qScript cDNA Synthesis Kit

Economical 2-component kit ideally suited for high throughput gene-expression studies

Features & Benefits

Economical 2-component kit ideally suited for high throughput expression studies
Optimized, double pre-primed master mix component ensures equal representation of 5′ and 3′ RNA sequences ≤ 1kb for quantitative and conventional two-step RT-PCR
Broad linear dynamic detection range with limiting (10pg) or plentiful (1ug) samples of total RNA

qScript cDNA Synthesis Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

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qScript cDNA Synthesis Kit

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25 x 20 μL rxns (1 x 25 µL)

95047-025

$130.00

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100 x 20 μL rxns (1 x 100 µL)

95047-100

$322.00

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500 x 20 μL rxns (1 x 500 µL)

95047-500

$1,353.00

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Description

The qScript cDNA Synthesis Kit is a sensitive and easy-to-use solution for RNA quantification using two step RT-qPCR. The novel qScript Reaction Mix provides all the necessary components for cDNA synthesis except enzyme and RNA template. qScript reverse transcriptase is a mixture of an engineered MMLV RT and a ribonuclease inhibitor protein. This economical, highly stable stabilize, 2-component reagent system has been rigorously optimized to ensure sensitive and linear RNA detection with a wide-range of input RNA and relative abundance. Reagent performance is unaffected by repetitive freeze/thaw cycling (>20X), conferring greater ease-of-use and assay performance consistency. Oligo (dT) and random primers are pre-blended in a precise ratio to provide equal representation of 5' and 3'-sequences for accurate gene expression quantification. The resulting cDNA product is directly compatible with all qPCR chemistries or conventional end-point RT-PCR of amplified fragments ≤1 kb in length.

For gene-specific priming (GSP) or two-step RT-PCR of RNA exceeding 1kb in total length, see our qScript® Flex cDNA Kit.

Details

Contents

5X concentrated master mix containing: titered primer blend (oligo dT(20) and random hexamer), qPCR-optimized dNTP blend and flexible magnesium titration
20X concentrated qScript reverse transcriptase
Nuclease-free water

Storage & Handling

Performance Data

Resource Panel

Details

Contents

5X concentrated master mix containing: titered primer blend (oligo dT(20) and random hexamer), qPCR-optimized dNTP blend and flexible magnesium titration
20X concentrated qScript reverse transcriptase
Nuclease-free water

Storage & Handling

Performance Data

Consistent cDNA Synthesis Efficiency - cDNA serial dilution vs. total RNA serial dilution

Broad Linear Dynamic Range

Resources

CofA (PSFs)

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SDSs

FAQs

Does the qScript™ cDNA Synthesis Kit include an RNase Inhibitor like the qScript™ cDNA SuperMix?

For first strand cDNA synthesis, is it better to use oligo(dT), random hexamers, gene specific primer (GSP) or combination of these primers?

What is the smallest quantity of RNA detectable by the qScript™ cDNA Synthesis Kit?

What is the RNA input for the qScript cDNA Synthesis Kit?

Publications

Transcriptional Targets of TWIST1 in Human Mesenchymal Stem/Stromal Cells Mechanistically Link Stem/Progenitor and Paracrine Functions

Christopher L. Haga - 2023

Abstract

Despite extensive clinical testing, mesenchymal stem/stromal cell (MSC)-based therapies continue to underperform with respect to efficacy, which reflects the paucity of biomarkers that predict potency prior to patient administration. Previously, we reported that TWIST1 predicts inter-donor differences in MSC quality attributes that confer potency. To define the full spectrum of TWIST1 activity in MSCs, the present work employed integrated omics-based profiling to identify a high confidence set of TWIST1 targets, which mapped to cellular processes related to ECM structure/organization, skeletal and circulatory system development, interferon gamma signaling, and inflammation. These targets are implicated in contributing to both stem/progenitor and paracrine activities of MSCs indicating these processes are linked mechanistically in a TWIST1 dependent manner. Targets implicated in extracellular matrix dynamics further implicate TWIST1 in modulating cellular responses to niche remodeling. Novel TWIST1 regulated genes identified herein may be prioritized for future mechanistic and functional studies.

The Effects of Bisphenol A on the Expression of microRNA-21 and miR-21 Mediated Pathways Including Epigenetic Regulation and Apoptosis in Bovine Granulosa Cells

Reem Sabry - 2023

Abstract

This research investigates the mechanism of action of microRNA-21 (miR-21) in bovine oocytes and granulosa cells exposed to the endocrine-disrupting compound, bisphenol A (BPA). miR-21 has been extensively reported as one of the microRNAs most affected by BPA, exhibiting significant upregulation in bovine oocytes and granulosa cells. Therefore, miR-21 serves as a promising candidate for further evaluation in the context of BPA-induced reproductive toxicity. The involvement of miR-21 in reproduction encompasses diverse pathways, including wellestablished as well as novel and less elucidated processes. Notably, miR-21 is known to regulate apoptosis, a form of programmed cell death, which is also a recognized effect of BPA in various cell types, including granulosa cells. Consequently, this study aimed to investigate a novel interplay between miR-21 and other epigenetic regulatory mechanisms. DNA methylation, a critical epigenetic mechanism, can be modulated by microRNAs, although its specific role in the reproductive context remains largely unexplored. Hence, elucidating the regulation of DNA methylation by miR-21 in bovine oocytes and granulosa cells constituted a parallel focus of this research. To comprehensively examine both established and novel mechanisms involving miR-21, it was crucial to establish a functional knockdown model for miR-21, thereby enabling further investigation of these pathways. Thus, the primary objective of this research was to establish an efficient knockdown of miR-21 in bovine granulosa cells and oocytes. Chapter 2 of this thesis iii outlines the establishment of a functional miR-21 knockdown model in bovine granulosa cells, while Chapter 3 utilizes this model to investigate the role of miR-21 in BPA-induced apoptosis. Chapter 4 expands the investigation to both the knockdown model and oocytes, examining miR21's involvement in BPA-induced aberrant DNA methylation. Overall, this study demonstrates that BPA induces apoptosis and aberrant DNA methylation in bovine oocytes and granulosa cells, while providing mechanistic insights into the independent and dependent effects of BPA on apoptosis and DNA methylation, respectively, through miR-21 signaling. By shedding light on the epigenetic mechanisms of action employed by BPA to induce reproductive toxicity, this research lays the groundwork for a better understanding of the underlying processes.

Influence of different heat stress models on nutrient digestibility and markers of stress, inflammation, lipid, and protein metabolism in broilers

J.R. Teyssier - 2023

Abstract

This experiment determined the effects of different HS models and pair-feeding (PF) on nutrient digestibility and markers of stress, inflammation, and metabolism in broilers. Birds (720 total) were allocated into 12 environmentally controlled chambers and reared under thermoneutral conditions until 20 d. Until 41 d birds were exposed to 4 treatments, including: thermoneutral at 24°C (TN-al), daily cyclic HS (12 h at 24 and 12 h at 35°C; cyHS), constant HS at 35°C (coHS), and PF birds maintained at 24°C and fed to equalize FI with coHS birds (TN-coPF). At d 41, ileal digesta were collected to determine nutrient apparent ileal digestibility (AID). Blood, liver, and breast tissues were collected from 8 birds per treatment to determine the mRNA expression of stress, inflammation, and metabolism markers. An additional 8 TN-al birds were sampled after acute HS exposure at 35°C for 4 h (aHS), and 8 cyHS birds were sampled either right before or 4 h after HS initiation. Data were analyzed by one-way ANOVA and means were separated using Tukey's HSD test. Compared with TN-al birds, AID of nitrogen and ether extract were reduced in coHS birds, and both cyHS and coHS reduced (P < 0.05) AID of total essential amino acids. TNFα and SOD2 expression were increased (P < 0.05) under aHS, coHS, and TN-coPF conditions. IL6 and HSP70 were increased (P < 0.05) under coHS and aHS, respectively. Expression of lipogenic enzymes ACCα and FASN were reduced by coHS and TN-coPF, while coHS increased the lipolytic enzyme ATGL (P < 0.05). IGF1 was lowered in coHS birds, and p70S6K and MyoG were reduced under coHS and TN-coPF (P < 0.05). Interestingly, MuRF1 and MAFbx were increased (P < 0.05) under coHS only. Overall, these results indicate that coHS has a greater impact on nutrient digestibility and metabolism than aHS and cyHS. Interestingly, increased protein degradation during HS appears to be mostly driven by HS per se and not the reduced FI.

Amphiregulin couples IL1RL1+ regulatory T cells and cancer-associated fibroblasts to impede antitumor immunity

Runzi Sun - 2023

Abstract

Regulatory T (Treg) cells and cancer-associated fibroblasts (CAFs) jointly promote tumor immune tolerance and tumorigenesis. The molecular apparatus that drives Treg cell and CAF coordination in the tumor microenvironment (TME) remains elusive. Interleukin 33 (IL-33) has been shown to enhance fibrosis and IL1RL1+ Treg cell accumulation during tumorigenesis and tissue repair. We demonstrated that IL1RL1 signaling in Treg cells greatly dampened the antitumor activity of both IL-33 and PD-1 blockade. Whole tumor single-cell RNA sequencing (scRNA-seq) analysis and blockade experiments revealed that the amphiregulin (AREG)–epidermal growth factor receptor (EGFR) axis mediated cross-talk between IL1RL1+ Treg cells and CAFs. We further demonstrated that the AREG/EGFR axis enables Treg cells to promote a profibrotic and immunosuppressive functional state of CAFs. Moreover, AREG mAbs and IL-33 concertedly inhibited tumor growth. Our study reveals a previously unidentified AREG/EGFR-mediated Treg/CAF coupling that controls the bifurcation of fibroblast functional states and is a critical barrier for cancer immunotherapy.

Epigenetic and molecular coordination between HDAC2 and SMAD3-SKI regulates essential brain tumour stem cell characteristics

Ravinder K. Bahia - 2023

Abstract

Histone deacetylases are important epigenetic regulators that have been reported to play essential roles in cancer stem cell functions and are promising therapeutic targets in many cancers including glioblastoma. However, the functionally relevant roles of specific histone deacetylases, in the maintenance of key self-renewal and growth characteristics of brain tumour stem cell (BTSC) sub-populations of glioblastoma, remain to be fully resolved. Here, using pharmacological inhibition and genetic loss and gain of function approaches, we identify HDAC2 as the most relevant histone deacetylase for re-organization of chromatin accessibility resulting in maintenance of BTSC growth and self-renewal properties. Furthermore, its specific interaction with the transforming growth factor-β pathway related proteins, SMAD3 and SKI, is crucial for the maintenance of tumorigenic potential in BTSCs in vitro and in orthotopic xenograft models. Inhibition of HDAC2 activity and disruption of the coordinated mechanisms regulated by the HDAC2-SMAD3-SKI axis are thus promising therapeutic approaches for targeting BTSCs.

A conserved signaling axis integrates conflicting environmental drought and heat signals to control stomatal aperture in plants

Ive De Smet - 2023

Abstract

Plants continuously respond to changing environmental conditions to prevent damage and maintain optimal performance. To regulate gas exchange with the environment and to control abiotic stress relief, plants have pores in their leaf epidermis, called stomata 1. Multiple environmental signals affect the opening and closing of these stomata 2. Heat promotes stomatal opening (to cool down) and drought induces stomatal closing (to prevent water loss). Coinciding stress conditions, however, may evoke conflicting stomatal responses, but the cellular mechanisms to resolve these conflicts are unknown. Here, we demonstrate that the high temperature-associated kinase TARGET OF TEMPERATURE 3 (TOT3) directly controls the activity of plasma membrane H+-ATPases to induce stomatal opening. This TOT3 activity is directly antagonized by OPEN STOMATA 1 (OST1), to prevent water loss during drought stress. This signaling axis harmonizes conflicting heat and drought signals to regulate stomatal aperture. In the context of global climate change, understanding how conflicting stress signals converge on stomatal regulation allows the development of climate change-ready crops.

Short heat shock has a long-term effect on mesenchymal stem cells’ transcriptome

Ivana Ribarski-Chorev - 2023

Abstract

The adverse effects of heat stress (HS) on physiological systems are well documented, yet the underlying molecular mechanisms behind it remain poorly understood. To address this knowledge gap, we conducted a comprehensive investigation into the impact of HS on mesenchymal stem cells (MSCs), focusing on their morphology, phenotype, proliferative capacity, and fate determination. Our indepth analysis of the MSCs’ transcriptome revealed a significant influence of HS on the transcriptional landscape. Notably, even after a short period of stress, we observed a persistent alteration in cell identity, potentially mediated by the activation of bivalent genes. Furthermore, by comparing the differentially expressed genes following short HS with their transcriptional state after recovery, we identified the transient upregulation of MLL and other histone modifiers, providing a potential mechanistic explanation for the stable activation of bivalent genes. This could be used to predict and modify the long-term effect of HS on cell identity.

Dietary fish oil sparing with blended vegetable oils on growth, fatty acid composition and lipid-metabolism-related genes expression in juvenile rainbow trout (Oncorhynchus mykiss)

Alireza Ghaedi - 2023

Abstract

A nutritional study was conducted to evaluate the influence of dietary fish oil (FO) sparing by using a mixture of vegetal oils (VO) on rainbow trout (Oncorhynchus mykiss) juveniles (30.0 g). A basal diet was supplemented with the experimental oils at a 10% level, including FO (FO diet), a blend of VO (sunflower, soybean and, rapeseed oils with a 1:1:1 ratio), and a mixture (1:1 ratio) of FO and the blends of VO (MIX). In a raceway system, four hundred and fifty fish were stocked in nine rectangular concrete tanks (50 fish. tank-1 ). Three experimental diets, with three replicate each, were offered to fish up to visual satiation for eight weeks. Growth performance did not change among groups (P>0.05). The highest and lowest proportions of n-3 polyunsaturated fatty acids (PUFA), n-3 long chain-PUFA, especially docosahexaenoic acid, and n-3/n-6 PUFA ratio were in the whole body of FO and the VO groups, respectively (P<0.05). Serum catalase and glutathione peroxidase activities in VO group were higher than others. Serum superoxide dismutase activity in fish-fed FO and VO diets was higher than MIX group (P<0.05). VO group had higher serum lysozyme, alternative complement pathway, and total immunoglobulin levels than other experimental groups. MIX group had highest serum triglyceride, cholesterol, and lowdensity lipoproteins. Serum high-density lipoproteins, aspartate aminotransferase, and alanine aminotransferase levels in VO treatment were in the lowest level compared to others (P<0.05). The highest relative transcription levels of fatty acid synthase, delta-6 fatty acyl desaturase, and apolipoprotein b-100 genes were in the liver of fish fed the MIX diet. The relative transcription level of the fatty acid-binding protein1 gene did not change (P>0.05). In summary, the results of this study revealed that dietary FO sparing with VO did not adversely affect the growth and health indices of rainbow trout; nevertheless, it had adverse effects on its nutritional values by reducing whole-body n-3 LC-PUFA content.

Fertility restoration in mice with chemotherapy induced ovarian failure using differentiated iPSCs

Kevin M. Elias - 2023

Abstract

Background Treatment options for premature ovarian insufficiency (POI) are limited to hormone replacement and donor oocytes. A novel induced pluripotent stem cell (iPSC) transplant paradigm in a mouse model has potential translational applications for management of POI. Methods Mouse ovarian granulosa cell derived-iPSCS were labelled with green fluorescent protein (GFP) reporter and differentiated in vitro into oocytes. Differentiated cells were assayed for estradiol and progesterone secretion by enzyme-linked immunosorbent assays. After Fluorescence-Activated Cell Sorting (FACS) for the cell surface marker anti-Mullerian hormone receptor (AMHR2), enriched populations of differentiated cells were surgically transplanted into ovaries of mice that had POI secondary to gonadotoxic pre-treatment with alkylating agents. A total of 100 mice were used in these studies in five separate experiments with 56 animals receiving orthotopic ovarian injections of either FACS sorted or unsorted differentiated iPSCSs and the remaining animals receiving sham injections of PBS diluent. Following transplantation surgery, mice were stimulated with gonadotropins inducing oocyte development and underwent oocyte retrieval. Nine transplanted mice were cross bred with wild-type mice to assess fertility. Lineage tracing of resultant oocytes, F1 (30 pups), and F2 (42 pups) litters was interrogated by GFP expression and validation by short tandem repeat (STR) lineage tracing. Findings [1] iPSCs differentiate into functional oocytes and steroidogenic ovarian cells which [2] express an ovarian (GJA1) and germ cell (ZP1) markers. [3] Endocrine function and fertility were restored in mice pretreated with gonadotoxic alkylating agents via orthotopic transplantation of differentiated iPSCS, thus generating viable, fertile mouse pups. Interpretation iPSC-derived ovarian tissue can reverse endocrine and reproductive sequelae of POI. Funding Center for Infertility and Reproductive Surgery Research Award, Siezen Foundation award (RMA). Reproductive Scientist Development Program, Marriott Foundation, Saltonstall Foundation, Brigham Ovarian Cancer Research Fund (K.E).

Glycopeptide-Based Supramolecular Hydrogels Induce Differentiation of Adipose Stem Cells into Neural Lineages

Vânia I.B. Castro - 2023

Abstract

We applied a bottom-up approach to develop biofunctional supramolecular hydrogels from an aromatic glycodipeptide. The self-assembly of the glycopeptide was induced by either temperature manipulation (heating–cooling cycle) or solvent (DMSO to water) switch. The sol–gel transition was salt-triggered in cell culture media and resulted in gels with the same chemical compositions but different mechanical properties. Human adipose derived stem cells (hASCs) cultured on these gels under basal conditions (i.e., without differentiation factors) overexpressed neural markers, such as GFAP, Nestin, MAP2, and βIII-tubulin, confirming the differentiation into neural lineages. The mechanical properties of the gels influenced the number and distribution of the adhered cells. A comparison with gels obtained from the nonglycosylated peptide showed that glycosylation is crucial for the biofunctionality of the hydrogels by capturing and preserving essential growth factors, e.g., FGF-2.

The plant cytosolic m6 A RNA methylome stabilizes photosynthesis in the cold

Vicente, A.M., Manavski - 2023

Abstract

The sessile lifestyle of plants requires an immediate response to environmental stressors that affect photosynthesis, growth, and crop yield. Here, we showed that three abiotic perturbations - heat, cold, and high light - triggered considerable changes in the expression signature of 42 epitranscriptomic factors (writers, erasers, and readers) with putative chloroplast-associated functions that formed clusters of commonly expressed genes. The expression changes under all conditions were reversible upon de-acclimation, identifying epitranscriptomic players as modulators in acclimation processes. Chloroplast dysfunctions, particularly those induced by the oxidative stress-inducing norflurazon in a largely GENOME UNCOUPLED (GUN)-independent manner, triggered retrograde signals to remodel chloroplast-associated epitranscriptomic expression patterns. N6 - methyladenosine (m6 42 A) is known as the most prevalent RNA modification and impacts numerous developmental and physiological functions in living organisms. During cold treatment, expression of components of the primary nuclear m6 A methyltransferase complex was upregulated, accompanied by a significant increase in cellular m6 A mRNA marks. In the cold, the presence of FIP37, a core component of the writer complex, plays an important role in the positive regulation of thylakoid structure, photosynthetic functions, and accumulation of photosystem (PS)I, Cytb6f complex, cyclic electron transport (CET) proteins, and Curvature Thylakoid1 (CurT1) but not that of PSII components and the chloroplast ATP synthase. Downregulation of FIP37 affects abundance, polysomal loading, and translation of cytosolic transcripts related to photosynthesis in the cold suggesting m6 A-dependent translational regulation of chloroplast functions. In summary, we identified multifaceted roles of the cellular m6 A RNA methylome in coping with cold predominantly associated with chloroplasts to stabilize photosynthesis.

An Efficient Humanized Mouse Model for Oral Anti-Retroviral Administration

Amber K. Virdi - 2023

Abstract

HIV anti-retrovirals (ARVs) have vastly improved the life expectancy of people living with HIV (PLWH). However, toxic effects attributed to long-term ARV use also contribute to HIV-related co-morbidities such as heart disease, bone loss and HIV-associated neurocognitive disorders (HAND). Unfortunately, mouse models used to study the effects of ARVs on viral suppression, toxicity and HIV latency/tissue reservoirs have not been widely established. Here, we demonstrate an effective mouse model utilizing immune-compromised mice, reconstituted with infected human peripheral blood mononuclear cell (PBMCs). ARV is incorporated into mouse chow and administered daily. Combination ARV regimens included Atripla (efavirenz, tenofovir disoproxil fumarate, and emtricitabine) and Triumeq (abacavir, dolutegravir and lamivudine). Our model measures HIV-infected human cell trafficking, and ARV penetration throughout most relevant HIV organs and plasma, with a large amount of trafficking to the secondary lymphoid organs. Furthermore, te HIV viral load within each organ and the plasma was reduced in ARVs treated vs. untreated control. Overall, we have demonstrated a mouse model that is relatively easy and affordable to establish and utilize to study ARVs’ effect on various tissues, including the co-morbid conditions associated with PLWH, such as HAND, and other toxic effects.

Sea transfer and net pen cleaning induce changes in stress-related gene expression in commercial Atlantic salmon (Salmo salar) gill tissue

Kine Samset Hoem - 2023

Abstract

Stress is a major concern in aquaculture production and more knowledge is needed on physiological responses towards different operational events. Few studies have been performed on fish reared in an actual commercial setting. Transferring salmon from hatchery to sea involves handling, crowding, pumping, transport as well as adjusting to a new environment. This case study investigates the relative expression of selected stress related-genes in farmed Atlantic salmon (Salmo salar) post-smolts that were relocated from land-based breeding tanks with few environmental stressors to sea phase with numerous environmental stressors and major management operations e.g. net pen cleaning. Gill tissue for analysis (n = 60) was harvested at four distinct time points: before, during, and 3 and 6 weeks after sea transfer. RT-qPCR was performed on a panel of 12 genes involved in different cellular pathways (alox5, cyp1α, hif1α, il4/13a, muc2, muc5, muc18, nrf2, pcna, phb, p38 and tnfα). While the transport process itself did not appear to induce notable stress levels, metabolic gene markers showed significant changes in expression after transfer to sea, implying cellular adaptations to sea phase. The occurrence of net pen cleaning induced a strong upregulation of pro-inflammatory markers (alox5, tnfα) and mucins (muc2, muc5 and muc18), suggesting their gene products to be relevant during this operational event. As p38 expression was significantly elevated during transport and after cage cleaning, we cautiously propose p38 as an interesting stress marker for future exploration. The study provides insight into the lives of farmed Atlantic salmon and demonstrates that timing of major operations is crucial to avoid accumulation of stress.

SAPS3 subunit of protein phosphatase 6 is an AMPK inhibitor and controls metabolic homeostasis upon dietary challenge in male mice

Ying Yang - 2023

Abstract

Inhibition of AMPK is tightly associated with metabolic perturbations upon over nutrition, yet the molecular mechanisms underlying are not clear. Here, we demonstrate the serine/threonine-protein phosphatase 6 regulatory subunit 3, SAPS3, is a negative regulator of AMPK. SAPS3 is induced under high fat diet (HFD) and recruits the PP6 catalytic subunit to deactivate phosphorylated-AMPK, thereby inhibiting AMPK-controlled metabolic pathways. Either whole-body or liver-specific deletion of SAPS3 protects male mice against HFD-induced detrimental consequences and reverses HFD-induced metabolic and transcriptional alterations while loss of SAPS3 has no effects on mice under balanced diets. Furthermore, genetic inhibition of AMPK is sufficient to block the protective phenotype in SAPS3 knockout mice under HFD. Together, our results reveal that SAPS3 is a negative regulator of AMPK and suppression of SAPS3 functions as a guardian when metabolism is perturbed and represents a potential therapeutic strategy to treat metabolic syndromes.

Sea transfer and net pen cleaning induce changes in stress-related gene expression in commercial Atlantic salmon (Salmo salar) gill tissue

Kine Samset Hoem - 2023

Abstract

Disruption of day-to-night changes in circadian gene expression with chronic tendinopathy

Ching-Yan Chloé Yeung - 2023

Abstract

Overuse injury in tendon tissue (tendinopathy) is a frequent and costly musculoskeletal disorder and represents a major clinical problem with unsolved pathogenesis. Studies in mice have demonstrated that circadian clock-controlled genes are vital for protein homeostasis and important in the development of tendinopathy. We performed RNA sequencing, collagen content and ultrastructural analyses on human tendon biopsies obtained 12 hours apart in healthy individuals to establish whether human tendon is a peripheral clock tissue and we performed RNA sequencing on patients with chronic tendinopathy to examine the expression of circadian clock genes in tendinopathic tissues. We found time-dependent expression of 280 RNAs including 11 conserved circadian clock genes in healthy tendons and markedly fewer (23) differential RNAs with chronic tendinopathy. Further, the expression of COL1A1 and COL1A2 was reduced at night but was not circadian rhythmic in synchronised human tenocyte cultures. In conclusion, day-to-night changes in gene expression in healthy human patellar tendons indicate a conserved circadian clock as well as the existence of a night-reduction in collagen I expression.

PTPN2 regulates bacterial clearance in a mouse model of enteropathogenic and enterohemorrhagic E. coli infection

Marianne R. Spalinger - 2023

Abstract

Macrophages intimately interact with intestinal epithelial cells, but the consequences of defective macrophage–epithelial cell interactions for protection against enteric pathogens are poorly understood. Here, we show that in mice with a deletion in protein tyrosine phosphatase nonreceptor type 2 (PTPN2) in macrophages, infection with Citrobacter rodentium, a model of enteropathogenic and enterohemorrhagic E. coli infection in humans, promoted a strong type 1/IL-22–driven immune response, culminating in accelerated disease but also faster clearance of the pathogen. In contrast, deletion of PTPN2 specifically in epithelial cells rendered the epithelium unable to upregulate antimicrobial peptides and consequently resulted in a failure to eliminate the infection. The ability of PTPN2-deficient macrophages to induce faster recovery from C. rodentium was dependent on macrophage-intrinsic IL-22 production, which was highly increased in macrophages deficient in PTPN2. Our findings demonstrate the importance of macrophage-mediated factors, and especially macrophage-derived IL-22, for the induction of protective immune responses in the intestinal epithelium, and show that normal PTPN2 expression in the epithelium is crucial to allow for protection against enterohemorrhagic E. coli and other intestinal pathogens.

The Senolytic Drug Fisetin Attenuates Bone Degeneration in the Zmpste24−/− Progeria Mouse Model

William S. Hambright - 2023

Abstract

Aging leads to several geriatric conditions including osteoporosis (OP) and associated frailty syndrome. Treatments for these conditions are limited and none target fundamental drivers of pathology, and thus identifying strategies to delay progressive loss of tissue homeostasis and functional reserve will significantly improve quality of life in elderly individuals. A fundamental property of aging is the accumulation of senescent cells. Senescence is a cell state defined by loss of proliferative capacity, resistance to apoptosis, and the release of a proinflammatory and anti-regenerative senescence-associated secretory phenotype (SASP). The accumulation of senescent cells and SASP factors is thought to significantly contribute to systemic aging. Senolytics—compounds which selectively target and kill senescent cells—have been characterized to target and inhibit anti-apoptotic pathways that are upregulated during senescence, which can elicit apoptosis in senescent cells and relieve SASP production. Senescent cells have been linked to several age-related pathologies including bone density loss and osteoarthritis in mice. Previous studies in murine models of OP have demonstrated that targeting senescent cells pharmacologically with senolytic drugs can reduce symptomology of the disease. Here, we demonstrate the efficacy of senolytic drugs (dasatinib, quercetin, and fisetin) to improve age-associated degeneration in bone using the Zmpste24−/− (Z24−/−) progeria murine system for Hutchinson–Gilford progeria syndrome (HGPS). We found that the combination of dasatinib plus quercetin could not significantly mitigate trabecular bone loss although fisetin administration could reduce bone density loss in the accelerated aging Z24−/− model. Furthermore, the overt bone density loss observed in the Z24−/− model reported herein highlights the Z24 model as a translational model to recapitulate alterations in bone density associated with advanced age. Consistent with the “geroscience hypothesis,” these data demonstrate the utility of targeting a fundamental driver of systemic aging (senescent cell accumulation) to alleviate a common condition with age, bone deterioration.

Topical phage therapy in a mouse model of Cutibacterium acnes-induced acne-like lesions

Amit Rimon - 2023

Abstract

Acne vulgaris is a common neutrophil-driven inflammatory skin disorder in which Cutibacterium acnes (C. acnes) is known to play a key role. For decades, antibiotics have been widely employed to treat acne vulgaris, inevitably resulting in increased bacterial antibiotic resistance. Phage therapy is a promising strategy to combat the growing challenge of antibiotic-resistant bacteria, utilizing viruses that specifically lyse bacteria. Herein, we explore the feasibility of phage therapy against C. acnes. Eight novel phages, isolated in our laboratory, and commonly used antibiotics eradicate 100% of clinically isolated C. acnes strains. Topical phage therapy in a C. acnes-induced acne-like lesions mouse model affords significantly superior clinical and histological scores. Moreover, the decrease in inflammatory response was reflected by the reduced expression of chemokine CXCL2, neutrophil infiltration, and other inflammatory cytokines when compared with the infected-untreated group. Overall, these findings indicate the potential of phage therapy for acne vulgaris as an additional tool to conventional antibiotics.

ADO-MEDIATED SYNTHESIS OF TAURINE ALTERS THE CHROMATIN LANDSCAPE OF INGUINAL ADIPOSE TISSUE TO ENHANCE NONSHIVERING THERMOGENESIS

Pei-Yin Tsai - 2023

Abstract

Non-shivering thermogenesis (NST) has strong potential to combat obesity, however, a safe molecular approach to activate this process has not yet been identified. The sulfur amino acid taurine has the ability to safely activate NST and confer protection against obesity and metabolic disease in both mice and humans, but the mechanism of action is unknown. In this study, we discover that a suite of taurine biosynthetic enzymes, especially that of cysteamine dioxygenase (ADO), significantly increases in response to b3 adrenergic signaling in inguinal tissues (IWAT) in order increase intracellular concentrations of taurine. We further show that ADO is critical for thermogenic mitochondrial function as its ablation in thermogenic adipocytes significantly reduces taurine levels which lead to declines in mitochondrial oxygen consumption rates. Finally, we demonstrate via assay for transposase-accessible chromatin with sequencing (ATAC-Seq) that taurine supplementation has the ability to remodel the chromatin landscape to increase the chromatin accessibility and transcription of genes, such as glucose-6-phosphate isomerase 1 (Gpi1), that are critical for NST. Taken together, our studies highlight a potential mechanism for taurine in the activation of NST that can be leveraged toward the treatment of obesity and metabolic disease.

DUSP15 expression is reduced in the hippocampus of Myrf knock-out mice but attention and object recognition memory remain intact

Florence Rawlings-Mortimer - 2023

Abstract

The atypical protein tyrosine phosphatase enzyme, dual-specificity phosphate 15 (DUSP15) is thought to be activated by myelin regulatory factor (MyRF) and to have a role in oligodendrocyte differentiation. Here, we assess whether Dusp15 is reduced in the hippocampus of mice with conditional knock-out of Myrf in oligodendrocyte precursor cells. Using quantitative polymerase chain reaction (qPCR) we found that Dusp15 expression was indeed lower in these mice. Alterations in myelin have been associated with Alzheimer’s disease (AD), autism spectrum disorder (ASD) and attention deficit/hyperactivity disorder (ADHD). Symptoms of these disorders can include impairments of object recognition and attention. We, therefore tested the mice in the object recognition task (ORT) and 5-choice serial reaction time task (5CSRTT). However, we did not find behavioural impairments indicating that attentional abilities and object recognition are not impacted by reduced oligodendrogenesis and hippocampal Dusp15 expression. Gaining insight into the role of newly formed oligodendrocytes and Dusp15 expression is helpful for the development of well targeted treatments for myelin dysregulation.

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.

Covariation between glucocorticoid levels and receptor expression modulates embryo development and postnatal phenotypes in gulls

Francisco Ruiz-Raya - 2023

Abstract

The hypothalamic-pituitary-adrenocortical axis can translate, through glucocorticoid secretion, the prenatal environment to development to produce phenotypes that match prevailing environmental conditions. However, whether developmental plasticity is modulated by the interaction between circulating glucocorticoids and receptor expression remains unclear. Here, we tested whether covariation between plasma corticosterone (CORT) and glucocorticoid receptor gene (Nr3c1) expression in blood underlies embryonic developmental programming in yellow-legged gulls (Larus michahellis). We examined variations in circulating levels of CORT and the expression and DNA methylation patterns of Nr3c1 in response to two ecologically relevant prenatal factors: adult alarm calls (a cue of predator presence) and changes in prenatal light environment (a cue of competitive disadvantage). We then determined whether embryonic development and postnatal phenotypes were associated with CORT levels and Nr3c1 expression, and explored direct and indirect relationships between the prenatal environment, hormone-receptor covariation, and postnatal phenotypes. Prenatal exposure to alarm calls increased CORT levels and up-regulated Nr3c1 expression in gull chicks, while exposure to light cues reduced both hormone levels and receptor expression. Chicks prenatally exposed to alarm calls showed altered DNA methylation profiles in the Nr3c1 regulatory region, but patterns varied throughout the breeding season and between years. Moreover, our results suggest a negative relationship between DNA methylation and expression in Nr3c1 , at least at specific CpG sites. The interplay between circulating CORT and Nr3c1 expression affected embryo developmental timing and vocalizations, as well as hatchling mass and fitness-relevant behaviours. These findings provide a link between prenatal inputs, glucocorticoid function and phenotypic outcomes, suggesting that hormone-receptor interaction may underlie developmental programming in free-living animals.

All together now: Geographically coordinated miticide treatment benefits honey bee health

Luke Woodford - 2023

Abstract

Deformed wing virus (DWV) is a pathogenic virus of honey bees transmitted by the ectoparasitic mite Varroa destructor. Annual overwintering colony losses, accounting for ~25% of all colonies, are associated with high levels of Varroa-DWV infestation. Effective miticide treatments are available to control Varroa. However, the absence of coordinated treatment means environmental transmission of mites continues unchecked. We aimed to determine whether rational, coordinated treatment is beneficial, and characterized the DWV population as an indicator of colony health. This study uses coordinated treatment of Varroa in a geographically isolated environment (Isle of Arran, Scotland) over 3 years. The study area contained 50–84 colonies managed by ~20 amateur beekeepers. Sampling and virus analysis to assess strain diversity and viral loads were conducted before and after treatments, and changes in population diversity were quantified by sequence analysis. Over the 3 years analysis of the virus population revealed that the dominant DWV variant shifted from Type A to Type B in all apiaries, regardless of mite levels or proximity to other colonies. During this period the number of managed colonies increased by 47% (57–84 colonies), but despite this, we estimate total mite numbers decreased by 58%. Synthesis and applications. In this study, the beekeepers in Arran significantly improved the number of colonies they managed, without importing any bees onto the island, indicating that an improved focus on management techniques, through the combination of a coordinated miticide programme and an improved understanding of bee diseases, could yield positive results for bee health and sustainability.

All together now: geographically coordinated miticide treatment benefits honey bee health

Luke Woodford - 2023

Abstract

Deformed wing virus (DWV) is a pathogenic virus of honey bees tra ns mit te d by theectoparasitic miteVarroa destructor. Annual overwinter ing colony losses, accountingfo r ~25 % of a ll c o lo nie s , are associated with high le ve ls of Varroa-DWV infestatio n.Effect ive mitic id e treatments are a va ila b le to control Varroa. However, the absence of coordinatedtreatmentmeans environme nta ltra ns mis s io n of mite s continue s unchecked. We a ime d to d e te rmine whether ra tio na l, coordinated treatment is b e ne fic ia l, and characterised the DWV population as an ind ic a to r of colony health.2.This study uses coordinated treatment of Varroa in a geographica l ly is o la t e denvironment (Isle of Arran, Scotland) over 3 years. The study area contained 50-84 c o lo nie s managed by ~20 amateur beekeepers. S a mp lin g and virus a na lys is to assesss tra in d ive rs it y and vira l loads were conducted before and after treatments, and change sin population d ive rs it y were quantified by sequence a na lys is .3.O ver the three years a na lys is of the virus population revealed that the d o mina nt DWVvariant s hifte d fro m Type A to Type B in a ll apiaries, regardless of mite le ve ls or p ro ximit y to other c o lo nie s . During this period the number of managed colonies This article has been accepted for publication and undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article as doi:10.1111/1365-2664.14367This article is protected by copyright. All rights reserved.Journal of Applied Ecologyincreased by 47 % (57 – 84 c o lo nie s ),but despite this , we e s timate total mite numb e r sdecreased by 58 %.4.Synthesis and applications. In this study the beekeepers on Arran significa nt l yimp ro ve d the number of c o lo nie s they managed, without importing any bees onto theis la nd , ind ic a t in g that an imp ro ve d fo c us on manageme nt technique s, through thec o mb ina t io n of a coordinated mitic id e p ro gra mme and an imp ro ve d understanding of bee diseases, c o uld yie ld p o s itive re s ults fo r bee health and sustainab ility.

Long-term exposure to house dust mites accelerates lung cancer development in mice

Dongjie Wang - 2023

Abstract

Background Individuals with certain chronic inflammatory lung diseases have a higher risk of developing lung cancer (LC). However, the underlying mechanisms remain largely unknown. Here, we hypothesized that chronic exposure to house dust mites (HDM), a common indoor aeroallergen associated with the development of asthma, accelerates LC development through the induction of chronic lung inflammation (CLI). Methods The effects of HDM and heat-inactivated HDM (HI-HDM) extracts were evaluated in two preclinical mouse models of LC (a chemically-induced model using the carcinogen urethane and a genetically-driven model with oncogenic KrasG12D activation in lung epithelial cells) and on murine macrophages in vitro. Pharmacological blockade or genetic deletion of the Nod-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome, caspase-1, interleukin-1β (IL-1β), and C–C motif chemokine ligand 2 (CCL2) or treatment with an inhaled corticosteroid (ICS) was used to uncover the pro-tumorigenic effect of HDM. Results Chronic intranasal (i.n) instillation of HDM accelerated LC development in the two mouse models. Mechanistically, HDM caused a particular subtype of CLI, in which the NLRP3/IL-1β signaling pathway is chronically activated in macrophages, and made the lung microenvironment conducive to tumor development. The tumor-promoting effect of HDM was significantly decreased by heat treatment of the HDM extract and was inhibited by NLRP3, IL-1β, and CCL2 neutralization, or ICS treatment. Conclusions Collectively, these data indicate that long-term exposure to HDM can accelerate lung tumorigenesis in susceptible hosts (e.g., mice and potentially humans exposed to lung carcinogens or genetically predisposed to develop LC).

Cannabidiol Modulates Alterations in PFC microRNAs in a Rat Model of Depression

Uri Bright - 2023

Abstract

Cannabidiol (CBD) is a potential antidepressant agent. We examined the association between the antidepressant effects of CBD and alterations in brain microRNAs in the unpredictable chronic mild stress (UCMS) model for depression. UCMS male rats were injected with vehicle or CBD (10 mg/kg) and tested for immobility time in the forced swim test. Alterations in miRNAs (miR16, miR124, miR135a) and genes that encode for the 5HT1a receptor, the serotonergic transporter SERT, β-catenin, and CB1 were examined. UCMS increased immobility time in a forced swim test (i.e., depressive-like behavior) and altered the expression of miRNAs and mRNA in the ventromedial prefrontal cortex (vmPFC), raphe nucleus, and nucleus accumbens. Importantly, CBD restored UCMS-induced upregulation in miR-16 and miR-135 in the vmPFC as well as the increase in immobility time. CBD also restored the UCMS-induced decrease in htr1a, the gene that encodes for the serotonergic 5HT1a receptor; using a pharmacological approach, we found that the 5HT1a receptor antagonist WAY100135 blocked the antidepressant-like effect of CBD on immobility time. Our findings suggest that the antidepressant effects of CBD in a rat model for depression are associated with alterations in miR-16 and miR-135 in the vmPFC and are mediated by the 5HT1a receptor.

Melanoma brain colonization involves the emergence of a brain-adaptive phenotype.

Vigdis Nygaard - 2014

Abstract

The brain offers a unique microenvironment that plays an important role in the establishment and progression of metastasis. However, the molecular determinants that promote development of melanoma brain metastases are largely unknown. Utilizing two species of immune-compromised animals, with in vivo cultivated metastatic tissues along with their corresponding host tissues in a metastasis model, we here identify molecular events associated with melanoma brain metastases. We find that the transcriptional changes in the melanoma cells, as induced by the brain-microenvironment in both host species, reveal the opportunistic nature of melanoma in this biological context in rewiring the molecular framework of key molecular players with their associated biological processes. Specifically, we identify the existence of a neuron-like melanoma phenotype, which includes synaptic characteristics and a neurotransmission-like circuit involving glutamate. Regulation of gene transcription and neuron-like plasticity by Ca2+-dependent signaling appear to occur through glutamate receptor activation. The brain-adaptive phenotype was found as more prominent in the early metastatic growth phases compared to a later phase, emphasizing a temporal requirement of critical events in the successful colonization of the brain. Analysis of the host tissue uncovered a cooperative inflammatory microenvironment formed by activated host cells that permitted melanoma growth at the expense of the host organism. Combined experimental and computational approaches clearly highlighted genes and signaling pathways being shared with neurodegenerative diseases. Importantly, the identification of essential molecular networks that operate to promote the brain-adaptive phenotype is of clinical relevance, as they represent leads to urgently needed therapeutic targets.

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.

Sex-specific roles of hippocampal microRNAs in stress vulnerability and resilience

Maayan Krispil-Alon - 2022

Abstract

Contrary to intuition, most individuals are resilient to psychological trauma and only a minority is vulnerable. Men and women are known to respond differently to trauma exposure, however, mechanisms underlying the relationship between sex differences and trauma resilience and vulnerability are not yet fully understood. Taking advantage of the Behavioral Profiling approach, which enables differentiating between ‘affected’ and ‘unaffected’ individuals, we examined sex-associated differences in stress exposure effects on hippocampal expression of selected stress-related GABA-A receptor targeting miRNAs. Levels of the miRNA-144 and miRNA-33 were measured in male and female affected (vulnerable, e.g., higher freezing time) and unaffected (resilient) rats. In male rats, increased levels of miRNA-144 and miRNA-33 were observed in the dorsal dentate gyrus (dDG) and ventral dentate gyrus (vDG) respectively, of stress-exposed but unaffected animals. In females, we observed an increased expression of miRNA-144 and miRNA-33 in the ventral cornu ammonis 1 (vCA1) of affected animals. Accordingly, we inhibited miRNAs expression selectively in hippocampal subregions using oligonucleotides containing locked nucleic acid bases, to examine the miRNAs’ causal contribution to either vulnerability or resilience to stress in each sex. Inhibition of miRNA-144 in dDG and miRNA-33 in vDG in males resulted in an increased prevalence of vulnerable animals, while inhibition of miRNA-144 and miRNA-33 in vCA1 in females increased the proportion of resilient animals. The current findings reveal a critical sex-associated difference in the role of miRNAs in stress vulnerability and resilience. This novel understanding of sex-associated epigenetic involvement in the mechanism of stress-related psychopathologies may help improve gender-specific diagnosis and effective treatment.

AHL-Priming Protein 1 mediates N-3-oxo-tetradecanoyl-homoserine lactone priming in Arabidopsis

Abhishek Shrestha - 2022

Abstract

Background N-3-oxo-tetradecanoyl-L-homoserine lactone (oxo-C14-HSL) is one of the N-acyl homoserine lactones (AHL) that mediate quorum sensing in Gram-negative bacteria. In addition to bacterial communication, AHL are involved in interactions with eukaryotes. Short-chain AHL are easily taken up by plants and transported over long distances. They promote root elongation and growth. Plants typically do not uptake hydrophobic long sidechain AHL such as oxo-C14-HSL, although they prime plants for enhanced resistance to biotic and abiotic stress. Many studies have focused on priming effects of oxo-C14-HSL for enhanced plant resistance to stress. However, specific plant factors mediating oxo-C14-HSL responses in plants remain unexplored. Here, we identify the Arabidopsis protein ALI1 as a mediator of oxo-C14-HSL-induced priming in plants. Results We compared oxo-C14-HSL-induced priming between wild-type Arabidopsis Col-0 and an oxo-C14-HSL insensitive mutant ali1. The function of the candidate protein ALI1 was assessed through biochemical, genetic, and physiological approaches to investigate if the loss of the ALI1 gene resulted in subsequent loss of AHL priming. Through different assays, including MAP kinase activity assay, gene expression and transcriptome analysis, and pathogenicity assays, we revealed a loss of AHL priming in ali1. This phenomenon was reverted by the reintroduction of ALI1 into ali1. We also investigated the interaction between ALI1 protein and oxo-C14-HSL using biochemical and biophysical assays. Although biophysical assays did not reveal an interaction between oxo-C14-HSL and ALI1, a pull-down assay and an indirect method employing biosensor E. coli LuxCDABE support such interaction. We expressed fluorescently tagged ALI1 in tobacco leaves to assess the localization of ALI1 and demonstrate that ALI1 colocalizes with the plasma membrane, tonoplast, and endoplasmic reticulum. Conclusions These results suggest that the candidate protein ALI1 is indispensable for oxo-C14-HSL-dependent priming for enhanced resistance in Arabidopsis and that the ALI1 protein may interact with oxo-C14-HSL. Furthermore, ALI1 protein is localized in the cell periphery. Our findings advance the understanding of interactions between plants and bacteria and provide an avenue to explore desired outcomes such as enhanced stress resistance, which is useful for sustainable crop protection.

omparisons of a sodium selenite and a seleniumenriched Spirulina supplementation after a selenium deficiency on growth, tissue selenium concentrations, antioxidant activities and selenoprotein expression in rat

Thomas Castel - 2022

Abstract

Selenium contributes to physiological functions through its incorporation into selenoproteins. It is involved in oxidative stress defense. A selenium deficiency results in the onset or aggravation of pathologies. Following a deficiency, the repletion of selenium leads to a selenoprotein expression hierarchy still misunderstood. Moreover, spirulina, a microalgae, exhibits antioxidant properties and can be enriched in selenium during its cultivation. Our objective was to determine the effects of a sodium selenite or seleniumenriched spirulina supplementation. Thirty-two female wistar rats were fed for 12 weeks with a seleniumdeficient diet. After 8 weeks, rats were divided into 4 groups of 8 rats and were fed with water, sodium selenite (20µg Se/kg body weight), spirulina (3 g/kg bw) or selenium-enriched spirulina (20µg Se/kg bw + 3g spirulina/kg bw). In parallel, another group of 8 rats were fed with normal diet during 12 weeks. Selenium concentration and antioxidant enzyme activities (GPx, SOD, CAT) were measured in plasma, urines, liver, brain, kidney, heart and soleus. Expression of GPx (1, 3), Sel (P, S, T, W), SEPHS2, TrxR1, ApoER2 and Megalin were quantified in liver, kidney, brain and heart. Our results showed that a selenium deficiency leads to a growth retardation, reversed by selenium supplementation. All tissues displayed a decrease in selenium concentration following deficiency. Brain seemed protected. Our results demonstrated a hierarchy in selenium distribution and selenoprotein expression depending on selenium supplementation form. A supplementation of sodium selenite improved GPx activities and selenoprotein expression while a seleniumenriched spirulina was more effective to restore selenium concentration.

HIF1 inhibitor Acriflavine Rescues Early-Onset Preeclampsia Phenotype in Mice Lacking Placental Prolyl Hydroxylase Domain Protein-2

Julien Sallais - 2022

Abstract

Preeclampsia is a serious pregnancy disorder that lacks effective treatments other than delivery. Improper sensing of oxygen changes during placentation by prolyl hydroxylases (PHD), specifically PHD2, causes placental Hypoxia-Inducible Factor-1 (HIF1) buildup and abnormal downstream signaling in early-onset preeclampsia; yet therapeutic targeting of HIF1 has never been attempted. Here we generated a conditional (placenta-specific) knockout of Phd2 in mice (Phd2-/-cKO) to reproduce HIF1 excess and to assess anti-HIF therapy. Conditional deletion of Phd2 in the junctional zone (JZ) during pregnancy increased placental HIF1 content, resulting in abnormal placentation, impaired remodeling of the uterine spiral arteries, and fetal growth restriction. Pregnant dams developed new 47 onset hypertension at mid-gestation (E9.5) in addition to proteinuria and renal and cardiac pathology, hallmarks of severe preeclampsia in humans. Daily injection of acriflavine, a small-molecule inhibitor of HIF1, to pregnant Phd2-/-cKO mice from E7.5 (prior to hypertension) or E10.5 (after hypertension has been established) to E14.5 corrected placental dysmorphologies and improved fetal growth. Moreover, it reduced maternal blood pressure and reverted renal and myocardial pathology. Thus, therapeutic targeting of the HIF pathway may improve placental development and function, as well as maternal and fetal health, in preeclampsia.

Critical Sites on Ostreolysin Are Responsible for Interaction with Cytoskeletal Proteins

Nastacia Adler Berke - 2022

Abstract

We explored the structural features of recombinant ostreolysin A (rOlyA), a protein produced by Pleurotus ostreatus and responsible for binding to α/β-tubulin. We found that rOlyA cell internalization is essential for the induction of adipocyte-associated activity, which is mediated by the interaction of rOlyA and microtubule proteins. We created different point mutations at conserved tryptophan (W) sites in rOlyA and analyzed their biological activity in HIB-1B preadipocytes. We demonstrated that the protein’s cell-internalization ability and the differentiated phenotype induced, such as small lipid-droplet formation and gene expression of mitogenesis activity, were impaired in point-mutated proteins W96A and W28A, where W was converted to alanine (A). We also showed that an rOlyA homologue, OlyA6 complexed with mCherry, cannot bind to β-tubulin and does not induce mitochondrial biosynthesis-associated markers, suggesting that the OlyA6 region masked by mCherry is involved in β-tubulin binding. Protein–protein docking simulations were carried out to investigate the binding mode of rOlyA with β-tubulin. Taken together, we identified functional sites in rOlyA that are essential for its binding to β-tubulin and its adipocyte-associated biological activity.

Per- and polyfluoroalkyl substances enhance Staphylococcus aureus pathogenicity and impair host immune response

Jagadish Chandra Kumar Mangu - 2022

Abstract

Per- and Poly-fluoroalkyl substances (PFAS) are one of the major persistent environmental contaminants. Epidemiological studies have linked PFAS exposures to altered immunity and increased occurrence of infections in children. However, the mechanisms leading to immune susceptibility to bacterial infections remains unclear. To elucidate the mechanism, transcriptional alteration in the Caenorhabditis elegans model caused by a PFAS contaminated environmental water and two reconstituted PFAS solutions were evaluated using RNA-sequencing. PFAS affected the expression of several genes involved in C. elegans immune surveillance to Gram-positive bacteria (cpr-2, tag-38, spp-1, spp-5, clec-7, clec-172). The combined exposure to PFAS and Staphylococcus aureus significantly reduced C. elegans survival and increased intestinal membrane permeability. Furthermore, the growth of S. aureus in the presence of PFAS increased the expression of virulence genes, specifically, the virulence gene regulator saeR and α-hemolysin, hla, which resulted in increased hemolytic activity. The present study demonstrated that PFAS exposure not only increased C. elegans susceptibility to pathogens by reducing host immunity and increasing intestinal membrane permeability, but also increased bacteria virulence. This presents a broader implication for humans and other animals, where environmental contaminants simultaneously reduce host resilience, while, increasing microbial pathogenicity.

The mismatch recognition protein MutSα promotes nascent strand degradation at stalled replication forks

Junqiu Zhang - 2022

Abstract

Mismatch repair (MMR) is a replication-coupled DNA repair mechanism and plays multiple roles at the replication fork. The well-established MMR functions include correcting misincorporated nucleotides that have escaped the proofreading activity of DNA polymerases, recognizing nonmismatched DNA adducts, and triggering a DNA damage response. In an attempt to determine whether MMR regulates replication progression in cells expressing an ultramutable DNA polymerase E (PolE), carrying a proline-to-arginine substitution at amino acid 286 (PolE-P286R), we identified an unusual MMR function in response to hydroxyurea (HU)-induced replication stress. PolE-P286R cells treated with hydroxyurea exhibit increased MRE11-catalyzed nascent strand degradation. This degradation by MRE11 depends on the mismatch recognition protein MutSα and its binding to stalled replication forks. Increased MutSα binding at replication forks is also associated with decreased loading of replication fork protection factors FANCD2 and BRCA1, suggesting blockage of these fork protection factors from loading to replication forks by MutSα. We find that the MutSα-dependent MRE11-catalyzed fork degradation induces DNA breaks and various chromosome abnormalities. Therefore, unlike the well-known MMR functions of ensuring replication fidelity, the newly identified MMR activity of promoting genome instability may also play a role in cancer avoidance by eliminating rogue cells.

he P2X7 Receptor Antagonist AZ10606120 Does Not Alter Graft-VersusHost Disease Development and Increases Serum Human Interferon-γ in a Humanized Mouse Model

Nicholas J. Geraghty - 2022

Abstract

Allogeneic hematopoietic stem cell transplantation is a curative therapy for hematological malignancies, but its efficacy is limited by graft-versus-host disease (GVHD). This lifethreatening disorder develops when donor (graft) immune cells cause inflammatory damage to recipient (host) tissues. The immune cell receptor channel P2X7 and its ligand adenosine 5’-triphosphate (ATP) have been implicated in GVHD pathogenesis. Therefore, this signaling axis represents a potential therapeutic target. This study aimed to investigate if the specific P2X7 antagonist AZ10606120 (AZ10) could prevent GVHD development in a preclinical, humanized mouse model, in which NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice are injected with human peripheral blood mononuclear cells(hPBMCs). Flow cytometric measurements of ATP-induced cation dye uptake revealed that AZ10 blocked P2X7 activity in human RPMI 8226 multiple myeloma cells (IC50 of 1 ± 1 nM) and murine RAW 264.7 macrophages (IC50 of 3 ± 1 nM), as well as primary donor CD4+ and CD8+ T cells. However, AZ10 (2 mg/kg), injected intraperitonealy (i.p.) daily for the first 10 days post-hPBMC injection, did not reduce clinical or histological GVHD development in mice. AZ10 did not impact engraftment of human leukocytes, predominantly CD4+ and CD8+ T cells. However, AZ10 increased serum human interferon gamma (hIFN-γ) concentrations, with CD8+ T cells being the main hIFN-γ producing T cell subset. In conclusion, this study suggests a role for P2X7 activation in impairing hIFN-γ production during GVHD pathogenesis, with the use of P2X7 blockade as a therapeutic strategy warranting further investigation.

Engineered Exosomes Containing Cathelicidin/LL-37 Exhibit Multiple Biological Functions

Yajuan Su - 2022

Abstract

Exosomes show great potential in diagnostic and therapeutic applications. Inspired by human innate immune defense, herein, we report engineered exosomes-derived from monocytic cells treated with immunomodulating compounds 1,25-dihydroxyvitamin D3 and CYP24A1 inhibitor VID400 which are slowly released from electrospun nanofiber matrices. These engineered exosomes contain significantly more cathelicidin/LL-37 when compared with exosomes-derived from either untreated cells or Cathelicidin Human Tagged ORF Clone transfected cells. In addition, such exosomes exhibit multiple biological functions evidenced by killing bacteria, facilitating human umbilical vein endothelial cell tube formation, and enhancing skin cell proliferation and migration. Taken together, the engineered exosomes developed in this study could be used as therapeutics alone or in combination with other biomaterials for effective infection management, wound healing, and tissue regeneration.

Characterizing Novel Kras/p53 Derived NSCLC Cell Lines

Quincyn Perry - 2022

Abstract

Lung cancer is the leading cause of cancer-related death. It is the second most common cancer in both men and women. Non-small cell lung cancer (NSCLC) is the most common lung cancer at ~85%; with 40% adenocarcinomas and of these 25% are caused by a KRas mutation. The lethality of lung cancer is due to its propensity to metastasize. At time of diagnosis most patients present with late stage cancer which has already metastasized.

High Levels of Progesterone Receptor B in MCF‐7 Cells Enable Radical Anti‐Tumoral and Anti‐Estrogenic Effect of Progestin

Natasa Bajalovic - 2022

Abstract

The widely reported conflicting effects of progestin on breast cancer suggest that the progesterone receptor (PR) has dual functions depending on the cellular context. Cell models that enable PR to fully express anti‐tumoral properties are valuable for the understanding of molecular determinant(s) of the anti‐tumoral property. This study evaluated whether the expression of high levels of PR in MCF‐7 cells enabled a strong anti‐tumoral response to progestin. MCF‐7 cells were engineered to overexpress PRB by stable transfection. A single dose of Promegestone (R5020) induced an irreversible cell growth arrest and senescence‐associated secretory phenotype in MCF‐7 cells with PRB overexpression (MCF‐7PRB cells) but had no effect on MCF‐7 cells with PRA overexpression. The growth‐arresting effect was associated with downregulations of cyclin A2 and B1, CDK2, and CDK4 despite an initial upregulation of cyclin A2 and B1. R5020 also induced an evident activation of Nuclear Factor κB (NF‐κB) and upregulation of interleukins IL‐1α, IL‐1β, and IL‐8. Although R5020 caused a significant increase of CD24+CD44+ cell population, R5020‐treated MCF‐7PRB cells were unable to form tumorspheres and underwent massive apoptosis, which is paradoxically associated with marked downregulations of the pro‐apoptotic proteins BID, BAX, PARP, and Caspases 7 and 8, as well as diminution of anti‐apoptotic protein BCL‐2. Importantly, R5020‐activated PRB abolished the effect of estrogen. This intense anti‐estrogenic effect was mediated by marked downregulation of ERα and pioneer factor FOXA1, leading to diminished chromatin‐associated ERα and FOXA1 and estrogen‐induced target gene expression. In conclusion, high levels of agonist‐activated PRB in breast cancer cells can be strongly anti‐tumoral and anti‐estrogenic despite the initial unproductive cell cycle acceleration. Repression of ERα and FOXA1 expression is a major mechanism for the strong anti‐estrogenic effect.

Evaluation of transplacental transfer of mRNA vaccine products and functional antibodies during pregnancy and infancy

Mary Prahl - 2022

Abstract

Studies are needed to evaluate the safety and effectiveness of mRNA SARS-CoV-2 vaccination during pregnancy, and the levels of protection provided to their newborns through placental transfer of antibodies. Here, we evaluate the transplacental transfer of mRNA vaccine products and functional anti-SARS-CoV-2 antibodies during pregnancy and early infancy in a cohort of 20 individuals vaccinated during late pregnancy. We find no evidence of mRNA vaccine products in maternal blood, placenta tissue, or cord blood at delivery. However, we find time-dependent efficient transfer of IgG and neutralizing antibodies to the neonate that persists during early infancy. Additionally, using phage immunoprecipitation sequencing, we find a vaccine-specific signature of SARS-CoV-2 Spike protein epitope binding that is transplacentally transferred during pregnancy. Timing of vaccination during pregnancy is critical to ensure transplacental transfer of protective antibodies during early infancy.

Dor Rafael - 2022

Abstract

Latent infection is a characteristic feature of herpesviruses’ life cycle. Herpes simplex virus 1 is a common human pathogen that establishes lifelong latency in peripheral neurons. Symptomatic or asymptomatic periodic reactivations from the latent state allow the virus to replicate and spread among individuals. The latent viral genomes are found as several quiescent episomes inside the infected nuclei; however, it is not clear if and how many latent genomes are able to reactivate together. To address this question, we developed a quiescent infection assay, which provides a quantitative analysis of the number of genomes reactivating per cell, in cultured immortalized fibroblasts. We found that, almost always, only one viral genome reactivates per cell. We showed that different timing of entry to quiescence did not result in a significant change in the probability of reactivating. Reactivation from this quiescent state allowed only limited intergenomic recombination between two viral strains compared to lytic infection. Following coinfection with a mutant that is unable to reactivate, only coreactivation with a reactivation-proficient recombinant can provide the opportunity for the mutant to reactivate. We speculate that each individual quiescent viral genome has a low and stochastic chance to reactivate in each cell, an assumption that can explain the limited number of genomes reactivating per cell. IMPORTANCE Herpesviruses are highly prevalent and cause significant morbidity in the human and animal populations. Most individuals who are infected with herpes simplex virus (HSV-1), a common human pathogen, will become lifelong carriers of the virus, as HSV-1 establishes latent (quiescent) infections in the host cells. Reactivation from the latent state leads to many of the viral symptoms and to the spread of the virus among individuals. While many triggers for reactivation were identified, how many genomes reactivate from an individual cell and how are these genomes selected remain understudied. Here, we identify that, in most cases, only one genome per cell reactivates. Mutated HSV-1 genomes require coinfection with another strain to allow coreactivation. Our findings suggest that the decision to reactivate is determined for each quiescent genome separately and support the notion that reactivation preferences occur at the single-genome level.

The primary macrophage chemokine, CCL2, is not necessary after a peripheral nerve injury for macrophage recruitment and activation or for conditioning lesion enhanced peripheral regeneration

Aaron D. Talsma - 2022

Abstract

Background Peripheral nerve injuries stimulate the regenerative capacity of injured neurons through a neuroimmune phenomenon termed the conditioning lesion (CL) response. This response depends on macrophage accumulation in affected dorsal root ganglia (DRGs) and peripheral nerves. The macrophage chemokine CCL2 is upregulated after injury and is allegedly required for stimulating macrophage recruitment and pro-regenerative signaling through its receptor, CCR2. In these tissues, CCL2 is putatively produced by neurons in the DRG and Schwann cells in the distal nerve. Methods Ccl2fl/fl mice were crossed with Advillin-Cre, P0-Cre, or both to create conditional Ccl2 knockouts (CKOs) in sensory neurons, Schwann cells, or both to hypothetically remove CCL2 and macrophages from DRGs, nerves or both. CCL2 was localized using Ccl2–RFPfl/fl mice. CCL2–CCR2 signaling was further examined using global Ccl2 KOs and Ccr2gfp knock-in/knock-outs. Unilateral sciatic nerve transection was used as the injury model, and at various timepoints, chemokine expression, macrophage accumulation and function, and in vivo regeneration were examined using qPCR, immunohistochemistry, and luxol fast blue staining. Results Surprisingly, in all CKOs, DRG Ccl2 gene expression was decreased, while nerve Ccl2 was not. CCL2–RFP reporter mice revealed CCL2 expression in several cell types beyond the expected neurons and Schwann cells. Furthermore, macrophage accumulation, myelin clearance, and in vivo regeneration were unaffected in all CKOs, suggesting CCL2 may not be necessary for the CL response. Indeed, Ccl2 global knockout mice showed normal macrophage accumulation, myelin clearance, and in vivo regeneration, indicating these responses do not require CCL2. CCR2 ligands, Ccl7 and Ccl12, were upregulated after nerve injury and perhaps could compensate for the absence of Ccl2. Finally, Ccr2gfp knock-in/knock-out animals were used to differentiate resident and recruited macrophages in the injured tissues. Ccr2gfp/gfp KOs showed a 50% decrease in macrophages in the distal nerve compared to controls with a relative increase in resident macrophages. In the DRG there was a small but insignificant decrease in macrophages. Conclusions CCL2 is not necessary for macrophage accumulation, myelin clearance, and axon regeneration in the peripheral nervous system. Without CCL2, other CCR2 chemokines, resident macrophage proliferation, and CCR2-independent monocyte recruitment can compensate and allow for normal macrophage accumulation.

The splicing factor RBM17 drives leukemic stem cell maintenance by evading nonsense-mediated decay of pro-leukemic factors

Lina Liu - 2022

Abstract

Chemo-resistance in acute myeloid leukemia (AML) patients is driven by leukemic stem cells (LSCs) resulting in high rates of relapse and low overall survival. Here, we demonstrate that upregulation of the splicing factor, RBM17 preferentially marks and sustains LSCs and directly correlates with shorten patient survival. RBM17 knockdown in primary AML cells leads to myeloid differentiation and impaired colony formation and in vivo engraftment. Integrative multi-omics analyses show that RBM17 repression leads to inclusion of poison exons and production of nonsense-mediated decay (NMD)-sensitive transcripts for pro-leukemic factors and the translation initiation factor, EIF4A2. We show that EIF4A2 is enriched in LSCs and its inhibition impairs primary AML progenitor activity. Proteomic analysis of EIF4A2-depleted AML cells shows recapitulation of the RBM17 knockdown biological effects, including pronounced suppression of proteins involved in ribosome biogenesis. Overall, these results provide a rationale to target RBM17 and/or its downstream NMD-sensitive splicing substrates for AML treatment.

In Vivo Genome-Wide PGR Binding in Pregnant Human Myometrium Identifies Potential Regulators of Labor

Arial J. Dotts - 2022

Abstract

The alterations in myometrial biology during labor are not well understood. The myometrium is the contractile portion of the uterus and contributes to labor, a process that may be regulated by the steroid hormone progesterone. Thus, human myometrial tissues from term pregnant in-active-labor (TIL) and term pregnant not-in-labor (TNIL) subjects were used for genome-wide analyses to elucidate potential future preventive or therapeutic targets involved in the regulation of labor. Using myometrial tissues directly subjected to RNA sequencing (RNA-seq), progesterone receptor (PGR) chromatin immunoprecipitation sequencing (ChIP-seq), and histone modification ChIP-seq, we profiled genome-wide changes associated with gene expression in myometrial smooth muscle tissue in vivo. In TIL myometrium, PGR predominantly occupied promoter regions, including the classical progesterone response element, whereas it bound mainly to intergenic regions in TNIL myometrial tissue. Differential binding analysis uncovered over 1700 differential PGR-bound sites between TIL and TNIL, with 1361 sites gained and 428 lost in labor. Functional analysis identified multiple pathways involved in cAMP-mediated signaling enriched in labor. A three-way integration of the data for ChIP-seq, RNA-seq, and active histone marks uncovered the following genes associated with PGR binding, transcriptional activation, and altered mRNA levels: ATP11A, CBX7, and TNS1. In vitro studies showed that ATP11A, CBX7, and TNS1 are progesterone responsive. We speculate that these genes may contribute to the contractile phenotype of the myometrium during various stages of labor. In conclusion, we provide novel labor-associated genome-wide events and PGR-target genes that can serve as targets for future mechanistic studies.

O-GlcNAc transferase maintains metabolic homeostasis in response to CDK9 inhibition

Aishwarya Gondane - 2022

Abstract

Co-targeting of O-GlcNAc transferase (OGT) and the transcriptional kinase CDK9 is toxic to prostate cancer cells. As OGT is an essential glycosyltransferase, identifying an alternative target showing similar effects is of great interest. Here, we used a multiomics approach (transcriptomics, metabolomics and proteomics) to better understand the mechanistic basis of the combinatorial lethality between OGT and CDK9 inhibition. CDK9 inhibition preferentially affected transcription. In contrast, depletion of OGT activity predominantly remodeled the metabolome. Using an unbiased systems biology approach (weighted gene correlation network analysis), we discovered that CDK9 inhibition alters mitochondrial activity / flux, and high OGT activity is essential to maintain mitochondrial respiration when CDK9 activity is depleted. Our metabolite profiling data revealed that pantothenic acid (vitamin B5) is the metabolite that is most robustly induced by both OGT and OGT+CDK9 inhibitor treatments, but not by CDK9 inhibition alone. Finally, supplementing prostate cancer cell lines with vitamin B5 in the presence of CDK9 inhibitor mimics the effects of co-targeting OGT and CDK9.

Prebiotic Modulation of Intestinal Permeability

Rebecca Zasloff - 2022

Abstract

The maintenance of a functional intestinal barrier is critical to overall health, as a “leaky gut”, often a result of age-associated defects in intestinal barrier integrity, can lead to systemic health problems, including dysregulated inflammation and infections. The gut barrier is modulated by host factors, including mucin and tight junction proteins, and by members of the gut microbiota that may influence the expression of genes associated with intestinal permeability. While use of dietary prebiotics is becoming a popular method to beneficially modulate the gut microbiota, it is unclear how these complex carbohydrates can improve gut physiology. In this study, we characterized the effects of dietary prebiotics galactooligosaccharides (GOS) and LacNAc-enriched galacto-oligosaccharides (humanized, hGOS) on intestinal permeability in mice. FITC-dextran assays showed that GOS and hGOS restored intestinal barrier function in aging animals. However, a histology approach to visualize and quantify mucin within intestinal sections, revealed that GOS-fed animals had more mucus than hGOS or control-fed animals. Finally, reverse transcription qPCR assays were performed to determine gene expression of permeability associated genes, including mucin and tight junction proteins in prebiotic-fed animals. This work provides valuable insights into how intestinal permeability can be modulated by dietary prebiotics.

https://assets.researchsquare.com/files/rs-1636760/v1/9ba3bf53-9c02-4287-b663-6069e3e160c5.pdf?c=1652215844

Tito Habib - 2022

Abstract

Background Natural products are considered the most successful source of potential drug leads. Accordingly, the present study investigated the potential antidiabetic effect of the Egyptian honey bee venom fraction known as bradykinin potentiating factor (BPF) in streptozotocin-induced (STZ) diabetic rats. Materials & Methods An in vivo study was performed on fifty albino male rats that were divided into five groups. (G1): vehicle control animals, (G2): diabetic STZ-induced group, (G3): nondiabetic BPF-treated group, (G4): BPFinjected animals and post-treated with STZ, (G5): STZ-injected animals and post-treated with BPF. Plasma glucose levels and ALT, AST, C reactive protein (C-RP), apelin, and resistin gene expression in BPFtreated rats were evaluated and compared to STZ-treated diabetic rats and vehicle control rats. The plasma protein profile of the five animal groups was investigated by sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE). Results The data indicated that the STZ-treated (G2) group showed a highly significant increase in the levels of plasma glucose, ALT, and AST compared to the BPF-treated (G3, G4, G5), and nondiabetic control (G1) groups. Quantitative reverse transcription PCR (RT-qPCR) was carried out to amplify the apelin and resistin genes with an internal reference gene (18S rRNA) using a real-time PCR system. The concentrations of C-RP (28.3 kDa) and apelin (16 kDa) proteins observed by SDS-PAGE were higher than those of apelin, and resistin gene expression was revealed by RT-qPCR in STZ-treated (G2) rats compared with BPF-treated (G4, G5) and negative control (G1) rats. Conclusion The study concluded the importance of BPF, which has therapeutic and protective effects against STZ-induced diabetes complications. The hypoglycemic effect is revealed by the improvement of the biochemical and genetic markers, which may be attributed to BPF against diabetes complications.

The Role of Mediator Kinases CDK8/19 in the Acquired Resistance to CDK4/6 Targeting Drugs in Breast Cancer

Kaitlyn Digsby - 2022

Abstract

Breast cancer remains the highest cause of worldwide cancer-related mortality. Unfortunately, acquired therapeutic resistance occurs in the majority of cases of estrogen receptor-positive breast cancer. An effort to discover drugs which act synergistically with Palbociclib and prevent the development of resistance and tumor growth is essential to improving patient outcomes. Cyclin dependent kinases (CDK) 8 and 19 are Mediator complex proteins. The Mediator complex is an enzymatic module regulating transcription. The goal of the research is to elucidate the mechanism by which CDK8/19 inhibitor, SNX631, prevents acquired resistance to CDK4/6 inhibitor, Palbociclib. The present study aims to elucidate the role of the senescence-associated secretory phenotype (SASP) in Palbociclib resistance, and furthermore, to investigate if SASP is mitigated by a novel CDK8/19 inhibitor, SNX631. It was determined that SASP was suppressed by Palbociclib and therefore not a contributor to resistance. TGF-B, a member of SASP, was identified as a key modulator of both tumorigenesis and cell cycle arrest in breast cancer. The possibility that SNX631 suppresses TGF-B signaling to prevent Palbociclib resistance was explored. Overall, it was determined that SNX631 suppresses TGF-B/Smad3 signalingpreventing changes to TGF-B signaling that occur throughout Palbociclib treatment. Lastly, alternative mechanisms of Palbociclib resistance are present, and they should be further explored in the context of SNX631.

CO2 induced seawater acidification impacts survival and development of European eel embryos

Daniela E. Sganga - 2022

Abstract

Fish embryos may be vulnerable to seawater acidification resulting from anthropogenic carbon dioxide (CO2) emissions or from excessive biological CO2 production in aquaculture systems. This study investigated CO2 effects on embryos of the European eel (Anguilla anguilla), a catadromous fish that is considered at risk from climate change and that is targeted for hatchery production to sustain aquaculture of the species. Eel embryos were reared in three independent recirculation systems with different pH/CO2 levels representing “control” (pH 8.1, 300 μatm CO2), end-of-century climate change (“intermediate”, pH 7.6, 900 μatm CO2) and “extreme” aquaculture conditions (pH 7.1, 3000 μatm CO2). Sensitivity analyses were conducted at 4, 24, and 48 hours post-fertilization (hpf) by focusing on development, survival, and expression of genes related to acute stress response (crhr1, crfr2), stress/repair response (hsp70, hsp90), water and solute transport (aqp1, aqp3), acid-base regulation (nkcc1a, ncc, car15), and inhibitory neurotransmission (GABAAα6b, Gabra1). Results revealed that embryos developing at intermediate pH showed similar survival rates to the control, but egg swelling was impaired, resulting in a reduction in egg size with decreasing pH. Embryos exposed to extreme pH had 0.6-fold decrease in survival at 24 hpf and a 0.3-fold change at 48 compared to the control. These observed effects of acidification were not reflected by changes in expression of any of the here studied genes. On the contrary, differential expression was observed along embryonic development independent of treatment, indicating that the underlying regulating systems are under development and that embryos are limited in their ability to regulate molecular responses to acidification. In conclusion, exposure to predicted end-of-century ocean pCO2 conditions may affect normal development of this species in nature during sensitive early life history stages with limited physiological response capacities, while extreme acidification will negatively influence embryonic survival and development under hatchery conditions.

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.

CO2 induced seawater acidification impacts survival and development of European eel embryos

Daniela E. Sganga - 2022

Abstract

Sulforaphane alleviates hypoxic vestibular vertigo (HVV) by increasing NO production via upregulating the expression of NRF2

Liyuan Zhou - 2022

Abstract

Sulforaphane (SFP) treatment represses oxidative stress by activating NRF2. Meanwhile, SFP may also increase the production of nitric oxide (NO) and activate the signaling pathway of cyclic guanosine monophosphate (cGMP), which is involved in the pathogenesis of hypoxic vestibular vertigo (HVV). However, it remains unknown as whether SFP plays a therapeutic role in the treatment of HVV. A rat model of HVV was established to measure the levels of escape latency, malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD) in the aorta tissues. Quantitative real-time PCR was performed to evaluate the expression of NRF2 mRNA, and Western blot and immunohistochemistry were carried out to analyze the expression of NRF2 protein. ELISA was used to examine the production of NO and cGMP. SFP treatment helped to maintain the escape latency and MDA, GSH, SOD concentrations in the brain of HVV rats, and recovered the expression of NRF2 inhibited in the brain of HVV rats. SFP treatment also elevated NO and cGMP production that was down-regulated in the brain of HVV rats. On the cellular level, SFP enhanced the expression of NRF2, reduced the concentrations of MDA, GSH and SOD, and promoted the production of NO and cGMP in a dose-dependent manner. In this study, we treated an animal model of HVV with SFP to investigate its effect on NO production and oxidative stress. Our work provided a mechanistic insight into the therapeutic effect of SFP on the treatment of HVV.

A disease-driver population within interstitial cells of human calcific aortic valves identified via single-cell and proteomic profiling

Julius L. Decano - 2022

Abstract

Cellular heterogeneity of aortic valves complicates the mechanistic evaluation of the calcification processes in calcific aortic valve disease (CAVD), and animal disease models are lacking. In this study, we identify a disease-driver population (DDP) within valvular interstitial cells (VICs). Through stepwise single-cell analysis, phenotype-guided omic profiling, and network-based analysis, we characterize the DDP fingerprint as CD44highCD29+CD59+CD73+CD45low and discover potential key regulators of human CAVD. These DDP-VICs demonstrate multi-lineage differentiation and osteogenic properties. Temporal proteomic profiling of DDP-VICs identifies potential targets for therapy, including MAOA and CTHRC1. In vitro loss-of-function experiments confirm our targets. Such a stepwise strategy may be advantageous for therapeutic target discovery in other disease contexts.

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.

Bioreactor and Small Molecule Drug Applications in Hair Cell Differentiation

Sylvia Yip - 2021

Abstract

Hair cells are mechanoreceptors of the inner ear that convert sounds into electrical signals to be perceived by the brain. The gradual loss of hair cells is common after a lifetime of chronic exposure to loud noises. Mammalian hair cells lack the ability to regenerate after embryonic development. Advancements in regenerative medicine strategies have been employed to restore function to damaged organs. The use of stem cells can regenerate and replace damaged hair cells. Understanding how stem cells differentiate into hair cells will provide insight into regeneration. To study hair cell regeneration, we employed an inner ear organoid system. Immortalized multipotent otic progenitor (iMOP) cells that differentiate into spiral ganglion neurons, supporting cells, and hair cells were used to generate organoids. This study tests whether a bioreactor facilitates sensory epithelial differentiation. The bioreactor agitates the medium to promote nutrient diffusion in the cultures. After ten days of differentiation, iMOP-derived organoids were collected, and relative changes in transcripts and protein markers that correspond to neurons, supporting cells, and hair cells were determined. The use of a bioreactor increased the percentage of cells expressing MYO6, a hair cell marker. The percentage of TUBB3 labeled neurons also increased while GFAP labeled supporting cells remained the same. Relative levels of Myo6 mRNA did not increase in any of the culture conditions while relative levels of Tubb3 mRNA significantly increased in all cultures compared to control samples. Although molecular mechanisms are unknown, the study shows that use of the bioreactor improves hair cell differentiation in otic progenitor-derived organoids.

Carbonic anhydrase and soluble adenylate cyclase regulation of cystic fibrosis cellular phenotypes

Kathleen Boyne - 2022

Abstract

Several aspects of the cell biology of cystic fibrosis (CF) epithelial cells are altered including impaired lipid regulation, disrupted intracellular transport, and impaired microtubule regulation. It is unclear how the loss of cystic fibrosis transmembrane conductance regulator (CFTR) function leads to these differences. It is hypothesized that the loss of CFTR function leads to altered regulation of carbonic anhydrase (CA) activity resulting in cellular phenotypic changes. In this study, it is demonstrated that CA2 protein expression is reduced in CF model cells, primary mouse nasal epithelial (MNE) cells, excised MNE tissue, and primary human nasal epithelial cells (p<0.05). This corresponds to a decrease in CA2 RNA expression measured by qPCR as well as an overall reduction in CA activity in primary CF MNEs. The addition of CFTR inhibitor-172 to WT MNE cells for ≥24 h mimics the significantly lower protein expression of CA2 in CF cells. Treatment of CF cells with L-Phenylalanine (L-Phe), an activator of CA activity, restores endosomal transport through an effect on microtubule regulation in a manner dependent on soluble adenylate cyclase (sAC). This effect can be blocked with the CA2- selective inhibitor dorzolamide. These data suggest the loss of CFTR function leads to the decreased expression of CA2 resulting in the downstream cell signaling alterations observed in CF.

Microbial pathogens induce neurodegeneration in Alzheimer’s disease mice: protection by microglial regulation

Tal Ganz - 2022

Abstract

Background Neurodegeneration is considered the consequence of misfolded proteins’ deposition. Little is known about external environmental effects on the neurodegenerative process. Infectious agent-derived pathogen-associated molecular patterns (PAMPs) activate microglia, key players in neurodegenerative diseases. We hypothesized that systemic microbial pathogens may accelerate neurodegeneration in Alzheimer’s disease (AD) and that microglia play a central role in this process. Methods We examined the effect of an infectious environment and of microbial Toll-like receptor (TLR) agonists on cortical neuronal loss and on microglial phenotype in wild type versus 5xFAD transgenic mice, carrying mutated genes associated with familial AD. Results We examined the effect of a naturally bred environment on the neurodegenerative process. Earlier and accelerated cortical neuron loss occurred in 5xFAD mice housed in a natural (“dirty”) environment than in a specific-pathogen-free (SPF) environment, without increasing the burden of Amyloid deposits and microgliosis. Neuronal loss occurred in a microglia-rich cortical region but not in microglia-poor CA regions of the hippocampus. Environmental exposure had no effect on cortical neuron density in wild-type mice. To model the neurodegenerative process caused by the natural infectious environment, we injected systemically the bacterial endotoxin lipopolysaccharide (LPS), a TLR4 agonist PAMP. LPS caused cortical neuronal death in 5xFAD, but not wt mice. We used the selective retinoic acid receptor α agonist Am580 to regulate microglial activation. In primary microglia isolated from 5xFAD mice, Am580 markedly attenuated TLR agonists-induced iNOS expression, without canceling their basic immune response. Intracerebroventricular delivery of Am580 in 5xFAD mice reduced significantly the fraction of (neurotoxic) iNOS + microglia and increased the fraction of (neuroprotective) TREM2 + microglia. Furthermore, intracerebroventricular delivery of Am580 prevented neurodegeneration induced by microbial TLR agonists. Conclusions Exposure to systemic infections causes neurodegeneration in brain regions displaying amyloid pathology and high local microglia density. AD brains exhibit increased susceptibility to microbial PAMPs’ neurotoxicity, which accelerates neuronal death. Microglial modulation protects the brain from microbial TLR agonist PAMP-induced neurodegeneration.

Osteogenic lithium-doped brushite cements for bone regeneration

K. Hurle - 2021

Abstract

This study investigated the osteogenic performance of new brushite cements obtained from Li+-doped β-tricalcium phosphate as a promising strategy for bone regeneration. Lithium (Li+) is a promising trace element to encourage the migration and proliferation of adipose-derived stem cells (hASCs) and the osteogenic differentiation-related gene expression, essential for osteogenesis. In-situ X-ray diffraction (XRD) and in-situ 1H nuclear magnetic resonance (1H NMR) measurements proved the precipitation of brushite, as main phase, and monetite, indicating that Li+ favored the formation of monetite under certain conditions. Li+ was detected in the remaining pore solution in significant amounts after the completion of hydration. Isothermal calorimetry results showed an accelerating effect of Li+, especially for low concentration of the setting retarder (phytic acid). A decrease of initial and final setting times with increasing amount of Li+ was detected and setting times could be well adjusted by varying the setting retarder concentration. The cements presented compressive mechanical strength within the ranges reported for cancellous bone. In vitro assays using hASCs showed normal metabolic and proliferative levels. The immunodetection and gene expression profile of osteogenic-related markers highlight the incorporation of Li+ for increasing the in vivo bone density. The osteogenic potential of Li-doped brushite cements may be recommended for further research on bone defect repair strategies.

The Non-Erythropoietic EPO Analogue Cibinetide Inhibits Osteoclastogenesis In Vitro and Increases Bone Mineral Density in Mice

Zamzam Awida - 2021

Abstract

The two erythropoietin (EPO) receptor forms mediate different cellular responses to erythropoietin. While hematopoiesis is mediated via the homodimeric EPO receptor (EPOR), tissue protection is conferred via a heteromer composed of EPOR and CD131. In the skeletal system, EPO stimulates osteoclast precursors and induces bone loss. However, the underlying molecular mechanisms are still elusive. Here, we evaluated the role of the heteromeric complex in bone metabolism in vivo and in vitro by using Cibinetide (CIB), a non-erythropoietic EPO analogue that exclusively binds the heteromeric receptor. CIB is administered either alone or in combination with EPO. One month of CIB treatment significantly increased the cortical (~5.8%) and trabecular (~5.2%) bone mineral density in C57BL/6J WT female mice. Similarly, administration of CIB for five consecutive days to female mice that concurrently received EPO on days one and four, reduced the number of osteoclast progenitors, defined by flow cytometry as Lin−CD11b−Ly6Chi CD115+, by 42.8% compared to treatment with EPO alone. In addition, CIB alone or in combination with EPO inhibited osteoclastogenesis in vitro. Our findings introduce CIB either as a stand-alone treatment, or in combination with EPO, as an appealing candidate for the treatment of the bone loss that accompanies EPO treatment

Colonic Medium-Chain Fatty Acids Act as a Source of Energy and for Colon Maintenance but Are Not Utilized to Acylate Ghrelin

András Gregor - 2021

Abstract

The capacity of microbiota to produce medium-chain fatty acids (MCFA) and related consequences for the gastrointestinal (GI) tract have never been reported before. We verified the impact of nutrition-related factors on fatty acid (FAs) production and found that caloric restriction decreased levels of most of MCFAs in the mouse cecum, whereas overnight fasting reduced the levels of acetate and butyrate but increased propionate and laurate. A diet high in soluble fibre boosted the production of short-chain fatty acids (SCFA) and caproate whereas a high-cellulose diet did not have an effect or decreased the levels of some of the FAs. Rectal infusion of caprylate resulted in its rapid metabolism for energy production. Repeated 10-day MCFA infusion impacted epididymal white adipose tissue (eWAT) weight and lipid accumulation. Repeated infusion of caprylate rectally tended to increase the concentration of active ghrelin in mice plasma; however, this increase was not statistically significant. In Caco-2 cells, caprylate increased the expression of Fabp2, Pdk4, Tlr3, and Gpr40 genes as well as counteracted TNFα-triggered downregulation of Pparγ, Occludin, and Zonulin mRNA expression. In conclusion, we show that colonic MCFAs can be rapidly utilized as a source of energy or stored as a lipid supply. Further, locally produced caprylate may impact metabolism and inflammatory parameters in the colon.

Dual role of neutrophils in modulating liver injury and fibrosis during development and resolution of diet-induced murine steatohepatitis

Andrea D. Kim - 2021

Abstract

Inflammatory changes in the liver represent a key feature of non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease (NAFLD). Innate immune activation including hepatic neutrophilic infiltration acts as an important inflammatory trigger as well as a potential mediator of inflammation resolution. In this study, we dissected the effects of neutrophil depletion via anti-lymphocyte antigen 6 complex locus G6D (Ly6G) antibodies administration during ongoing high fat-fructose-cholesterol (FFC) diet-induced murine NASH and during inflammation resolution by switching into a low-fat control diet. During NASH progression, protective effects were shown as HSC activation, cell infiltration and activation of pro-inflammatory macrophages were ameliorated. Furthermore, these changes were contrasted with the effects observed when neutrophil depletion was performed during the resolution phase. Impaired resolving mechanisms, such as a failure to balance the pro and anti-inflammatory cytokines ratio, deficient macrophage phenotypic switch into a pro-restorative profile, and defective repair and remodeling processes were observed when neutrophils were depleted in this scenario. This study described phase-dependent contrasting roles of neutrophils as triggers and pro-resolutive mediators of liver injury and fibrosis associated with diet-induced NASH in mice. These findings have important translational implications at the time of designing NASH therapeutic strategies.

Human and mouse muscle transcriptomic analyses identify insulin receptor mRNA downregulation in hyperinsulinemia-associated insulin resistance

Haoning Howard Cen - 2021

Abstract

Hyperinsulinemia is commonly viewed as a compensatory response to insulin resistance, yet studies have demonstrated that chronically elevated insulin may also drive insulin resistance. The molecular mechanisms underpinning this potentially cyclic process remain poorly defined, especially on a transcriptome-wide level. Transcriptomic meta-analysis in >450 human samples demonstrated that fasting insulin reliably and negatively correlated with INSR mRNA in skeletal muscle. To establish causality and study the direct effects of prolonged exposure to excess insulin in muscle cells, we incubated C2C12 myotubes with elevated insulin for 16 h, followed by 6 h of serum starvation, and established that acute AKT and ERK signaling were attenuated in this model of in vitro hyperinsulinemia. Global RNA-sequencing of cells both before and after nutrient withdrawal highlighted genes in the insulin receptor (INSR) signaling, FOXO signaling, and glucose metabolism pathways indicative of ‘hyperinsulinemia’ and ‘starvation’ programs. Consistently, we observed that hyperinsulinemia led to a substantial reduction in Insr gene expression, and subsequently a reduced surface INSR and total INSR protein, both in vitro and in vivo. Bioinformatic modeling combined with RNAi identified SIN3A as a negative regulator of Insr mRNA (and JUND, MAX, and MXI as positive regulators of Irs2 mRNA). Together, our analysis identifies mechanisms which may explain the cyclic processes underlying hyperinsulinemia-induced insulin resistance in muscle, a process directly relevant to the etiology and disease progression of type 2 diabetes.

Anti-Biofilm Activity of Cannabigerol against Streptococcus mutans

Muna Aqawi - 2021

Abstract

Streptococcus mutans is a common cariogenic bacterium in the oral cavity involved in plaque formation. Previous studies showed that Cannabigerol (CBG) has bacteriostatic and bacteriocidic activity against S. mutans. The aim of the present study was to study its effect on S. mutans biofilm formation and dispersion. S. mutans was cultivated in the presence of CBG, and the resulting biofilms were examined by CV staining, MTT assay, qPCR, biofilm tracer, optical profilometry, and SEM. Gene expression was determined by real-time qPCR, extracellular polysaccharide (EPS) production was determined by Congo Red, and reactive oxygen species (ROS) were determined using DCFH-DA. CBG prevented the biofilm formation of S. mutans shown by reduced biofilm biomass, decreased biofilm thickness, less EPS production, reduced DNA content, diminished metabolic activity, and increased ROS levels. CBG altered the biofilm roughness profile, resulting in a smoother biofilm surface. When treating preformed biofilms, CBG reduced the metabolic activity of S. mutans with a transient effect on the biomass. CBG reduced the expression of various genes involved in essential metabolic pathways related to the cariogenic properties of S. mutans biofilms. Our data show that CBG has anti-biofilm activities against S. mutans and might be a potential drug for preventive treatment of dental caries.

A Novel Spectral Annotation Strategy Streamlines Reporting of mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-y

Shiori Kuraoka - 2021

Abstract

Mass spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4-hour exposure to systemic IFN-. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method.

Nuclear Organization during Hepatogenesis in Zebrafish Requires Uhrf1

Bhavani P. Madakashira - 2021

Abstract

Acquisition of cellular fate during development is initiated and maintained by well-coordinated patterns of gene expression that are dictated by the epigenetic landscape and genome organization in the nucleus. While the epigenetic marks that mediate developmental gene expression patterns during organogenesis have been well studied, less is known about how epigenetic marks influence nuclear organization during development. This study examines the relationship between nuclear structure, chromatin accessibility, DNA methylation, and gene expression during hepatic outgrowth in zebrafish larvae. We investigate the relationship between these features using mutants that lack DNA methylation. Hepatocyte nuclear morphology was established coincident with hepatocyte differentiation at 80 h post-fertilization (hpf), and nuclear shape and size continued to change until the conclusion of outgrowth and morphogenesis at 120 hpf. Integrating ATAC-Seq analysis with DNA methylation profiling of zebrafish livers at 120 hpf showed that closed and highly methylated chromatin occupies most transposable elements and that open chromatin correlated with gene expression. DNA hypomethylation, due to mutation of genes encoding ubiquitin-like, containing PHD and RING Finger Domains 1 (uhrf1) and DNA methyltransferase (dnmt1), did not block hepatocyte differentiation, but had dramatic effects on nuclear organization. Hepatocytes in uhrf1 mutants have large, deformed nuclei with multiple nucleoli, downregulation of nucleolar genes, and a complete lack of the nuclear lamina. Loss of lamin B2 staining was phenocopied by dnmt1 mutation. Together, these data show that hepatocyte nuclear morphogenesis coincides with organ morphogenesis and outgrowth, and that DNA methylation directs chromatin organization, and, in turn, hepatocyte nuclear shape and size during liver development.

Food Chemistry: Molecular Sciences 3 (2021) 100034Available online 13 July 20212666-5662/© 2021 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Direct effects of p

Oren Hadaya - 2021

Abstract

We assessed the potential of Pistacia lentiscus (lentisk) phenolic compounds to enhance production of milk composition in lactating goats and caprine primary mammary epithelial cells (MEC). Damascus goats were given a lentisk infusion (LI) or fresh water (FW) to drink, in a crossover design. Milk from LI vs. FW goats was 0.43% richer in fat and 30% in omega 3 fatty acids. Lentisk infusion enhanced antioxidant capacity of plasma and milk by 37% and 30% respectively, and induced transcriptional activation of antioxidant genes. To assess the direct effect of polyphenols on milk quality in terms of composition and antioxidant capacity, we used plasma collected from goats fed hay (HP) or browsed on phenolic compounds-rich pasture (primarily lentisk; PP) as a conditioning medium for primary culture of MEC. PP increased 2-fold cellular triglyceride content and 2.4-fold intracellular casein, and increased ATP production and non-mitochondrial oxygen consumption. Taken together, the results imply that lentisk phenolic compounds affect blood, MEC and milk oxidative status, which increase fat pro-duction by the mammary gland.

Time-restricted feeding normalizes hyperinsulinemia to inhibit breast cancer in obese postmenopausal mouse models

Manasi Das - 2021

Abstract

Accumulating evidence indicates that obesity with its associated metabolic dysregulation, including hyperinsulinemia and aberrant circadian rhythms, increases the risk for a variety of cancers including postmenopausal breast cancer. Caloric restriction can ameliorate the harmful metabolic effects of obesity and inhibit cancer progression but is difficult to implement and maintain outside of the clinic. In this study, we aim to test a time-restricted feeding (TRF) approach on mouse models of obesity-driven postmenopausal breast cancer. We show that TRF abrogates the obesity-enhanced mammary tumor growth in two orthotopic models in the absence of calorie restriction or weight loss. TRF also reduces breast cancer metastasis to the lung. Furthermore, TRF delays tumor initiation in a transgenic model of mammary tumorigenesis prior to the onset of obesity. Notably, TRF increases whole-body insulin sensitivity, reduces hyperinsulinemia, restores diurnal gene expression rhythms in the tumor, and attenuates tumor growth and insulin signaling. Importantly, inhibition of insulin secretion with diazoxide mimics TRF whereas artificial elevation of insulin through insulin pumps implantation reverses the effect of TRF, suggesting that TRF acts through modulating hyperinsulinemia. Our data suggest that TRF is likely to be effective in breast cancer prevention and therapy.

The Expression Levels and Cellular Localization of Pigment Epithelium Derived Factor (PEDF) in Mouse Testis: Its Possible Involvement in the Differentiation of Spermatogonial Cells

Noy Bagdadi - 2021

Abstract

Pigment epithelium derived factor (PEDF) is a multifunctional secretory soluble glycoprotein that belongs to the serine protease inhibitor (serpin) family. It was reported to have neurotrophic, anti-angiogenic and anti-tumorigenic activity. Recently, PEDF was found in testicular peritubular cells and it was assumed to be involved in the avascular nature of seminiferous tubules. The aim of this study was to determine the cellular origin, expression levels and target cells of PEDF in testicular tissue of immature and adult mice under physiological conditions, and to explore its possible role in the process of spermatogenesis in vitro. Using immunofluorescence staining, we showed that PEDF was localized in spermatogenic cells at different stages of development as well as in the somatic cells of the testis. Its protein levels in testicular homogenates and Sertoli cells supernatant showed a significant decrease with age. PEDF receptor (PEDF-R) was localized within the seminiferous tubule cells and in the interstitial cells compartment. Its RNA expression levels showed an increase with age until 8 weeks followed by a decrease. RNA levels of PEDF-R showed the opposite trend of the protein. Addition of PEDF to cultures of isolated cells from the seminiferous tubules did not changed their proliferation rate, however, a significant increase was observed in number of meiotic/post meiotic cells at 1000 ng/mL of PEDF; indicating an in vitro differentiation effect. This study may suggest a role for PEDF in the process of spermatogenesis.

Heterogeneity in PHGDH protein expression potentiates cancer cell dissemination and metastasis

Matteo Rossi - 2021

Abstract

Cancer metastasis requires the transient activation of cellular programs enabling dissemination and seeding in distant organs. Genetic, transcriptional and translational intra-tumor heterogeneity contributes to this dynamic process. Beyond this, metabolic intra-tumor heterogeneity has also been observed, yet its role for cancer progression remains largely elusive. Here, we discovered that intra-tumor heterogeneity in phosphoglycerate dehydrogenase (PHGDH) protein expression drives breast cancer cell dissemination and metastasis formation. Specifically, we observed intra-tumor heterogeneous PHGDH expression in primary breast tumors, with low PHGDH expression being indicative of metastasis in patients. In mice, Phgdh protein, but not mRNA, expression is low in circulating tumor cells and early metastatic lesions, leading to increased dissemination and metastasis formation. Mechanistically, low PHGDH protein expression induces an imbalance in glycolysis that can activate sialic acid synthesis. Consequently, cancer cells undergo a partial EMT and show increased p38 as well as SRC phosphorylation, which activate cellular programs of dissemination. In turn, inhibition of sialic acid synthesis through knock-out of cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) counteracts the increased cancer cell dissemination and metastasis induced by low PHGDH expression. In conclusion, we find that heterogeneity in PHGDH protein expression promotes cancer cell dissemination and metastasis formation

Evaluation of PC12 Cell Neural Differentiation on Graphene Coated ITO Microchips

Tansu Golcez - 2020

Abstract

In this study, the impact of graphene on neuronal differentiation of PC12 cells into neuron-like cells was evaluated in conjunction with electrical stimuli. First, an ITO (Indium Tin Oxide) microchip with a certain number of electrodes was fabricated using photolithography and then a chemically synthesized graphene was coated on the microchip. The electrical stimulation was applied through the ITO-microchip. Following optimization of neuronal differentiation conditions, the effect of AC and DC electrical stimulation on both bare and graphenecoated ITO-microchips for neuronal differentiation was investigated. According to the results, it was observed that electrical stimulation with direct current for 30 minutes caused a large degree of neuronal cell differentiation on the graphene coated ITO-microchips. The results were also verified by real-time qPCR.

Transition between canonical to non-canonical Wnt signaling during interactions between mesenchymal stem cells and osteosarcomas

Masha Asulin - 2020

Abstract

Background: Wnt signaling pathways are taking a part in regulation of cell fate decisions in normal and cancerous cells. In some cancer types, a transition from canonical to non-canonical Wnt signaling pathways was identifi ed, a phenomenon, that in return led to increase proliferation, invasiveness and metastasis. Methods: In the current in vitro study we investigated the infl uence of MSCs, co-cultured in direct and indirect contact with OS cells, on the role of Wnt signaling pathways and tumor aggressiveness. Sub-populations were separated using Boyden chambers. Gene expression profi les were determined by qPCR. Results: The results revealed that interactions with MSCs increased migration and invasion capacities along with OS proliferation. Moreover, canonical Wnt signaling activity was low in OS, and co-culture with MSC. However, MSCs did not trigger a switch between the canonical to the no-canonical Wnt pathways. In addition, a more aggressive OS sub-population tend to undergo a transition towards the non-canonical pathway. Moreover, this aggressive subtype presented cancer stem-cells like characteristic. Conclusions: We submit that the progression in OS aggressiveness is attributed to a transition in Wnt signaling from canonical to non-canonical pathways, although MSCs are likely to take a part during the tumor progression, in the case of OS, they did not affect the Wnt switch. These complex tumor promoting interactions may be found in the natural and tumorigenic bone microenvironment. A better understanding of the molecular signaling mechanisms involved in the tumor development and metastasis may contribute to development of new cancer therapies.

Choroid plexus LAT2 and SNAT3 as partners in CSF amino acid homeostasis maintenance

Elena Dolgodilina - 2020

Abstract

Background Cerebrospinal fluid (CSF) is mainly produced by the choroid plexus (CP) located in brain ventricles. Although derived from blood plasma, it is nearly protein-free (~ 250-fold less) and contains about 2–20-fold less free amino acids, with the exception of glutamine (Gln) which is nearly equal. The aim of this study was to determine which amino acid transporters are expressed in mouse CP epithelium in order to gain understanding about how this barrier maintains the observed amino acid concentration gradient. Methods Expression of amino acid transporters was assessed in isolated choroid plexuses (CPs) by qRT-PCR followed by localization studies using immunofluorescence with specific antibodies. The impact of LAT2 (Slc7a8) antiporter deletion on CSF amino acids was determined. Results The purity of isolated choroid plexuses was tested on the mRNA level using specific markers, in particular transthyretin (Ttr) that was enriched 330-fold in CP compared to cerebral tissue. In a first experimental round, 14 out of 32 Slc amino acid transporters tested on the mRNA level by qPCR were selected for further investigation. Out of these, five were considered highly expressed, SNAT1 (Slc38a1), SNAT3 (Slc38a3), LAT2 (Slc7a8), ASC1 (Slc7a10) and SIT1 (Slc6a20b). Three of them were visualized by immunofluorescence: SNAT1 (Slc38a1), a neutral amino acid-Na+ symporter, found at the blood side basolateral membrane of CP epithelium, while SNAT3 (Slc38a3), an amino acid-Na+ symporter and H+ antiporter, as well as LAT2 (Slc7a8), a neutral amino acid antiporter, were localized at the CSF-facing luminal membrane. In a LAT2 knock-out mouse model, CSF Gln was unchanged, whereas other amino acids normally 2–20-fold lower than in plasma, were increased, in particular the LAT2 uptake substrates leucine (Leu), valine (Val) and tryptophan (Trp) and some other amino acids such as glutamate (Glu), glycine (Gly) and proline (Pro). Conclusion These results suggest that Gln is actively transported by SNAT1 from the blood into CP epithelial cells and then released luminally into CSF via SNAT3 and LAT2. Its efflux via LAT2 may drive the reuptake from the CSF of essential amino acid substrates of this antiporter and thereby participates to maintaining the amino acid gradient between plasma and CSF.

Conserved statin-mediated activation of the p38-MAPK pathway protects Caenorhabditis elegans from the cholesterol-independent effects of statins

Irina Langier Goncalves - 2020

Abstract

Objective Statins are a group of medications that reduce cholesterol synthesis by inhibiting the activity of HMG-CoA reductase, a key enzyme in the mevalonate pathway. The clinical use of statins to lower excess cholesterol levels has revolutionized the cardiovascular field and increased the survival of millions, but some patients have adverse side effects. A growing body of data suggests that some of the beneficial and adverse effects of statins, including their anti-inflammatory, anti-tumorigenic, and myopathic activities, are cholesterol-independent. However, the underlying mechanisms for these effects of statins are not well defined. Methods Because Caenorhabditis elegans (C. elegans) lacks the cholesterol synthesis branch of the mevalonate pathway, this organism is a powerful system to unveil the cholesterol-independent effects of statins. We used genetic and biochemical approaches in C. elegans and cultured macrophage-derived murine cells to study the cellular response to statins. Results We found that statins activate a conserved p38-MAPK (p38) cascade and that the protein geranylgeranylation branch of the mevalonate pathway links the effect of statins to the activation of this p38 pathway. We propose that the blockade of geranylgeranylation impairs the function of specific small GTPases we identified as upstream regulators of the p38 pathway. Statin-mediated p38 activation in C. elegans results in the regulation of programs of innate immunity, stress, and metabolism. In agreement with this regulation, knockout of the p38 pathway results in the hypersensitivity of C. elegans to statins. Treating cultured mammalian cells with clinical doses of statins results in the activation of the same p38 pathway, which upregulates the COX-2 protein, a major regulator of innate immunity in mammals. Conclusions Statins activate an evolutionarily conserved p38 pathway to regulate metabolism and innate immunity. Our results highlight the cytoprotective role of p38 activation under statin treatment in vivo and propose that this activation underlies many of the critical cholesterol-independent effects of statins.

Introduction of a green algal squalene synthase enhances squalene accumulation in a strain of Synechocystis sp. PCC 6803

Bagmi Pattanaik - 2020

Abstract

Squalene is a triterpene which is produced as a precursor for a wide range of terpenoid compounds in many organisms. It has commercial use in food and cosmetics but could also be used as a feedstock for production of chemicals and fuels, if generated sustainably on a large scale. We have engineered a cyanobacterium, Synechocystis sp. PCC 6803, for production of squalene from CO2. In this organism, squalene is produced via the methylerythritol-phosphate (MEP) pathway for terpenoid biosynthesis, and consumed by the enzyme squalene hopene cyclase (Shc) for generation of hopanoids. The gene encoding Shc in Synechocystis was inactivated (Δshc) by insertion of a gene encoding a squalene synthase from the green alga Botryococcus braunii, under control of an inducible promoter. We could demonstrate elevated squalene generation in cells where the algal enzyme was induced. Heterologous overexpression of genes upstream in the MEP pathway further enhanced the production of squalene, to a level three times higher than the Δshc background strain. During growth in flat panel bioreactors, a squalene titer of 5.1 mg/L of culture was reached.

Blimp-1 is essential for allergen-induced asthma and Th2 cell development in the lung

Kun He - 2020

Abstract

A Th2 immune response is central to allergic airway inflammation, which afflicts millions worldwide. However, the mechanisms that augment GATA3 expression in an antigen-primed developing Th2 cell are not well understood. Here, we describe an unexpected role for Blimp-1, a transcriptional repressor that constrains autoimmunity, as an upstream promoter of GATA3 expression that is critical for Th2 cell development in the lung to inhaled but not systemically delivered allergens but is dispensable for TFH function and IgE production. Mechanistically, Blimp-1 acts through Bcl6, leading to increased GATA3 expression in lung Th2 cells. Surprisingly, the anti-inflammatory cytokine IL-10, but not the pro-inflammatory cytokines IL-6 or IL-21, is required via STAT3 activation to up-regulate Blimp-1 and promote Th2 cell development. These data reveal a hitherto unappreciated role for an IL-10–STAT3–Blimp-1 circuit as an initiator of an inflammatory Th2 response in the lung to allergens. Thus, Blimp-1 in a context-dependent fashion can drive inflammation by promoting rather than terminating effector T cell responses.

Molecular Mechanisms Underlying the Absorption of Aglycone and Glycosidic Flavonoids in a Caco-2 BBe1 Cell Model

Hua Zhang - 2020

Abstract

The mechanisms of cellular absorption and transport underlying the differences between flavonoid aglycones and glycosides and the effect of the structural feature are not well established. In this study, aglycone, mono-, and diglycosides of quercetin and cyanidin were selected to examine the effects of the structural feature on the bioavailability of flavonoids using hexose transporters SGLT1 and GLUT2 in a Caco-2 BBe1 cell model. Cellular uptake and transport of all glycosides were significantly different. The glycosides also significantly inhibited cellular uptake of d-glucose, indicating the involvement of the two hexose transporters SGLT1 and GLUT2 in the absorption, and the potential of the glycosides in lowering the blood glucose level. The in silico prediction model also supported these observations. The absorption of glycosides, especially diglycosides but not the aglycones, was significantly blocked by SGLT1 and GLUT2 inhibitors (phloridzin and phloretin) and further validated in SGLT1 knockdown Caco-2 BBe1 cells.

Extrahepatic cholangiocyte obstruction is mediated by decreased glutathione, Wnt and Notch signaling pathways in a toxic model of biliary atresia

Sophia Fried - 2020

Abstract

Biliary atresia is a neonatal liver disease with extrahepatic bile duct obstruction and progressive liver fibrosis. The etiology and pathogenesis of the disease are unknown. We previously identified a plant toxin, biliatresone, responsible for biliary atresia in naturally-occurring animal models, that causes cholangiocyte destruction in in-vitro models. Decreases in reduced glutathione (GSH) mimic the effects of biliatresone, and agents that replenish cellular GSH ameliorate the effects of the toxin. The goals of this study were to define signaling pathways downstream of biliatresone that lead to cholangiocyte destruction and to determine their relationship to GSH. Using cholangiocyte culture and 3D cholangiocyte spheroid cultures, we found that biliatresone and decreases in GSH upregulated RhoU/Wrch1, a Wnt signaling family member, which then mediated an increase in Hey2 in the NOTCH signaling pathway, causing downregulation of the transcription factor Sox17. When these genes were up- or down-regulated, the biliatresone effect on spheroids was phenocopied, resulting in lumen obstruction. Biopsies of patients with biliary atresia demonstrated increased RhoU/Wrch1 and Hey2 expression in cholangiocytes. We present a novel pathway of cholangiocyte injury in a model of biliary atresia, which is relevant to human BA and may suggest potential future therapeutics.

Expanding the search for genetic biomarkers of Parkinson's disease into the living brain

Simon M. Benoit - 2020

Abstract

Altered gene expression related to Parkinson's Disease (PD) has not been described in the living brain, yet this information may support novel discovery pertinent to disease pathophysiology and treatment. This study compared the transcriptome in brain biopsies obtained from living PD and Control patients. To evaluate the novelty of this data, a comprehensive literature review also compared differentially expressed gene (DEGs) identified in the current study with those reported in PD cadaveric brain and peripheral tissues. RNA was extracted from rapidly cryopreserved frontal lobe specimens collected from PD and Control patients undergoing neurosurgical procedures. RNA sequencing (RNA-Seq) was performed and validated using quantitative polymerase chain reaction. DEG data was assessed using bioinformatics and subsequently included within a comparative analysis of PD RNA-Seq studies. 370 DEGs identified in living brain specimens reflected diverse gene groups and included key members of trophic signaling, apoptosis, inflammation and cell metabolism pathways. The comprehensive literature review yielded 7 RNA-Seq datasets generated from blood, skin and cadaveric brain but none from a living brain source. From the current dataset, 123 DEGs were identified only within the living brain and 267 DEGs were either newly found or had distinct directional change in living brain relative to other tissues. This is the first known study to analyze the transcriptome in brain tissue from living PD and Control patients. The data produced using these methods offer a unique, unexplored resource with potential to advance insight into the genetic associations of PD.

Embryonic development and secondary axis induction in the Brazilian white knee tarantula Acanthoscurria geniculata, C. L. Koch, 1841 (Araneae; Mygalomorphae; Theraphosidae)

Matthias Pechmann - 2020

Abstract

Tarantulas represent some of the heaviest and most famous spiders. However, there is little information about the embryonic development of these spiders or their relatives (infraorder Mygalomorphae) and time-lapse recording of the embryonic development is entirely missing. I here describe the complete development of the Brazilian white knee tarantula, Acanthoscurria geniculata, in fixed and live embryos. The establishment of the blastoderm, the formation, migration and signalling of the cumulus and the shape changes that occur in the segment addition zone are analysed in detail. In addition, I show that there might be differences in the contraction process of early embryos of different theraphosid spider species. A new embryonic reference transcriptome was generated for this study and was used to clone and analyse the expression of several important developmental genes. Finally, I show that embryos of A. geniculata are amenable to tissue transplantation and bead insertion experiments. Using these functional approaches, I induced axis duplication in embryos via cumulus transplantation and ectopic activation of BMP signalling. Overall, the mygalomorph spider A. geniculata is a useful laboratory system to analyse evolutionary developmental questions, and the availability of such a system will help understanding conserved and divergent aspects of spider/chelicerate development.

Improving synthetic methylotrophy via dynamic formaldehyde regulation of pentose phosphate pathway genes and redox perturbation

Julia Rohlhill - 2020

Abstract

Escherichia coli is an ideal choice for constructing synthetic methylotrophs capable of utilizing the non-native substrate methanol as a carbon and energy source. All current E. coli-based synthetic methylotrophs require co-substrates. They display variable levels of methanol-carbon incorporation due to a lack of native regulatory control of biosynthetic pathways, as E. coli does not recognize methanol as a proper substrate despite its ability to catabolize it. Here, using the E. coli formaldehyde-inducible promoter Pfrm, we implement dynamic expression control of select pentose-phosphate genes in response to the formaldehyde produced upon methanol oxidation. Genes under Pfrm control exhibited 8- to 30-fold transcriptional upregulation during growth on methanol. Formaldehyde-induced episomal expression of the B. methanolicus rpe and tkt genes involved in the regeneration of ribulose 5-phosphate required for formaldehyde fixation led to significantly improved methanol assimilation into intracellular metabolites, including a 2-fold increase of 13C-methanol into glutamate. Using a simple strategy for redox perturbation by deleting the E. coli NAD-dependent malate dehydrogenase gene maldh, we demonstrate 5-fold improved biomass formation of cells growing on methanol in the presence of a small concentration of yeast extract. Further improvements in methanol utilization are achieved via adaptive laboratory evolution and heterologous rpe and tkt expression. A short-term in vivo 13C-methanol labeling assay was used to determine methanol assimilation activity for Δmaldh strains, and demonstrated dramatically higher labeling in intracellular metabolites, including a 6-fold and 1.8-fold increase in glycine labeling for the rpe/tkt and evolved strains, respectively. The combination of formaldehyde-controlled pentose phosphate pathway expression and redox perturbation with the maldh knock-out greatly improved both growth benefit with methanol and methanol carbon incorporation into intracellular metabolites.

Catestatin improves insulin sensitivity by attenuating endoplasmic reticulum stress: In vivo and in silico validation

Abhijit Dasgupta - 2020

Abstract

Obesity is characterized by a state of chronic, unresolved inflammation in insulin-targeted tissues. Obesity-induced inflammation causes accumulation of proinflammatory macrophages in adipose tissue and liver. Proinflammatory cytokines released from tissue macrophages inhibits insulin sensitivity. Obesity also leads to inflammation-induced endoplasmic reticulum (ER) stress and insulin resistance. In this scenario, based on the data (specifically patterns) generated by our in vivo experiments on both diet-induced obese (DIO) and normal chow diet (NCD) mice, we developed an in silico state space model to integrate ER stress and insulin signaling pathways. Computational results successfully followed the experimental results for both DIO and NCD conditions. Chromogranin A (CgA) peptide catestatin (CST: hCgA352−372) improves obesity-induced hepatic insulin resistance by reducing inflammation and inhibiting proinflammatory macrophage infiltration. We reasoned that the anti-inflammatory effects of CST would alleviate ER stress. CST decreased obesity-induced ER dilation in hepatocytes and macrophages. On application of Proportional-Integral-Derivative (PID) controllers on the in silico model, we checked whether the reduction of phosphorylated PERK resulting in attenuation of ER stress, resembling CST effect, could enhance insulin sensitivity. The simulation results clearly pointed out that CST not only decreased ER stress but also enhanced insulin sensitivity in mammalian cells. In vivo experiment validated the simulation results by depicting that CST caused decrease in phosphorylation of UPR signaling molecules and increased phosphorylation of insulin signaling molecules. Besides simulation results predicted that enhancement of AKT phosphorylation helps in both overcoming ER stress and achieving insulin sensitivity. These effects of CST were verified in hepatocyte culture model.

Acute Synovitis after Trauma Precedes and is Associated with Osteoarthritis Onset and Progression

Lifan Liao - 2020

Abstract

Osteoarthritis (OA) is a whole-joint disease characterized by cartilage destruction, subchondral bone sclerosis, osteophyte formation, and synovitis. However, it remains unclear which part of the joint undergoes initial pathological changes that drives OA onset and progression. In the present study, we investigated the longitudinal alterations of the entire knee joint using a surgically-induced OA mouse model. Histology analysis showed that synovitis occurred as early as 1 week after destabilization of the medial meniscus (DMM), which preceded the events of cartilage degradation, subchondral sclerosis and osteophyte formation. Importantly, key pro-inflammatory cytokines such as IL-1β, IL-6, TNFα, and Ccl2, major matrix degrading enzymes including Adamts4, Mmp3 and Mmp13, as well as nerve growth factor (NGF), all increased significantly in both synovium and articular cartilage. It is notable that the inductions of these factors in synovium are far more extensive than those in articular cartilage. Results from behavioral tests demonstrated that sensitization of knee joint pain developed after 8 weeks, later than histological and molecular changes. In addition, the nanoindentation modulus of the medial tibiae decreased 4 weeks after DMM surgery, simultaneous with histological OA signs, which is also later than appearance of synovitis. Collectively, our data suggested that synovitis precedes and is associated with OA, and thus synovium may be an important target to intervene in OA treatment.

The Δ133p53β isoform promotes an immunosuppressive environment leading to aggressive prostate cancer

Marina Kazantseva - 2019

Abstract

Prostate cancer is the second most common cancer in men, for which there are no reliable biomarkers or targeted therapies. Here we demonstrate that elevated levels of Δ133TP53β isoform characterize prostate cancers with immune cell infiltration, particularly T cells and CD163+ macrophages. These cancers are associated with shorter progression-free survival, Gleason scores ≥ 7, and an immunosuppressive environment defined by a higher proportion of PD-1, PD-L1 and colony-stimulating factor 1 receptor (CSF1R) positive cells. Consistent with this, RNA-seq of tumours showed enrichment for pathways associated with immune signalling and cell migration. We further show a role for hypoxia and wild-type p53 in upregulating Δ133TP53 levels. Finally, AUC analysis showed that Δ133TP53β expression level alone predicted aggressive disease with 88% accuracy. Our data identify Δ133TP53β as a highly accurate prognostic factor for aggressive prostate cancer.

Targeted exon skipping with AAV-mediated split adenine base editors

Jackson Winter - 2019

Abstract

Techniques for exclusion of exons from mature transcripts have been applied as gene therapies for treating many different diseases. Since exon skipping has been traditionally accomplished using technologies that have a transient effect, it is particularly important to develop new techniques that enable permanent exon skipping. We have recently shown that this can be accomplished using cytidine base editors for permanently disabling the splice acceptor of target exons. We now demonstrate the application of CRISPR-Cas9 adenine deaminase base editors to disrupt the conserved adenine within splice acceptor sites for programmable exon skipping. We also demonstrate that by altering the amino acid sequence of the linker between the adenosine deaminase domain and the Cas9-nickase or by coupling the adenine base editor with a uracil glycosylase inhibitor, the DNA editing efficiency and exon-skipping rates improve significantly. Finally, we developed a split base editor architecture compatible with adeno-associated viral packaging. Collectively, these results represent significant progress toward permanent in vivo exon skipping through base editing and, ultimately, a new modality of gene therapy for the treatment of genetic diseases.

Retinoids induce antagonism between FOXO3A and FOXM1 transcription factors in human oral squamous cell carcinoma (OSCC) cells

Kwame Osei-Sarfo - 2019

Abstract

To gain a greater understanding of oral squamous cell carcinoma (OSCC) we investigated the actions of all-trans-retinoic acid (RA; a retinoid), bexarotene (a pan-RXR agonist), and forkhead box (FOX) transcription factors in human OSCC-derived cell lines. RA and bexarotene have been shown to limit several oncogenic pathways in many cell types. FOXO proteins typically are associated with tumor suppressive activities, whereas FOXM1 acts as an oncogene when overexpressed in several cancers. RA and/or bexarotene increased the transcript levels of FOXO1, FOXO3A, and TRAIL receptors; reduced the transcript levels of FOXM1, Aurora kinase B (AURKB), and vascular endothelial growth factor A (VEGFA); and decreased the proliferation of OSCC-derived cell lines. Also, RA and/or bexarotene influenced the recruitment of FOXO3A and FOXM1 to target genes. Additionally, FOXM1 depletion reduced cell proliferation, decreased transcript levels of downstream targets of FOXM1, and increased transcript levels of TRAIL receptors. Overexpression of FOXO3A decreased proliferation and increased binding of histone deacetylases (HDACs) 1 and 2 at the FOXM1, AURKB, and VEGFA promoters. This research suggests novel influences of the drugs RA and bexarotene on the expression of FOXM1 and FOXO3A in transcriptional regulatory pathways of human OSCC.

Defining fallopian tube‐derived miRNA cancer signatures

Selam B. Dejene - 2019

Abstract

Background MicroRNAs have recently emerged as promising circulating biomarkers in diverse cancer types, including ovarian cancer. We utilized conditional, doxycycline‐induced fallopian tube (FT)‐derived cancer models to identify changes in miRNA expression in tumors and plasma, and further validated the murine findings in high‐grade ovarian cancer patient samples. Methods We analyzed 566 biologically informative miRNAs in doxycycline‐induced FT and metastatic tumors as well as plasma samples derived from murine models bearing inactivation of Brca, Tp53, and Pten genes. We identified miRNAs that showed a consistent pattern of dysregulated expression and validated our results in human patient serum samples. Results We identified six miRNAs that were significantly dysregulated in doxycycline‐induced FTs (P < .05) and 130 miRNAs differentially regulated in metastases compared to normal fallopian tissues (P < .05). Furthermore, we validated miR‐21a‐5p, miR‐146a‐5p, and miR‐126a‐3p as dysregulated in both murine doxycycline‐induced FT and metastatic tumors, as well as in murine plasma and patient serum samples. Conclusions In summary, we identified changes in miRNA expression that potentially accompany tumor development in murine models driven by commonly found genetic alterations in cancer patients. Further studies are required to test both the function of these miRNAs in driving the disease and their utility as potential biomarkers for diagnosis and/or disease progression.

Redox-regulation and life-history trade-offs: scavenging mitochondrial ROS improves growth in a wild bird

Alberto Velando - 2019

Abstract

It has been proposed that animals usually restrain their growth because fast growth leads to an increased production of mitochondrial reactive oxygen species (mtROS), which can damage mitochondrial DNA and promote mitochondrial dysfunction. Here, we explicitly test whether this occurs in a wild bird by supplementing chicks with a mitochondria-targeted ROS scavenger, mitoubiquinone (mitoQ), and examining growth rates and mtDNA damage. In the yellow-legged gull Larus michahellis, mitoQ supplementation increased the early growth rate of chicks but did not reduce mtDNA damage. The level of mtDNA damage was negatively correlated with chick mass, but this relationship was not affected by the mitoQ treatment. We also found that chick growth was positively correlated with both mtDNA copy number and the mitochondrial enzymatic activity of citrate synthase, suggesting a link between mitochondrial content and growth. Additionally, we found that MitoQ supplementation increased mitochondrial content (in males), altered the relationship between mtDNA copy number and damage, and downregulated some transcriptional pathways related to cell rejuvenation, suggesting that scavenging mtROS during development enhanced growth rates but at the expense of cellular turnover. Our study confirms the central role of mitochondria modulating life-history trade-offs during development by other mechanisms than mtROS-inflicted damage.

Selective inhibition of N-linked glycosylation impairs receptor tyrosine kinase processing

Elsenoor Klaver - 2019

Abstract

Global inhibition of N-linked glycosylation broadly reduces glycan occupancy on glycoproteins, but identifying how this inhibition functionally impacts specific glycoproteins is challenging. This limits our understanding of pathogenesis in the congenital disorders of glycosylation (CDG). We used selective exo-enzymatic labeling of cells deficient in the two catalytic subunits of oligosaccharyltransferase – STT3A and STT3B – to monitor the presence and glycosylation status of cell surface glycoproteins. We show reduced abundance of two canonical tyrosine receptor kinases – the insulin receptor and insulinlike growth factor 1 receptor (IGF-1R) – at the cell surface in STT3A-null cells, due to decreased N-linked glycan site occupancy and proteolytic processing in combination with increased endoplasmic reticulum localization. Providing cDNA for Golgi-resident proprotein convertase subtilisin/kexin type 5a (PCSK5a) and furin cDNA to wild-type and mutant cells produced under-glycosylated forms of PCSK5a, but not furin, in cells lacking STT3A. Reduced glycosylation of PCSK5a in STT3A-null cells or cells treated with the oligosaccharyltransferase inhibitor NGI-1 corresponded with failure to rescue receptor processing, implying that alterations in the glycosylation of this convertase have functional consequences. Collectively, our findings show that STT3A-dependent inhibition of N-linked glycosylation on receptor tyrosine kinases and their convertases combines to impair receptor processing and surface localization. These results provide new insight into CDG pathogenesis and highlight how the surface abundance of some glycoproteins can be dually impacted by abnormal glycosylation.

Mast Cell-Specific Expression of Human Siglec-8 in Conditional Knock-in Mice

Yadong Wei - 2019

Abstract

Sialic acid-binding Ig-like lectin 8 (Siglec-8) is expressed on the surface of human eosinophils, mast cells, and basophils—cells that participate in allergic and other diseases. Ligation of Siglec-8 by specific glycan ligands or antibodies triggers eosinophil death and inhibits mast cell degranulation; consequences that could be leveraged as treatment. However, Siglec-8 is not expressed in murine and most other species, thus limiting preclinical studies in vivo. Based on a ROSA26 knock-in vector, a construct was generated that contains the CAG promoter, a LoxP-floxed-Neo-STOP fragment, and full-length Siglec-8 cDNA. Through homologous recombination, this Siglec-8 construct was targeted into the mouse genome of C57BL/6 embryonic stem (ES) cells, and chimeric mice carrying the ROSA26-Siglec-8 gene were generated. After cross-breeding to mast cell-selective Cre-recombinase transgenic lines (CPA3-Cre, and Mcpt5-Cre), the expression of Siglec-8 in different cell types was determined by RT-PCR and flow cytometry. Peritoneal mast cells (dual FcεRI+ and c-Kit+) showed the strongest levels of surface Siglec-8 expression by multicolor flow cytometry compared to expression levels on tissue-derived mast cells. Siglec-8 was seen on a small percentage of peritoneal basophils, but not other leukocytes from CPA3-Siglec-8 mice. Siglec-8 mRNA and surface protein were also detected on bone marrow-derived mast cells. Transgenic expression of Siglec-8 in mice did not affect endogenous numbers of mast cells when quantified from multiple tissues. Thus, we generated two novel mouse strains, in which human Siglec-8 is selectively expressed on mast cells. These mice may enable the study of Siglec-8 biology in mast cells and its therapeutic targeting in vivo.

Propionic fermentation by the probiotic Propionibacterium freudenreichii to functionalize whey

Song Huang - 2019

Abstract

A new probiotic functionalized sweet whey was evaluated. Weaned healthy piglets consumed sweet whey (SW), unmodified or fermented by P. freudenreichii CIRM-BIA 129 (PF-SW). Fecal short chain fatty acids amounts remained unchanged. Bifidobacteria were enhanced in the PF-SW group, and so was the expression of T-bet, which orchestrates Th1 differentiation of T lymphocytes, in mesenteric lymph nodes immune cells (MLNC). This was consistent with ex vivo increased TNF-α secretion by MLNC in response to lipopolysaccharide (LPS). The consumption of the functionalized whey induced a different response in peripheral blood mononuclear cells (PBMC) to ex vivo stimulations, as the inhibition of TNF-α secretion in response to concanavalin A stimulation. Thus, by cultivating a probiotic GRAS bacterium in concentrated whey, prior to spray drying, it is possible to transform this by-product into a functional ingredient. This opens new avenues for the development of functional ingredients through enhanced valorisation of whey.

Tissue-specific decellularized endometrial substratum mimicking different physiological conditions influences in vitro embryo development in a rabbit model

Hannes Campo - 2019

Abstract

In the last decades, the decellularization (DC) of organs has become an established technique in the fieldof regenerative medicine to yield complex and vascularized bioscaffolds. Furthermore, it has beendemonstratedin vitrothat these decellularized scaffolds retain their native tissue-specificity. This is alsothe case when this tissue-specific extracellular matrix (ECM) is solubilized and used as hydrogels or coat-ings to create a biomimetic environment. In this study we investigated if this specificity not only remainswhen applied to distinct tissues but even more, that these differences can be distinguished within thesame tissue at different stages of proliferation. To address this question, a sensitivein vitroanimal modelwas used: rabbit embryos at the third day of development were cultured on coatings made from acellularendometrium that was non-proliferating (non-synchronous, NS) and proliferating (synchronous with theembryo, S) and their development was compared.For this, we obtained whole NS and S rabbit uteri and subjected them to an adapted decellularizationprotocol. The acellular endometrium was carefully separated by microdissection and converted into apre-gel solution to be used as hydrogels and coatings forin vitroassays. First, the characteristics of theseNS and S hydrogels were investigated by proteomic analysis, electron microscopy and gelling kinetics.When used as substrata for day 3 embryos culture, it became apparent that only the acellular ECM fromsynchronous endometrial coating achieved similar results to the gold standard culture protocols and con-ditions, possibly because of the slow release of growth factors present in the synchronous/proliferatingendometrium.

The Role of Lactate Metabolism in Prostate Cancer Progression and Metastases Revealed by Dual-Agent Hyperpolarized 13C MRSI

Robert Bok - 2019

Abstract

This study applied a dual-agent, 13C-pyruvate and 13C-urea, hyperpolarized 13C magnetic resonance spectroscopic imaging (MRSI) and multi-parametric (mp) 1H magnetic resonance imaging (MRI) approach in the transgenic adenocarcinoma of mouse prostate (TRAMP) model to investigate changes in tumor perfusion and lactate metabolism during prostate cancer development, progression and metastases, and after lactate dehydrogenase-A (LDHA) knock-out. An increased Warburg effect, as measured by an elevated hyperpolarized (HP) Lactate/Pyruvate (Lac/Pyr) ratio, and associated Ldha expression and LDH activity were significantly higher in high- versus low-grade TRAMP tumors and normal prostates. The hypoxic tumor microenvironment in high-grade tumors, as measured by significantly decreased HP 13C-urea perfusion and increased PIM staining, played a key role in increasing lactate production through increased Hif1α and then Ldha expression. Increased lactate induced Mct4 expression and an acidic tumor microenvironment that provided a potential mechanism for the observed high rate of lymph node (86%) and liver (33%) metastases. The Ldha knockdown in the triple-transgenic mouse model of prostate cancer resulted in a significant reduction in HP Lac/Pyr, which preceded a reduction in tumor volume or apparent water diffusion coefficient (ADC). The Ldha gene knockdown significantly reduced primary tumor growth and reduced lymph node and visceral metastases. These data suggested a metabolic transformation from low- to high-grade prostate cancer including an increased Warburg effect, decreased perfusion, and increased metastatic potential. Moreover, these data suggested that LDH activity and lactate are required for tumor progression. The lactate metabolism changes during prostate cancer provided the motivation for applying hyperpolarized 13C MRSI to detect aggressive disease at diagnosis and predict early therapeutic response.

A Protective Role for the Lectin CD169/Siglec-1 against a Pathogenic Murine Retrovirus

Pradeep D. Uchil - 2019

Abstract

Lymph- and blood-borne retroviruses exploit CD169/Siglec-1-mediated capture by subcapsular sinus and marginal zone metallophilic macrophages for trans-infection of permissive lymphocytes. However, the impact of CD169-mediated virus capture on retrovirus dissemination and pathogenesis in vivo is unknown. In a murine model of the splenomegaly-inducing retrovirus Friend virus complex (FVC) infection, we find that while CD169 promoted draining lymph node infection, it limited systemic spread to the spleen. At the spleen, CD169-expressing macrophages captured incoming blood-borne retroviruses and limited their spread to the erythroblasts in the red pulp where FVC manifests its pathogenesis. CD169-mediated retroviral capture activated conventional dendritic cells 1 (cDC1s) and promoted cytotoxic CD8+ T cell responses, resulting in efficient clearing of FVC-infected cells. Accordingly, CD169 blockade led to higher viral loads and accelerated death in susceptible mouse strains. Thus, CD169 plays a protective role during FVC pathogenesis by reducing viral dissemination to erythroblasts and eliciting an effective cytotoxic T lymphocyte response via cDC1s.

Blocking IL-1β reverses the immunosuppression in mouse breast cancer and synergizes with anti–PD-1 for tumor abrogation

Irena Kaplanov - 2019

Abstract

Interleukin-1β (IL-1β) is abundant in the tumor microenvironment, where this cytokine can promote tumor growth, but also antitumor activities. We studied IL-1β during early tumor progression using a model of orthotopically introduced 4T1 breast cancer cells. Whereas there is tumor progression and spontaneous metastasis in wild-type (WT) mice, in IL-1β–deficient mice, tumors begin to grow but subsequently regress. This change is due to recruitment and differentiation of inflammatory monocytes in the tumor microenvironment. In WT mice, macrophages heavily infiltrate tumors, but in IL-1β–deficient mice, low levels of the chemokine CCL2 hamper recruitment of monocytes and, together with low levels of colony-stimulating factor-1 (CSF-1), inhibit their differentiation into macrophages. The low levels of macrophages in IL-1β– deficient mice result in a relatively high percentage of CD11b+ dendritic cells (DCs) in the tumors. In WT mice, IL-10 secretion from macrophages is dominant and induces immunosuppression and tumor progression; in contrast, in IL-1β–deficient mice, IL-12 secretion by CD11b+ DCs prevails and supports antitumor immunity. The antitumor immunity in IL-1β–deficient mice includes activated CD8+ lymphocytes expressing IFN-γ, TNF-α, and granzyme B; these cells infiltrate tumors and induce regression. WT mice with 4T1 tumors were treated with either anti–IL-1β or anti–PD-1 Abs, each of which resulted in partial growth inhibition. However, treating mice first with anti–IL-1β Abs followed by anti– PD-1 Abs completely abrogated tumor progression. These data define microenvironmental IL-1β as a master cytokine in tumor progression. In addition to reducing tumor progression, blocking IL-1β facilitates checkpoint inhibition.

Epiregulin is released from intervertebral disks and induces spontaneous activity in pain pathways

Mette Kongstop - 2019

Abstract

Introduction: Lumbar radicular pain after disk herniation is associated with local release of many inflammatory molecules from nucleus pulposus (NP) cells leaking out of the intervertebral disk. Here, we have used a rat model to investigate the role of epiregulin (EREG), a member of the epidermal growth factor (EGF) family, in this process. Methods: A protein immunoassay was chosen to confirm the release of EREG from the NP tissue. Single unit recordings were used to demonstrate the effect of recombinant EREG applied onto the dorsal nerve roots in vivo. Intracellular responses induced by recombinant EREG were studied in cultured dorsal root ganglion (DRG) cells by phosphoprotein assay. Changes in EGF receptor expression induced by NP in the DRG were examined by quantitative polymerase chain reaction. Results: The protein immunoassay showed that EREG was released from the NP tissue. Moreover, application of EREG onto the spinal dorsal nerve roots induced a decrease in the evoked responses, but an increase in spontaneous activity in the dorsal horn neurons. Interestingly, the EREG activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in the DRG, a pathway previously linked to cellular growth, proliferation, and tissue regeneration. An NP-induced upregulation of the EGF receptor HER3 in the DRG was also revealed. Conclusion: Taken together, the present observations indicate that EREG may induce changes in the DRG and spontaneous activity in the pain pathways. We suggest that EREG signaling may be involved in the pathophysiological process leading to sensory deficits and neuropathic pain in patients after disk herniation.

CSDC2, a cold shock domain RNA‐binding protein in decidualization

Griselda Vallejo - 2019

Abstract

RNA‐binding proteins (RBPs) have been described for cancer cell progression and differentiation, although there is still much to learn about their mechanisms. Here, using in vivo decidualization as a model, we describe the role of RBP cold shock domain containing C2 (CSDC2) in the endometrium. Csdc2 messenger RNA expression was differentially regulated depending on time and areas of decidua development, with the most variation in antimesometrium (AM) and, to a lesser degree, in the junctional zone (JZ). Immunohistochemistry of CSDC2 showed a preferentially cytoplasmic localization at AM and JZ, and nuclear localization in underneath myometrium and mesometrium (M). Cytoplasmic localization coincided with differentiated, DESMIN‐marked areas, while nuclear localization coincides with proliferative zones. Uterine suppression of CSDC2 through intrauterine‐injected‐specific small interfering RNA (siRNA) led to abnormal decidualization in early pregnancy, with more extended antimesometrial area and with poor M development if compared with control siRNA‐injected animals. These results suggest that CSDC2 could be a regulator during decidua development.

Eukaryotic initiation factor 5B (eIF5B) provides a critical cell survival switch to glioblastoma cells via regulation of apoptosis

Joseph A. Ross - 2019

Abstract

Physiological stress conditions attenuate global mRNA translation via modifications of key eukaryotic initiation factors. However, non-canonical translation initiation mechanisms allow cap-independent translation of certain mRNAs. We have previously demonstrated that eIF5B promotes cap-independent translation of the mRNA encoding the antiapoptotic factor, XIAP, during cellular stress. Here, we show that depletion of eIF5B sensitizes glioblastoma multiforme cells to TRAIL-induced apoptosis by a pathway involving caspases-8, −9, and −7, with no significant effect on cell cycle progression. eIF5B promotes evasion of apoptosis by promoting the translation of several IRES-containing mRNAs, encoding the antiapoptotic proteins XIAP, Bcl-xL, cIAP1, and c-FLIPS. We also show that eIF5B promotes translation of nuclear factor erythroid 2-related factor 2 and suggest that reactive oxygen species contribute to increased apoptosis under conditions of eIF5B depletion. Finally, eIF5B depletion leads to decreased activation of the canonical NF-κB pathway. Taken together, our data suggest that eIF5B represents a regulatory node, allowing cancer cells to evade apoptosis by promoting the translation of pro-survival proteins from IRES-containing mRNAs.

Specific loss of adipocyte CD248 improves metabolic health via reduced white adipose tissue hypoxia, fibrosis and inflammation

Paul Petrus - 2019

Abstract

Background A positive energy balance promotes white adipose tissue (WAT) expansion which is characterized by activation of a repertoire of events including hypoxia, inflammation and extracellular matrix remodelling. The transmembrane glycoprotein CD248 has been implicated in all these processes in different malignant and inflammatory diseases but its potential impact in WAT and metabolic disease has not been explored. Methods The role of CD248 in adipocyte function and glucose metabolism was evaluated by omics analyses in human WAT, gene knockdowns in human in vitro differentiated adipocytes and by adipocyte-specific and inducible Cd248 gene knockout studies in mice. Findings CD248 is upregulated in white but not brown adipose tissue of obese and insulin-resistant individuals. Gene ontology analyses showed that CD248 expression associated positively with pro-inflammatory/pro-fibrotic pathways. By combining data from several human cohorts with gene knockdown experiments in human adipocytes, our results indicate that CD248 acts as a microenvironmental sensor which mediates part of the adipose tissue response to hypoxia and is specifically perturbed in white adipocytes in the obese state. Adipocyte-specific and inducible Cd248 knockouts in mice, both before and after diet-induced obesity and insulin resistance/glucose intolerance, resulted in increased microvascular density as well as attenuated hypoxia, inflammation and fibrosis without affecting fat cell volume. This was accompanied by significant improvements in insulin sensitivity and glucose tolerance. Interpretation CD248 exerts detrimental effects on WAT phenotype and systemic glucose homeostasis which may be reversed by suppression of adipocyte CD248. Therefore, CD248 may constitute a target to treat obesity-associated co-morbidities.

Impaired HDL Function Amplifies Systemic Inflammation in Common Variable Immunodeficiency

Magnhild E. Macpherson - 2019

Abstract

Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency, characterized by inadequate antibody responses and recurrent bacterial infections. Paradoxically, a majority of CVID patients have non-infectious inflammatory and autoimmune complications, associated with systemic immune activation. Our aim was to explore if HDL, known to have anti-inflammatory properties, had impaired function in CVID patients and thereby contributed to their inflammatory phenotype. We found reduced HDL cholesterol levels in plasma of CVID patients compared to healthy controls, particularly in patients with inflammatory and autoimmune complications, correlating negatively with inflammatory markers CRP and sCD25. Reverse cholesterol transport capacity testing showed reduced serum acceptance capacity for cholesterol in CVID patients with inflammatory and autoimmune complications. They also had reduced cholesterol efflux capacity from macrophages to serum and decreased expression of ATP-binding cassette transporter ABCA1. Human HDL suppressed TLR2-induced TNF release less in blood mononuclear cells from CVID patients, associated with decreased expression of transcriptional factor ATF3. Our data suggest a link between impaired HDL function and systemic inflammation in CVID patients, particularly in those with autoimmune and inflammatory complications. This identifies HDL as a novel therapeutic target in CVID as well as other more common conditions characterized by sterile inflammation or autoimmunity.

Single-Cell Fluorescence Analysis of Pseudotemporal Ordered Cells Provides Protein Expression Dynamics for Neuronal Differentiation

Zhichao Song - 2019

Abstract

Stem cell replacement therapy is a potential method for repopulating lost spiral ganglion neurons (SGNs) in the inner ear. Efficacy of cell replacement relies on proper differentiation. Defining the dynamic expression of different transcription factors essential for neuronal differentiation allows us to monitor the progress and determine when the protein functions in differentiating stem cell cultures. Using immortalized multipotent otic progenitors (iMOPs) as a cellular system for SGN differentiation, a method for determining dynamic protein expression from heterogeneous cultures was developed. iMOP-derived neurons were identified and ordered by increasing neurite lengths to create a pseudotime course that reflects the differentiation trajectory. The fluorescence intensities of transcription factors SOX2 and NEUROD1 from individual pseudotemporally ordered cells were measured. Individual cells were grouped by K-means clustering and the mean fluorescence intensity for each cluster determined. Curve fit of the mean fluorescence represented the protein expression dynamics in differentiating cells. The method provides information about protein expression dynamics in differentiating stem cell cultures.

The unfolded protein response modulators GSK2606414 and KIRA6 are potent KIT inhibitors

Mohamed Mahameed - 2019

Abstract

IRE1, PERK, and ATF6 are the three transducers of the mammalian canonical unfolded protein response (UPR). GSK2606414 is a potent inhibitor of PERK, while KIRA6 inhibits the kinase activity of IRE1. Both molecules are frequently used to probe the biological roles of the UPR in mammalian cells. In a direct binding assay, GSK2606414 bound to the cytoplasmic domain of KIT with dissociation constants (Kd) value of 664 ± 294 nM whereas KIRA6 showed a Kd value of 10.8 ± 2.9 µM. In silico docking studies confirmed a compact interaction of GSK2606414 and KIRA6 with KIT ATP binding pocket. In cultured cells, GSK2606414 inhibited KIT tyrosine kinase activity at nanomolar concentrations and in a PERK-independent manner. Moreover, in contrast to other KIT inhibitors, GSK2606414 enhanced KIT endocytosis and its lysosomal degradation. Although KIRA6 also inhibited KIT at nanomolar concentrations, it did not prompt KIT degradation, and rescued KIT from GSK2606414-mediated degradation. Consistent with KIT inhibition, nanomolar concentrations of GSK2606414 and KIRA6 were sufficient to induce cell death in a KIT signaling-dependent mast cell leukemia cell line. Our data show for the first time that KIT is a shared target for two seemingly unrelated UPR inhibitors at concentrations that overlap with PERK and IRE1 inhibition. Furthermore, these data underscore discrepancies between in vitro binding measurements of kinase inhibitors and inhibition of the tyrosine kinase receptors in living cells.

LARGE-SCALE SECRETOME ANALYSES UNVEIL THE SUPERIOR IMMUNOSUPPRESSIVE PHENOTYPE OF UMBILICAL CORD STROMAL CELLS AS COMPARED TO OTHER ADULT MESENCHYMAL STROMAL CELLS

A. Islam - 2019

Abstract

Mesenchymal stromal cells (MSCs), given their regenerative potential, are being investigated as a potential therapeutic tool for cartilage lesions. MSCs express several bioactive molecules which act in a paracrine fashion to modulate the tissue microenvironment. Yet, little is known about the divergence of these signalling molecules in different MSC populations. The present study investigated secretomes of stromal cells harvested from Hoffa’s fat pad (HFPSCs), synovial membrane (SMSCs), umbilical cord (UCSCs) and cartilage (ACs) by quantitative liquid chromatography-mass spectrometry (LC-MS/MS) proteomics. Also, multiplex protein arrays and functional assays were performed to compare the constitutive immunomodulatory capabilities of different MSCs. Proteins involved in extracellular matrix degradation and inflammation, such as matrix metalloproteinases (MMPs), interleukin (IL)-17 and complement factors, were downregulated in UCSCs as compared to adult cell sources. Additionally, secretion of transforming growth factor (TGF)-β1 and prostaglandin E2 (PGE2) was enhanced in UCSC supernatants. UCSCs were superior in inhibiting peripheral blood mononuclear cell (PBMC) proliferation, migration and cytokine secretion as compared to adult stromal cells. SMSCs significantly suppressed the proliferation of PBMCs only if they were primed with pro-inflammatory cytokines. Although all cell types repressed human leukocyte antigen-DR isotype (HLADR) surface expression and cytokine release by activated macrophages, only UCSCs significantly blocked IL-6 and IL-12 production. Furthermore, UCSCs supernatants increased aggrecan gene expression in twodimensional chondrocyte cultures. The data demonstrated that UCSCs displayed superior anti-inflammatory and immunosuppressive properties than stromal cells from adult tissues. This allogeneic cell source could potentially be considered as an adjuvant therapy for articular cartilage repair

A Ubiquitous Platform for Bacterial Nanotube Biogenesis

Saurabh Bhattacharya - 2019

Abstract

We have previously described the existence of membranous nanotubes, bridging adjacent bacteria, facilitating intercellular trafficking of nutrients, cytoplasmic proteins, and even plasmids, yet components enabling their biogenesis remain elusive. Here we reveal the identity of a molecular apparatus providing a platform for nanotube biogenesis. Using Bacillus subtilis (Bs), we demonstrate that conserved components of the flagellar export apparatus (FliO, FliP, FliQ, FliR, FlhB, and FlhA), designated CORE, dually serve for flagellum and nanotube assembly. Mutants lacking CORE genes, but not other flagellar components, are deficient in both nanotube production and the associated intercellular molecular trafficking. In accord, CORE components are located at sites of nanotube emergence. Deleting COREs of distinct species established that CORE-mediated nanotube formation is widespread. Furthermore, exogenous COREs from diverse species could restore nanotube generation and functionality in Bs lacking endogenous CORE. Our results demonstrate that the CORE-derived nanotube is a ubiquitous organelle that facilitates intercellular molecular trade across the bacterial kingdom.

LACC1 Regulates TNF and IL-17 in Mouse Models of Arthritis and Inflammation

Cara Skon-Hegg - 2019

Abstract

Both common and rare genetic variants of laccase domain-containing 1 (LACC1, previously C13orf31) are associated with inflammatory bowel disease, leprosy, Behcet disease, and systemic juvenile idiopathic arthritis. However, the functional relevance of these variants is unclear. In this study, we use LACC1-deficient mice to gain insight into the role of LACC1 in regulating inflammation. Following oral administration of Citrobacter rodentium, LACC1 knockout (KO) mice had more severe colon lesions compared with wildtype (WT) controls. Immunization with collagen II, a collagen-induced arthritis (CIA) model, resulted in an accelerated onset of arthritis and significantly worse arthritis and inflammation in LACC1 KO mice. Similar results were obtained in a mannan-induced arthritis model. Serum and local TNF in CIA paws and C. rodentium colons were significantly increased in LACC1 KO mice compared with WT controls. The percentage of IL-17A–producing CD4+ T cells was elevated in LACC1 KO mice undergoing CIA as well as aged mice compared with WT controls. Neutralization of IL-17, but not TNF, prevented enhanced mannan-induced arthritis in LACC1 KO mice. These data provide new mechanistic insight into the function of LACC1 in regulating TNF and IL-17 during inflammatory responses. We hypothesize that these effects contribute to immune-driven pathologies observed in individuals carrying LACC1 variants.

Serine protease inhibitors rich Coccinia grandis (L.) Voigt leaf extract induces protective immune responses in murine visceral leishmaniasis

Asmita Pramanik - 2019

Abstract

Leishmaniasis is a parasite-mediated tropical disease affecting millions of individuals worldwide. The available antileishmanial chemotherapeutic modalities exhibit adverse toxicity, exorbitant price and advent of drug-resistant parasites. Hence, plant-derived products are an alternative preference for the emergence of novel and effective antileishmanial agents that rejuvenate the host immunity with limited toxicity. The present work is complementary to our previous report that revealed the in vitro antileishmanial and immunomodulatory activity of Coccinia grandis (L.) Voigt leaf extract (Cg-Ex) rich in serine protease inhibitors. Thus, preliminary objectives of the study were to elucidate the leishmanicidal activity and host effector mechanism in Leishmania donovani infected BALB/c mice treated with Cg-Ex. Oral administration of Cg-Ex significantly reduced the spleen and liver parasite burden at dose-dependently. The parasite elimination was associated with generation of ROS and NO that are interrelated with up-regulation of disease-suppressing Th1 cytokines and down-regulation of disease-promoting Th2 cytokines at both protein and mRNA level. Moreover, Cg-Ex augmented the delayed-type hypersensitivity (DTH) response and serum IgG2a level which are correlated with the diminution of parasite burden with no hepatic and renal toxicity. Additionally, histological analysis of spleen depicted the improvement of structural disorganization of white and red pulp after Cg-Ex treatment. Therefore, our intriguing findings have presented the first indication of in vivo antileishmanial efficacy through activation of pro-inflammatory immune responses of the host by a natural plant leaf extract (Cg-Ex) containing serine protease inhibitors which could have a role as a potential immunomodulator against visceral leishmaniasis.

Kinetic studies on clinical and immunological modulations by intramuscular injection of Escherichia coli LPS in laying hens

Wendy Leirmann - 2019

Abstract

The present study investigated clinical and immunological modulations due to intramuscular injection of Escherichia coli LPS in 49-wk-old laying hens over 48 h post injection (p.i.). LPS induced characteristic sickness behavior but no significant body temperature alterations (P > 0.05). During experimental period decreases in blood albumin, calcium, phosphorus and tryptophan concentrations, hyperglycemia, increased plasma nitrite concentrations, leucopenia, decreased thrombocyte counts, lymphopenia, heterophilia and an increased heterophilic granulocyte/lymphocyte (H/L) ratio were observed after LPS administration. Time-dependent effects were shown on T and B cell subsets in caecal tonsils (CT) and on splenic CD3+/CD4+/CD8+ proportions, on IL-1β and -10 and inducible NO synthase mRNA expression in peripheral blood lymphocytes (PBL), liver, spleen and CT, and on the mRNA expression of the TLR4 in PBL, liver and spleen p.i. (P < 0.05). The main responding period of mentioned alterations due to LPS appears to include the period from 2 until 8 h p.i. According to the H/L ratio, the most stressful phase was 5 h p.i. T and B cell subsets in CT, the IL-1β and TLR4 mRNA expression in liver and plasma nitrite concentrations seemed to be affected for a longer period.

A novel nonosteocytic regulatory mechanism of bone modeling

Lior Ofer - 2019

Abstract

Osteocytes, cells forming an elaborate network within the bones of most vertebrate taxa, are thought to be the master regulators of bone modeling, a process of coordinated, local bone-tissue deposition and removal that keeps bone strains at safe levels throughout life. Neoteleost fish, however, lack osteocytes and yet are known to be capable of bone modeling, although no osteocyte-independent modeling regulatory mechanism has so far been described. Here, we characterize a novel, to our knowledge, bone-modeling regulatory mechanism in a fish species (medaka), showing that although lacking osteocytes (i.e., internal mechanosensors), when loaded, medaka bones model in mechanically directed ways, successfully reducing high tissue strains. We establish that as in mammals, modeling in medaka is regulated by the SOST gene, demonstrating a mechanistic link between skeletal loading, SOST down-regulation, and intense bone deposition. However, whereas mammalian SOST is expressed almost exclusively by osteocytes, in both medaka and zebrafish (a species with osteocytic bones), SOST is expressed by a variety of nonosteocytic cells, none of which reside within the bone bulk. These findings argue that in fishes (and perhaps other vertebrates), nonosteocytic skeletal cells are both sensors and responders, shouldering duties believed exclusive to osteocytes. This previously unrecognized, SOST-dependent, osteocyte-independent mechanism challenges current paradigms of osteocyte exclusivity in bone-modeling regulation, suggesting the existence of multivariate feedback networks in bone modeling—perhaps also in mammalian bones—and thus arguing for the possibility of untapped potential for cell targets in bone therapeutics.

Effects of a synthetic di‐phosphoserine peptide (SS‐2) on gene expression profiling against TNF‐α induced inflammation

Hua Zhang - 2019

Abstract

It has been showed bioactive di‐phosphoserine peptide (SS‐2) possesses functions to ameliorate oxidative stress in vitro. This study aimed to substantiate the role of bioactive di‐phosphoserine peptide (SS‐2) in modulating inflammatory responses in TNF‐α‐stimulated HT‐29 cells, and its mechanism of action. SS‐2 significantly reduced IL‐8 secretion in TNF‐α‐induced HT‐29 cells, and also suppressed pro‐inflammatory cytokines, including IL‐8, IL‐12, MCP‐1 and TNF‐α. Moreover, SS‐2 inhibited TNF‐α initiated signalling cascades by suppressing phosphorylation of the ERK1/2, JNK, P38 and IκB those culminate in above cellular inflammatory responses. Differentially expressed genes analysis within NF‐κB signalling pathway revealed that SS‐2 blocks multiple sites of upstream NF‐κB signalling cascade, including FADD and MyD88, thereby preventing the signalling transduction involved in cellular inflammatory response. These results provide a new insight into molecular mechanism for anti‐inflammatory action of SS‐2, suggesting SS‐2 is a potential alternative approach to treat IBD by particular targeting TNF‐α driven inflammatory event.

In vitro chondrogenic potency of surplus chondrocytes from autologous transplantation procedures does not predict short-term clinical outcomes

Ashraful Islam - 2019

Abstract

Background Autologous chondrocyte implantation (ACI) has been used over the last two decades to treat focal cartilage lesions aiming to delay or prevent the onset of osteoarthritis; however, some patients do not respond adequately to the procedure. A number of biomarkers that can forecast the clinical potency of the cells have been proposed, but evidence for the relationship between in vitro chondrogenic potential and clinical outcomes is missing. In this study, we explored if the ability of cells to make cartilage in vitro correlates with ACI clinical outcomes. Additionally, we evaluated previously proposed chondrogenic biomarkers and searched for new biomarkers in the chondrocyte proteome capable of predicting clinical success or failure after ACI. Methods The chondrogenic capacity of chondrocytes derived from 14 different donors was defined based on proteoglycans staining and visual histological grading of tissues generated using the pellet culture system. A Lysholm score of 65 two years post-ACI was used as a cut-off to categorise “success” and “failure” clinical groups. A set of predefined biomarkers were investigated in the chondrogenic and clinical outcomes groups using flow cytometry and qPCR. High-throughput proteomics of cell lysates was used to search for putative biomarkers to predict chondrogenesis and clinical outcomes. Results Visual histological grading of pellets categorised donors into “high” and “low” chondrogenic groups. Direct comparison between donor-matched in vitro chondrogenic potential and clinical outcomes revealed no significant associations. Comparative analyses of selected biomarkers revealed that expression of CD106 and TGF-β-receptor-3 was enhanced in the low chondrogenic group, while expression of integrin-α1 and integrin-β1 was significantly upregulated in the high chondrogenic group. Additionally, increased surface expression of CD166 was observed in the clinical success group, while the gene expression of cartilage oligomeric matrix protein was downregulated. High throughput proteomics revealed no differentially expressed proteins from success and failure clinical groups, whereas seven proteins including prolyl-4-hydroxylase 1 were differentially expressed when comparing chondrogenic groups. Conclusion In our limited material, we found no correlation between in vitro cartilage-forming capacity and clinical outcomes, and argue on the limitations of using the chondrogenic potential of cells or markers for chondrogenesis as predictors of clinical outcomes.

Cellulose nanocrystals modulate alveolar macrophage phenotype and phagocytic function

Johanna Samulin Erdem - 2019

Abstract

Nanocellulose is a promising bio-nanomaterial with attractive properties suitable for multiple industrial applications. The increased use of nanocellulose may lead to occupational exposure and negative health outcomes. However, knowledge on its health effects is limited, and while nanocellulose exposure may induce acute inflammatory responses in the lung, the underlying mechanisms are unknown. Alveolar macrophages are key cells in alveolar particle clearance. Their activation and function may be affected by various particles. Here, we investigated the uptake of pristine cellulose nanocrystals (CNC), and their effects on alveolar macrophage polarization and biological function. CNC uptake enhanced the secretion of several cytokines but did not on its own induce a complete macrophage polarization. In presence of macrophage activators, such as LPS/IFNG and IL4/IL13, CNC exposure enhanced the expression of M1 phenotype markers and the secretion of pro-inflammatory cytokines and chemokines, while decreasing M2 markers. CNC exposure also affected the function of activated alveolar macrophages resulting in a prominent cytokine burst and altered phagocytic activity. In conclusion, CNC exposure may result in dysregulation of macrophage activation and function that are critical in inflammatory responses in the lung.

Defective IgA response to atypical intestinal commensals in IL-21 receptor deficiency reshapes immune cell homeostasis and mucosal immunity

Hyeson Cho - 2019

Abstract

Despite studies indicating the effects of IL-21 signaling in intestinal inflammation, its roles in intestinal homeostasis and infection are not yet clear. Here, we report potent effects of commensal microbiota on the phenotypic manifestations of IL-21 receptor deficiency. IL-21 is produced highly in the small intestine and appears to be critical for mounting an IgA response against atypical commensals such as segmented filamentous bacteria and Helicobacter, but not to the majority of commensals. In the presence of these atypical commensals, IL-21R-deficient mice exhibit reduced numbers of germinal center and IgA+ B cells and expression of activation-induced cytidine deaminase in Peyer’s patches as well as a significant decrease in small intestine IgA+ plasmablasts and plasma cells, leading to higher bacterial burdens and subsequent expansion of Th17 and Treg cells. These microbiota-mediated secondary changes in turn enhance T cell responses to an oral antigen and strikingly dampen Citrobacter rodentium-induced immunopathology, demonstrating a complex interplay between IL-21-mediated mucosal immunity, microbiota, and pathogens.

S-Nitrosylation of α1-Antitrypsin Triggers Macrophages Toward Inflammatory Phenotype and Enhances Intra-Cellular Bacteria Elimination

Ziv Kaner - 2019

Abstract

Background: Human α1-antitrypsin (hAAT) is a circulating anti-inflammatory serine-protease inhibitor that rises during acute phase responses. in vivo, hAAT reduces bacterial load, without directly inhibiting bacterial growth. In conditions of excess nitric-oxide (NO), hAAT undergoes S-nitrosylation (S-NO-hAAT) and gains antibacterial capacity. The impact of S-NO-hAAT on immune cells has yet to be explored. Aim: Study the effects of S-NO-hAAT on immune cells during bacterial infection. Methods: Clinical-grade hAAT was S-nitrosylated and then compared to unmodified hAAT, functionally, and structurally. Intracellular bacterial clearance by THP-1 macrophages was assessed using live Salmonella typhi. Murine peritoneal macrophages were examined, and signaling pathways were evaluated. S-NO-hAAT was also investigated after blocking free mambranal cysteine residues on cells. Results: S-NO-hAAT (27.5 uM) enhances intracellular bacteria elimination by immunocytes (up to 1-log reduction). S-NO-hAAT causes resting macrophages to exhibit a pro-inflammatory and antibacterial phenotype, including release of inflammatory cytokines and induction of inducible nitric oxide synthase (iNOS) and TLR2. These pro-inflammatory effects are dependent upon cell surface thiols and activation of MAPK pathways. Conclusions: hAAT duality appears to be context-specific, involving S-nitrosylation in a nitric oxide rich environment. Our results suggest that S-nitrosylation facilitates the antibacterial activity of hAAT by promoting its ability to activate innate immune cells. This pro-inflammatory effect may involve transferring of nitric oxide from S-NO-hAAT to a free cysteine residue on cellular targets.

B cell activation and proliferation increase intracellular zinc levels

Johanna Ollig - 2019

Abstract

Zinc ions serve as second messengers in major cellular pathways, including the regulation pathways of proliferation and their proper regulation is necessary for homeostasis and a healthy organism. Accordingly, expression of zinc transporters can be altered in various cancer cell lines and is often involved in producing elevated intracellular zinc levels. In this study, human B cells were infected with Epstein–Barr virus (EBV) to generate immortalized cells, which revealed traits of tumor cells, such as high proliferation rates and an extended lifespan. These cells showed differentially altered zinc transporter expression with ZIP7 RNA and protein expression being especially increased as well as a corresponding increased phosphorylation of ZIP7 in EBV-transformed B cells. Accordingly, free zinc levels were elevated within these cells. To prove whether the observed changes resulted from immortalization or rather high proliferation, free zinc levels in in vitro activated B cells and in freshly isolated B cells expressing the activation marker CD69 were determined. Here, comparatively increased zinc levels were found, suggesting that activation and proliferation, but not immortalization, act as crucial factors for the elevation of intracellular free zinc.

MiR-135 suppresses glycolysis and promotes pancreatic cancer cell adaptation to metabolic stress by targeting phosphofructokinase-1

Ying Yang - 2019

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers. It thrives in a nutrient-poor environment; however, the mechanisms by which PDAC cells undergo metabolic reprogramming to adapt to metabolic stress are still poorly understood. Here, we show that microRNA-135 is significantly increased in PDAC patient samples compared to adjacent normal tissue. Mechanistically, miR-135 accumulates specifically in response to glutamine deprivation and requires ROS-dependent activation of mutant p53, which directly promotes miR-135 expression. Functionally, we found miR-135 targets phosphofructokinase-1 (PFK1) and inhibits aerobic glycolysis, thereby promoting the utilization of glucose to support the tricarboxylic acid (TCA) cycle. Consistently, miR-135 silencing sensitizes PDAC cells to glutamine deprivation and represses tumor growth in vivo. Together, these results identify a mechanism used by PDAC cells to survive the nutrient-poor tumor microenvironment, and also provide insight regarding the role of mutant p53 and miRNA in pancreatic cancer cell adaptation to metabolic stresses.

Co-administering Melatonin With an Estradiol-Progesterone Menopausal Hormone Therapy Represses Mammary Cancer Development in a Mouse Model of HER2-Positive Breast Cancer

Balasunder R. Dodda - 2019

Abstract

Melatonin has numerous anti-cancer properties reported to influence cancer initiation, promotion, and metastasis. With the need for effective hormone therapies (HT) to treat menopausal symptoms without increasing breast cancer risk, co-administration of nocturnal melatonin with a natural, low-dose HT was evaluated in mice that develop primary and metastatic mammary cancer. Individually, melatonin (MEL) and estradiol-progesterone therapy (EPT) did not significantly affect mammary cancer development through age 14 months, but, when combined, the melatonin-estradiol-progesterone therapy (MEPT) significantly repressed tumor formation. This repression was due to effects on tumor incidence, but not latency. These results demonstrate that melatonin and the HT cooperate to decrease the mammary cancer risk. Melatonin and EPT also cooperate to alter the balance of the progesterone receptor (PR) isoforms by significantly increasing PRA protein expression only in MEPT mammary glands. Melatonin significantly suppressed amphiregulin transcripts in MEL and MEPT mammary glands, suggesting that amphiregulin together with the higher PRA:PRB balance and other factors may contribute to reducing cancer development in MEPT mice. Melatonin supplementation influenced mammary morphology by increasing tertiary branching in the mouse mammary glands and differentiation in human mammary epithelial cell cultures. Uterine weight in the luteal phase was elevated after long-term exposure to EPT, but not to MEPT, indicating that melatonin supplementation may reduce estrogen-induced uterine stimulation. Melatonin supplementation significantly decreased the incidence of grossly-detected lung metastases in MEL mice, suggesting that melatonin delays the formation of metastatic lesions and/or decreases aggressiveness in this model of HER2+ breast cancer. Mammary tumor development was similar in EPT and MEPT mice until age 8.6 months, but after 8.6 months, only MEPT continued to suppress cancer development. These data suggest that melatonin supplementation has a negligible effect in young MEPT mice, but is required in older mice to inhibit tumor formation. Since melatonin binding was significantly decreased in older mammary glands, irrespective of treatment, melatonin supplementation may overcome reduced melatonin responsiveness in the aged MEPT mice. Since melatonin levels are known to decline near menopause, nocturnal melatonin supplementation may also be needed in aging women to cooperate with HT to decrease breast cancer risk.

Autophagy Regulation of Metabolism Is Required for CD8+ T Cell Anti-tumor Immunity

Lindsay DeVorkin - 2019

Abstract

Autophagy is a cell survival process essential for the regulation of immune responses to infections. However, the role of T cell autophagy in anti-tumor immunity is less clear. Here, we demonstrate a cell-autonomous role for autophagy in the regulation of CD8+ T-cell-mediated control of tumors. Mice deficient for the essential autophagy genes Atg5, Atg14, or Atg16L1 display a dramatic impairment in the growth of syngeneic tumors. Moreover, T cells lacking Atg5 have a profound shift to an effector memory phenotype and produce greater amounts of interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α). Mechanistically, Atg5−/− CD8+ T cells exhibit enhanced glucose metabolism that results in alterations in histone methylation, increases in H3K4me3 density, and transcriptional upregulation of both metabolic and effector target genes. Nonetheless, glucose restriction is sufficient to suppress Atg5-dependent increases in effector function. Thus, autophagy-dependent changes in CD8+ T cell metabolism directly regulate anti-tumor immunity.

O-GlcNAc Transferase Inhibition Differentially Affects Breast Cancer Subtypes

Anna Barkovskaya - 2019

Abstract

Post-translational modification of intracellular proteins with a single N-acetylglucosamine sugar (O-GlcNAcylation) regulates signaling, proliferation, metabolism and protein stability. In breast cancer, expression of the enzyme that catalyzes O-GlcNAcylation – O-GlcNAc-transferase (OGT), and the extent of protein O-GlcNAcylation, are upregulated in tumor tissue, and correlate with cancer progression. Here we compare the significance of O-GlcNAcylation in a panel of breast cancer cells of different phenotypes. We find a greater dependency on OGT among triple-negative breast cancer (TNBC) cell lines, which respond to OGT inhibition by undergoing cell cycle arrest and apoptosis. Searching for the cause of this response, we evaluate the changes in the proteome that occur after OGT inhibition or knock-down, employing a reverse-phase protein array (RPPA). We identify transcriptional repressor - hairy and enhancer of split-1 (HES1) - as a mediator of the OGT inhibition response in the TNBC cells. Inhibition of OGT as well as the loss of HES1 results in potent cytotoxicity and apoptosis. The study raises a possibility of using OGT inhibition to potentiate DNA damage in the TNBC cells.

p53 Promotes Cancer Cell Adaptation to Glutamine Deprivation by Upregulating Slc7a3 to Increase Arginine Uptake

Xazmin H. Lowman - 2019

Abstract

Cancer cells heavily depend on the amino acid glutamine to meet the demands associated with growth and proliferation. Due to the rapid consumption of glutamine, cancer cells frequently undergo glutamine starvation in vivo. We and others have shown that p53 is a critical regulator in metabolic stress resistance. To better understand the molecular mechanisms by which p53 activation promotes cancer cell adaptation to glutamine deprivation, we identified p53-dependent genes that are induced upon glutamine deprivation by using RNA-seq analysis. We show that Slc7a3, an arginine transporter, is significantly induced by p53. We also show that increased intracellular arginine levels following glutamine deprivation are dependent on p53. The influx of arginine has minimal effects on known metabolic pathways upon glutamine deprivation. Instead, we found arginine serves as an effector for mTORC1 activation to promote cell growth in response to glutamine starvation. Therefore, we identify a p53-inducible gene that contributes to the metabolic stress response.

The Modulatory Role of MicroRNA-873 in the Progression of KRAS-Driven Cancers

Hamada A. Mokhlis - 2019

Abstract

KRAS is one of the most frequently mutated proto-oncogenes in pancreatic ductal adenocarcinoma (PDAC) and aberrantly activated in triple-negative breast cancer (TNBC). A profound role of microRNAs (miRNAs) in the pathogenesis of human cancer is being uncovered, including in cancer therapy. Using in silico prediction algorithms, we identified miR-873 as a potential regulator of KRAS, and we investigated its role in PDAC and TNBC. We found that reduced miR-873 expression is associated with shorter patient survival in both cancers. miR-873 expression is significantly repressed in PDAC and TNBC cell lines and inversely correlated with KRAS levels. We demonstrate that miR-873 directly bound to the 3′ UTR of KRAS mRNA and suppressed its expression. Notably, restoring miR-873 expression induced apoptosis; recapitulated the effects of KRAS inhibition on cell proliferation, colony formation, and invasion; and suppressed the activity of ERK and PI3K/AKT, while overexpression of KRAS rescued the effects mediated by miR-873. Moreover, in vivo delivery of miR-873 nanoparticles inhibited KRAS expression and tumor growth in PDAC and TNBC tumor models. In conclusion, we provide the first evidence that miR-873 acts as a tumor suppressor by targeting KRAS and that miR-873-based gene therapy may be a therapeutic strategy in PDAC and TNBC.

Dietary Tryptophan Induces Opposite Health-Related Responses in the Senegalese Sole (Solea senegalensis) Reared at Low or High Stocking Densities With Implications in Disease Resistance

Rita Azeredo - 2019

Abstract

High rearing densities are typical conditions of both inland and onshore intensive aquaculture units. Despite obvious drawbacks, this strategy is nonetheless used to increase production profits. Such conditions inflict stress on fish, reducing their ability to cope with disease, bringing producers to adopt therapeutic strategies. In an attempt to overcome deleterious effects of chronic stress, Senegalese sole, Solea senegalensis, held at low (LD) or high density (HD) were fed tryptophan-supplemented diets with final tryptophan content at two (TRP2) or four times (TRP4) the requirement level, as well as a control and non-supplemented diet (CTRL) for 38 days. Fish were sampled at the end of the feeding trial for evaluation of their immune status, and mortalities were recorded following intra-peritoneal infection with Photobacterium damselae subsp. piscicida. Blood was collected for analysis of the hematological profile and innate immune parameters in plasma. Pituitary and hypothalamus were sampled for the assessment of neuro-endocrine-related gene expression. During the feeding trial, fish fed TRP4 and held at LD conditions presented higher mortalities, whereas fish kept at HD seemed to benefit from this dietary treatment, as disease resistance increased over that of CTRL-fed fish. In accordance, cortisol level tended to be higher in fish fed both supplemented diets at LD compared to fish fed CTRL, but was lower in fish fed TRP4 than in those fed TRP2 under HD condition. Together with lower mRNA levels of proopiomelanocortin observed with both supplementation levels, these results suggest that higher levels of tryptophan might counteract stress-induced cortisol production, thereby rendering fish better prepared to cope with disease. Data regarding sole immune status showed no clear effects of tryptophan on leucocyte numbers, but TRP4-fed fish displayed inhibited alternative complement activity (ACH50) when held at LD, as opposed to their HD counterparts whose ACH50 was higher than that of CTRL-fed fish. In conclusion, while dietary tryptophan supplementation might have harmful effects in control fish, it might prove to be a promising strategy to overcome chronic stress-induced disease susceptibility in farmed Senegalese sole.

The autism/neuroprotection-linked ADNP/NAP regulate the excitatory glutamatergic synapse

Schlomo Sragovich - 2019

Abstract

Activity-dependent neuroprotective protein (ADNP), essential for brain formation, was discovered as a leading de novo mutated gene causing the autism-like ADNP syndrome. This syndrome is phenotypically characterized by global developmental delays, intellectual disabilities, speech impediments, and motor dysfunctions. The Adnp haploinsufficient mouse mimics the human ADNP syndrome in terms of synapse density and gene expression patterns, as well as in developmental, motor, and cognitive abilities. Peripheral ADNP was also discovered as a biomarker for Alzheimer’s disease and schizophrenia, with nasal administration of the ADNP snippet peptide NAP (enhancing endogenous ADNP activity) leading to partial cognitive and functional protection at the cellular, animal and clinical settings. Here, a novel formulation for effective delivery of NAP is provided with superior brain penetration capabilities. Also provided are methods for treating pertinent clinical implications such as autism, cognitive impairments, olfactory deficits, and muscle strength using the formulation in the Adnp haploinsufficient mouse. Results showed a dramatically specific increase in brain/body bioavailability with the new formulation, without breaching the blood brain barrier. Additional findings included improvements using daily intranasal treatments with NAP, at the behavioral and brain structural levels, diffusion tensor imaging (DTI), translatable to clinical practice. Significant effects on hippocampal and cerebral cortical expression of the presynaptic Slc17a7 gene encoding vesicular excitatory glutamate transporter 1 (VGLUT1) were observed at the RNA and immunohistochemical levels, explaining the DTI results. These findings tie for the first time a reduction in presynaptic glutamatergic synapses with the autism/Alzheimer’s/schizophrenia-linked ADNP deficiency coupled with amelioration by NAP (CP201).

Curcumin and o-Vanillin Exhibit Evidence of Senolytic Activity in Human IVD Cells In Vitro

Hosni Cherif - 2019

Abstract

Curcumin and o-Vanillin cleared senescent intervertebral disc (IVD) cells and reduced the senescence-associated secretory phenotype (SASP) associated with inflammation and back pain. Cells from degenerate and non-mildly-degenerate human IVD were obtained from organ donors and from patients undergoing surgery for low back pain. Gene expression of senescence and SASP markers was evaluated by RT-qPCR in isolated cells, and protein expression of senescence, proliferation, and apoptotic markers was evaluated by immunocytochemistry (ICC). The expression levels of SASP factors were evaluated by enzyme-linked immunosorbent assay (ELISA). Matrix synthesis was verified with safranin-O staining and the Dimethyl-Methylene Blue Assay for proteoglycan content. Western blotting and ICC were used to determine the molecular pathways targeted by the drugs. We found a 40% higher level of senescent cells in degenerate compared to non-mildly-degenerate discs from unrelated individuals and a 10% higher level in degenerate compared to non-mildly-degenerate discs from the same individual. Higher levels of senescence were associated with increased SASP. Both drugs cleared senescent cells, and treatment increased the number of proliferating as well as apoptotic cells in cultures from degenerate IVDs. The expression of SASP factors was decreased, and matrix synthesis increased following treatment. These effects were mediated through the Nrf2 and NFkB pathways

Transport and Recovery of Gilthead Seabream (Sparus aurata L.) Sedated With Clove Oil and MS-222: Effects on Stress Axis Regulation and Intermediary Metabolism

Ismael Jerez-Cepa - 2019

Abstract

Transport processes between aquaculture facilities activate the stress response in fish. To deal with these situations, the hypothalamic-pituitary-interrenal (HPI) axis releases cortisol, leading to an increase in circulating energy resources to restore homeostasis. However, if the allostatic load generated exceeds fish tolerance limits, stress-related responses will compromise health and welfare of the animals. In this context, anesthetics have arisen as potential agents aiming to reduce negative effects of stress response. Here we assessed the effects of a sedative dose of clove oil (CO) and MS-222 on hallmarks involved in HPI axis regulation and energy management after simulated transport, and further recovery, in gilthead seabream (Sparus aurata L.) juveniles. Fish were placed in a mobile setup of water tanks where transport conditions were simulated for 6 h. Sedation doses of either CO (2.5 mg L−1) or MS-222 (5 mg L−1) were added in the water tanks. A control group without anesthetics was also included in the setup. Half of the animals (n = 12 per group) were sampled immediately after transport, while remaining animals were allowed to recover for 18 h in clean water tanks and then sampled. Our results showed that the HPI axis response was modified at peripheral level, with differences depending on the anesthetic employed. Head kidney gene-expressions related to cortisol production (star and cyp11b1) matched concomitantly with increased plasma cortisol levels immediately after transport in CO-sedated fish, but these levels remained constant in MS-222-sedated fish. Differential changes in the energy management of carbohydrates, lipids and amino acids, depending on the anesthetic employed, were also observed. The use of CO stimulated amino acids catabolism, while MS-222-sedated fish tended to consume liver glycogen and mobilize triglycerides. Further studies, including alternative doses of both anestethics, as well as the assessment of time-course HPI activation and longer recovery periods, are necessary to better understand if the use of clove oil and MS-222 is beneficial for S. aurata under these circumstances.

Identification of traits associated with barley yield performance using contrasting nitrogen fertilizations and genotypes

Ruben Vicente - 2019

Abstract

Much attention has been paid to understanding the traits associated with crop performance and the associated underlying physiological mechanisms, with less effort done towards combining different plant scales, levels of observation, or including hybrids of autogamous species. We aim to identify mechanisms at canopy, leaf and transcript levels contributing to crop performance under contrasting nitrogen supplies in three barley genotypes, two hybrids and one commercial line. High nitrogen fertilization did not affect photosynthetic capacity on a leaf area basis and lowered nitrogen partial factor productivity past a certain point, but increased leaf area and biomass accumulation, parameters that were closely tracked using various different high throughput remote sensing based phenotyping techniques. These aspects, together with a larger catabolism of leaf nitrogen compounds amenable to sink translocation, contributed to higher crop production. Better crop yield and growth in hybrids compared to the line was linked to a nitrogen-saving strategy in source leaves to the detriment of larger sink size, as indicated by the lower leaf nitrogen content and downregulation of nitrogen metabolism and aquaporin genes. While these changes did not reduce photosynthesis capacity on an area basis, they were related with better nitrogen use in the hybrids compared with the line.

Bulky DNA adducts, microRNA profiles, and lipid biomarkers in Norwegian tunnel finishing workers occupationally exposed to diesel exhaust

Iselin Rynning - 2019

Abstract

Objectives This study aimed to assess the biological impact of occupational exposure to diesel exhaust (DE) including DE particles (DEP) from heavy-duty diesel-powered equipment in Norwegian tunnel finishing workers (TFW). Methods TFW (n=69) and referents (n=69) were investigated for bulky DNA adducts (by 32P-postlabelling) and expression of microRNAs (miRNAs) (by small RNA sequencing) in peripheral blood mononuclear cells (PBMC), as well as circulating free arachidonic acid (AA) and eicosanoid profiles in plasma (by liquid chromatography–tandem mass spectrometry). Results PBMC from TFW showed significantly higher levels of DNA adducts compared with referents. Levels of DNA adducts were also related to smoking habits. Seventeen miRNAs were significantly deregulated in TFW. Several of these miRNAs are related to carcinogenesis, apoptosis and antioxidant effects. Analysis of putative miRNA-gene targets revealed deregulation of pathways associated with cancer, alterations in lipid molecules, steroid biosynthesis and cell cycle. Plasma profiles showed higher levels of free AA and 15-hydroxyeicosatetraenoic acid, and lower levels of prostaglandin D2 and 9-hydroxyoctadecadienoic acid in TFW compared with referents. Conclusion Occupational exposure to DE/DEP is associated with biological alterations in TFW potentially affecting lung homoeostasis, carcinogenesis, inflammation status and the cardiovascular system. Of particular importance is the finding that tunnel finishing work is associated with an increased level of DNA adducts formation in PBMC. This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial

The heterogeneity and complexity of Cannabis extracts as antitumor agents

Liran Baram - 2019

Abstract

The Cannabis plant contains over 100 phytocannabinoids and hundreds of other components. The biological effects and interplay of these Cannabis compounds are not fully understood and yet influence the plant’s therapeutic effects. Here we assessed the antitumor effects of whole Cannabis extracts, which contained significant amounts of differing phytocannabinoids, on different cancer lines from various tumor origins. We first utilized our novel electrospray ionization liquid chromatography mass spectrometry method to analyze the phytocannabinoid contents of 124 Cannabis extracts. We then monitored the effects of 12 chosen different Cannabis extracts on 12 cancer cell lines. Our results show that specific Cannabis extracts impaired the survival and proliferation of cancer cell lines as well as induced apoptosis. Our findings showed that pure (-)-Δ9-trans-tetrahydrocannabinol (Δ9-THC) did not produce the same effects on these cell lines as the whole Cannabis extracts. Furthermore, Cannabis extracts with similar amounts of Δ9-THC produced significantly different effects on the survival of specific cancer cells. In addition, we demonstrated that specific Cannabis extracts may selectively and differentially affect cancer cells and differing cancer cell lines from the same organ origin. We also found that cannabimimetic receptors were differentially expressed among various cancer cell lines and suggest that this receptor diversity may contribute to the heterogeneous effects produced by the differing Cannabis extracts on each cell line. Our overall findings indicate that the effect of a Cannabis extract on a specific cancer cell line relies on the extract’s composition as well as on certain characteristics of the targeted cells.

The Hematopoietic Oxidase NOX2 Regulates Self-Renewal of Leukemic Stem Cells

Biniam Adane - 2019

Abstract

The NADPH-dependent oxidase NOX2 is an important effector of immune cell function, and its activity has been linked to oncogenic signaling. Here, we describe a role for NOX2 in leukemia-initiating stem cell populations (LSCs). In a murine model of leukemia, suppression of NOX2 impaired core metabolism, attenuated disease development, and depleted functionally defined LSCs. Transcriptional analysis of purified LSCs revealed that deficiency of NOX2 collapses the self-renewal program and activates inflammatory and myeloid-differentiation-associated programs. Downstream of NOX2, we identified the forkhead transcription factor FOXC1 as a mediator of the phenotype. Notably, suppression of NOX2 or FOXC1 led to marked differentiation of leukemic blasts. In xenotransplantation models of primary human myeloid leukemia, suppression of either NOX2 or FOXC1 significantly attenuated disease development. Collectively, these findings position NOX2 as a critical regulator of malignant hematopoiesis and highlight the clinical potential of inhibiting NOX2 as a means to target LSCs.

Multi‐omics identify xanthine as a pro‐survival metabolite for nematodes with mitochondrial dysfunction

Anna Gioran - 2019

Abstract

Aberrant mitochondrial function contributes to the pathogenesis of various metabolic and chronic disorders. Inhibition of insulin/IGF‐1 signaling (IIS) represents a promising avenue for the treatment of mitochondrial diseases, although many of the molecular mechanisms underlying this beneficial effect remain elusive. Using an unbiased multi‐omics approach, we report here that IIS inhibition reduces protein synthesis and favors catabolism in mitochondrial deficient Caenorhabditis elegans. We unveil that the lifespan extension does not occur through the restoration of mitochondrial respiration, but as a consequence of an ATP‐saving metabolic rewiring that is associated with an evolutionarily conserved phosphoproteome landscape. Furthermore, we identify xanthine accumulation as a prominent downstream metabolic output of IIS inhibition. We provide evidence that supplementation of FDA‐approved xanthine derivatives is sufficient to promote fitness and survival of nematodes carrying mitochondrial lesions. Together, our data describe previously unknown molecular components of a metabolic network that can extend the lifespan of short‐lived mitochondrial mutant animals.

Broad and Protective Influenza B Virus Neuraminidase Antibodies in Humans after Vaccination and their Clonal Persistence as Plasma Cells

Michael S. Piepenbrink - 2019

Abstract

Although most seasonal inactivated influenza vaccines (IIV) contain neuraminidase (NA), the extent and mechanisms of action of protective human NAspecific humoral responses induced by vaccination are poorly resolved. Due to the propensity of influenza virus for antigenic drift and shift and its tendency to elicit predominantly strain-specific antibodies, humanity remains susceptible to waves of new strains of seasonal viruses and is at risk from viruses with pandemic potential for which limited or no immunity may exist. Here we demonstrate that the use of IIV results in increased levels of influenza B virus (IBV) NA-specific serum antibodies. Detailed analysis of the IBV NA B cell response indicates concurrent expansion of IBV NA-specific peripheral blood plasmablasts 7 days after IIV immunization which express monoclonal antibodies with broad and potent antiviral activity against both IBV Victoria and Yamagata lineages and prophylactic and therapeutic activity in mice. These IBV NA-specific B cell clonal lineages persisted in CD138 long-lived bone marrow plasma cells. These results represent the first demonstration that IIVinduced NA human antibodies can protect and treat influenza virus infection in vivo and suggest that IIV can induce a subset of IBV NA-specific B cells with broad protective potential, a feature that warrants further study for universal influenza vaccine development. IMPORTANCE Influenza virus infections continue to cause substantial morbidity and mortality despite the availability of seasonal vaccines. The extensive genetic variability in seasonal and potentially pandemic influenza strains necessitates new vaccine strategies that can induce universal protection by focusing the immune response on generating protective antibodies against conserved targets such as regions within the influenza neuraminidase protein. We have demonstrated that seasonal immunization stimulates neuraminidase-specific antibodies in humans that are broad and potent in their protection from influenza B virus when tested in mice. These antibodies further persist in the bone marrow, where they are expressed by long-lived antibody-producing cells, referred to here as plasma cells. The significance in our research is the demonstration that seasonal influenza immunization can induce a subset of neuraminidase-specific B cells with broad protective potential, a process that if further studied and enhanced could aid in the development of a universal influenza vaccine.

Hybrid nanocarriers incorporating mechanistically distinct drugs for lymphatic CD4+ T cell activation and HIV-1 latency reversal

Shijie Cao - 2019

Abstract

A proposed strategy to cure HIV uses latency-reversing agents (LRAs) to reactivate latent proviruses for purging HIV reservoirs. A variety of LRAs have been identified, but none has yet proven effective in reducing the reservoir size in vivo. Nanocarriers could address some major challenges by improving drug solubility and safety, providing sustained drug release, and simultaneously delivering multiple drugs to target tissues and cells. Here, we formulated hybrid nanocarriers that incorporate physicochemically diverse LRAs and target lymphatic CD4+ T cells. We identified one LRA combination that displayed synergistic latency reversal and low cytotoxicity in a cell model of HIV and in CD4+ T cells from virologically suppressed patients. Furthermore, our targeted nanocarriers selectively activated CD4+ T cells in nonhuman primate peripheral blood mononuclear cells as well as in murine lymph nodes, and substantially reduced local toxicity. This nanocarrier platform may enable new solutions for delivering anti-HIV agents for an HIV cure.

Functional Screening Identifies MicroRNAs as Multi-Cellular Regulators of Heart Failure

Robin Verjans - 2019

Abstract

Heart failure (HF) is the leading cause of death in the Western world. Pathophysiological processes underlying HF development, including cardiac hypertrophy, fibrosis and inflammation, are controlled by specific microRNAs (miRNAs). Whereas most studies investigate miRNA function in one particular cardiac cell type, their multicellular function is poorly investigated. The present study probed 194 miRNAs –differentially expressed in cardiac inflammatory disease – for regulating cardiomyocyte size, cardiac fibroblasts collagen content, and macrophage polarization. Of the tested miRNAs, 13%, 26%, and 41% modulated cardiomyocyte size, fibroblast collagen production, and macrophage polarization, respectively. Seventeen miRNAs affected all three cellular processes, including miRNAs with established (miR-210) and unknown roles in cardiac pathophysiology (miR-145-3p). These miRNAs with a multi-cellular function commonly target various genes. In-depth analysis in vitro of previously unstudied miRNAs revealed that the observed phenotypical alterations concurred with changes in transcript and protein levels of hypertrophy-, fibrosis- and inflammation-related genes. MiR-145-3p and miR-891a-3p were identified to regulate the fibrotic response, whereas miR-223-3p, miR-486-3p, and miR-488-5p modulated macrophage activation and polarisation. In conclusion, miRNAs are multi-cellular regulators of different cellular processes underlying cardiac disease. We identified previously undescribed roles of miRNAs in hypertrophy, fibrosis, and inflammation, and attribute new cellular effects to various well-known miRNAs.

Krt5+/Krt15+ foregut basal progenitors give rise to cyclooxygenase-2-dependent tumours in response to gastric acid stress

Hyeongsun Moon - 2019

Abstract

The effective prevention of tumor initiation, especially for potentially inoperable tumors, will be beneficial to obtain an overall higher quality of our health and life. Hence, thorough understanding of the pathophysiological mechanisms of early tumor formation arising from identifiable cellular origins is required to develop efficient preventative and early treatment options for each tumor type. Here, using genetically engineered mouse models, we provide preclinical experimental evidence for a long-standing open question regarding the pathophysiological potential of a microenvironmental and physiological stressor in tumor development, gastric acid-mediated regional microscopic injury in foregut squamous epithelia. This study demonstrates the association of gastric acid stress with Cyclooxygenase-2-dependent tumor formation originating from tumor-competent Krt5+/Krt15+ foregut basal progenitor cells. Our findings suggest that clinical management of microenvironmental stressor-mediated microscopic injury may be important in delaying tumor initiation from foregut basal progenitor cells expressing pre-existing tumorigenic mutation(s) and genetic alteration(s).

A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA[Ser]Sec Gene

Caroline Vindry - 2019

Abstract

The translation of selenoprotein mRNAs involves a non-canonical ribosomal event in which an in-frame UGA is recoded as a selenocysteine (Sec) codon instead of being read as a stop codon. The recoding machinery is centered around two dedicated RNA components: The selenocysteine insertion sequence (SECIS) located in the 3′ UTR of the mRNA and the selenocysteine-tRNA (Sec-tRNA[Ser]Sec). This translational UGA-selenocysteine recoding event by the ribosome is a limiting stage of selenoprotein expression. Its efficiency is controlled by the SECIS, the Sec-tRNA[Ser]Sec and their interacting protein partners. In the present work, we used a recently developed CRISPR strategy based on murine leukemia virus-like particles (VLPs) loaded with Cas9-sgRNA ribonucleoproteins to inactivate the Sec-tRNA[Ser]Sec gene in human cell lines. We showed that these CRISPR-Cas9-VLPs were able to induce efficient genome-editing in Hek293, HepG2, HaCaT, HAP1, HeLa, and LNCaP cell lines and this caused a robust reduction of selenoprotein expression. The alteration of selenoprotein expression was the direct consequence of lower levels of Sec-tRNA[Ser]Sec and thus a decrease in translational recoding efficiency of the ribosome. This novel strategy opens many possibilities to study the impact of selenoprotein deficiency in hard-to-transfect cells, since these CRISPR-Cas9-VLPs have a wide tropism.

In vitro effects of resistin on epithelial to mesenchymal transition (EMT) in MCF-7 and MDA-MB-231 breast cancer cells – qRT-PCR and westen blot analyses data

Dimiter Avtanski - 2019

Abstract

Resistin is an adipokine produced by the white adipocytes and adipose-derived macrophages, which mediates inflammation and insulin resistance [1], [2] Huang et al., 1997 and Renehan et al., 2008 Feb. Here, we provide data on the effect of resistin on epithelial to mesenchymal transition (EMT) in breast cancer cells in vitro. As model systems, we used human MCF-7 (low-metastatic) and MDA-MB-231 (high-metastatic) breast cancer cell lines. To optimize experimental conditions, we treated the cells with various concentrations of resistin (12.5, 25 and 50 ng/ml) for different time intervals (6 and 24 hours), and measured SOCS3 mRNA expression by using qRT-PCR analysis. Further, we used qRT-PCR and Western blot analyses to measure the expression of various epithelial (E-cadherin, claudin-1) and mesenchymal (SNAIL, SLUG, ZEB1, TWIST1, fibronectin, and vimentin) markers after resistin treatment. This data article is part of a study Avtanski et al., 2019 May, where detailed interpretation and discussion.

A truncating MEIOB mutation responsible for familial primary ovarian insufficiency abolishes its interaction with its partner SPATA22 and their recruitment to DNA double-strand breaks

Sandrine Caburet - 2019

Abstract

Background Primary Ovarian Insufficiency (POI), a major cause of infertility, affects about 1–3% of women under forty years of age. Although there is a growing list of causal genetic alterations, POI remains mostly idiopathic. Methods We performed exome sequencing (WES) of two sisters affected with POI, one unaffected sister and their mother from a consanguineous family. We assessed the impact of the identified MEIOB variant with a minigene assay and by sequencing illegitimate transcripts from the proband's leukocytes. We studied its functional impact on the interaction between MEIOB with its partner SPATA22 and their localization to DNA double-strand breaks (DSB). Findings We identified a homozygous variant in the last base of exon 12 of MEIOB, which encodes a factor essential for meiotic recombination. This variant was predicted to strongly affect MEIOB pre-mRNA splicing. Consistently, a minigene assay showed that the variant induced exon 12 skipping, which was confirmed in vivo in the proband's leukocytes. Aberrant splicing leads to the production of a C-terminally truncated protein that cannot interact with SPATA22, abolishing their recruitment to DSBs. Interpretation This truncating MEIOB variant is expected to provoke meiotic defects and a depleted follicular stock, as in Meiob−/− mice. This is the first molecular defect reported in a meiosis-specific single-stranded DNA-binding protein (SSB) responsible for POI. We hypothesise that alterations in other SSB proteins could explain cases of syndromic or isolated ovarian insufficiency. Fund Université Paris Diderot, Fondation pour la Recherche Médicale, Fondation ARC contre le cancer, Commissariat à l'Energie Atomique and Institut Universitaire de France.

Insights from transcriptome profiling on the non-photosynthetic and stomatal signaling response of maize carbonic anhydrase mutants to low CO2

Allison R. Kolbe - 2019

Abstract

Carbonic anhydrase (CA) catalyzes the hydration of CO2 in the first biochemical step of C4 photosynthesis, and has been considered a potentially rate-limiting step when CO2 availability within a leaf is low. Previous work in Zea mays (maize) with a double knockout of the two highest-expressed β-CA genes, CA1 and CA2, reduced total leaf CA activity to less than 3% of wild-type. Surprisingly, this did not limit photosynthesis in maize at ambient or higher CO2concentrations. However, the ca1ca2 mutants exhibited reduced rates of photosynthesis at sub-ambient CO2, and accumulated less biomass when grown under sub-ambient CO2 (9.2 Pa). To further clarify the importance of CA for C4 photosynthesis, we assessed gene expression changes in wild-type, ca1 and ca1ca2 mutants in response to changes in pCO2 from 920 to 9.2 Pa. Results Leaf samples from each genotype were collected for RNA-seq analysis at high CO2 and at two time points after the low CO2 transition, in order to identify early and longer-term responses to CO2 deprivation. Despite the existence of multiple isoforms of CA, no other CA genes were upregulated in CA mutants. Although photosynthetic genes were downregulated in response to low CO2, differential expression was not observed between genotypes. However, multiple indicators of carbon starvation were present in the mutants, including amino acid synthesis, carbohydrate metabolism, and sugar signaling. In particular, multiple genes previously implicated in low carbon stress such as asparagine synthetase, amino acid transporters, trehalose-6-phosphate synthase, as well as many transcription factors, were strongly upregulated. Furthermore, genes in the CO2 stomatal signaling pathway were differentially expressed in the CA mutants under low CO2. Conclusions Using a transcriptomic approach, we showed that carbonic anhydrase mutants do not compensate for the lack of CA activity by upregulating other CA or photosynthetic genes, but rather experienced extreme carbon stress when grown under low CO2. Our results also support a role for CA in the CO2 stomatal signaling pathway. This study provides insight into the importance of CA for C4 photosynthesis and its role in stomatal signaling.

High OGT activity is essential for MYC-driven proliferation of prostate cancer cells

Harri M. Itkonen - 2019

Abstract

O-GlcNAc transferase (OGT) is overexpressed in aggressive prostate cancer. OGT modifies intra-cellular proteins via single sugar conjugation (O-GlcNAcylation) to alter their activity. We recently discovered the first fast-acting OGT inhibitor OSMI-2. Here, we probe the stability and function of the chromatin O-GlcNAc and identify transcription factors that coordinate with OGT to promote proliferation of prostate cancer cells. Methods: Chromatin immunoprecipitation (ChIP) coupled to sequencing (seq), formaldehyde-assisted isolation of regulatory elements, RNA-seq and reverse-phase protein arrays (RPPA) were used to study the importance of OGT for chromatin structure and transcription. Mass spectrometry, western blot, RT-qPCR, cell cycle analysis and viability assays were used to establish the role of OGT for MYC-related processes. Prostate cancer patient data profiled for both mRNA and protein levels were used to validate findings. Results: We show for the first time that OGT inhibition leads to a rapid loss of O-GlcNAc chromatin mark. O-GlcNAc ChIP-seq regions overlap with super-enhancers (SE) and MYC binding sites. OGT inhibition leads to down-regulation of SE-dependent genes. We establish the first O-GlcNAc chromatin consensus motif, which we use as a bait for mass spectrometry. By combining the proteomic data from oligonucleotide enrichment with O-GlcNAc and MYC ChIP-mass spectrometry, we identify host cell factor 1 (HCF-1) as an interaction partner of MYC. Inhibition of OGT disrupts this interaction and compromises MYC's ability to confer androgen-independent proliferation to prostate cancer cells. We show that OGT is required for MYC-mediated stabilization of mitotic proteins, including Cyclin B1, and/or the increased translation of their coding transcripts. This implies that increased expression of mRNA is not always required to achieve increased protein expression and confer aggressive phenotype. Indeed, high expression of Cyclin B1 protein has strong predictive value in prostate cancer patients (p=0.000014) while mRNA does not. Conclusions: OGT promotes SE-dependent gene expression. OGT activity is required for the interaction between MYC and HCF-1 and expression of MYC-regulated mitotic proteins. These features render OGT essential for the androgen-independent, MYC-driven proliferation of prostate cancer cells. Androgen-independency is the major mechanism of prostate cancer progression, and our study identifies OGT as an essential mediator in this process.

Impaired LXRα Phosphorylation Attenuates Progression of Fatty Liver Disease

Natalia Becares - 2019

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a very common indication for liver transplantation. How fat-rich diets promote progression from fatty liver to more damaging inflammatory and fibrotic stages is poorly understood. Here, we show that disrupting phosphorylation at Ser196 (S196A) in the liver X receptor alpha (LXRα, NR1H3) retards NAFLD progression in mice on a high-fat-high-cholesterol diet. Mechanistically, this is explained by key histone acetylation (H3K27) and transcriptional changes in pro-fibrotic and pro-inflammatory genes. Furthermore, S196A-LXRα expression reveals the regulation of novel diet-specific LXRα-responsive genes, including the induction of Ces1f, implicated in the breakdown of hepatic lipids. This involves induced H3K27 acetylation and altered LXR and TBLR1 cofactor occupancy at the Ces1f gene in S196A fatty livers. Overall, impaired Ser196-LXRα phosphorylation acts as a novel nutritional molecular sensor that profoundly alters the hepatic H3K27 acetylome and transcriptome during NAFLD progression placing LXRα phosphorylation as an alternative anti-inflammatory or anti-fibrotic therapeutic target.

Insulin Receptor deletion in S100a4-lineage cells accelerates age-related bone loss

Valentina Studentsova - 2019

Abstract

Type I and Type II Diabetes dramatically impair skeletal health. Altered Insulin Receptor (IR) signaling is a common feature of both diseases, and insulin has potent bone anabolic functions. Several previous studies have demonstrated that loss of IR in bone cells results in disrupted bone homeostasis during early post-natal growth. Here we have deleted IR in S100a4-lineage cells (IRcKOS100a4) and assessed the effects on bone homeostasis in both young (15 weeks) and older adult (48 weeks) mice. S100a4-Cre has previously been shown to target the perichondrium during bone development, and here we show that S100a4 is expressed by adult trabecular and cortical bone cells, and that S100a4-Cre effectively targets adult bone, resulting in efficient deletion of IRβ. Deletion of IRβ in S100a4-lineage cells does not affect initial bone acquisition or homeostasis with no changes in cortical, trabecular or mechanical properties at 15-weeks of age, relative to wild type (WT) littermates. However, by 48-weeks of age, IRcKOS100a4 mice display substantial declines in trabecular bone volume, bone volume fraction and torsional rigidity, relative to age-matched WT controls. This work establishes the utility of using S100a4-cre to target bone and demonstrates that IRβ in S100a4-lineage cells is required for maintenance of bone homeostasis in adult mice.

Standardization of Human Calcific Aortic Valve Disease in vitro Modeling Reveals Passage-Dependent Calcification

Lang H. Lee - 2019

Abstract

Aortic valvular interstitial cells (VICs) isolated from patients undergoing valve replacement are commonly used as in vitro models of calcific aortic valve disease (CAVD). Standardization of VIC calcification, however, has not been implemented, which impairs comparison of results from different studies. We hypothesized that different culture methods impact the calcification phenotype of human VICs. We sought to identify the key parameters impacting calcification in primary human VICs to standardize CAVD in vitro research. Here we report that in calcification media containing organic phosphate, termed osteogenic media (OM), primary human VICs exhibited a passage-dependent decrease in calcification potential, which was not observed in calcification media containing inorganic phosphate, termed pro-calcifying media (PM). We used Alizarin red staining to compare the calcification potential of VICs cultured in OM and PM between the first and fourth passages after cell isolation from human CAVD tissues. Human VICs showed consistent Alizarin red stain when cultured with PM in a passage-independent manner. VICs cultured in OM did not exhibit consistent calcification potential between donors in early passages and consistently lacked positive Alizarin red stain in late passages. We performed whole cell, cytoplasmic and nuclear fractionation proteomics to identify factors regulating VIC passage-dependent calcification in OM. Proteomics cluster analysis identified tissue non-specific alkaline phosphatase (TNAP) as a regulator of passage-dependent calcification in OM. We verified an association of TNAP activity with calcification potential in VICs cultured in OM, but not in PM in which VICs calcified independent of TNAP activity. This study demonstrates that media culture conditions and cell passage impact the calcification potential of primary human VICs and should be taken into consideration in cell culture models of CAVD. Our results help standardize CAVD modeling as part of a greater effort to identify disease driving mechanisms and therapeutics for this unmet medical need.

Development and validation of an in vitro 3D model of NASH with severe fibrotic phenotype

Sumanta Mukherjee - 2019

Abstract

Nonalcoholic steatohepatitis represents a significant and rapidly growing unmet medical need. The development of novel therapies has been hindered in part, by the limitations of existing preclinical models. There is a strong need for physiologically relevant in vivo and in vitro liver fibrosis models that are characterized by better translational predictability. In this study, we used the InSphero 3D InSightTM three-dimensional (3D) human liver microtissue (3D-hLMT) system prepared by co-culturing primary human hepatocytes with hepatic stellate cells, Kupffer cells and endothelial cells to develop a model of NASH with a severe fibrotic phenotype. In our model, palmitic acid (PA) induced a robust proinflammatory and profibrogenic phenotype in the 3D-hLMT. PA significantly increased several markers of the inflammatory and profibrotic process including gene expression of collagens, α-sma, tissue inhibitor of matrix metalloprotease 1 (timp1) and the stellate cell activation marker pdgfrβ as well as secreted CXCL8 (IL8) levels. We also observed TGFβ pathway activation, increase in active collagen synthesis and significant overall increase in tissue damage in the 3D-hLMTs. Immunohistochemistry analysis demonstrated the upregulation of collagen, cleaved caspase 3 as well as of the PDGFRβ protein. We further validated the model using a phase 3 clinical compound, GS-4997, an apoptosis signal-regulating kinase 1 (ASK-1) inhibitor and showed that GS-4997 significantly decreased PA induced profibrotic and proinflammatory response in the 3D-hLMTs with decreases in apoptosis and stellate cell activation in the microtissues. Taken together we have established and validated an in vitro 3D-hLMT NASH model with severe fibrotic phenotype that can be a powerful tool to investigate experimental compounds for the treatment of NASH.

Dysregulation of Circadian Rhythm Gene Expression in Cystic Fibrosis Mice

Eric Barbato - 2019

Abstract

Cystic fibrosis (CF) is autosomal recessive disease that affects multiple body systems. CF patients often experience sleep disturbances, altered sleep patterns, and sleep apnea. Sleep in mammals is controlled in part by circadian clock genes, including Clock, Bmal1, Period1, Period2, Cryptochrome1, and Cryptochrome2. The purpose of this study was to gain a better understanding of the biological underpinnings of disordered sleep experienced in CF. To accomplish this, we evaluated circadian clock gene expression profiles in CF and wildtype mice, divided into two subgroups each based on sleep condition. One subgroup of each genotype was permitted to maintain their sleep-wake cycle while the other was deprived of sleep for six hours prior to sacrifice. Brain, skeletal muscle, jejunum, colon, lung and adipose tissues were collected from each mouse. Quantitative polymerase chain reaction (PCR) was used to quantify expression of Clock, Bmal1, Period1, Period2, Cryptochrome1 and Cryptochrome2, and expression levels were compared between study groups. Our comparisons showed distinct differences between the CF groups and the wildtype groups under both sleep conditions. Additionally, we found the CF mice that had been sleep deprived had severely dysregulated expression of all measured genes in the lung apart from Cry1. Our findings suggest that (1) disordered sleep in CF may be caused by circadian system dysregulation and (2) the loss of the cystic fibrosis transmembrane conductance regulator (CFTR) is a causative factor in the dysregulated circadian clock gene expression profiles of CF mice.

Isolation and profiling of plasma microRNAs: Biomarkers for asthma and allergic rhinitis

Ronaldo P. Panganiban - 2019

Abstract

Chronic inflammatory diseases can be particularly challenging to diagnose and characterize, as inflammatory changes in tissue may not be present in blood. There is a crucial need to develop non-invasive biomarkers that would be useful in diagnosing disease and selecting medical therapies. For example, there are no blood tests to diagnose asthma, a common inflammatory lung disease. MicroRNA (miRNA) expression profiling in blood is emerging as a potentially sensitive and useful biomarker of many diseases. In particular, we have characterized a cost-effective PCR-based array technology to measure and profile circulating miRNAs in the plasma of patients with allergic rhinitis and asthma. Here, we describe the methods to isolate, quantify, and analyze miRNAs in the plasma of human subjects as well as ways to determine their diagnostic utility.

Glucocorticoid-driven transcriptomes in human airway epithelial cells: commonalities, differences and functional insight from cell lines and primary cells

Mahmoud M. Mostafa - 2019

Abstract

Background Glucocorticoids act on the glucocorticoid receptor (GR; NR3C1) to resolve inflammation and, as inhaled corticosteroids (ICS), are the cornerstone of treatment for asthma. However, reduced efficacy in severe disease or exacerbations indicates a need to improve ICS actions. Methods Glucocorticoid-driven transcriptomes were compared using PrimeView microarrays between primary human bronchial epithelial (HBE) cells and the model cell lines, pulmonary type II A549 and bronchial epithelial BEAS-2B cells. Results In BEAS-2B cells, budesonide induced (≥2-fold, P ≤ 0.05) or, in a more delayed fashion, repressed (≤0.5-fold, P ≤ 0.05) the expression of 63, 133, 240, and 257 or 15, 56, 236, and 344 mRNAs at 1, 2, 6, and 18 h, respectively. Within the early-induced mRNAs were multiple transcriptional activators and repressors, thereby providing mechanisms for the subsequent modulation of gene expression. Using the above criteria, 17 (BCL6, BIRC3, CEBPD, ERRFI1, FBXL16, FKBP5, GADD45B, IRS2, KLF9, PDK4, PER1, RGCC, RGS2, SEC14L2, SLC16A12, TFCP2L1, TSC22D3) induced and 8 (ARL4C, FLRT2, IER3, IL11, PLAUR, SEMA3A, SLC4A7, SOX9) repressed mRNAs were common between A549, BEAS-2B and HBE cells at 6 h. As absolute gene expression change showed greater commonality, lowering the cut-off (≥1.25 or ≤ 0.8-fold) within these groups produced 93 induced and 82 repressed genes in common. Since large changes in few mRNAs and/or small changes in many mRNAs may drive function, gene ontology (GO)/pathway analyses were performed using both stringency criteria. Budesonide-induced genes showed GO term enrichment for positive and negative regulation of transcription, signaling, proliferation, apoptosis, and movement, as well as FOXO and PI3K-Akt signaling pathways. Repressed genes were enriched for inflammatory signaling pathways (TNF, NF-κB) and GO terms for cytokine activity, chemotaxis and cell signaling. Reduced growth factor expression and effects on proliferation and apoptosis were highlighted. Conclusions While glucocorticoids repress mRNAs associated with inflammation, prior induction of transcriptional activators and repressors may explain longer-term responses to these agents. Furthermore, positive and negative effects on signaling, proliferation, migration and apoptosis were revealed. Since many such gene expression changes occurred in human airways post-ICS inhalation, the effects observed in cell lines and primary HBE cells in vitro may be relevant to ICS in vivo.

CDK9 Inhibition Induces a Metabolic Switch that Renders Prostate Cancer Cells Dependent on Fatty Acid Oxidation

Harri M. Itkonen - 2019

Abstract

Cyclin-dependent kinase 9 (CDK9), a key regulator of RNA-polymerase II, is a candidate drug target for cancers driven by transcriptional deregulation. Here we report a multi-omics-profiling of prostate cancer cell responses to CDK9 inhibition to identify synthetic lethal interactions. These interactions were validated using live-cell imaging, mitochondrial flux-, viability- and cell death activation assays. We show that CDK9 inhibition induces acute metabolic stress in prostate cancer cells. This is manifested by a drastic down-regulation of mitochondrial oxidative phosphorylation, ATP depletion and induction of a rapid and sustained phosphorylation of AMP-activated protein kinase (AMPK), the key sensor of cellular energy homeostasis. We used metabolomics to demonstrate that inhibition of CDK9 leads to accumulation of acyl-carnitines, metabolic intermediates in fatty acid oxidation (FAO). Acyl-carnitines are produced by carnitine palmitoyltransferase enzymes 1 and 2 (CPT), and we used both genetic and pharmacological tools to show that inhibition of CPT-activity is synthetically lethal with CDK9 inhibition. To our knowledge this is the first report to show that CDK9 inhibition dramatically alters cancer cell metabolism.

MicroRNA‑223 attenuates LPS‑induced inflammation in an acute lung injury model via the NLRP3 inflammasome and TLR4/NF‑κB signaling pathway via RHOB

Yurong Yan - 2019

Abstract

Acute lung injury (ALI) and the more severe acute respiratory distress syndrome are common and complex inflammatory lung diseases. MicroRNAs (miRs) have emerged as novel gene regulatory molecules, serving a crucial role in a variety of complex diseases, including ALI. In the present study, the anti‑inflammatory action of miR‑223 on inflammation in ALI was demonstrated and the possible mechanism was further examined. In lipopolysaccharide‑induced ALI, the expression of miR‑223 was reduced compared with that in the control normal group. An in vitro model was used to analyze the effect of miR‑223 downregulation on an ALI model, which increased inflammation, and induced the activation of the NACHT, LRR and PYD domains‑containing protein 3 (NLRP3) inflammasome and Toll‑like receptor 4 (TLR4)/nuclear factor (NF)‑κB signaling pathway via rho‑related GTP‑binding protein RhoB (RHOB). In addition, the overexpression of miR‑223 reduced inflammation and suppressed the NLRP3 inflammasome and TLR4/NF‑κB signaling pathway via RHOB in the in vitro model. Furthermore, TLR4 inhibitor or NLRP3 inhibitor reduced the pro‑inflammatory effect of miR‑223 downregulation in ALI. In conclusion, the results of the present study indicated that miR‑223 functioned as a biological indicator by regulating inflammation in ALI, and may represent a novel potential therapeutic target and prognostic marker of ALI.

Reduced expression of the IL7Ra signaling pathway in Neuromyelitis optica

Livnat Brill - 2018

Abstract

Neuromyelitis optica (NMO) is a chronic inflammatory demyelinating autoimmune disease of the central nervous system that most commonly affects the optic nerves and spinal cord. To characterize the immunological pathways involved in NMO, whole blood RNA expression array was performed using Nanostring nCounter technology. Two major clusters of genes were found associated with NMO: T cell-associated genes and the TNF/NF-kB signaling pathway. Analysis of the genes within the first cluster confirmed significantly reduced expression of IL7Ra (CD127) in the peripheral blood of NMO patients vs that in healthy controls. IL7Ra upstream transcription factors and its downstream survival signaling pathway were also markedly reduced. In line with the essential role of IL7Ra in T cell maturation and survival, a significantly lower number of naïve T cells, and reduced T cell survival signaling mediated by increased BID (BH3-interacting domain death agonist) expression and increased apoptosis was observed. Cumulatively, these findings indicate that the IL7Ra signaling pathway may play a role in the autoimmune process in NMO.

Small adipose stores in cystic fibrosis mice are characterized by reduced cell volume, not cell number

Ilya R. Bederman - 2018

Abstract

Cystic fibrosis (CF) is a lethal genetic disorder that affects many organ systems of the body, including various endocrine and exocrine tissues. Health and survival positively associate with body mass and as a consequence, CF clinical care includes high-fat, high-calorie diets to maintain and increase adipose tissue stores. Such strategies have been implemented without a clear understanding of the cause and effect relationship between body mass and patients' health. Here, we use CF mouse models, which display small adipose stores, to begin examining body fat as a prelude into mechanistic studies of low body growth in CF, so that optimal therapeutic strategies can be developed. We reasoned that low adiposity must result from reduced number and/or volume of adipocytes. To determine relative contribution of either mechanism, we quantified volume of intraperitoneal and subcutaneous adipocytes. We found smaller, but not fewer, adipocytes in CF compared to wildtype (WT) animals. Specifically, intraperitoneal CF adipocytes were half the volume of WT cells, whereas subcutaneous cells were less affected by the Cftr genotype. No differences were found in cell types between CF and WT adipose tissues. Adipose tissue CFTR mRNA was detected and we found greater CFTR expression in intraperitoneal depots as compared with subcutaneous samples. RNA sequencing revealed that CF adipose tissue exhibited lower expression of several key genes of adipocyte function (Lep, Pck1, Fas, Jun), consistent with low triglyceride storage. The data indicate that CF adipocytes contain less triglycerides than WT cells and a role for CFTR in these cells is proposed.

CETP Inhibition Improves HDL Function but Leads to Fatty Liver and Insulin Resistance in CETP-Expressing Transgenic Mice on a High-Fat Diet

Lin Zhu - 2018

Abstract

In clinical trials inhibition of cholesteryl ester transfer protein (CETP) raises HDL cholesterol levels but doesn’t robustly improve cardiovascular outcomes. About 2/3 of trial participants were obese. Lower plasma CETP activity is associated with increased cardiovascular risk in human studies, and protective aspects of CETP have been observed in mice fed a high-fat diet (HFD) with regard to metabolic outcomes. To define if CETP inhibition has different effects depending on the presence of obesity, we performed short-term anacetrapib treatment in chow- and HFD-fed CETP-transgenic mice. Anacetrapib raised HDL cholesterol and improved aspects of HDL functionality including reverse cholesterol transport and HDL’s anti-oxidative capacity in HFD-fed mice better than in chow-fed mice. Anacetrapib worsened the anti-inflammatory capacity of HDL in HFD-fed mice. The HDL proteome was markedly different with anacetrapib treatment in HFD-fed vs. chow-fed mice. Despite benefits on HDL, anacetrapib led to liver triglyceride accumulation and insulin resistance in HFD-fed mice. Overall, our results support a physiologic importance of CETP in protecting from fatty liver, and demonstrate a context-selectivity of CETP inhibition that might be important in obese subjects.

Induction of early growth response gene 1 (EGR1) by endoplasmic reticulum stress is mediated by the extracellular regulated kinase (ERK) arm of the MAPK pathways

Jixiu Shan - 2018

Abstract

Endoplasmic reticulum (ER) stress activates three principal signaling pathways, collectively known as the unfolded protein response, leading to translational and transcriptional control mechanisms that dictate the cell's response as adaptive or apoptotic. The present study illustrates that for HepG2 human hepatocellular carcinoma cells the signaling pathways triggered by ER stress extend beyond the three principal pathways to include mitogen-activated protein kinase (MAPK) signaling, leading to activation of transcription from the early growth response 1 (EGR1) gene. Analysis provided evidence for a SRC-RAS-RAF-MEK-ERK cascade mechanism that leads to enhanced phosphorylation of the transcription factor ELK1. ELK1 and serum response factor (SRF) are constitutively bound to the EGR1 promoter and are phosphorylated by nuclear localized ERK. The promoter abundance of both phospho-SRF and phopsho-ELK1 was increased by ER stress, but the SRF phosphorylation was transient. Knockdown of ELK1 had little effect on the basal EGR1 mRNA content, but completely blocked the increase in response to ER stress. Conversely, knockdown of SRF suppressed basal EGR1 mRNA content, but had only a small effect on the induction by ER stress. This research highlights the importance of MAPK signaling in response to ER stress and identifies ELK1 as a transcriptional mediator and the EGR1 gene as a target.

Treatment with Lecinoxoids Attenuates Focal and Segmental Glomerulosclerosis Development in Nephrectomized Rats

Niva Yacov - 2018

Abstract

Focal segmental glomerulosclerosis (FSGS) is a scarring process associated with chronic low-grade inflammation ascribed to toll-like receptor (TLR) activation and monocyte migration. We developed synthetic, small-molecule lecinoxoids, VB-201 and VB-703, that differentially inhibit TLR-2- and TLR-4-mediated activation and monocyte migration. The efficacy of anti-inflammatory lecinoxoid treatment on FSGS development was explored using a 5/6 nephrectomy rat model. Five-sixths nephrectomized rats were treated with lecinoxoids VB-201, VB-703 or PBS, for 7 weeks. Upon sacrifice, albumin/creatinine ratio, glomerulosclerosis, fibrosis-related gene expression, and the number of glomerular and interstitial monocyte were evaluated. Treatment of nephrectomized rats with lecinoxoids ameliorated glomerulosclerosis. The percent of damaged glomeruli, glomerular sclerosis and glomeruli fibrotic score were significantly reduced following VB-201 and VB-703 treatment. VB-703 attenuated the expression of fibrosis hallmark genes collagen, fibronectin (FN), and transforming growth factor β (TGF-β) in kidneys and improved albumin/creatinine ratio with higher efficacy than did VB-201, but only VB-201 significantly reduced the number of glomerular and interstitial monocytes. These results indicate that treatment with TLR-2, and more prominently, TLR-4 antagonizing lecinoxioids, is sufficient to significantly inhibit FSGS. Moreover, inhibiting monocyte migration can also contribute to treatment of FSGS. Our data demonstrate that targeting TLR2-4 and/or monocyte migration, directly affects the priming phase of fibrosis and may consequently perturb disease parthogenesis. This article is protected by copyright. All rights reserved.

Myeloperoxidase deficiency attenuates systemic and dietary Iron-induced adverse effects

Xia Xiao - 2018

Abstract

Iron deficiency is routinely treated with oral or systemic iron supplements, which is highly reactive and could induce oxidative stress via augmenting the activity of proinflammatory enzyme, myeloperoxidase (MPO). To investigate the extent to which MPO is involved in iron-induced toxicity, acute (24 h) iron toxicity was induced by intraperitoneal administration of FeSO4 (25 mg/kg body weight) to MPO deficient (MpoKO) mice and their WT littermates. Acute iron-toxicity was also assessed in WT mice pretreated with a MPO inhibitor, 4-aminobenzoic acid hydrazide (ABAH). Systemic iron administration upregulated circulating MPO, neutrophil elastase and elevated systemic inflammatory and organ damage markers in WT mice. However, genetic deletion of MPO or its inhibition significantly reduced iron-induced organ damage and systemic inflammatory responses. In contrast to the acute model, 8 weeks of 2% carbonyl iron diet feeding to WT mice did not change the levels of circulating MPO and neutrophil elastase but promoted their accumulation in the liver. Even though both MpoKO and WT mice displayed similar levels of diet-induced hyperferremia, MpoKO mice showed significantly reduced inflammatory response and oxidative stress than the WT mice. In addition, WT bone marrow-derived neutrophils (BMDN) generated more reactive oxygen species than MPO deficient BMDN upon iron stimulation. Altogether, genetic deficiency or pharmacologic inhibition of MPO substantially attenuated acute and chronic iron-induced toxicity. Our results suggest that targeting MPO during iron supplementation is a promising approach to reduce iron-induced toxicity/side effects in vulnerable population.

Humoral and cellular immune response of European sea bass (Dicentrarchus labrax(vaccinated with heat-killed Mycobacterium marinum (iipA::kan mutant)

N. Ziklo - 2018

Abstract

No vaccine is yet commercially available against Mycobacterium marinum, the etiological agent of “fish tuberculosis”. The mycobacterial gene responsible for invasion and intracellular persistence, iipA, is known to moderate M. marinum pathology in zebrafish. Two doses of heat-killed, wild type virulent M. marinum and two doses of a heat-killed avirulent M. marinum iipA::kan mutant strain were used in parallel to vaccinate sea bass (Dicentrarchus labrax). The fish were then challenged with live virulent M. marinum and the pathogenesis of the infection was monitored. High specific IgM response and increase in cytokine TNF-α mRNA expression levels were observed in all vaccinated fish. One month post-challenge, TNF-α expression levels increased in spleen tissues of fish vaccinated with the virulent type and of unvaccinated fish, whereas in the head-kidney expression up-regulated only in unvaccinated fish. The expression then decreased and two months post-challenge appeared similar in all vaccination types. Highest survival rate (75%) was recorded in the group of fish vaccinated with high dose of iipA::kan avirulent mutant. The iipA::kan mutant induced a strong immune response accompanied by only modest tissue disruption. Coupled with an effective program of booster treatments, the iipA::kan mutant vaccine may be developed into a powerful preventive measure against fish mycobacteriosis. This article is protected by copyright. All rights reserved.

Resident brain neural precursor cells develop age-dependent loss of therapeutic functions in Alzheimer's mice

Nina Fainstein - 2018

Abstract

There is vast knowledge on pathogenic mechanisms in Alzheimer's disease, but very little on means by which the brain protects itself from disease. A major candidate in providing neuroprotection is the resident brain neural stem/precursor cell (NPC) pool. Transplanted NPCs possess powerful immune-modulatory and trophic properties in vivo and in vitro, underscoring the question whether resident brain NPCs have any role in regulating disease pathology in Alzheimer's disease, and particularly whether they fail to protect the brain from degeneration. To evaluate brain NPC function in relation to disease pathology, we first characterized the pathological properties of 5xFAD transgenic mouse model of Alzheimer's disease at different ages. We found that age seven months is a critical time point of heavy amyloid deposition and gliosis but prior to neurodegeneration and a normal basal rate of NPC turnover in the subventricular zone (SVZ) of 5xFAD mice as compared to wild type mice. Analysis of NPC functional properties showed that despite preserved rate of turnover, there was substantial SVZ NPC dysfunction as indicated by both ex vivo and in vivo assays. Freshly isolated NPCs from seven months old 5xFAD mice exhibited reduced expansion rate, and diminished immune-modulatory and trophic properties. Moreover, there was slowed recovery of SVZ NPCs after Cytosine-arabinoside insult and markedly reduced migratory response following a Lysolecithine-induced lesion in the Corpus-Callosum in vivo. Importantly, these functions were fully preserved in two-months old 5xFAD mice, a time-point prior to Alzheimer's-specific pathological changes. There was reduced expression of key genes involved in NPC proliferative and migratory response in NPCs derived from seven months old 5xFAD mice. The dysfunctional properties and down-regulation of gene expression were reversible in NPCs derived from seven-months old 5xFAD mice following in-vitro expansion and were reproduced in wild type NPC by addition of amyloid beta peptide. Thus, there is age-dependent acquired NPC dysfunction, with loss of immune-modulatory and neurotrophic properties, which is induced by the pathological Alzheimer's brain environment at a critical time point prior to neurodegeneration. We suggest that failure of resident NPC to provide tissue support may be involved in promoting neurodegeneration.

CSDC2, a cold shock domain RNA-binding protein in decidualization

Griselda Vallejo - 2018

Abstract

RNA-binding proteins (RBPs) have been described for cancer cell progression and differentiation, although there is still much to learn about their mechanisms. Here, using in vivo decidualization as a model, we describe the role of RBP cold shock domain containing C2 (CSDC2) in the endometrium. Csdc2 messenger RNA expression was differentially regulated depending on time and areas of decidua development, with the most variation in antimesometrium (AM) and, to a lesser degree, in the junctional zone (JZ). Immunohistochemistry of CSDC2 showed a preferentially cytoplasmic localization at AM and JZ, and nuclear localization in underneath myometrium and mesometrium (M). Cytoplasmic localization coincided with differentiated, DESMIN-marked areas, while nuclear localization coincides with proliferative zones. Uterine suppression of CSDC2 through intrauterine-injected-specific small interfering RNA (siRNA) led to abnormal decidualization in early pregnancy, with more extended antimesometrial area and with poor M development if compared with control siRNA-injected animals. These results suggest that CSDC2 could be a regulator during decidua development.

Deletion of Axin1 in condylar chondrocytes leads to osteoarthritis-like phenotype in temporomandibular joint via activation of β-catenin and FGF signaling

Yachuan Zhou - 2018

Abstract

Osteoarthritis (OA) in the temporomandibular joint (TMJ) is a degenerative disease in the adult, which is characterized by the pathological degeneration of condylar cartilage. Axin1 plays a critical role in the regulation of cartilage development and homeostasis. To determine the role of Axin1 in TMJ tissue at the adult stage, we generated Axin1Agc1ER mice, in which Axin1 was deleted in aggrecan-expressing chondrocytes at 2 months of age. Histology, histomorphometry, and immunostaining analyses were performed using TMJ tissues harvested from 4- and 6-month-old mice after tamoxifen administration. Total RNA isolated from TMJ cartilage of 6-month-old mice was used for gene expression analysis. Progressive OA-like degeneration was observed in condylar cartilage in Axin1 knockout (KO) mice with loss of surface continuity and the formation of vertical fissures. In addition, reduced alcian blue staining in condylar cartilage was also found in Axin1 KO mice. Immunostaining and reverse transcription quantitative polymerase chain reaction (qRT-PCR) assays revealed disturbed homeostasis in condylar cartilage with increased expressions of MMP13 and Adamts5 and decreased lubricin expression in Axin1-deficient chondrocytes. Less proliferative cells with increased hypertrophic and apoptotic activities were presented in the condylar cartilage of Axin1Agc1ER KO mice. As a scaffolding protein, the deletion of Axin1 stimulated not only the β-catenin but also the fibroblast growth factor (FGF) signaling via extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activation. The qRT-PCR results showed an increased expression of Fgfr1 in Axin1 KO cartilage. Overall, the deletion of Axin1 in condylar chondrocytes altered the β-catenin and FGF/ERK1/2 signaling pathways, thus cooperatively contribute to the cartilage degeneration.

Assessment of Sep1virus interaction with stationary cultures by transcriptional and flow cytometry studies

Luís D. R. Melo - 2018

Abstract

The majority of phage infection studies are performed in bacteria that are growing exponentially, although in nature, phages usually interact also with non-replicating cells. These stationary-phase cells differ from exponential cells morphologically, physiologically and metabolically. The interaction of a Sep1virus with Staphylococcus epidermidis stationary and exponential phase cells was explored. Phage SEP1 efficiently infected both cell culture states, without the addition of any fresh nutrients to stationary cultures. Phage-host interactions, analysed by flow cytometry, showed stationary-phase cells response to phage immediately after SEP1 addition. Quantitative PCR experiments corroborate that phage genes are expressed within 5 min of contact with stationary phase cells. The increase of host RNA polymerase transcripts in stationary cells, suggests that SEP1 infection leads to upregulation of host machinery fundamental for completion of its lytic life cycle. SEP1 infection and replication process highlights its potential clinical interest targeting stationary phase cells.

Lactobacillus casei BL23 modulates the innate immune response in Staphylococcus aureus-stimulated bovine mammary epithelial cells

R.f.s. Souza - 2018

Abstract

Probiotics have been adopted to treat and prevent various diseases in humans and animals. They were notably shown to be a promising alternative to prevent mastitis in dairy cattle. This inflammation of the mammary gland is generally of infectious origin and generates extensive economic losses worldwide. In a previous study, we found that Lactobacillus casei BL23 was able to inhibit the internalisation of Staphylococcus aureus, one of the major pathogens involved in mastitis, into bovine mammary epithelial cells (bMEC). In this study, we further explored the capacity of this strain to modulate the innate immune response of bovine mammary epithelial cells during S. aureus infection. L. casei BL23 was able to decrease the expression of several pro-inflammatory cytokines, including interleukins 6, 8, 1α and 1β and tumour necrosis factor alpha, in S. aureus-stimulated bMEC, 8 h post-infection. On the other hand, L. casei did not impair the induction of defensins, such as lingual antimicrobial peptide and defensin β1 in the presence of S. aureus, and even slightly increased the induction of tracheal antimicrobial peptide during S. aureus infection. Finally, this strain did not alter the expression of the pattern recognition receptor nucleotide-binding oligomerisation domain proteins (NOD2). This study demonstrates that L. casei BL23 displayed anti-inflammatory properties on S. aureus-stimulated bMEC. These results open the way to further characterisation of the BL23 probiotic potential in a bovine mammary gland context and to a better understanding of how all these beneficial properties combine in vivo to combat mastitis pathogens.

The von Hippel Lindau tumour suppressor gene is a novel target of E2F4-mediated transcriptional repression in preeclampsia

Sruthi Alahari - 2018

Abstract

The von Hippel Lindau tumour suppressor (VHL) protein is essential for proper placental development and is downregulated in preeclampsia (PE), a devastating disorder of pregnancy typified by chronic hypoxia. To date, knowledge on VHL genetic and epigenetic regulation is restricted to inactivating mutations and loss-of-heterozygosity in renal cell carcinomas. Herein, we sought to examine whether VHL DNA is subject to differential methylation, and if so, whether it is altered in early-onset PE (E-PE). Sodium bisulfite modification and methylation-specific PCR analysis revealed that VHL is subject to extensive methylation in a CpG-rich region within its promoter in the human placenta. Notably, we detected significant differences in methylation in E-PE placentae relative to normotensive age-matched controls at key transcription factor binding sites, including that of the transcriptional repressor E2F4. Treatment of JEG3 cells with 5-Aza-2′-deoxycytidine, revealed that loss of DNA methylation promoted VHL transcription by attenuating VHL association with E2F4. RNAi knockdown of E2F4 in vitro confirmed its function on VHL repression. Exposure of JEG3 cells to transforming growth factor beta (TGFβ) downregulated VHL mRNA. In line with elevated levels of TGFβ3 in E-PE, chromatin immunoprecipitation assays revealed that E2F4-VHL association was enhanced upon TGFβ3 treatment, indicative of VHL transcriptional inhibition. In line with decreased VHL expression in E-PE, augmented E2F4-VHL association was also observed in E-PE placental tissue relative to controls. In conclusion, we demonstrate for the first time that hypomethylation of VHL DNA at a key transcription factor binding site has significant consequences for its transcriptional repression in early-onset preeclampsia.

A STAT4 variant increases liver fibrosis risk in Caucasian patients with chronic hepatitis B

R. El Sharkawy - 2018

Abstract

Background Host genetic modifiers of the natural history of chronic hepatitis B (CHB) remain poorly understood. Recently, a genome-wide association study (GWAS)-identified polymorphism in the STAT4 gene that contributes to the risk for hepatocellular carcinoma (HCC) was shown to be associated with the full spectrum of hepatitis B virus (HBV) outcomes in Asian patients. However, the functional mechanisms for this effect are unknown and the role of the variant in modulating HBV disease in Caucasians has not been investigated. Aims To determine whether STAT4 genetic variation is associated with liver injury in Caucasian patients with CHB and to investigate potential mechanisms mediating this effect. Methods STAT4 rs7574865 was genotyped in 1085 subjects (830 with CHB and 255 healthy controls). STAT4 expression in liver, PBMCs and NK cells, STAT4 phosphorylation and secretion of interferon-gamma (IFN-γ) according to STAT4 genetic variation was examined. Results STAT4 rs7574865 genotype was independently associated with hepatic inflammation (OR: 1.42, 95% CI: 1.07-2.06, P = 0.02) and advanced fibrosis (OR: 1.83, 95% CI: 1.19-2.83, P = 0.006). The minor allele frequency of rs7574865 was significantly lower than that in healthy controls. rs7574865 GG risk carriers expressed lower levels of STAT4 in liver, PBMCs and in NK cells, while NK cells from patients with the risk genotype had impaired STAT4 phosphorylation following stimulation with IL-12/IL-18 and a reduction in secretion of IFN-γ. Conclusion Genetic susceptibility to HBV persistence, hepatic inflammation and fibrosis in Caucasians associates with STAT4 rs7574865 variant. Downstream effects on NK cell function through STAT4 phosphorylation-dependent IFN-γ production likely contribute to these effects.

Development of Escherichia coli based gene expression profiling of sewage sludge leachates

Manish Goswami - 2018

Abstract

Aims The impact of municipal waste on pathogenic microorganisms released into the environment is a public health concern. The present study aims to evaluate the effects of sewage sludge and antibiotic contaminants on stress response, virulence and antibiotic resistance in a pathogenic Escherichia coli. Methods and Results The effects of sewage sludge leachates on uropathogenic E. coli CFT073 were determined by monitoring the expression of 45 genes associated with antibiotic/metal resistance, stress response and virulence using RT-qPCR. The E. coli gene expression was validated using sub-inhibitory concentrations of tetracycline and ciprofloxacin. E. coli exposed to sewage sludge or sewage sludge-fly ash leachates altered the expression of 5 antibiotic and metal resistance, 3 stress response and 2 virulence associated genes. When antibiotics were combined with sludge or sludge-fly ash the antibiotic-associated gene expression was altered. Conclusions E. coli treated with two sludge leachates had distinct gene expression patterns that were altered when the sludge leachates were combined with tetracycline, although to a lesser extent with ciprofloxacin. Significance and Impact of Study The E. coli multigene expression analysis is a potential new tool for assessing the effects of pollutants on pathogenic microbes in environmental waters for improved risk assessment. This article is protected by copyright. All rights reserved.

High MUC2 Mucin Biosynthesis in Goblet Cells Impedes Restitution and Wound Healing by Elevating Endoplasmic Reticulum Stress and Altered Production of Growth Factors

Adelaide Tawiah - 2018

Abstract

Intestinal epithelial cell wound healing involves cell migration, proliferation, and differentiation. Although numerous studies have analyzed the migration of absorptive epithelial cells during wound healing, it remains unclear how goblet cells restitute and how MUC2 mucin production affects this process. In this study, we examined the role of high MUC2 production in goblet cell migration during wound healing and demonstrated that during high MUC2 output, goblet cells migrated slower because of impaired production of wound healing factors and endoplasmic reticulum (ER) stress. Two goblet cell lines, HT29-H and HT29-L, that produced high and low MUC2 mucin, respectively, were used. HT29-L healed wounds faster than HT29-H cells by producing significantly higher amounts of fibroblast growth factor (FGF) 1, FGF2, vascular endothelial growth factor-C, and matrix metallopeptidase 1. Predictably, treatment of HT29-H cells with recombinant FGF2 significantly enhanced migration and wound healing. High MUC2 biosynthesis in HT29-H cells induced ER stress and delayed migration that was abrogated by inhibiting ER stress with tauroursodeoxycholic acid and IL-22. FGF2- and IL-22–induced wound repair was dependent on STAT1 and STAT3 signaling. During wound healing after dextran sulfate sodium–induced colitis, restitution of Math1M1GFP+ goblet cells occurred earlier in the proximal colon, followed by the mid and then distal colon, where ulceration was severe. We conclude that high MUC2 output during colitis impairs goblet cell migration and wound healing by reducing production of growth factors critical in wound repair.

Roflumilast promotes memory recovery and attenuates white matter injury in aged rats subjected to chronic cerebral hypoperfusion

Amanda Santiago - 2018

Abstract

Chronic cerebral hypoperfusion (CCH) has been associated with aging-related vascular dementia, including Alzheimer's disease. It can be induced by the four-vessel occlusion/internal carotid artery (4VO/ICA) model in aged rats, resulting in persistent memory deficits, white matter injury, and significant neuronal loss in the hippocampus and cerebral cortex. The phosphodiesterase type 4 inhibitor (PDE4-I) roflumilast has been reported to have pro-cognitive effects in several behavioral paradigms. The present study evaluated the effects of repeated roflumilast treatment in aged rats that were subjected to CCH. After surgery, roflumilast (0.003 and 0.01 mg/kg) was administered intraperitoneally once per day for 29 days. Memory performance was assessed in the aversive radial maze (AvRM) 7, 14, and 21 days after CCH. The effects of roflumilast on hippocampal neurodegeneration and white matter injury were investigated using Nissl and Kluver-Barrera staining, respectively. Western blot and RT-qPCR were used to explore microglial polarization using M1 (Iba-1 and iNOS) and M2 (Arginase-1) markers. Chronic cerebral hypoperfusion caused persistent memory deficits, hippocampal neurodegeneration, and vacuolization and fiber disarrangement in white matter. Repeated roflumilast treatment restored CCH-induced cognitive impairments in aged rats but in the absence of the rescue of hippocampal neurons. Attenuation of white matter injury was detected in the optic tract in aged CCH rats that were treated with roflumilast. In vitro, roflumilast increased Arg-1 gene expression in myelin-laden primary microglia. The present data suggest that roflumilast might be useful for the treatment of cognitive sequelae associated with CCH.

Elevation of the TP53 isoform Δ133p53β in glioblastomas: an alternative to mutant p53 in promoting tumour development

Marina Kazantseva - 2018

Abstract

As tumour protein 53 (p53) isoforms have tumour promoting, migration and inflammatory properties, this study investigated whether p53 isoforms contributed to glioblastoma progression. The expression levels of full-length TP53α (TAp53α) and six TP53 isoforms were quantitated by RT-qPCR in 89 glioblastomas and correlated with TP53 mutation status, tumour-associated macrophage content and various immune cell markers. Elevated levels of Δ133p53β mRNA characterised glioblastomas with increased CD163-positive macrophages and wild-type TP53. In situ based analyses found Δ133p53β expression localised to malignant cells in areas with increased hypoxia, and in cells with the monocyte chemoattractant protein C-C motif chemokine ligand 2 (CCL2) expressed. Tumours with increased Δ133p53β had increased numbers of cell positive for macrophage colony stimulating factor 1 receptor (CSF1R) and programmed death ligand 1 (PDL1). In addition, cells expressing a murine ‘mimic’ of Δ133p53 (Δ122p53) were resistant to temozolomide treatment and oxidative stress. Our findings suggest elevated Δ133p53β is an alternative pathway to TP53 mutation in glioblastoma that aids tumour progression by promoting an immunosuppressive and chemoresistant environment. Adding Δ133p53β to a TP53 signature along with TP53 mutation status will better predict treatment resistance in glioblastoma. This article is protected by copyright. All rights reserved.

Isolation and profiling of plasma microRNAs: Biomarkers for asthma and allergic rhinitis

Ronaldo P. Panganiban - 2018

Abstract

Gemcitabine resistance mediated by ribonucleotide reductase M2 in lung squamous cell carcinoma is reversed by GW8510 through autophagy induction

Ping Chen - 2018

Abstract

Although chemotherapeutic regimen containing gemcitabine is the first-line therapy for advanced lung squamous cell carcinoma (LSCC), gemcitabine resistance remains an important clinical problem. Some studies suggest that overexpressions of ribonucleotide reductase subunit M2 (RRM2) may be involved in gemcitabine resistance. We used a novel RRM2 inhibitor, GW8510, as a gemcitabine sensitization agent to investigate the therapeutic utility in reversing gemcitabine resistance in LSCC. Results showed that the expressions of RRM2 were increased in gemcitabine intrinsic resistant LSCC cells upon gemcitabine treatment. GW8510 not only suppressed LSCC cell survival, but also sensitized gemcitabine-resistant cells to gemcitabine through autophagy induction mediated by RRM2 downregulation along with decreases of deoxyribonucleotide triphosphate (dNTP) levels. The combination of GW8510 and gemcitabine produced a synergistic effect on killing LSCC cells. The synergism of the two agents was impeded by addition of autophagy inhibitors chloroquine or bafilomycin A1, or knockdown of the autophagy gene BECN1. Moreover, GW8510-caused LSCC cell sensitization to gemcitabine through autophagy induction was parallel with impairment of DNA double strand break repair and marked increase of cell apoptosis, revealing a crosstalk between autophagy and DNA damage repair, and an interplay between autophagy and apoptosis. Finally, gemcitabine sensitization mediated by autophagy induction through GW8510-caused RRM2 downregulation was demonstrated in vivo gemcitabine-resistant LSCC tumor xenograft, further indicating that the sensitization is dependent on autophagy activation. In conclusions, GW8510 can reverses gemcitabine resistance in LSCC cells through RRM2 downregulation-mediated autophagy induction, and GW850 may be a promising therapeutic agent against LSCC as it combined with gemcitabine.

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