Hypersensitive Region Research Articles

Heat stress causes chromatin accessibility and related gene expression changes in crown tissues of barley (Hordeum vulgare).

Plant responses to stress caused by high temperatures involve changes occurring at the molecular, metabolic, and physiological levels. Understanding the mechanisms by which plants recognize signals to activate this response is a prerequisite for identifying key genes and signaling pathways and for obtaining heat-tolerant plants. We demonstrated the first implementation of an assay for transposase-accessible chromatin to identify open chromatin regions (OCRs) in crown tissues of barley using three genotypes carrying different allelic forms of the sdw1 gene encoding gibberellin 20-oxidase subjected to elevated temperatures. In parallel, we performed gene expression analysis, which allowed us to relate changes in chromatin state to changes in transcriptional activity. The obtained data revealed that the hypersensitive chromatin regions within the genes were more repeatable than those outside the gene intervals. We observed that prolonged exposure to high temperatures increased chromatin accessibility. Genes with OCRs in their regulatory regions were involved in stress signaling and tolerance, including calcium-dependent protein kinase, mitogen-activated protein kinase (MAPK3), receptor-like cytoplasmic kinase (RLK), TIFY domain-containing transcriptional regulator, bZIP transcription factor, and regulatory protein NPR1. The effect of genotype on gene expression was not as pronounced as that of temperature. By combining results from the differential analysis of chromatin accessibility and expression profiles, we identified genes with high temperature-induced changes in chromatin accessibility associated with expression alterations. Importantly, our data revealed a relationship between the loss of chromatin accessibility in response to heat and the downregulation of genes related to gibberellin signaling.

Brain-wide mapping of c-Fos expression in nitroglycerin-induced models of migraine

BackgroundMigraine is a neurological disorder characterized by complex, widespread, and sudden attacks with an unclear pathogenesis, particularly in chronic migraine (CM). Specific brain regions, including the insula, amygdala, thalamus, and cingulate, medial prefrontal, and anterior cingulate cortex, are commonly activated by pain stimuli in patients with CM and animal models. This study employs fluorescence microscopy optical sectioning tomography (fMOST) technology and AAV-PHP.eB whole-brain expression to map activation patterns of brain regions in CM mice, thus enhancing the understanding of CM pathogenesis and suggesting potential treatment targets.MethodsBy repeatedly administering nitroglycerin (NTG) to induce migraine-like pain in mice, a chronic migraine model (CMM) was established. Olcegepant (OLC) was then used as treatment and its effects on mechanical pain hypersensitivity and brain region activation were observed. All mice underwent mechanical withdrawal threshold, light-aversive, and elevated plus maze tests. Viral injections were administered to the mice one month prior to modelling, and brain samples were collected 2 h after the final NTG/vehicle control injection for whole-brain imaging using fMOST.ResultsIn the NTG-induced CMM, mechanical pain threshold decreased, photophobia, and anxiety-like behavior were observed, and OLC was found to improve these manifestations. fMOST whole-brain imaging results suggest that the isocortex-cerebral cortex plate region, including somatomotor areas (MO), somatosensory areas (SS), and main olfactory bulb (MOB), appears to be the most sensitive area of activation in CM (P < 0.05). Other brain regions such as the inferior colliculus (IC) and intermediate reticular nucleus (IRN) were also exhibited significant activation (P < 0.05). The improvement in migraine-like symptoms observed with OLC treatment may be related to its effects on these brain regions, particularly SS, MO, ansiform lobule (AN), IC, spinal nucleus of the trigeminal, caudal part (Sp5c), IRN, and parvicellular reticular nucleus (PARN) (P < 0.05).ConclusionsfMOST whole-brain imaging reveals c-Fos + cells in numerous brain regions. OLC improves migraine-like symptoms by modulating brain activity in some brain regions. This study demonstrates the activation of the specific brain areas in NTG-induced CMM and suggests some regions as a potential treatment mechanism according to OLC.Graphical

Genetic variant rs11568818 of matrix metalloproteinase MMP7 associated with newborn weight in pregnant women with fetal growth restriction

Background: Fetal growth restriction is a common complication of pregnancy that has been associated with a variety of adverse perinatal outcomes. The condition carries significant risks of neonatal mortality, major and minor morbidity, and long term health sequelae. The aim of the study: The aim of the study was to study the associations of matrix metalloproteinases gene polymorphism with newborn weight in pregnant women with fetal growth restriction and to evaluate their functional effects. Materials and methods: 98 cases of women with fetal growth restriction were selected as the experimental group. All pregnant women were performed genotyping of 5 SNPs of genes of matrix metalloproteinase (rs1799750 MMP-1, rs243865 MMP-2, rs3025058 MMP-3, rs11568818 MMP-7, rs17577 MMP-9). Results: We found associations of the rs11568818 polymorphism of the MMP-7 gene with newborn weight in women with fetal growth restriction within a recessive model (β = 0.32±0.13, p=0.016 pperm=0.022). This polymorphic locus possesses important functional effects. It is localized in an evolutionarily conserved region, a binding site for regulatory proteins (TBP, CFOS, CJUN), a region of DNase hypersensitivity, and a site of modified histones marking enhancers and promoters in mesenchymal and hematopoietic stem cells, osteoblast cells, adipocytes, and fibroblast cell line lungs, various parts of the brain, lungs, etc., determines the sensitivity of DNA to four transcription factors (Foxa, GR, PLZF, Pou5f1), is associated with the level of mRNA expression of the MMP-7 gene in the lungs, liver, and skeletal muscles. Conclusion: The rs11568818 polymorphism of the MMP-7 gene is associated with a newborn weight.

Association between acetaminophen metabolites and CYP2E1 DNA methylation level in neonate cord blood in the Boston Birth Cohort

BackgroundAcetaminophen is a commonly used medication by pregnant women and is known to cross the placenta. However, little is known about the biological mechanisms that regulate acetaminophen in the developing offspring. Cytochrome 2E1 (CYP2E1) is the primary enzyme responsible for the conversion of acetaminophen to its toxic metabolite. Ex vivo studies have shown that the CYP2E1 gene expression in human fetal liver and placenta is largely controlled by DNA methylation (DNAm) at CpG sites located in the gene body of CYP2E1 at the 5’ end. To date, no population studies have examined the association between acetaminophen metabolite and fetal DNAm of CYP2E1 at birth.MethodsWe utilized data from the Boston Birth Cohort (BBC) which represents an urban, low-income, racially and ethnically diverse population in Boston, Massachusetts. Acetaminophen metabolites were measured in the cord plasma of newborns enrolled in BBC between 2003 and 2013 using liquid chromatography-tandem mass spectrometry. DNAm at 28 CpG sites of CYP2E1 was measured by Illumina Infinium MethylationEPIC BeadChip. We used linear regression to identify differentially methylated CpG sites and the “DiffVar” method to identify differences in methylation variation associated with the detection of acetaminophen, adjusting for cell heterogeneity and batch effects. The false discovery rate (FDR) was calculated to account for multiple comparisons.ResultsAmong the 570 newborns included in this study, 96 (17%) had detectable acetaminophen in cord plasma. We identified 7 differentially methylated CpGs (FDR < 0.05) associated with the detection of acetaminophen and additional 4 CpGs showing a difference in the variation of methylation (FDR < 0.05). These CpGs were all located in the gene body of CYP2E1 at the 5’ end and had a 3–6% lower average methylation level among participants with detectable acetaminophen compared to participants without. The CpG sites we identified overlap with previously identified DNase hypersensitivity and open chromatin regions in the ENCODE project, suggesting potential regulatory functions.ConclusionsIn a US birth cohort, we found detection of cord biomarkers of acetaminophen was associated with DNAm level of CYP2E1 in cord blood. Our findings suggest that DNA methylation of CYP2E1 may be an important regulator of acetaminophen levels in newborns.

Contribution of intergenic interactions of polymorphic variants of candidate genes to the development of gastric ulcer

Introduction: Gastric ulcer is a chronic disease with a recurrent course. The morphological substrate during periods of exacerbation are ulcers of the gastric mucosa. Peptic ulcer disease has a high prevalence among the adult population and is often characterized by a complicated course. Hereditary predisposition, along with other external and internal risk factors, plays a role in the etiopathogenesis of the disease. The aim of the study: To evaluate the effect of polymorphic variants of cell adhesion molecule genes on the development of Helicobacter pylori-negative gastric ulcer (GU). Materials and methods: 119 patients with Helicobacter pylori-negative GU and 347 individuals of the control group were examined. The regulatory potential of 7 polymorphic loci of genes of cell adhesion molecules pathogenetically significant for the development of gastric ulcer (rs6136 of the SELP gene, rs8176720, rs2519093, rs507666 of the ABO gene, rs651007, rs579459, rs649129 of the ABO/RF00019 gene) was evaluated using the HaploReg v4.1, PolyPhen-2, GTEx Portal Internet resources. DNA samples isolated from peripheral blood were genotyped by PCR. The analysis of associations was carried out by the method of logistic regression in the framework of allelic, additive, dominant and recessive genetic models. Results: The T allele of the RF00019/ABO gene (rs651007) is a protective factor in the development of H. pylori-negative GU (OR=0.14). This polymorphism is located in the region of histones marking promoters, regions of hypersensitivity to DNAse and the HNF4 regulatory motif, is associated with the expression of the ABO and SURF1 genes and alternative splicing of the ABO and LCN1P1 genes in various organs (tissues), including in the organs of the digestive and nervous systems.

Mer-tyrosine kinase: a novel susceptibility gene for SLE related end-stage renal disease

ObjectiveLupus nephritis (LN) is a common and severe manifestation of SLE. The genetic risk for nephritis and progression to end-stage renal disease (ESRD) in patients with LN remains unclear. Herein,...

Генетические маркеры тяжелого течения преэклампсии

In modern medicine, close attention is paid to the issues of reducing maternal morbidity and mortality, to the structure of which hypertensive disorders of gestation, especially preeclampsia, make a significant contribution. The complex pathomorphological mechanisms underlying the etiopathogenesis of this pregnancy complication occur long before the manifestation of pronounced clinical signs, which complicates the early diagnosis of preeclampsia and determines the relevance of the search for new preeclampsia-specific markers, including genetic ones. The aim of the study: To evaluate the associations of polymorphic markers of GWAS-significant candidate genes of hypertension with the development of severe preeclampsia. Materials and methods: The sample of women with moderate preeclampsia included 145 individuals, and the sample of women with severe preeclampsia included 72 patients. All subjects underwent genotyping of four polymorphic loci (rs8068318 TBX2, rs2681472 ATP2B1, rs4387287 OBFC1, rs1799945 HFE). The empirical distribution of genotypes and its correspondence to the theoretically expected one within the framework of the Hardy-Weinberg regularity are studied. Logistic regression analysis was carried out and associations of polymorphic loci with the development of severe and moderate preeclampsia were studied according to four genetic models, with the introduction of corrections for covariates. Results: It was found that rs8068318 of the TBX2 gene is associated with the development of severe preeclampsia in the framework of allelic (OR = 0.45; рperm = 0.004), additive (OR = 0.46; рperm = 0.002), dominant (OR = 0.42; рperm = 0.005) and recessive (OR = 0.22; рperm = 0.04) genetic models. The polymorphic locus rs8068318 of the TBX2 gene is localized in the region of hypersensitivity to DNase, the region of DNA regulatory motifs to four transcription factors, the region of histone tags marking enhancers and promoters in various organs and tissues, negatively regulates the expression of the TBX2-AS1 gene in adipose tissue and brain, the TBX2 gene in the thyroid gland, and is associated with the level of alternative splicing of TBX2-AS1 and RP11-332H18.5 genes in various tissues. Conclusion: The rs8068318 polymorphic marker of the TBX2 gene is associated with the development of severe preeclampsia in the population of the Central Chernozem region of the Russian Federation.

Genomic map of candidate human imprint control regions: the imprintome

ABSTRACT Imprinted genes – critical for growth, metabolism, and neuronal function – are expressed from one parental allele. Parent-of-origin-dependent CpG methylation regulates this expression at imprint control regions (ICRs). Since ICRs are established before tissue specification, these methylation marks are similar across cell types. Thus, they are attractive for investigating the developmental origins of adult diseases using accessible tissues, but remain unknown. We determined genome-wide candidate ICRs in humans by performing whole-genome bisulphite sequencing (WGBS) of DNA derived from the three germ layers and from gametes. We identified 1,488 hemi-methylated candidate ICRs, including 19 of 25 previously characterized ICRs (https://humanicr.org/). Gamete methylation approached 0% or 100% in 332 ICRs (178 paternally and 154 maternally methylated), supporting parent-of-origin-specific methylation, and 65% were in well-described CTCF-binding or DNaseI hypersensitive regions. This draft of the human imprintome will allow for the systematic determination of the role of early-acquired imprinting dysregulation in the pathogenesis of human diseases and developmental and behavioural disorders.

A β‐catenin‐TCF‐sensitive Locus Control Region Mediates GUCY2C Ligand Loss in Colorectal Cancer

BackgroundColorectal cancer most commonly arises from mutations in the tumor suppressor APC, or its downstream degradation target β‐catenin, initiating an oncogenic program of β‐catenin/TCF‐dependent nuclear transcription. Mechanisms coupling these mutations to tumorigenesis continue to be refined. The tumor suppressor axis regulated by the intestinal receptor guanylyl cyclase C (GUCY2C) and its paracrine ligands, guanylin and uroguanylin, contributes to intestinal homeostasis by regulating the proliferation, migration, and differentiation programs that maintain intestinal epithelial architecture. Furthermore, GUCY2C agonists oppose transformation in multiple mouse models of intestinal tumorigenesis. This signaling axis is among the earliest pathways silenced in tumorigenesis, reflecting an evolutionarily conserved pattern of GUCY2C retention, but ligand loss, in tumors. These observations suggest a pathophysiological model in which transformation orphans the receptor, silencing its signaling and lifting a block on tumorigenesis. Here, we examined the hypothesis that GUCY2C ligand loss arises from transcriptional silencing by β‐catenin/TCF.MethodsWe mapped the β‐catenin/TCF‐transcriptional program by RNA‐seq in four human colon cancer cell models. We then performed a comparative analysis of orthogonal approaches, including luciferase reporters, ChIP‐seq, CRISPR Cas9 knockout, and CRISPR epigenome editing to identify gene enhancers mediating GUCY2C ligand loss.ResultsRNA‐seq revealed a core β‐catenin/TCF‐transcriptional program of 1,289 genes, of which guanylin and uroguanylin were the seventh and twelfth most sensitive transcripts, reflecting transcriptional silencing. ChIP‐seq analyses of colon tissue revealed an evolutionarily conserved genomic locus encompassing the GUCY2C ligand genes, containing multiple regions of DNase hypersensitivity and H3K27ac enrichment, hallmarks of genetic enhancer loci. These markers were observed in normal colon, but not cancer tissue, consistent with enhancer inactivation and transcriptional silencing during transformation. ChIP‐seq in a colon cancer cell line revealed RNA Polymerase II and H3K27ac recruitment to the regions identified in human tissue, reflecting poised transcriptional machinery. Incorporation of the putative enhancer DNA into luciferase reporter constructs revealed a 2,683 bp locus control region (LCR) responsible for coordinated β‐catenin/TCF‐sensitive control of both GUCY2C ligand promoters. Both CRISPR‐Cas9 deletion of the LCR and epigenetic inactivation with a dCas9.KRAB repressor construct abolished β‐catenin/TCF‐sensitivity of GUCY2C ligand expression. Finally, LCR activation with a dCas9.VP64 transcriptional activator construct reconstituted GUCY2C ligand expression, overcoming gene silencing by β‐catenin/TCF.ConclusionsThis study reveals DNA elements regulating co‐repression of GUCY2C ligand transcription by β‐catenin/TCF, reflecting a novel step in tumorigenesis, and offers unique strategies to re‐establish hormone expression to oppose transformation.

Functional Magnetic Resonance Connectivity in Patients With Temporomadibular Joint Disorders.

Myofascial pain in the masticatory region, generally referred to as headache, is a common temporomandibular disorder (TMD) characterized by the hypersensitive regions of the contracted skeletal muscle fibers. A correct clinical treatment of myofascial pain has the potential to modify the functional activation of cerebral networks associated with pain and unconscious teeth clenching, specifically the pain network (PN) and default mode network (DMN). In this study, research is presented as a case series of five patients with myofascial pain: three were diagnosed with intra- and extra-articular disorders, and two were diagnosed with only extra-articular disorders. All five patients received gnathological therapy consisting of passive splints and biofeedback exercises for tongue–palatal vault coordination. Before and after treatment, patients underwent pain assessments (through measures of visual analog scales and muscular palpation tests), nuclear magnetic resonance of the temporomandibular joint, and functional nuclear magnetic resonance of the brain. In each patient, temporomandibular joint nuclear magnetic resonance results were similar before and after the gnathological treatment. However, the treatment resulted in a considerable reduction in pain for all patients, according to the visual analog scales and the palpation test. Furthermore, functional nuclear magnetic resonance of the brain clearly showed a homogeneous modification in cerebral networks associated with pain (i.e., PN and DMN), in all patients. In conclusion, gnathological therapy consisting of passive aligners and biofeedback exercises improved myofascial pain in all five patients. Most importantly, this study showed that all five patients had a homogeneous functional modification of pain and default mode networks. Using passive splints in combination with jaw exercises may be an effective treatment option for patients with TMD. This research could be a starting point for future investigations and for clinicians who want to approach similar situations.

IDHS-Deep: an integrated tool for predicting DNase I hypersensitive sites by deep neural network.

DNase I hypersensitive site (DHS) refers to the hypersensitive region of chromatin for the DNase I enzyme. It is an important part of the noncoding region and contains a variety of regulatory elements, such as promoter, enhancer, and transcription factor-binding site, etc. Moreover, the related locus of disease (or trait) are usually enriched in the DHS regions. Therefore, the detection of DHS region is of great significance. In this study, we develop a deep learning-based algorithm to identify whether an unknown sequence region would be potential DHS. The proposed method showed high prediction performance on both training datasets and independent datasets in different cell types and developmental stages, demonstrating that the method has excellent superiority in the identification of DHSs. Furthermore, for the convenience of related wet-experimental researchers, the user-friendly web-server iDHS-Deep was established at http://lin-group.cn/server/iDHS-Deep/, by which users can easily distinguish DHS and non-DHS and obtain the corresponding developmental stage ofDHS.

Mapping of functional elements of the Fab-6 boundary involved in the regulation of the Abd-B hox gene in Drosophilamelanogaster

The autonomy of segment-specific regulatory domains in the Bithorax complex is conferred by boundary elements and associated Polycomb response elements (PREs). The Fab-6 boundary is located at the junction of the iab-5 and iab-6 domains. Previous studies mapped it to a nuclease hypersensitive region 1 (HS1), while the iab-6 PRE was mapped to a second hypersensitive region HS2 nearly 3 kb away. To analyze the role of HS1 and HS2 in boundary we generated deletions of HS1 or HS1 + HS2 that have attP site for boundary replacement experiments. The 1389 bp HS1 deletion can be rescued by a 529 bp core Fab-6 sequence that includes two CTCF sites. However, Fab-6 HS1 cannot rescue the HS1 + HS2 deletion or substitute for another BX-C boundary – Fab-7. For this it must be combined with a PRE, either Fab-7 HS3, or Fab-6 HS2. These findings suggest that the boundary function of Fab-6 HS1 must be bolstered by a second element that has PRE activity.

Analysis of the functional role of polymorphism in the CDKN2B-AS1 gene GWAS-significant for primary open-angle glaucoma (an in-silico study)

To study in silico the functional significance of the CDKN2B-AS1 gene polymorphism GWAS-significant for primary open-angle glaucoma. The in-silico analysis was based on data from the GWAS catalog, five polymorphic loci of the CDKN2B-AS1 gene (rs1063192, rs7865618, rs2157719, rs944800, rs4977756) associated with POAG were selected. The study evaluated the regulatory potential, the relationship with the expression and alternative splicing of genes of the CDKN2B-AS1 gene polymorphism using modern databases for functional genomics - HaploReg and GTExportal. An important functional significance of the polymorphic loci rs1063192, rs7865618, rs2157719, rs944800, rs4977756 of the CDKN2B-AS1 gene was revealed. These loci are located in the region of histones marking enhancers and in the region of hypersensitivity to DNAse-1, can be found in more than ten different organs and tissues, in the regions of regulatory DNA motifs to five transcription factors (AIRE, GATA, Tgif1, Pou2f2, and Zfp187), and are associated with expression of three genes (CDKN2B-AS1, CDKN2B, CDKN2A) and alternative splicing of transcripts of two genes (CDKN2B-AS1 and RP11-149I2.4) in cell cultures, organs and tissues with pathogenic significance for glaucoma development. Polymorphism of the CDKN2B-AS1 gene (rs1063192, rs7865618, rs2157719, rs944800, rs4977756) has significant regulatory potential and is associated with the expression and alternative splicing of genes, which possibly underlies its association with primary open-angle glaucoma.

Study of the functional significance of polymorphic loci of the LOXL1 gene associated with glaucoma according to genome-wide studies (in silico analysis)

Using the catalog of genome-wide studies (GWAS) of the National Human Genome Research Institute (https://www.genome.gov/gwastudies/), three polymorphic loci of the LOXL1 gene (rs2165241, rs4886776, rs893818) associated with glaucoma (pseudoexfoliation glaucoma/syndrome) were chosen for the study. Using modern databases on functional genomics (SIFT, PolyPhen-2, HaploReg, GTExportal), the functional significance of these polymorphic loci was assessed (nonsynonymous substitutions, epigenetic effects, association with gene expression, associations with alternative splicing of gene transcripts). The work establishes the important functional significance of the rs2165241, rs4886776 and rs893818 polymorphic loci of the LOXL1 gene. They demonstrate significant epigenetic effects (affect the affinity to five transcription factors, are located in the region of promoters and enhancers, in the region of hypersensitivity to DNase-1), are associated with the expression and alternative splicing of three genes (LOXL1, LOXL1-AS1, RP11-24D15.1) in cell cultures, organs and tissues pathogenetically significant for development of glaucoma, are strongly linked to the rs1048661 polymorphism, which causes the replacement of the Arg141Leu amino acid in the LOXL1 polypeptide. Polymorphic loci of the LOXL1 gene (rs2165241, rs4886776, and rs893818) are of great functional importance (epigenetic, eQTL, and sQTL), which may be the biomedical basis of their associations with glaucoma.

Exposure to extracellular vesicles from Pseudomonas aeruginosa result in loss of DNA methylation at enhancer and DNase hypersensitive site regions in lung macrophages

ABSTRACT Various pathogens use differing strategies to evade host immune response including modulating the host’s epigenome. Here, we investigate if EVs secreted from P. aeruginosa alter DNA methylation in human lung macrophages, thereby potentially contributing to a dysfunctional innate immune response. Using a genome-wide DNA methylation approach, we demonstrate that P. aeruginosa EVs alter certain host cell DNA methylation patterns. We identified 1,185 differentially methylated CpGs (FDR < 0.05), which were significantly enriched for distal DNA regulatory elements including enhancer regions and DNase hypersensitive sites. Notably, all but one of the 1,185 differentially methylated CpGs were hypomethylated in association with EV exposure. Significantly hypomethylated CpGs tracked to genes including AXL, CFB and CCL23. Gene expression analysis identified 310 genes exhibiting significantly altered expression 48 hours post P. aeruginosa EV treatment, with 75 different genes upregulated and 235 genes downregulated. Some CpGs associated with cytokines such as CSF3 displayed strong negative correlations between DNA methylation and gene expression. Our infection model illustrates how secreted products (EVs) from bacteria can alter DNA methylation of the host epigenome. Changes in DNA methylation in distal DNA regulatory regions in turn can modulate cellular gene expression and potential downstream cellular processes.

Proteomic analysis of 2-chloroethanol extracts of rice (Oryza sativa L.) seeds

Ethanol-soluble proteins, including prolamins, are one of the most important seed proteins in rice (Oryza sativa L.). However, little is known about the proteomic profile of ethanol-soluble protein fraction extracted from rice grain. In this work, the differential profile of ethanol-soluble proteins extracted by 2-chloroethanol and ethanol has been documented. Proteome analysis utilizing LC-MS/MS identified a total of 64 unique proteins in the 2-chloroethanol extract of rice seeds. The majority of these proteins had low molecular weight ranging from 10 to 25 kD and isoelectric point (pI) in mid-acidic (pH 5–pH 7) and mid-basic (pH 7–pH 9) ranges. Database searches combined with transmembrane domain (TMD) analysis revealed that >70% of identified proteins were hydrophobic, i.e., had at least one TMD. Gene ontology classification and enrichment analysis showed that the identified proteins were involved in13 types of biological processes, 5 types of cell components, and 17 types of molecular functions. These results were significant based on the hyper p-value of <0.05. The most frequent categories of biological processes, cell components, and molecular functions were, respectively, type I hypersensitivity, extracellular space and extracellular region, and serine-type endopeptidase inhibitor activity. Interestingly, in addition to seed storage proteins such as prolamins and glutelins, certain allergen proteins, protease inhibitors, and lipid transfer proteins were identified in the extracts. Together, the collected data provide novel insights into the protein profile of 2-chloroethanol extract of rice seeds.

Mid-Infrared Imaging Is Able to Characterize and Separate Cancer Cell Lines

Malignancies are still responsible for a large share of lethalities. Macroscopical evaluation of the surgical resection margins is uncertain. Big data based imaging approaches have emerged in the recent decade (mass spectrometry, two-photon microscopy, infrared and Raman spectroscopy). Indocianine green labelled MS is the most common approach, however, label free mid-infrared imaging is more promising for future practical application. We aimed to identify and separate different transformed (A-375, HT-29) and non-transformed (CCD986SK) cell lines by a label-free infrared spectroscopy method. Our approach applied a novel set-up for label-free mid-infrared range classification method. Transflection spectroscopy was used on aluminium coated glass slides. Both whole range spectra (4000–648 cm−1) and hypersensitive fingerprint regions (1800–648 cm−1) were tested on the imaged areas of cell lines fixed in ethanol. Non-cell spectra were possible to be excluded based on mean transmission values being above 90%. Feasibility of a mean transmission based spectra filtering method with principal component analysis and linear discriminant analysis was shown to separate cell lines representing different tissue types. Fingerprint region resulted the best separation of cell lines spectra with accuracy of 99.84% at 70–75 mean transmittance range. Our approach in vitro was able to separate unique cell lines representing different tissues of origin. Proper data handling and spectra processing are key steps to achieve the adaptation of this dye-free technique for intraoperative surgery. Further studies are urgently needed to test this novel, marker-free approach.

Gene-environment interactions between polymorphic loci of MMPs and obesity in essential hypertension in women.

The prevalence of essential hypertension (ЕH) is increasing every year, both in Russia and around the world. Genetic and environmental risk factors are involved in the development of hypertension, and obesity plays an important role. Therefore, the study of gene-ecological interactions in the development of hypertension seems to be relevant. to study the gene-environment interactions between polymorphic loci of MMP and obesity in essential hypertension in women. The study was conducted in a case-control design. The sample included 584 subjects: 375 patients with EH and 209 women in the control group. All individuals included in the study were genotyped for eight polymorphic loci of MMPs. The study of the gene-environmental interactions during the formation of hypertension was performed using the GMDR method (Generalized Multifactor Dimensionality Reduction, http://www.ssg.uab.edu/gmdr). rs11568818 MMР7 and rs11225395 MMР8 polymorphic loci were found to be involved in the development of arterial hypertension in women without obesity (p&lt;0.050). Fifteen three-, four-, and five-factor models of gene-environmental interactions of 8 MMPs with obesity, associated with EH (p=0.01), were found. It is shown that the analyzed SNPs are located in the DNA regions that bind to histones, marking promoters and enhancers, in the region of hypersensitivity to DNAse-1, in the binding sites of regulatory proteins and transcription factors. The loci of MMPs rs17577, rs11568818, rs1320632 and rs11225395 have cis-eQTL-value. They affecting the expression of the genes of MMP7, SNX21, SLC12A5 and RP11-817J15.3. SNP rs11568818 MMP7 and rs11225395 MMP8 and gene-environmental interactions of MMPs rs1799750, rs243865, rs3025058, rs11568818, rs1320632, rs11225395, rs17577, rs652438 with obesity are involved in the development of essential hypertension in women.

The role of obesity in the implementation of genetic predisposition to the development of essential hypertension in men

BACKGROUND: Obesity is considered a non-infectious pandemic, and the increase in its spread is a serious medical and social problem. High values of body mass index closely correlate with arterial hypertension and its complications, but the effect of obesity on the realization of hereditary susceptibility to essential hypertension (EH) remains poorly understood.&#x0D; AIMS: To study the associations of polymorphic loci of MMPs with the development of EH in men depending on the presence of obesity.&#x0D; MATERIALS AND METHODS: The study was conducted in a case-control design. Surveyed 821 men 564 patients with hypertension and 257 patients of the control group. Groups of patients and controls were divided into subgroups depending on the presence of obesity. All men were genotyped for eight polymorphic loci of MMPs. Nonsynonymous SNPs were detected using the software SIFT (https://sift.bii.a-star.edu.sg/). The regulatory potential was studied using the HaploReg service (https://pubs.broadinstitute.org/mammals/haploreg/haploreg.php). The association of SNPs with the expression level was detected using GTEx-portal (http://www.gtexportal.org).&#x0D; RESULTS: It was found that in obese men allele A (OR=2.01; p=0.01) and genotype GG (OR=0.42, p=0.01) of rs11568818 MMP7 are associated with the essential hypertension. In men without obesity allele 6A (OR=1.32; p=0.04) of rs3025058 MMР3 and genotypes GG (OR=1.52; p=0.04) and GA (OR=0.63; p=0.03)) of rs17577 MMP9 are associated with the development of the disease. These SNPs located in region of promoter and enhancer histone marks, in the region of hypersensitivity to DNAse-1, in binding sites of regulatory proteins and transcription factors. These SNPs associated with the level of gene expression.&#x0D; CONCLUSIONS: In this study we established associations with the development of EH of SNP rs11568818 MMP7 in obese men and of SNPs rs3025058 MMР3 and rs17577 MMP9 in non-obese men.

TMIC-32. PARALLEL SINGLE CELL NUCLEOSOME OCCUPANCY AND RNA SEQUENCING ON RECURRENT GBM

Abstract Nearly all patients diagnosed with Glioblastoma (GBM) will experience a fatal recurrence of the tumor. Thought to be in part driven by immense tumor heterogeneity it is important to explore the small subpopulations of tumor cells with in depth single cell sequencing pre and post therapy in order to understand recurrence. To this end, we created an experimental in vivo pipeline that allows us profile not only gene expression changes during therapy, but also epigenetic shifts due to methylation across the genome at the single cell level from a single tumor. Single Cell NomE-Seq libraries were created and correctly identified characteristic CTCF binding regions within DNase1 hypersensitivity regions when compared back to representative encode data sets. Primary tumors analyzed by single cell RNA sequencing showed enrichment for on average 5 significant principle components (St. Dev &gt;2 verified by Elbow Plot). Comparatively, recurrent temozolomide (TMZ) exposed tumors averaged 10 significant principle components (St. Dev &gt;2 verified by Elbow Plot) suggesting TMZ therapy causes tumors to diversify their gene expression profiles. For direct comparison samples were merged into a continuous data set and dimensional analysis with TSNE reduction (15 dimensions used) identified 6 unique clusters, with one cluster being over-represented by cells from recurrent, TMZ exposed mice. Gene Ontology analysis on expression values contained within in this cluster demonstrated significant enrichment for ribosome biogenesis/ribosomal structure, (p=2.87e-17) and mRNA catabolic processes (p=2.47e-30). Further, comparison to a similar data set gathered using cells in vitro culture conditions demonstrates superior intraturmoral heterogeneity from cells isolated freshly from the brains of mice as opposed to tissue culture, indicating the effectiveness of our model for assaying heterogeneity. Ultimately, these data illuminate possible new mechanisms for TMZ induced ribosomal modifications underlying intraturmoral heterogeneity in GBM and provide an expandable technique for further experimentation.