drosophila we identified, p21-activated kinase 3 (Pak3) as a modulator of ATXN1 levels. Loss-of-function of fly Pak3 or Pak1, whose mammalian homologs belong to Group I of PAK proteins, reduces ATXN1 levels, and accordingly, improves disease pathology in a Drosophila model of SCA1. Knockdown of PAK1 potently reduces ATXN1 levels in mammalian cells independent of the well-characterized S776 phosphorylation site (known to stabilize ATXN1) thus revealing a novel molecular pathway that regulates ATXN1 levels. Furthermore, pharmacological inhibition of PAKs decreases ATXN1 levels in a mouse model of SCA1. To explore the potential of using PAK inhibitors in combination therapy, we combined the pharmacological inhibition of PAK with MSK1, a previously identified modulator of ATXN1, and examined their effects on ATXN1 levels. We found that inhibition of both pathways results in an additive decrease in ATXN1 levels. Together, this study identifies PAK signaling as a distinct molecular pathway that regulates ATXN1 levels and presents a promising opportunity to pursue for developing potential therapeutics for SCA1.
    The distribution and detection of grapevine red blotch virus in its host depends on time of sampling and tissue type
    Felicia J Setiono - 2018
    Abstract
    Grapevine red blotch virus (GRBV) is the causal agent of grapevine red blotch, an emerging disease that affects cultivated grapevine such as Vitis vinifera. The ability to detect viruses in grapevine is often hindered by low virus titers compounded by a variable distribution in the plant and seasonal variations. In order to examine these two variables in relation to GRBV, we developed a quantitative qPCR method that incorporates both internal and external references to enhance assay robustness. In greenhouse-grown vines infected with GRBV, qPCR identified highest virus titers in the petioles of fully expanded leaves and significantly reduced levels of virus in the shoot extremities. In vineyard-grown vines infected with GRBV, the virus titer in July and October 2016 followed a similar pattern to that found for the greenhouse-grown plants, but most strikingly close to half (44%) of the samples analyzed in June 2015 tested negative for infection. The technique presented and results obtained highlight the variability of virus distribution in its host and provide a useful guide for selecting the best tissues for optimal GRBV diagnosis.
    Porcine circovirus 2 infection induces IFNβ expression through increased expression of genes involved in RIG-I and IRF7 signaling pathways
    Cheryl M. T.Dvorak - 2018
    Abstract
    Porcine circovirus-associated disease (PCVAD), caused by porcine circovirus 2 (PCV2), is characterized by a highly variable pathogenesis that is manifested by various disease syndromes and includes immune evasion. Hence, even though PCVAD is effectively controlled by vaccination, pigs and farms remain infected so that continued vaccination is necessary to control disease. We investigated the molecular interactions of PCV2 and its permissive VR1BL host cell for gene expression signatures that could provide insight into mechanisms leading towards disease. Molecular pathways involved in the innate immune response to PCV2 infection were examined to identify changes in gene expression associated with productive infection of VR1BL cells. RNA profiling from infected and uninfected cells showed that 139 genes were induced by infection and 43 genes were down-regulated, using a p value <0.05 and an absolute fold-change difference>2. A strong type 1 interferon response, including an increase in genes involved in the RIG-I/MDA5 pathway and downstream interferon induced genes, was observed. Key regulators involved in PCV2 infection were identified as IFNβ, DDX58 (RIG-I), and IRF7. PCV2 infection induces a strong interferon response which unexpectedly facilitates viral gene expression, perhaps due to the presence of an interferon-sensitive response element in the viral promoter. The findings suggest that PCV2 interventions that attenuate type 1 interferon responses at the cellular level might enhance immunity and eliminate persistent infection.
    Molecular network, pathway, and functional analysis of time-dependent gene changes related to cathepsin G exposure in neonatal rat cardiomyocytes
    Sanket Kumar Shukla - 2018
    Abstract
    The molecular pathways activated in response to acute cathepsin G (CG) exposure, as well as the mechanisms involved in activation of signaling pathways that culminate in myocyte detachment and apoptosis remain unclear. This study aimed to determine the changes in gene expression patterns associated with time dependent CG exposure to neonatal rat cardiomyocytes (NRCMs). Microarray analysis revealed a total of 451, 572 and 1127 differentially expressed genes after CG exposure at 1, 4 and 8 h respectively. A total of 54 overlapped genes at each time point were mapped by Ingenuity Pathway Analysis (IPA). The top up-regulated genes included Hamp, SMAD6, NR4A1, FOSL2, ID3 and SLAMF7, and down-regulated genes included CYR61, GDF6, Olr640, Vom2r36, DUSP6 and MMP20. Our data suggest that there are multiple deregulated pathways associated with cardiomyocyte death after CG exposure, including JAK/Stat signaling, IL-9 signaling and Nur77 signaling. In addition, we also generated the molecular network of expressed gene and found most of the molecules were connected to ERK1/2, caspase, BCR (complex) and Cyclins. Our study reveals the ability to assess time-dependent changes in gene expression patterns in NRCMs associated with CG exposure. The global gene expression profiles may provide insight into the cellular mechanism that regulates CG dependent myocyte apoptosis. In future, the pathways important in CG response, as well as the genes found to be differentially expressed might represent the therapeutic targets for myocyte survival in heart failure.
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