Decrease In Gene Transcription Research Articles

Water-soluble garlic polysaccharide (WSGP) improves ulcerative colitis by modulating the intestinal barrier and intestinal flora metabolites

WSGP has demonstrated significant potential for various bioactive effects. However, limited research has explored their anti-ulcerative colitis (UC) effects and mechanism on the colonic system and gut microbial metabolites. We evaluated the ameliorative effects of WSGP on the UC mice model. Using H&E to assess histological injury of colon morphology, AB-PAS staining to detect mucin secretion from goblet cells and the mucous layer, IF to evaluate the expression of intercellular tight junction proteins, ELISA to measure inflammatory factors, WB analysis to measure protein expression of inflammatory signaling pathways, RT-qPCR to quantify gene transcription of inflammatory factors, and LC-MS to analyze metabolites in mouse cecum contents. WSGP supplementation increased food intake, body weight, and colon length while reducing disease activity and histological scores in colitis-afflicted mice. WSGP mitigated colonic tissue damage and restored intestinal barrier integrity by suppressing NF-κB/STAT3 signaling, thereby decreasing gene transcription, protein expression of proinflammatory factors, and nitric oxide production. Additionally, WSGP improved UC by altering the variety of intestinal microbial metabolites. This study demonstrates that WSGP supplementation attenuates UC mice by suppressing the NF-κB/STAT3 signaling pathway, enhancing mucosal barrier function, reducing pro-inflammatory cytokines, and modulating gut microbial metabolites.

ASS1 metabolically contributes to the nuclear and cytosolic p53-mediated DNA damage response

Downregulation of the urea cycle enzyme argininosuccinate synthase (ASS1) in multiple tumors is associated with a poor prognosis partly because of the metabolic diversion of cytosolic aspartate for pyrimidine synthesis, supporting proliferation and mutagenesis owing to nucleotide imbalance. Here, we find that prolonged loss of ASS1 promotes DNA damage in colon cancer cells and fibroblasts from subjects with citrullinemia type I. Following acute induction of DNA damage with doxorubicin, ASS1 expression is elevated in the cytosol and the nucleus with at least a partial dependency on p53; ASS1 metabolically restrains cell cycle progression in the cytosol by restricting nucleotide synthesis. In the nucleus, ASS1 and ASL generate fumarate for the succination of SMARCC1, destabilizing the chromatin-remodeling complex SMARCC1–SNF5 to decrease gene transcription, specifically in a subset of the p53-regulated cell cycle genes. Thus, following DNA damage, ASS1 is part of the p53 network that pauses cell cycle progression, enabling genome maintenance and survival. Loss of ASS1 contributes to DNA damage and promotes cell cycle progression, likely contributing to cancer mutagenesis and, hence, adaptability potential.

Severe thermal and major traumatic injury results in elevated plasma concentrations of total heme that are associated with poor clinical outcomes and systemic immune suppression.

Traumatic and thermal injuries result in a state of systemic immune suppression, yet the mechanisms that underlie its development are poorly understood. Released from injured muscle and lysed red blood cells, heme is a damage associated molecular pattern with potent immune modulatory properties. Here, we measured plasma concentrations of total heme in over 200 traumatic and thermally-injured patients in order to examine its relationship with clinical outcomes and post-injury immune suppression. Blood samples were collected from 98 burns (≥15% total body surface area) and 147 traumatically-injured (injury severity score ≥8) patients across the ultra-early (≤1 hour) and acute (4-72 hours) post-injury settings. Pro-inflammatory cytokine production by lipopolysaccharide (LPS) challenged whole blood leukocytes was studied, and plasma concentrations of total heme, and its scavengers haptoglobin, hemopexin and albumin measured, alongside the expression of heme-oxygenase-1 (HO-1) in peripheral blood mononuclear cells (PBMCs). LPS-induced tumour necrosis factor-alpha (TNF-α) production by THP-1 cells and monocytes following in vitro heme treatment was also examined. Burns and traumatic injury resulted in significantly elevated plasma concentrations of heme, which coincided with reduced levels of hemopexin and albumin, and correlated positively with circulating levels of pro and anti-inflammatory cytokines. PBMCs isolated from trauma patients 4-12 and 48-72 hours post-injury exhibited increased HO-1 gene expression. Non-survivors of burn injury and patients who developed sepsis, presented on day 1 with significantly elevated heme levels, with a difference of 6.5 µM in heme concentrations corresponding to a relative 52% increase in the odds of post-burn mortality. On day 1 post-burn, heme levels were negatively associated with ex vivo LPS-induced TNF-α and interleukin-6 production by whole blood leukocytes. THP-1 cells and monocytes pre-treated with heme exhibited significantly reduced TNF-α production following LPS stimulation. This impairment was associated with decreased gene transcription, reduced activation of extracellular signal-regulated kinase 1/2 and an impaired glycolytic response. Major injury results in elevated plasma concentrations of total heme that may contribute to the development of endotoxin tolerance and increase the risk of poor clinical outcomes. Restoration of the heme scavenging system could be a therapeutic approach by which to improve immune function post-injury.

Role of 6-phosphogluconate dehydrogenase enzyme 1 in growth and virulence of Toxoplasma gondii and development of attenuated live vaccine.

Toxoplasma gondii is a ubiquitous pathogen that infects all warm-blooded animals, including humans, causing substantial socioeconomic and healthcare burdens. However, there is no ideal vaccine for toxoplasmosis. As metabolism is important in the growth and virulence of Toxoplasma, some key pathways are promising antiparasitic targets. Here, we identified 6-phosphogluconate dehydrogenase 1 (Tg6PGDH1) in the oxidative pentose phosphate pathway as a cytoplasmic protein that is dispensable for tachyzoite growth of T. gondii in vitro but critical for virulence and cyst formation in vivo. The depletion of Tg6PGDH1 causes decreased gene transcription involved in signal transduction, transcriptional regulation and virulence. Furthermore, we analysed the protective effect of the ME49Δ6pgdh1 mutant as an attenuated vaccine and found that ME49Δ6pgdh1 immunization stimulated strong protective immunity against lethal challenges and blocked cyst formation caused by reinfection. Furthermore, we showed that ME49Δ6pgdh1 immunization stimulated increased levels of interferon-gamma, tumour necrosis factor-alpha and Toxoplasma-specific IgG antibodies. These data highlight the role of Tg6PGDH1 in the growth and virulence of T. gondii and its potential as a target for the development of a live-attenuated vaccine.

Abstract A024: PRC2 loss drives MPNST metastasis and matrix remodeling

Abstract Epigenetic reprogramming alters gene transcription in response to different cellular cues and can occur at both the DNA level as well as histone post-translational modifications. Polycomb repressive complex 2 (PRC2) is a common epigenetic modifier that is responsible for adding methyl groups to the twenty-seventh lysine of histone 3. This modification is associated with decreased gene transcription and recruits complexes that further compact chromatin. PRC2 mutations have been implicated in the tumorigenesis of many cancer types; however, the role that PRC2 plays in tumorigenesis is complex and not well understood. PRC2 loss-of-function mutations have been identified in malignant peripheral nerve sheath tumors (MPNST), an aggressive sarcoma. PRC2 mutations are found in the EED and SUZ12 subunits in 70-92% of tumors. There are currently no effective therapies for MPNSTs resulting in a poor prognosis for individuals with inoperable tumors. Using a CRISPR/Cas9 system to delete EED or SUZ12, we developed in vitro and in vivo models to further study the biology of PRC2 loss in MPNSTs. Our data suggests that loss of PRC2 subunits increases the expression of extra-cellular matrix (ECM) remodeling genes, promotes cell migration in vitro, and increases pulmonary metastasis in vivo. We also observed that loss of either EED or SUZ12 increases collagen handling and a clustering metastatic phenotype. Additionally, loss of H3K27me3 was correlated with increased ECM and ECM remodeling genes as well as metastasis and overall survival in patient RNA-seq and tumor microarray data, respectively. Further characterization of the underlying mechanisms of PRC2-dependent metastasis will allow us to better understand the role of PRC2 in MPNST biology and test novel therapies for these patients. Citation Format: Qierra R. Brockman, Amanda Scherer, Gavin R. McGivney, Wade R. Gutierrez, Andrew Voigt, Alexandra Isaacson, Emily A. Laverty, Grace Roughton, Vickie Knepper-Adrian, Benjamin Darbro, Munir R. Tanas, Christopher Stipp, Rebecca D. Dodd. PRC2 loss drives MPNST metastasis and matrix remodeling. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A024.

Exportin-1 Connects Dynamic Transcription and Translation of Genotoxic Stress Genes in Diffuse Large B-Cell Lymphoma Patients

Exportin-1 (XPO1), that exports a subset of proteins from the nucleus to the cytoplasm, is amplified in 30% of DLBCL. Regardless of XPO1 gene status, DLBCLs require XPO1 to proliferate and survive as demonstrated in pre-clinical (Marullo et al. AACR 2015) and clinical (Kuruvilla at al. Blood 2017) studies with the covalent XPO1 inhibitor selinexor. However, the mechanism underlying such dependency remains unknown.We analyzed the expression of XPO1 protein in a cohort of 57 DLBCL patients by IHC and correlated with XPO1 gene status and clinical outcome. XPO1 increased copy number is associated with elevated protein expression (p = 0.03) and, importantly, patients with elevated XPO1 levels (XPO1 high) are less likely to respond to RCHOP as refractory/early relapse diseases (p = 0.005). This suggests that XPO1 high DLBCL could be biologically fitter to tolerate genotoxic stress. To explore this hypothesis, we compared gene expression profiles (RNA-sequencing) of XPO1 high (n = 15) vs. XPO1 low (n = 16) DLBCLs by DESeq2. XPO1 high DLBCLs were enriched (q<0.005) for genes regulating “cell proliferation”, “DNA damage response and repair”, “mRNA processing” and “RNA export”; whereas depleted (q<0.05) for genes regulating “immune response”. Gene Set Enrichment Analysis also revealed that XPO1 high were enriched for transcripts up-regulated in highly aneuploid tumors (FDR = 0, NES = 2.19). All these findings were validated in an independent cohort of 72 DLBCL patients.To gain insight into the mechanism by which XPO1 enhances genotoxic stress tolerance, we exposed a panel of 5 DLBCL cell lines to DNA damaging agents (etoposide, doxorubicin, mechloretamine) with or without XPO1 inhibition (selinexor). Comet assay experiments revealed that selinexor did not affect the amount of DNA damage induced by chemotherapy, but delayed cell's ability to repair the DNA damage (P>0.005). This translated into a significant increase of sensitivity toward otherwise sub-lethal effect of chemotherapy, as all the combinations tested showed synergistic effect on growth inhibition. To determine the existence of a therapeutic window to capitalize on this effect, we evaluated the combination of CHOP +/- selinexor in an XPO1 high DLBCL patient-derived xenograft mice model (n = 40). Combination treatment achieved higher anti-lymphoma effect compared to either selinexor or CHOP alone (p>0.005 and p= 0.0015, respectively). Importantly, no increase in toxicity was observed by biochemistry or pathological examination. Thus, proliferating DLBCL cells but not normal cells rely on XPO1 to survive exogenous genotoxic stress.To characterize the molecular consequences of XPO1 inhibition, we quantified the expression of proteins involved in DNA damage response and repair (immunoblotting). Exposure to selinexor decreased the expression of a subset of these proteins including CHEK1, BRCA1, BCL6 and RAD51. This reduction was neither due to decreased gene transcription (qRT-PCR) nor to increased protein degradation (cycloheximide chase). Instead, analysis of nascent proteins by L-azido-homo-alanine labeling revealed that XPO1 inhibition decreases the synthesis of specific DNA damage repair proteins whose increased turnover is required to survive genotoxic stress. Since XPO1 inhibition affected only a subset of proteins, the mechanism may involve selective decrease in nuclear export of mRNA templates. Thus, we quantified the nuclear/cytoplasmic ratio of mRNAs encoding for DNA damage repair proteins in DLBCL cells treated with selinexor. XPO1 inhibition resulted in nuclear retention of BRCA1, RAD51, BCL6 and CHEK1 mRNAs but not of β-actin. Since XPO1 is unable to directly bind mRNAs, we reasoned that its inhibition may impair the export of proteins carrying mRNA (ribonucleoproteins). Indeed, we identified eIF4E as one of these cargoes. Immunofluorescence experiments revealed that eIF4E is retained in the nucleus of DLBCL cells upon XPO1 inhibition and its nuclear retention is enhanced in etoposide-treated cells. Moreover, RIP-qPCR experiments revealed that eIF4E binds several DNA damage repair mRNAs such as CHEK1, BCL6 and BRCA1 in the nucleus of DLBCL cells and this binding increases significantly in response to DNA damage.Overall, our data demonstrate that XPO1, by regulating the selective export of mRNA-binding proteins, promotes genotoxic stress tolerance thus conferring a survival advantage to XPO1high DLBCLs. DisclosuresYang:Regeneron Pharmaceuticals: Employment. Cerchietti:Leukemia and Lymphoma Society: Research Funding; Lymphoma Research Foundation: Research Funding; Celgene: Research Funding; Weill Cornell Medicine - New York Presbyterian Hospital: Employment.

Structure-based analyses of Salmonella RcsB variants unravel new features of the Rcs regulon.

RcsB is a transcriptional regulator that controls expression of numerous genes in enteric bacteria. RcsB accomplishes this role alone or in combination with auxiliary transcriptional factors independently or dependently of phosphorylation. To understand the mechanisms by which RcsB regulates such large number of genes, we performed structural studies as well as in vitro and in vivo functional studies with different RcsB variants. Our structural data reveal that RcsB binds promoters of target genes such as rprA and flhDC in a dimeric active conformation. In this state, the RcsB homodimer docks the DNA-binding domains into the major groove of the DNA, facilitating an initial weak read-out of the target sequence. Interestingly, comparative structural analyses also show that DNA binding may stabilize an active conformation in unphosphorylated RcsB. Furthermore, RNAseq performed in strains expressing wild-type or several RcsB variants provided new insights into the contribution of phosphorylation to gene regulation and assign a potential role of RcsB in controlling iron metabolism. Finally, we delimited the RcsB box for homodimeric active binding to DNA as the sequence TN(G/A)GAN4TC(T/C)NA. This RcsB box was found in promoter, intergenic and intragenic regions, facilitating both increased or decreased gene transcription.

In This Issue

HomeCirculation ResearchVol. 127, No. 9In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published8 Oct 2020https://doi.org/10.1161/RES.0000000000000443Circulation Research. 2020;127:1119is related toEpigenomic and Transcriptomic Dynamics During Human Heart OrganogenesisEnhancement of ALK1 Signaling as a HHT Therapy (p 1122)Boosting activity of kinase ALK1 could be a treatment for hereditary hemorrhagic telangiectasia, say Kim et al.Download figureDownload PowerPointHereditary hemorrhagic telangiectasia (HHT) is a genetic disease characterized by vascular abnormalities called arteriovenous malformations (AVMs) in which arteries are directly connected to veins. Without intervening capillaries, blood pressure in the veins becomes excessive and can cause aneurysms and ruptures. Indeed, HHT patients often endure frequent bleeds and anemia, and sometimes suffer more severe events such as brain or pulmonary hemorrhages. HHT is caused by mutations in one of three genes—ALK1, ENG, or SMAD4—all of which encode components of a TGFβ-related growth factor signaling pathway. Because ninety percent of HHT cases are driven by ALK1 or ENG mutations, Kim et al focused on these two factors, examining how they might interact and compensate for each other. Using mice that conditionally lacked either ALK1 or ENG and had AVMs as a result, the team found that genetically increasing ALK1 production could prevent AVMs in the ENG- and ALK-lacking mice, but that increasing ENG production could only compensate for deficiency of ENG, not ALK1. Together these results suggest that ALK1 acts downstream of ENG in their shared signaling pathway and that augmenting ALK1 production could be a therapeutic approach for treating HHT.Periostin Is a Target to Treat PH (p 1138)Nie et al suggest periostin as a novel therapeutic target for treating pulmonary hypertension.Download figureDownload PowerPointPulmonary hypertension (PH) is a life threatening disease in which excess proliferation of vascular smooth muscle cells (VSMCs) and deposition of extracellular matrix (ECM) thickens the walls of the lung vasculature causing an increase in pulmonary blood pressure. Vasodilatory medications are used to treat the symptoms of the disease, but there are no available treatments that target the underlying pathologic remodeling. Nie and colleagues now suggest that drugs targeting the secreted ECM protein periostin might work. Periostin is unusually abundant in the arteries of PH patients’ lungs and is thought to be involved in cell adhesion and wound healing mechanisms such as proliferation and migration of VSMCs. The team has now confirmed the increased production of periostin in patient lungs and shown the same is true for mice with induced PH. Moreover they showed that genetic deletion of periostin attenuated PH in mice, while suppression of periostin via RNA inhibition could even reverse pathologic vessel thickening and right ventricle hypertrophy. The team went on to identify factors—HIF-1A and TrkB—that mediate periostin’s effects in cultured arterial cells, and suggest that blocking either these factors or periostin itself could be novel strategies for treating PH.Gene Regulatory Dynamics of Developing Human Heart (p e184)VanOudenhove et al examine the epigenomics and transcriptomics of the developing human heart.Download figureDownload PowerPointCongenital heart defects (CHDs) are a common form of birth abnormality. While some genes have been linked to CHDs, the majority—nearly sixty percent—have unknown etiologies. It’s thought that multiple genetic and environmental factors contribute to CHDs, one of which could be variations in regulatory regions of the genome. To find such heart-specific regulatory regions, VanOudenhove and colleagues examined heart tissue from human embryos obtained 4 to 8 weeks after conception. They performed chromatin immunoprecipitation (ChIP) experiments to scour the heart genomes for histone modifications associated with increased or decreased gene transcription. They also performed transcriptome analysis to see whether the genomic regions identified by ChIP corresponded with the activity status of nearby genes. In total, the team found more than 12 thousand previously unknown enhancers that were enriched for binding sites for heart-specific transcription factors (including GATA, MEF2 and NKX) and that tended to be close to genes activated in the heart. Many of the regions also contained sequence variations previously associated with atrial fibrillation. These newly identified sites are potential CHD candidate loci, say the authors, who have made their data freely available for study. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesEpigenomic and Transcriptomic Dynamics During Human Heart OrganogenesisJennifer VanOudenhove, et al. Circulation Research. 2020;127:e184-e209 October 9, 2020Vol 127, Issue 9Article InformationMetrics Download: 270 © 2020 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000443 Originally publishedOctober 8, 2020 PDF download

Integrating multiple omics to identify common and specific molecular changes occurring in Arabidopsis under chronic nitrate and sulfate limitations

Plants have fundamental dependences on nitrogen and sulfur and frequently have to cope with chronic limitations when their supply is sub-optimal. This study aimed at characterizing the metabolomic, proteomic, and transcriptomic changes occurring in Arabidopsis leaves under chronic nitrate (Low-N) and chronic sulfate (Low-S) limitations in order to compare their effects, determine interconnections, and examine strategies of adaptation. Metabolite profiling globally revealed opposite effects of Low-S and Low-N on carbohydrate and amino acid accumulations, whilst proteomic data showed that both treatments resulted in increases in catabolic processes, stimulation of mitochondrial and cytosolic metabolism, and decreases in chloroplast metabolism. Lower abundances of ribosomal proteins and translation factors under Low-N and Low-S corresponded with growth limitation. At the transcript level, the major and specific effect of Low-N was the enhancement of expression of defence and immunity genes. The main effect of chronic Low-S was a decrease in transcripts of genes involved in cell division, DNA replication, and cytoskeleton, and an increase in the expression of autophagy genes. This was consistent with a role of target-of-rapamycin kinase in the control of plant metabolism and cell growth and division under chronic Low-S. In addition, Low-S decreased the expression of several NLP transcription factors, which are master actors in nitrate sensing. Finally, both the transcriptome and proteome data indicated that Low-S repressed glucosinolate synthesis, and that Low-N exacerbated glucosinolate degradation. This showed the importance of glucosinolate as buffering molecules for N and S management.

The effect of the demyelinating agent cuprizone on binge-like eating of sweetened palatable food in female and male C57BL/6 substrains

Binge eating is a heritable symptom of eating disorders with an unknown genetic etiology. Rodent models for binge-like eating (BLE) of palatable food permit the study of genetic and biological mechanisms. We previously genetically mapped a coding mutation in Cyfip2 associated with increased BLE of sweetened palatable food in the C57BL/6NJ versus C57BL/6J substrain. The increase in BLE in C57BL/6NJ mice was associated with a decrease in transcription of genes enriched for myelination in the striatum. Here, we tested the hypothesis that decreasing myelin levels with the demyelinating agent cuprizone would enhance BLE. Mice were treated with a 0.3% cuprizone home cage diet for two weeks. Cuprizone induced similar weight loss in both substrains and sexes that recovered within 48 h after removal of cuprizone. Following a three-week recovery period, mice were trained for BLE in an intermittent, limited access procedure. Surprisingly, cuprizone significantly reduced BLE in male but not female C57BL/6NJ mice while having no effect in C57BL/6J mice. Cuprizone also reduced myelin basic protein (MBP) at seven weeks post-cuprizone removal while having no effect on myelin-associated glycoprotein at this time point. C57BL/6NJ mice also showed less MBP than C57BL/6J mice. There were no statistical interactions of Treatment with Sex on MBP levels, indicating that differences in MBP reduction are unlikely to account for sex differences in BLE. To summarize, cuprizone induced an unexpected, significant reduction in BLE in C57BL/6NJ males, which could indicate genotype-dependent sex differences in the biological mechanisms of BLE.

Activation of the Unfolded Protein Response via Inhibition of Protein Disulfide Isomerase Decreases the Capacity for DNA Repair to Sensitize Glioblastoma to Radiotherapy.

Patients with glioblastoma multiforme (GBM) survive on average 12 to 14 months after diagnosis despite surgical resection followed by radiotheraphy and temozolomide therapy. Intrinsic or acquired resistance to chemo- and radiotherapy is common and contributes to a high rate of recurrence. To investigate the therapeutic potential of protein disulfide isomerase (PDI) as a target to overcome resistance to chemoradiation, we developed a GBM tumor model wherein conditional genetic ablation of prolyl 4-hydroxylase subunit beta (P4HB), the gene that encodes PDI, can be accomplished. Loss of PDI expression induced the unfolded protein response (UPR) and decreased cell survival in two independent GBM models. Nascent RNA Bru-seq analysis of PDI-depleted cells revealed a decrease in transcription of genes involved in DNA repair and cell-cycle regulation. Activation of the UPR also led to a robust decrease in RAD51 protein expression as a result of its ubiquitination-mediated proteosomal degradation. Clonogenic survival assays demonstrated enhanced killing of GBM cells in response to a combination of PDI knockdown and ionizing radiation (IR) compared with either modality alone, which correlated with a decreased capacity to repair IR-induced DNA damage. Synergistic tumor control was also observed with the combination of PDI inhibition and IR in a mouse xenograft model compared with either single agent alone. These findings provide a strong rationale for the development of PDI inhibitors and their use in combination with DNA damage-inducing, standard-of-care therapies such as IR. SIGNIFICANCE: These findings identify PDIA1 as a therapeutic target in GBM by demonstrating efficacy of its inhibition in combination with radiotherapy through a novel mechanism involving downregulation of DNA repair genes.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2923/F1.large.jpg.

Expression and Functional Characteristics of Genes in the Globodera rostochiensis Woll. Potato Cyst Nematode at Different Stages of Its Life Cycle

The expression of the Ams-1, Pel-1, Eng-1.4, Cle, VAP-1, SPRYSEC-1, Tpx, and Gpx genes was studied in Globodera rostochiensis juveniles at early stages of the life cycle. Low gene activity was registered in juveniles inside the cyst. It was established that the transition to active life is accompanied by transcriptome activation. It was found that when susceptible plants (characterized by slow development of protective reactions) act as a host, gene expression is increased in the juveniles depending on the stage of development; this provides a successful passage of the nematode life cycle. A decreased gene transcription is registered in the juveniles in the case of plants treated with short-term temperature drops (DROP treatment); this can lead to the suppression of nematode development.

Molecular regulation of luteal progesterone synthesis in domestic ruminants

Regulation of progesterone secretion from the corpus luteum during the oestrous cycle requires the integration of multiple signals to achieve the appropriate amount of progesterone to maximize reproductive efficiency. Development of a mature corpus luteum capable of secreting sufficient amounts of progesterone is dependent upon the pituitary hormones LH and growth hormone (GH). Continued secretion of progesterone from the mature corpus luteum is also dependent upon pituitary hormones. If pregnancy does not occur, prostaglandin F2 alpha (PGF2 alpha) of uterine origin causes a precipitous decrease in progesterone secretion and demise of the corpus luteum. A major point of regulation of progesterone secretion by both luteotrophic and luteolytic hormones appears to be regulation of transport of cholesterol through the mitochondrial membranes to cytochrome P450scc. It is likely that both luteotrophic and luteolytic hormones regulate steroidogenic acute regulatory protein (StAR), which facilitates transport. Regulation may be occurring through increases or decreases in gene transcription, translation efficiency or post-translational modifications such as phosphorylation. Thus, although synthesis of progesterone is a complex process, both positive and negative regulation of the process appears to occur primarily at a single step (transport of cholesterol to the inner mitochondrial membrane) in the pathway.

Aberrant Transcriptional Regulation of Super-enhancers by RET Finger Protein-histone Deacetylase 1 Complex in Glioblastoma: Chemoresistance to Temozolomide.

Glioblastoma (GBM), the most common primary brain tumor, is the most aggressive human cancers, with a median survival rate of only 14.6 months. Temozolomide (TMZ) is the frontline chemotherapeutic drug in GBM. Drug resistance is the predominant obstacle in TMZ therapy. Drug resistance occurs via multiple pathways such as DNA mismatch repair and base excision repair systems, by which glioma cells acquire chemoresistance to some extent (5% and 95%, respectively). Histone3 Lysin27 residue-acetylation (H3K27ac) status regulates cis-regulatory elements, which increases the likelihood of gene transcription. Histone deacetylase (HDAC) complex deacetylate lysine residues on core histones, leading to a decrease in gene transcription. In cis-regulatory element regions, complexes with HDAC repress histones by H3K27ac deacetylation. The cis-regulating and three-dimensional transcriptional mechanism is called “super-enhancer”. RET finger protein (RFP) is a protein that is expressed in many kinds of cancer. RFP forms a protein complex with HDAC1. The disruption of the RFP–HDAC1 complex has resulted in increased drug sensitivity in other cancers. We conclude that the downregulation of RFP or the disruption of the RFP/HDAC1 complex leads to an increase in TMZ efficacy in glioblastoma by changing histone modifications which lead to changes in cell division, cell cycle and apoptosis.

Extracellular Acidic pH Inhibits Acetate Consumption by Decreasing Gene Transcription of the Tricarboxylic Acid Cycle and the Glyoxylate Shunt

Escherichia coli produces acetate during aerobic growth on various carbon sources. After consuming the carbon substrate, E. coli can further grow on the acetate. This phenomenon is known as the acetate switch, where cells transition from producing acetate to consuming it. In this study, we investigated how pH governs the acetate switch. When E. coli was grown on a glucose-supplemented medium initially buffered to pH 7, the cells produced and then consumed the acetate. However, when the initial pH was dropped to 6, the cells still produced acetate but were only able to consume it when little (<10 mM) acetate was produced. When significant acetate was produced in acidic medium, which occurs when the growth medium contains magnesium, amino acids, and sugar, the cells were unable to consume the acetate. To determine the mechanism, we characterized a set of metabolic mutants and found that those defective in the tricarboxylic acid (TCA) cycle or glyoxylate shunt exhibited reduced rates of acetate consumption. We further found that the expression of the genes in these pathways was reduced during growth in acidic medium. The expression of the genes involved in the AckA-Pta pathway, which provides the principal route for both acetate production and consumption, was also inhibited in acidic medium but only after glucose was depleted, which correlates with the acetate consumption phase. On the basis of these results, we conclude that growth in acidic environments inhibits the expression of the acetate catabolism genes, which in turn prevents acetate consumption.IMPORTANCE Many microorganisms produce fermentation products during aerobic growth on sugars. One of the best-known examples is the production of acetate by Escherichia coli during aerobic growth on sugars. In E. coli, acetate production is reversible: once the cells consume the available sugar, they can consume the acetate previously produced during aerobic fermentation. We found that pH affects the reversibility of acetate production. When the cells produce significant acetate during growth in acidic environments, they are unable to consume it. Unconsumed acetate may accumulate in the cell and inhibit the expression of pathways required for acetate catabolism. These findings demonstrate how acetate alters cell metabolism; they also may be useful for the design of aerobic fermentation processes.

Транскрипционная активность генов TP53 и MDM2 в отдаленный период у лиц, подвергшихся хроническому радиационному воздействию

Purpose: To study the levels of transcriptional activity of TP53 and MDM2 genes in the residents of the Techa riverside villages chronically exposed at a wide dose range. Material and methods: transcriptional activity of TP53 and MDM2 genes was assessed in 95 persons. The main study group included 80 people exposed to combined external and internal radiation (peripheral blood samples were taken 60–70 years after the beginning of chronic radiation exposure), mean accumulated dose to red bone marrow was 0.86 ± 0.08 Gy (doses varied in the range 0.1–3.65 Gy). The control group consisted of 15 people living in similar socio-economic conditions in the Southern Urals; the accumulated doses to red bone marrow did not exceed 0.07 Gy. Gene transcription activity profile was studied with real-time PCR assay. The data were analyzed using a comparative CT method with normalization to the “housekeeping” gene transcription in each sample. Statistical analysis was performed using the software PAST. Results and conclusion: In the course of the analysis we did not receive statistically significant differences between the study groups, but there was a tendency to a decrease in gene transcription in the group of exposed persons. The correlation analysis showed a weak negative dependence for TP53 and MDM2 genes, and this dependence was characterized not only by the accumulated dose value but was also associated with the age of the individuals under study. A tendency to a decrease in the transcription activity of the genes under study was noted when studying the effect of the dose. Statistically significant differences were shown for MDM2 gene in the group of individuals whose accumulated doses exceeded 2 Gy (p = 0.044). The analysis of age-peculiarities on gene transcription revealed a statistically significant decrease in TP53 gene transcription with increasing age of patients (p = 0.02). Non-radiation factors including smoking were also studied. The levels of gene transcription were compared between men and women of 2 main ethnicities (Bashkirs/Tartars and Slavs). Results of the study showed that neither sex nor ethnicity had any effect on the levels of TP53 and MDM2 gene transcription in the study groups. The effect of smoking on the activity of the genes under study was negligible.

Neurodegenerative Disease Proteinopathies Are Connected to Distinct Histone Post-translational Modification Landscapes.

Amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) are devastating neurodegenerative diseases involving the progressive degeneration of neurons. No cure is available for patients diagnosed with these diseases. A prominent feature of both ALS and PD is the accumulation of protein inclusions in the cytoplasm of degenerating neurons; however, the particular proteins constituting these inclusions vary: the RNA-binding proteins TDP-43 and FUS are most notable in ALS, while α-synuclein aggregates into Lewy bodies in PD. In both diseases, genetic causes fail to explain the occurrence of a large proportion of cases, and thus, both are considered mostly sporadic. Despite mounting evidence for a possible role of epigenetics in the occurrence and progression of ALS and PD, epigenetic mechanisms in the context of these diseases remain mostly unexplored. Here we comprehensively delineate histone post-translational modification (PTM) profiles in ALS and PD yeast proteinopathy models. Remarkably, we find distinct changes in histone modification profiles for each. We detect the most striking changes in the context of FUS aggregation: changes in several histone marks support a global decrease in gene transcription. We also detect more modest changes in histone modifications in cells overexpressing TDP-43 or α-synuclein. Our results highlight a great need for the inclusion of epigenetic mechanisms in the study of neurodegeneration. We hope our work will pave the way for the discovery of more effective therapies to treat patients suffering from ALS, PD, and other neurodegenerative diseases.

Suppression of Antimicrobial Resistance in MRSA Using CRISPR-dCas9

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are genetic elements that function with CRISPR-Associated (Cas) proteins as an adaptive immune system to foreign genetic material in prokaryotic organisms. The CRISPR-dCas9 system is modified to suppress gene transcription. Methicillin-Resistant Staphylococcus aureus (MRSA) is a dangerous human pathogen that is resistant to beta-lactam antibiotics. This is due to the mecA methicillin resistance gene coding for penicillin binding protein 2A (PBP 2A), which inhibits the activity of beta-lactam antibiotics. Two CRISPR-dCas9 systems were designed to target the promoter region of mecA in MRSA to suppress transcription of the gene. A cefoxitin disk diffusion test showed that the target on the coding strand significantly reduced antibiotic resistance in MRSA, whereas the target on the noncoding strand did not. An oxacillin microbroth serial dilution was used to confirm disk diffusion results. The CRISPR system targeting the coding strand was the only one to reduce antibiotic resistance and thus was chosen for continued testing. mecA gene expression levels were analyzed using Reverse Transcriptase Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR). Results showed that mecA gene expression in the CRISPR-treated sample was reduced to 0.230 fold of the value in the control, representing a 77% decrease in gene transcription. The 77% decrease in gene expression was not enough to make MRSA clinically susceptible to betalactam antibiotics. ABBREVIATIONS:BLAST - basic local alignment search tool, bp - basepairs, Cas - CRISPR-associated, cDNA - complementary DNA, CLSI - Clinical Laboratory Standards Institute, Cq - quantification cycle, CRISPRs - clustered regularly spaced short palindromic repeats, E.coli - Escherichia coli,M-MLV RT - Moloney Murine Leukemia Virus Reverse Transcriptase, MIC - minimum inhibitory concentration, MRSA - methicillin-resistant Staphylococcus aureus,NCBI - National Center for Biotechnology Information, PAM - protospacer adjacent motif, PBP 2A - penicillin binding protein 2A, RT-qPCR - reverse transcriptase quantitative real-time polymerase chain reaction, tracr - trans-activating CRISPR, UDG - uracil DNA glycosylase

In Vitro Effect of DFC-2 on Mycolic Acid Biosynthesis in Mycobacterium tuberculosis.

DFC-2, a methyl 5-[2-(dimethylamino)ethoxy]-7,12-dioxo-7,12-dihydrodinaphtho[1,2-b:2',3'-d]furan-6-carboxylate, is reported to have antitubercular effects against Mycobacterium tuberculosis. At concentrations ranging from 0.19 to 0.39 μg/ml, DFC-2 inhibited both drug-susceptible and -resistant strains of M. tuberculosis. Microarray analyses were employed to gain insights into the molecular mechanisms of DFC-2's action in M. tuberculosis. The most affected functional gene category was "lipid biosynthesis," which is involved in mycolic acid synthesis. The decrease in transcription of genes related to mycolic acid synthesis was confirmed by RT-PCR. Furthermore, we found that DFC-2 triggered a reduction in mycolic acid levels, showing a similar pattern to that of mycolic acid synthesis inhibitor isoniazid. These results may explain how this compound kills mycobacteria efficiently by inhibiting mycolic acid synthesis.

Anti-inflammaging effect of human alpha 1 antitrypsin

Abstract An important feature of aging is a low grade, persistent, systemic inflammation without overt infection which is defined as “inflammaging”. Inflammaging plays an important role in both morbidity and mortality of most age-related diseases. However, the mechanism of inflammaging is largely unknown, and no treatment has been discovered. Human alpha-1 antitrypsin (hAAT), with anti-inflammatory properties, holds potential to decelerate the aging process. To test the anti-aging effect of hAAT, we generate hAAT transgenic Drosophila lines and showed that over-expression of hAAT in Drosophila fat body (liver) significantly extended the life span. Importantly, real-time PCR and RNA sequencing analyses showed that age-related inflammatory genes (Relish and Diptericin), and immune responsive genes were significantly inhibited in hAAT transgenic lines. To further investigate this anti-inflammaging effect in human cells, we employed a widely used irradiation-induced senescence cell model and tested the effect of hAAT treatment on Senescence-associated Secretory Phenotype (SASP). Our results from this study showed that hAAT treatments significantly decreased gene transcription and protein secretion of IL-6 and IL-8 (the 2 major SASP factors). In contrast, no effect from hAAT on senescence formation was detected. Intriguingly, it is strongly suggesting the epigenetic effect of hAAT since the inhibitory effect can last at least 7 days after withdraw of the treatment. In conclusion, our results have shown that hAAT has the anti-inflammaging effects and may extend life span by inhibiting transcription of inflammatory factors of SASP. Hence, as a FDA-approved drug, hAAT is a promising candidate to combat aging and aging-related diseases.