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Bovine mastitis is a complex infectious disease that develops in the mammary gland, predominantly by a bacterial infection of mammary tissue. Mastitis is economically important in the context of a long-lasting decrease in milk yield (in the range of 5-25%), cost of treatment, and effect on cow’s health. Genetic variability of mastitis is well established and depends upon different quantitative trait loci (QTL) related to mastitis resistance or susceptibility. The susceptibility is often attributed to single nucleotide polymorphisms (SNPs) in the variable cow breed genomes. Several global investigative attempts have resulted in studies mapping mastitis to the variations in the relevant genes. Reports have been attributed to dramatic genetic expression changes in Toll Like Receptor 4 (TLR4) genes in mastitis-positive cows. However, the mechanism behind this variable genetic expression of TLR4 genes has been studied poorly. This study aims to investigate possible mechanisms behind this variable expression of TLR4 by comparative SNP evaluation and transcriptional factor profile mining. So that the important genetic mutations and effects thereof can be exploited in selecting specific breeds with higher mastitis resistance and milk yields, this paper identifies several mutations of prominence and almost sixteen SNPs falling in TLR4 promoter and 5` untranslated region (5`UTR) sequences in mastitis-positive genotypes compared to reference genomes. The study also investigates the potential transcriptional factor program deployed in response to variable mastitis development resistance. This paper combines the classic parameters of mastitis like somatic cell count (SCC), milk electric conductivity (EC), milk pH, California mastitis test (CMT) and attempts to attribute a genetic cause in relation to variable TLR4 promoter and 5`UTR sequence SNPs. The results of this paper have wide applicability for researchers developing mastitis-resistant breeding programs and the data generated may aid in the selection of better genetic breeds. The transcription factor binding profiles given in the paper will serve as concrete leads about the studies on bovine mastitis at the molecular level and may also aid global research groups working on transcription factor (TF) -based molecular pathology of mastitis.

Circulating free-cell miRNAs are increasingly important as potential non-invasive biomarkers due to the easy accessibility of clinical materials. Moreover, their epigenetic role may provide insight into the mechanisms of pathogenesis. Nevertheless, these aspects are mostly studied in the area of oncological diseases. Therefore, this research aimed to find the potential association of selected miRNAs in serum with the expression of Th17/Treg transcription factors and clinical features in RA patients. Accordingly, experiments was conducted on rheumatoid arthritis (RA), osteoarthritis (OA) and healthy subjects (HC). Analysis of miRNAs level in serum was performed using LNA miRNA PCR assays. mir-10 was detected only in RA patients. Furthermore, its expression was correlated with IL-35 serum concentration and the mRNA level of STAT5a in whole blood in RA. Additionally, a tendency of the raised level of miR-10 was noted in RA patients with high activity disease. miR-326 was significantly upregulated in RA patients with rheumatoid factor presence. In HC the correlation between miR-26 and IL-21 serum levels and expression of SMAD3 have been found. In OA patients, correlations between miR-126 and HIF1 expression and between miR-146 and RORc have been noted. The differential association of transcription factor expression with serum miRNA levels may be important in the diagnosis and progression of RA and OA.

Chronic venous disease (CVD) is an often underestimated inflammatory pathological condition that can have a serious impact on quality of life. Many therapies have been proposed to deal with CVD, but unfortunately the symptoms recur with increasing frequency and intensity as soon as treatments are stopped. Previous studies have shown that the common inflammatory transcription factor AP-1 (activator protein-1) and nuclear factor kappa-activated B-cell light chain enhancer (NF-kB) play key roles in the initiation and progression of this vascular dysfunction. The aim of this research was to develop a herbal product that acts simultaneously on different aspects of CVD-related inflammation. Based on the evidence that several natural components of plant origin are used to treat venous insufficiency and that magnolol has been suggested as a putative modulator of AP-1, two herbal preparations based on Ruscus aculeatus root extracts, and Vitis vinifera seed extracts, as well as diosmetin and magnolol, were established. A preliminary MTT-based evaluation of the possible cytotoxic effects of these preparations led to the selection of one of them, named DMRV-2, for further investigation. First, the anti-inflammatory efficacy of DMRV-2 was demonstrated by monitoring its ability to reduce cytokine secretion from endothelial cells subjected to LPS-induced inflammation. Furthermore, using a real-time PCR-based protocol, the effect of DMRV-2 on AP-1 expression and activity was also evaluated; the results obtained demonstrated that the incubation of the endothelial cells with this preparation almost completely nullified the effects exerted by the treatment with LPS on AP-1. Similar results were also obtained for NF-kB, whose activation was evaluated by monitoring its distribution between the cytosol and the nucleus of endothelial cells after the different treatments.

Simple Summary One of the most important health problems in the paediatric population, due to its prevalence worldwide, is the occurrence of congenital heart defects. The integration of multidisciplinary approaches to clinical diagnosis will allow us to detect this type of problem in patients in a timely way. The TBX5 gene has an important participation in cardiogenesis. Therefore, we performed a case-control study, that involved the DNA methylation assessment of the TBX5 gene promoter region in patients that were non-syndromic with congenital septal defects, to identify an epigenetic marker. Moreover, we evaluated the exposure to environmental factors during pregnancy in mothers of these patients. Additionally, we used bioinformatic tools to identify transcription factors binding to the TBX5 gene region of interest and to learn their possible functional effect. These results could help to clarify the mechanisms that regulate these pathologies and establish risk markers, which can be used in future in clinical practice. Abstract The TBX5 gene regulates morphological changes during heart development, and it has been associated with epigenetic abnormalities observed in congenital heart defects (CHD). The aim of this research was to evaluate the association between DNA methylation levels of the TBX5 gene promoter and congenital septal defects. DNA methylation levels of six CpG sites in the TBX5 gene promoter were evaluated using pyrosequencing analysis in 35 patients with congenital septal defects and 48 controls. Average methylation levels were higher in individuals with congenital septal defects than in the controls (p < 0.004). In five CpG sites, we also found higher methylation levels in patients than in the controls (p < 0.05). High methylation levels were associated with congenital septal defects (OR = 3.91; 95% CI = 1.02–14.8; p = 0.045). The analysis of Receiver Operating Characteristic (ROC) showed that the methylation levels of the TBX5 gene could be used as a risk marker for congenital septal defects (AUC = 0.68, 95% CI = 0.56–0.80; p = 0.004). Finally, an analysis of environmental factors indicated that maternal infections increased the risk (OR = 2.90; 95% CI = 1.01–8.33; p = 0.048) of congenital septal defects. Our data suggest that a high DNA methylation of the TBX5 gene could be associated with congenital septal defects.

Background Recent evidences indicated that shear stress is critical in orchestrating gene expression in cardiovascular disease. It is necessary to identify the mechanism of shear stress influencing gene expression in physiology and pathophysiology conditions. This paper aimed to identify candidate hub genes and its transcription factors with bioinformatics. Methods We analyzed microarray expression profile of GSE16706 to identify differentially expressed genes (DEGs) in low shear stress (1 dyne/cm2) treated human umbilical vein endothelial cells (HUVECs) compared with static condition for 24 h. Results 652 DEGs, including 333 up-regulated and 319 down-regulated DEGs, were screen out. Functional enrichment analysis indicated enrichment items mainly included cytokine-cytokine receptor interaction and cell cycle. Five hub genes (CDC20, CCNA2, KIF11, KIF2C and PLK1) and one significant module (score = 17.39) were identified through protein–protein interaction (PPI) analysis. Key transcriptional factor FOXC1 displayed close interaction with all the hub genes via gene-transcriptional factor network. Single-gene GSEA analysis indicated that CDC20 was linked to the G2M_CHECKPOINT pathway and cell cycle pathway. Conclusions By using integrated bioinformatic analysis, a new transcriptional factor and hub-genes network related to HUVECs treated with low shear stress were identified. The new regulation mechanism we discovered may be a promising potential therapeutic target for cardiovascular disease.

Different types of post-translational modifications are present in bacteria that play essential roles in bacterial metabolism modulation. Nevertheless, limited information is available on these types of modifications in actinobacteria, particularly on their effects on secondary metabolite biosynthesis. Recently, phosphorylation, acetylation, or phosphopantetheneylation of transcriptional factors and key enzymes involved in secondary metabolite biosynthesis have been reported. There are two types of phosphorylations involved in the control of transcriptional factors: (1) phosphorylation of sensor kinases and transfer of the phosphate group to the receiver domain of response regulators, which alters the expression of regulator target genes. (2) Phosphorylation systems involving promiscuous serine/threonine/tyrosine kinases that modify proteins at several amino acid residues, e.g., the phosphorylation of the global nitrogen regulator GlnR. Another post-translational modification is the acetylation at the epsilon amino group of lysine residues. The protein acetylation/deacetylation controls the activity of many short and long-chain acyl-CoA synthetases, transcriptional factors, key proteins of bacterial metabolism, and enzymes for the biosynthesis of non-ribosomal peptides, desferrioxamine, streptomycin, or phosphinic acid-derived antibiotics. Acetyltransferases catalyze acetylation reactions showing different specificity for the acyl-CoA donor. Although it functions as acetyltransferase, there are examples of malonylation, crotonylation, succinylation, or in a few cases acylation activities using bulky acyl-CoA derivatives. Substrates activation by nucleoside triphosphates is one of the central reactions inhibited by lysine acetyltransferases. Phosphorylation/dephosphorylation or acylation/deacylation reactions on global regulators like PhoP, GlnR, AfsR, and the carbon catabolite regulator glucokinase strongly affects the expression of genes controlled by these regulators. Finally, a different type of post-translational protein modification is the phosphopantetheinylation, catalized by phosphopantetheinyl transferases (PPTases). This reaction is essential to modify those enzymes requiring phosphopantetheine groups like non-ribosomal peptide synthetases, polyketide synthases, and fatty acid synthases. Up to five PPTases are present in S. tsukubaensis and S. avermitilis. Different PPTases modify substrate proteins in the PCP or ACP domains of tacrolimus biosynthetic enzymes. Directed mutations of genes encoding enzymes involved in the post-translational modification is a promising tool to enhance the production of bioactive metabolites.

Made available in DSpace on 2022-05-01T10:18:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-11-01 Bundesministerium für Bildung und Forschung American Historical Association Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Deutsche Forschungsgemeinschaft National Institutes of Health Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Adipose tissue has been classified based on its morphology and function as white, brown, or beige/brite. It plays an essential role as a regulator of systemic metabolism through paracrine and endocrine signals. Recently, multiple adipocyte subtypes have been revealed using RNA sequencing technology, going beyond simply defined morphology but also by their cellular origin, adaptation to metabolic stress, and plasticity. Here, we performed an in-depth analysis of publicly available single-nuclei RNAseq from adipose tissue and utilized a workflow template to characterize adipocyte plasticity, heterogeneity, and secretome profiles. The reanalyzed dataset led to the identification of different subtypes of adipocytes including three subpopulations of thermogenic adipocytes, and provided a characterization of distinct transcriptional profiles along the adipocyte trajectory under thermogenic challenges. This study provides a useful resource for further investigations regarding mechanisms related to adipocyte plasticity and trans-differentiation. Department of Genetics Ribeirão Preto Medical School University of São Paulo Center for Cell-Based Therapy (CEPID/FAPESP) National Institute of Science and Technology in Stem Cell and Cell Therapy (INCTC/CNPq) Regional Blood Center of Ribeirão Preto Institute for Cancer Research IPEC Department of Structural and Functional Biology Institute of Biosciences São Paulo State University (UNESP) Department of Translational Genomics Medical Faculty University of Cologne Department of Biochemistry School of Medicine Boston University Department of Integrated Biotechnology University of Mogi das Cruzes Department of Structural and Functional Biology Institute of Biosciences São Paulo State University (UNESP) Bundesministerium für Bildung und Forschung: 01ZX1901A American Historical Association: 17POST33660875 CNPq: 311319/2018-1 CNPq: 311530/2019-2 Deutsche Forschungsgemeinschaft: 413326622 CNPq: 870415/1997-2 National Institutes of Health: DK117161 National Institutes of Health: DK117163 National Institutes of Health: P30-DK-046200 Deutsche Forschungsgemeinschaft: SFB1399

Background Normal-weight polycystic ovary syndrome (PCOS) women exhibit adipose resistance in vivo accompanied by enhanced subcutaneous (SC) abdominal adipose stem cell (ASC) development to adipocytes with accelerated lipid accumulation per cell in vitro. The present study examines chromatin accessibility, RNA expression and fatty acid (FA) synthesis during SC abdominal ASC differentiation into adipocytes in vitro of normal-weight PCOS versus age- and body mass index-matched normoandrogenic ovulatory (control) women to study epigenetic/genetic characteristics as well as functional alterations of PCOS and control ASCs during adipogenesis. Results SC abdominal ASCs from PCOS women versus controls exhibited dynamic chromatin accessibility during adipogenesis, from significantly less chromatin accessibility at day 0 to greater chromatin accessibility by day 12, with enrichment of binding motifs for transcription factors (TFs) of the AP-1 subfamily at days 0, 3, and 12. In PCOS versus control cells, expression of genes governing adipocyte differentiation (PPARγ, CEBPα, AGPAT2) and function (ADIPOQ, FABP4, LPL, PLIN1, SLC2A4) was increased two–sixfold at days 3, 7, and 12, while that involving Wnt signaling (FZD1, SFRP1, and WNT10B) was decreased. Differential gene expression in PCOS cells at these time points involved triacylglycerol synthesis, lipid oxidation, free fatty acid beta-oxidation, and oxidative phosphorylation of the TCA cycle, with TGFB1 as a significant upstream regulator. There was a broad correspondence between increased chromatin accessibility and increased RNA expression of those 12 genes involved in adipocyte differentiation and function, Wnt signaling, as well as genes involved in the triacylglycerol synthesis functional group at day 12 of adipogenesis. Total content and de novo synthesis of myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), and oleic (C18:1) acid increased from day 7 to day 12 in all cells, with total content and de novo synthesis of FAs significantly greater in PCOS than controls cells at day 12. Conclusions In normal-weight PCOS women, dynamic chromatin remodeling of SC abdominal ASCs during adipogenesis may enhance adipogenic gene expression as a programmed mechanism to promote greater fat storage.

One common and reversible type of post-translational modification (PTM) is the addition of O-linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation), and its dynamic balance is controlled by O-GlcNAc transferase (OGT) and glycoside hydrolase O-GlcNAcase (OGA) through the addition or removal of O-GlcNAc groups. A large amount of research data confirms that proteins regulated by O-GlcNAcylation play a pivotal role in cells. In particularly, imbalanced levels of OGT and O-GlcNAcylation have been found in various types of cancers. Recently, increasing evidence shows that imbalanced O-GlcNAcylation directly or indirectly impacts the process of cancer metastasis. This review summarizes the current understanding of the influence of O-GlcNAc-proteins on the regulation of cancer metastasis. It will provide a theoretical basis to further elucidate of the molecular mechanisms underlying cancer emergence and progression.

Steroid microbial degradation plays a significant ecological role for biomass decomposition and removal/detoxification of steroid pollutants. In this study, the initial steps of cholesterol degradation and lithocholate bioconversion by a strain with enhanced 3-ketosteroid dehydrogenase (3-KSD) activity, Nocardioides simplex VKM Ac-2033D, were studied. Biochemical, transcriptomic, and bioinformatic approaches were used. Among the intermediates of sterol sidechain oxidation cholest-5-en-26-oic acid and 3-oxo-cholesta-1,4-dien-26-oic acid were identified as those that have not been earlier reported for N. simplex and related species. The transcriptomic approach revealed candidate genes of cholesterol and lithocholic acid (LCA) catabolism by the strain. A separate set of genes combined in cluster and additional 3-ketosteroid Δ1-dehydrogenase and 3-ketosteroid 9α-hydroxylases that might be involved in LCA catabolism were predicted. Bioinformatic calculations based on transcriptomic data showed the existence of a previously unknown transcription factor, which regulates cholate catabolism gene orthologs. The results contribute to the knowledge on diversity of steroid catabolism regulation in actinobacteria and might be used at the engineering of microbial catalysts for ecological and industrial biotechnology.

One common and reversible type of post-translational modification (PTM) is the addition of O-linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation), and its dynamic balance is controlled by O-GlcNAc transferase (OGT) and glycoside hydrolase O-GlcNAcase (OGA) through the addition or removal of O-GlcNAc groups. A large amount of research data confirms that proteins regulated by O-GlcNAcylation play a pivotal role in cells. In particularly, imbalanced levels of OGT and O-GlcNAcylation have been found in various types of cancers. Recently, increasing evidence shows that imbalanced O-GlcNAcylation directly or indirectly impacts the process of cancer metastasis. This review summarizes the current understanding of the influence of O-GlcNAc-proteins on the regulation of cancer metastasis. It will provide a theoretical basis to further elucidate of the molecular mechanisms underlying cancer emergence and progression.

Transcription factors (TFs) play a central role in the gene regulation associated with a plant's development and its response to the environmental factors. The work of TFs is well regulated at each stage of their activities. TFs usually consist of three protein domains required for DNA binding, dimerization, and transcriptional regulation. Alternative splicing (AS) produces multiple proteins with varying composition of domains. Recent studies have shown that AS of some TF genes form small proteins (small interfering peptide/small interfering protein, siPEP/siPRoT), which lack one or more domains and negatively regulate target TFs by the mechanism of protein interference (peptide interference/protein interference, PEPi/PROTi). The presence of an alternative form for the transcription factor CCA1 of Arabidopsis thaliana, has been shown to be involved in the regulation of the response to cold stress. For the PtFLC protein, one of the isoforms was found, which is formed as a result of alternative splicing and acts as a negative repressor, binding to the full-length TF PtFLC and therefore regulating the development of the Poncirus trifoliata. For A. thaliana, a FLM gene was found forming the FLM-б isoform, which acts as a dominant negative regulator and stimulates the development of the flower formation process due to the formation of a heterodimer with SVP TF. Small interfering peptides and proteins can actively participate in the regulation of gene expression, for example, in situations of stress or at different stages of plant development. Moreover, small interfering peptides and proteins can be used as a tool for fundamental research on the function of genes as well as for applied research for permanent or temporary knockout of genes. In this review, we have demonstrated recent studies related to siPEP/siPROT and their involvement in the response to various stresses, as well as possible ways to obtain small proteins.

Introduction: The programmed death receptor ligand 1 (PD-L1) immunohistochemistry (IHC) assay is a widely used selection method for pembrolizumab treatment in gastric cancer (GC) patients. PD-L1 is the main regulator of immunity in oncogenesis. Material and methods: The study included 38 patients with GC. The combined treatment consisted of neoadjuvant FOLFOX6, or FLOT, chemotherapy and surgery. PD-L1 + tumor status was recorded in 12 patients (CPS &gt, 5), with a negative status recorded in 26 patients. RT-PCR determined the expression of molecular markers. The level of LC3B protein was detected by Western Blotting analysis. Results: An overexpression of PD-1, PD-L2 in the tumor is associated with AKT/mTOR mRNA profile change and autophagy initiation in IHC PD-L1 positive GCs. NACT influences these biological features, modifying the expression of AKT/mTOR components and autophagic flux. In PD-L1 positive cancers, the effect of NACT and molecular markers rearrangements are essential compared to the PD-L1 negative cancers. Conclusion: The IHC PD-L1 status in gastric cancers is the significant marker of cancer progression, recovering the multiple inner mechanisms of cancer spreading and leading to ineffective therapy. Autophagy induction and angiogenesis are found in PD-L1 positive gastric cancers.

// Li Min 1, 2, 3, * , Cheng Zhang 1, * , Like Qu 1 , Jialiang Huang 2, 3 , Lan Jiang 2, 3 , Jiafei Liu 1 , Luca Pinello 2, 3 , Guo-Cheng Yuan 2, 3 , Chengchao Shou 1 1 Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Departments of Biochemistry and Molecular Biology, Peking University Cancer Hospital & Institute, Beijing 100036, P. R. China 2 Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA 3 Harvard T. H. Chan School of Public Heath, Boston, MA 02115, USA * These authors have contributed equally to this work Correspondence to: Chengchao Shou, email: scc@bjcancer.org Guo-Cheng Yuan, email: gcyuan@jimmy.harvard.edu Keywords: gene regulatory pattern, network analysis, transcriptional factors, TNBC Received: October 27, 2016 Accepted: January 10, 2017 Published: February 27, 2017 ABSTRACT Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype. Genome-scale molecular characteristics and regulatory mechanisms that distinguish TNBC from other subtypes remain incompletely characterized. Results: By combining gene expression analysis and PANDA network, we defined three different TF regulatory patterns. A core TNBC-Specific TF Activation Driven Pattern (TNBCac) was specifically identified in TNBC by computational analysis. The essentialness of core TFs (ZEB1, MZF1, SOX10) in TNBC was highlighted and validated by cell proliferation analysis. Furthermore, 13 out of 35 co-targeted genes were also validated to be targeted by ZEB1, MZF1 and SOX10 in TNBC cell lines by real-time quantitative PCR. In three breast cancer cohorts, non-TNBC patients could be stratified into two subgroups by the 35 co-targeted genes along with 5 TFs, and the subgroup that more resembled TNBC had a worse prognosis. Methods: We constructed gene regulatory networks in breast cancer by Passing Attributes between Networks for Data Assimilation (PANDA). Co-regulatory modules were specifically identified in TNBC by computational analysis, while the essentialness of core translational factors (TF) in TNBC was highlighted and validated by in vitro experiments. Prognostic effects of different factors were measured by Log-rank test and displayed by Kaplan-Meier plots. Conclusions: We identified a core co-regulatory module specifically existing in TNBC, which enabled subtype re-classification and provided a biologically feasible view of breast cancer.

Complications due to malaria are caused mostly by host immunological responses. Plasmodium falciparum subverts host immunity by various strategies, including modulation in the host immune responses by regulating cytokines. The transcriptional alterations of major cytokines and immunoregulators were analyzed in this study through gene expression profiling in clinically defined subgroups of P. falciparum patients. Malaria patients were included from Dhalai district hospital of Tripura with uncomplicated malaria (UC) and severe malaria (SM) and healthy controls from endemic and non-endemic areas of India. qPCR gene expression analysis was performed for all factors and they were grouped into three clusters based on their altered expressions. The first cluster was downregulated with an increased parasitic burden which included T-BET, GATA3, EOMES, TGF-β, STAT4, STAT6 and cytokines IFN-γ, IL-12, IL-4, IL-5, and IL-13. RANTES, IL-8, CCR8, and CXCR3 were decreased in the SM group. The second cluster was upregulated with severity and included TNF-α, IL-10, IL-1β and IL-7. PD-1 and BCL6 were increased in the SM group. The third cluster comprised of NF-κB and was not altered. The level of perforin was suppressed while GrB expression was elevated in SM. P. falciparum malaria burden is characterized by the modulation of host immunity via compromization of T cell-mediated responses and suppression of innate immune-regulators.

Nature has evolved exquisite sensing mechanisms to detect cellular and environmental signals surrounding living organisms. These biosensors have been widely used to sense small molecules, detect environmental cues and diagnose disease markers. Metabolic engineers and synthetic biologists have been able to exploit metabolites-responsive transcriptional factors (MRTFs) as basic tools to rewire cell metabolism, reprogram cellular activity as well as boost cell’s productivity. This is commonly achieved by integrating sensor-actuator systems with biocatalytic functions and dynamically allocating cellular resources to drive carbon flux toward the target pathway. Up to date, most of identified MRTFs are derived from bacteria. As an endeavor to advance intelligent biomanufacturing in yeast cell factory, we will summarize the opportunities and challenges to transfer the bacteria-derived MRTFs to expand the small-molecule sensing capability in eukaryotic cells. We will discuss the design principles underlying MRTF-based biosensors in eukaryotic cells, including the choice of reliable reporters and the characterization tools to minimize background noise, strategies to tune the sensor dynamic range, sensitivity and specificity, as well as the criteria to engineer activator and repressor-based biosensors. Due to the physical separation of transcription and protein expression in eukaryotes, we argue that nuclear import/export mechanism of MRTFs across the nuclear membrane plays a critical role in regulating the MRTF sensor dynamics. Precisely-controlled MRTF response will allow us to repurpose the vast majority of transcriptional factors as molecular switches to achieve temporal or spatial gene expression in eukaryotes. Uncovering this knowledge will inform us fundamental design principles to deliver robust cell factories and enable the design of reprogrammable and predictable biological systems for intelligent biomanufacturing, smart therapeutics or precision medicine in the foreseeable future.

Streptococcus pyogenes (group A streptococci; GAS) is an exclusively human pathogen. It causes a variety of suppurative and non-suppurative diseases in people of all ages worldwide. Not all can be successfully treated with antibiotics. A licensed vaccine, in spite of its global importance, is not yet available. GAS express an arsenal of virulence factors responsible for pathological immune reactions. The transcription of all these virulence factors is under the control of three types of virulence-related regulators: (i) two-component systems (TCS), (ii) stand-alone regulators, and (iii) non-coding RNAs. This review summarizes major TCS and stand-alone transcriptional regulatory systems, which are directly associated with virulence control. It is suggested that this treasure of knowledge on the genetics of virulence regulation should be better harnessed for new therapies and prevention methods for GAS infections, thereby changing its global epidemiology for the better.

Background Dextrorotatory borneol (D-borneol), a cyclic monoterpene, is widely used in traditional Chinese medicine as an efficient topical analgesic drug. Fresh leaves of Cinnamomum trees, e.g., C. burmannii and C. camphor, are the main sources from which D-borneol is extracted by steam distillation, yet with low yields. Insufficient supply of D-borneol has hampered its clinical use and production of patent remedies for a long time. Biological synthesis of D-borneol offers an additional approach; however, mechanisms of D-borneol biosynthesis remain mostly unresolved. Hence, it is important and necessary to elucidate the biosynthetic pathway of D-borneol. Results Comparative analysis on the gene expression patterns of different D-borneol production C. burmannii samples facilitates elucidation on the underlying biosynthetic pathway of D-borneol. Herein, we collected three different chemotypes of C. burmannii, which harbor different contents of D-borneol.A total of 100,218 unigenes with an N50 of 1,128 bp were assembled de novo using Trinity from a total of 21.21 Gb clean bases. We used BLASTx analysis against several public databases to annotate 45,485 unigenes (45.38%) to at least one database, among which 82 unigenes were assigned to terpenoid biosynthesis pathways by KEGG annotation. In addition, we defined 8,860 unigenes as differentially expressed genes (DEGs), among which 13 DEGs were associated with terpenoid biosynthesis pathways. One 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and two monoterpene synthase, designated as CbDXS9, CbTPS2 and CbTPS3, were up-regulated in the high-borneol group compared to the low-borneol and borneol-free groups, and might be vital to biosynthesis of D-borneol in C. burmannii. In addition, we identified one WRKY, two BHLH, one AP2/ERF and three MYB candidate genes, which exhibited the same expression patterns as CbTPS2 and CbTPS3, suggesting that these transcription factors might potentially regulate D-borneol biosynthesis. Finally, quantitative real-time PCR was conducted to detect the actual expression level of those candidate genes related to the D-borneol biosynthesis pathway, and the result showed that the expression patterns of the candidate genes related to D-borneol biosynthesis were basically consistent with those revealed by transcriptome analysis. Conclusions We used transcriptome sequencing to analyze three different chemotypes of C. burmannii, identifying three candidate structural genes (one DXS, two monoterpene synthases) and seven potential transcription factor candidates (one WRKY, two BHLH, one AP2/ERF and three MYB) involved in D-borneol biosynthesis. These results provide new insight into our understanding of the production and accumulation of D-borneol in C. burmannii.

Trigonella foenum-graecum L. (fenugreek) is a valuable resource of producing diosgenin which serves as a substrate for synthesizing more than two hundred kinds of steroidal drugs. Phytochemical analysis indicated that methyl jasmonate (MeJA) efficiently induced diosgenin biosynthesis in fenugreek seedlings. Though early steps up to cholesterol have recently been elucidated in plants, cytochrome P450 (CYP)- and glycosyltransferase (GT)-encoding genes involved in the late steps from cholesterol to diosgenin remain unknown. This study established comparative fenugreek transcriptome datasets from the MeJA-treated seedlings and the corresponding control lines. Differential gene expression analysis identified a number of MeJA-induced CYP and GT candidate genes. Further gene expression pattern analysis across a different MeJA-treating time points, together with a phylogenetic analysis, suggested specific family members of CYPs and GTs that may participate in the late steps during diosgenin biosynthesis. MeJA-induced transcription factors (TFs) that may play regulatory roles in diosgenin biosynthesis were also discussed. This study provided a valuable genetic resource to functionally characterize the genes involved in diosgenin biosynthesis, which will push forward the production of diosgenin in microbial organisms using a promising synthetic biology strategy.

The ability of Mycobacterium tuberculosis (M. tb) to survive and persist in the host for decades in an asymptomatic state is an important aspect of tuberculosis pathogenesis. Although adaptation to hypoxia is thought to play a prominent role underlying M. tb persistence, how the bacteria achieve this goal is largely unknown. Rv0081, a member of the DosR regulon, is induced at the early stage of hypoxia while Rv3334 is one of the enduring hypoxic response genes. In this study, we uncovered genetic interactions between these two transcription factors. RNA-seq analysis of ΔRv0081 and ΔRv3334 revealed that the gene expression profiles of these two mutants were highly similar. We also found that under hypoxia, Rv0081 positively regulated the expression of Rv3334 while Rv3334 repressed transcription of Rv0081. In addition, we demonstrated that Rv0081 formed dimer and bound to the promoter region of Rv3334. Taken together, these data suggest that Rv0081 and Rv3334 work in the same regulatory pathway and that Rv3334 functions immediately downstream of Rv0081. We also found that Rv3334 is a bona fide regulator of the enduring hypoxic response genes. Our study has uncovered a regulatory pathway that connects the early and the enduring hypoxic response, revealing a transcriptional cascade that coordinates the temporal response of M. tb to hypoxia.

Pancreatic cancer is one of the most lethal malignancies. Significant progresses have been made in understanding of pancreatic cancer pathogenesis, including appreciation of precursor lesions or premalignant pancreatic intraepithelial neoplasia (PanINs), description of sequential transformation from normal pancreatic tissue to invasive pancreatic cancer and identification of major genetic and epigenetic events and the biological impact of those events on malignant behavior. However, the currently used therapeutic strategies targeting tumor epithelial cells, which are potent in cell culture and animal models, have not been successful in the clinic. Presumably, therapeutic resistance of pancreatic cancer is at least in part due to its drastic desmoplasis, which is a defining hallmark for and circumstantially contributes to pancreatic cancer development and progression. Improved understanding of the dynamic interaction between cancer cells and the stroma is important to better understanding pancreatic cancer biology and to designing effective intervention strategies. This review focuses on the origination, evolution and disruption of stromal molecular and cellular components in pancreatic cancer, and their biological effects on pancreatic cancer pathogenesis.

Many phenolics are known to possess anti-obesity properties through mechanisms that inhibit adipogenesis, stimulate lipolysis or induce apoptosis. This study aimed to examine the anti-adipogenic effects of two phenolics, oxyresveratrol and cyanomaclurin in 3T3-L1 cells. At doses that they did not induce cytotoxicity (based on the lactate dehydrogenase assay), the exposure of oxyresveratrol (0–100 µM) or cyanomaclurin (0–600 µM) in the first three days of differentiation led to a dose-dependent decrease of triacylglycerol accumulation on day 9 post-confluent. Results of cell proliferation and cell cycle study demonstrated that both compounds inhibited differentiation through inducing cell cycle arrest by retaining the preadipocytes in the G1 phase during the first two days post-confluent. Their anti-adipogenic properties were also attributed to the down-regulation of protein expression of major transcriptional factors in adipogenesis, including peroxisome proliferator-activated receptor γ (PPAR γ) and CCAAT/Enhancer-binding proteins α (C/EBP α), and the induction of cell cycle arrest through regulating cyclin D1, cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase inhibitor 1B (P27/kip1) expression during the mitotic clonal expansion period.

Floral shape in higher plants typically requires genetic regulation through MADS transcription factors. In Japan, hundreds of azalea cultivars including flower shape mutations have been selected from the diversity of endogenous species and natural hybrids since the early 17th century, the Edo era (1603–1867). The long-lasting trait, known as “Misome-shō” in Japanese, has been identified in several species and cultivar groups of evergreen azaleas (Rhododendron L.) from three hundred years ago in Japan. However, the natural mutation conferring the long-lasting trait in azalea remains unknown. Here, we showed MADS-box gene mutations in long-lasting flowers, R. kaempferi ‘Nikkō-misome,’ R. macrosepalum ‘Kochō-zoroi,’ R. indicum ‘Chōjyu-hō,’ and R. × hannoense ‘Amagi-beni-chōjyu.’ All of the long-lasting flowers exhibited small-sized corollas with stomata during long blooming. In the long-lasting flowers, transcript of the APETALA3 (AP3)/DEFICIENS (DEF) homolog was reduced, and an LTR-retrotransposon was independently inserted into exons 1, 2, and 7 or an unknown sequence in exon 1 in gDNA of each cultivar. This insertion apparently abolished the normal mRNA sequence of the AP3/DEF homolog in long-lasting flowers. Also, long-lasting flowers were shown from F2 hybrids that had homozygous ap3/def alleles. Therefore, we concluded that the loss of function of the AP3/DEF homolog through a transposable element insertion may confer a stable long-lasting mutation in evergreen azaleas.

In agricultural production, abiotic stresses are known as the main disturbance leading to negative impacts on crop performance. Research on elucidating plant defense mechanisms against the stresses at molecular level has been addressed for years in order to identify the major contributors in boosting the plant tolerance ability. From literature, numerous genes from different species, and from both functional and regulatory gene categories, have been suggested to be on the list of potential candidates for genetic engineering. Noticeably, enhancement of plant stress tolerance by manipulating expression of Transcription Factors (TFs) encoding genes has emerged as a popular approach since most of them are early stress-responsive genes and control the expression of a set of downstream target genes. Consequently, there is a higher chance to generate novel cultivars with better tolerance to either single or multiple stresses. Perhaps, the difficult task when deploying this approach is selecting appropriate gene(s) for manipulation. In this review, on the basis of the current findings from molecular and post-genomic studies, our interest is to highlight the current understanding of the roles of TFs in signal transduction and mediating plant responses towards abiotic stressors. Furthermore, interactions among TFs within the stress-responsive network will be discussed. The last section will be reserved for discussing the potential applications of TFs for stress tolerance improvement in plants.

The panicle architecture of rice is an important characteristic that influences reproductive success and yield. It is largely determined by the number and length of the primary and secondary branches. The number of panicle branches is defined by the inflorescence meristem state between determinacy and indeterminacy; for example, the maize ramosa2 (ra2) mutant has more branches in its tassel through loss of spikelet determinacy. Some genes and factors influencing the number of primary and secondary branches have been studied, but little is known about the molecular mechanism underlying pedicel development, which also influences panicle architecture. We report here that rice OsRAMOSA2 (OsRA2) gene modifies panicle architecture through regulating pedicel length. Ectopic expression of OsRA2 resulted in a shortened pedicel while inhibition of OsRA2 through RNA interference produced elongated pedicel. In addition, OsRA2 influenced seed morphology. The OsRA2 protein localized to the nucleus and showed transcriptional activation in yeast; in accordance with its function in pedicel development, OsRA2 mRNA was enriched in the anlagen of axillary meristems, such as primary and secondary branch meristems and the spikelet meristems of young panicles. This indicates a conserved role of OsRA2 for shaping the initial steps of inflorescence architecture. Genetic analysis revealed that OsRA2 may control panicle architecture using the same pathway as that of the axillary meristem gene LAX1 (LAX PANICLE1). Moreover, OsRA2 acted downstream of RCN2 in regulating pedicel and branch lengths, but upstream of RCN2 for control of the number of secondary branches, indicating that branch number and length development in the panicle were respectively regulated using parallel pathway. Functional conservation between OsRA2 and AtLOB, and the conservation and diversification of RA2 in maize and rice are also discussed.

The 5' end or alpha-S1 casein promoter has a significant role in milk protein gene expression. The understanding of the translation process of alpha-S1 casein mutants will provide us an opportunity to make the best selection in livestock providing more proteins in milk. Blood samples were taken from three hundred of Naeinian goats and sheep, and DNA extraction was done using modified salting out method. Polymerase chain reactions (PCR) were carried out using a specific primer pairs for amplification a fragment of 1133 bp from part of 5'-UTR and exon 1 of alpha s1 casein gene. The AluI and HinfI restriction enzyme treatment of all samples provided the same homozygous AA genotype in both species. Subsequently, one sample of each species was selected and cloned, and the final sequences were analyzed by BioEdit, CLC genomic, Mega4 and DNASIS MAX software. Several polymorphisms are recognized between Naeinian goat and sheep that are presented on motif sites. In this research, the interested location, including exon I and a part of 5', was analyzed, and genetic element comparisons were done between Naeinian goat and sheep. The number and location of probable binding sites can have a crucial role as a result of antagonistic and synergistic effects on gene regulation activities.

Kinases and transcriptional regulators are fundamental components of cell signaling that are expressed on many types of immune cells which are involved in secretion of cytokines, cell proliferation, differentiation, and apoptosis. Both play important roles in biological responses in health as well as in illnesses such as the autoimmune diseases which comprise at least 80 disorders. These diseases are caused by complex genetic and environmental interactions that lead to a breakage of immunologic tolerance and a disruption of the balance between self-reactive cells and regulatory cells. Kinases or transcriptional regulatory factors often have an abnormal expression in the autoimmune cells that participate in the pathogenesis of autoimmune disease. These abnormally expressed kinases or transcriptional regulators can over-activate the function of self-reactive cells to produce inflammatory cytokines or down-regulate the activity of regulatory cells, thus causing autoimmune diseases. In this review we introduce five kinds of kinase and transcriptional regulator related to autoimmune diseases, namely, members of the Janus kinase (JAK) family (JAK3 and/or tyrosine kinase 2 (TYK2)), fork head box protein 3 (Foxp3), the retinoic acid-related orphan receptor gamma t (RORγt), and T-box expressed in T cells (T-bet) factors. We also provide a mechanistic insight into how these kinases and transcriptional regulators affect the function of the immune cells related to autoimmune diseases, as well as a description of a current drug design targeting these kinases and transcriptional regulators. Understanding their exact role helps offer new therapies for control of the inflammatory responses that could lead to clinical improvement of the autoimmune diseases.

Trigonella foenum-graecum L. (fenugreek) is a valuable resource of producing diosgenin which serves as a substrate for synthesizing more than two hundred kinds of steroidal drugs. Phytochemical analysis indicated that methyl jasmonate (MeJA) efficiently induced diosgenin biosynthesis in fenugreek seedlings. Though early steps up to cholesterol have recently been elucidated in plants, cytochrome P450 (CYP)- and glycosyltransferase (GT)-encoding genes involved in the late steps from cholesterol to diosgenin remain unknown. This study established comparative fenugreek transcriptome datasets from the MeJA-treated seedlings and the corresponding control lines. Differential gene expression analysis identified a number of MeJA-induced CYP and GT candidate genes. Further gene expression pattern analysis across a different MeJA-treating time points, together with a phylogenetic analysis, suggested specific family members of CYPs and GTs that may participate in the late steps during diosgenin biosynthesis. MeJA-induced transcription factors (TFs) that may play regulatory roles in diosgenin biosynthesis were also discussed. This study provided a valuable genetic resource to functionally characterize the genes involved in diosgenin biosynthesis, which will push forward the production of diosgenin in microbial organisms using a promising synthetic biology strategy.

Phytoalexins are antimicrobial substances of low molecular weight produced by plants in response to infection or stress, which form part of their active defense mechanisms. Starting in the 1950's, research on phytoalexins has begun with biochemistry and bio-organic chemistry, resulting in the determination of their structure, their biological activity as well as mechanisms of their synthesis and their catabolism by microorganisms. Elucidation of the biosynthesis of numerous phytoalexins has permitted the use of molecular biology tools for the exploration of the genes encoding enzymes of their synthesis pathways and their regulators. Genetic manipulation of phytoalexins has been investigated to increase the disease resistance of plants. The first example of a disease resistance resulting from foreign phytoalexin expression in a novel plant has concerned a phytoalexin from grapevine which was transferred to tobacco. Transformations were then operated to investigate the potential of other phytoalexin biosynthetic genes to confer resistance to pathogens. Unexpectedly, engineering phytoalexins for disease resistance in plants seem to have been limited to exploiting only a few phytoalexin biosynthetic genes, especially those encoding stilbenes and some isoflavonoids. Research has rather focused on indirect approaches which allow modulation of the accumulation of phytoalexin employing transcriptional regulators or components of upstream regulatory pathways. Genetic approaches using gain- or less-of functions in phytoalexin engineering together with modulation of phytoalexin accumulation through molecular engineering of plant hormones and defense-related marker and elicitor genes have been reviewed.

Aim: To clarify whether the activities of multiple CYPs associated with warfarin metabolism would be correlated with each other. Methods: Oral clearances (CLpo) of warfarin enantiomers were estimated in 378 Chinese, Caucasians and African–Americans. The partial metabolic clearances (CLm) for 7-hydroxywarfarin enantiomers were also measured. In addition, CLpo and CLm were determined in a patient on warfarin and rifampicin. Results: Correlations between CLpo for warfarin enantiomers existed across the three populations. In addition, there was a significant correlation between the CLm for 7-hydroxylation of warfarin enantiomers. Under induced conditions by rifampicin, there were significant correlations between the enantio- and regio-selective metabolisms of warfarin. Conclusion: Metabolic activities of CYP2C9, CYP1A2 and CYP3A4 may be regulated by common transcriptional mechanism(s).

Recent analyses suggest that transposable element-derived transcripts are processed to yield a variety of small RNA species that play critical functional roles in gene regulation and chromatin organization as well as genome stability and maintenance. Here we report a mass spectrometry analysis of an RNA-affinity complex isolation using a piRNA homologous sequence derived from Alu retrotransposal RNA. Our data point to potential roles for piALU RNAs in DNA repair, cell cycle and chromatin regulations.

Supplemental Digital Content is available in the text, End-stage renal disease (ESRD) is the final stage of chronic kidney disease in which the kidney is not sufficient to meet the needs of daily life. It is necessary to understand the role of genes expression involved in ESRD patient responses to nocturnal hemodialysis (NHD) and to improve the immunity responsiveness. The aim of this study was to investigate novel immune-associated genes that may play important roles in patients with ESRD. The microarray expression profiles of peripheral blood in patients with ESRD before and after NHD were analyzed by network-based approaches, and then using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway analysis to explore the biological process and molecular functions of differentially expressed genes. Subsequently, a transcriptional regulatory network of the core genes and the connected transcriptional regulators was constructed. We found that NHD had a significant effect on neutrophil activation and immune response in patients with ESRD. In addition, Our findings suggest that MAPKAPK3, RHOA, ARRB2, FLOT1, MYH9, PRKCD, RHOG, PTPN6, MAPK3, CNPY3, PI3KCG, and PYGL genes maybe potential targets regulated by core transcriptional factors, including ARNT, C/EBPalpha, CEBPA, CREB1, PSG1, DAND5, SP1, GATA1, MYC, EGR2, and EGR3.

We have previously shown a higher level of HIV-1 replication and gene expression in neonatal (cord) blood mononuclear cells (CBMC) compared with adult blood cells (PBMC), which could be due to differential expression of host factors. We performed the gene expression profile of CBMC and PBMC and found that 8013 genes were expressed at higher levels in CBMC than PBMC and 8028 genes in PBMC than CBMC, including 1181 and 1414 genes upregulated after HIV-1 infection in CBMC and PBMC, respectively. Several transcription factors (NF-kappaB, E2F, HAT-1, TFIIE, Cdk9, Cyclin T1), signal transducers (STAT3, STAT5A) and cytokines (IL-1beta, IL-6, IL-10) were upregulated in CBMC than PBMC, which are known to influence HIV-1 replication. In addition, a repressor of HIV-1 transcription, YY1, was down regulated in CBMC than PBMC and several matrix metalloproteinase (MMP-7, -12, -14) were significantly upregulated in HIV-1 infected CBMC than PBMC. Furthermore, we show that CBMC nuclear extracts interacted with a higher extent to HIV-1 LTR cis-acting sequences, including NF-kappaB, NFAT, AP1 and NF-IL6 compared with PBMC nuclear extracts and retroviral based short hairpin RNA (shRNA) for STAT3 and IL-6 down regulated their own and HIV-1 gene expression, signifying that these factors influenced differential HIV-1 gene expression in CBMC than PBMC.

The ERK/MAP kinase pathway is an important regulator of gene expression and differentiation in postmitotic cells. To understand how this pathway controls gene expression in bone, we examined the subnuclear localization of P-ERK in differentiating osteoblasts. Induction of differentiation was accompanied by increased ERK phosphorylation and expression of osteoblast-related genes, including osteocalcin (Bglap2) and bone sialoprotein (Ibsp). Confocal immunofluorescence microscopy revealed that P-ERK colocalized with the RUNX2 transcription factor in the nuclei of differentiating cells. Interestingly, a portion of this nuclear P-ERK was directly bound to the proximal promoter regions of Bglap2 and Ibsp. Furthermore, the level of P-ERK binding to chromatin increased with differentiation, whereas RUNX2 binding remained relatively constant. The P-ERK-chromatin interaction was seen only in RUNX2-positive cells, required intact RUNX2-selective enhancer sequences, and was blocked with MAPK inhibition. These studies show for the first time that RUNX2 specifically targets P-ERK to the chromatin of osteoblast-related genes, where it may phosphorylate multiple substrates, including RUNX2, resulting in altered chromatin structure and gene expression. © 2010 American Society for Bone and Mineral Research

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ., [Background]: Our prior characterization of RasGrf1 deficient mice uncovered significant defects in pancreatic islet count and size as well as beta cell development and signaling function, raising question about the mechanisms linking RasGrf1 to the generation of those >pancreatic> phenotypes. [Results]: Here, we compared the transcriptional profile of highly purified pancreatic islets from RasGrf1 KO mice to that of WT control animals using commercial oligonucleotide microarrays. RasGrf1 elimination resulted in differential gene expression of numerous components of MAPK- and Calcium-signaling pathways, suggesting a relevant contribution of this GEF to modulation of cellular signaling in the cell lineages integrating the pancreatic islets. Whereas the overall transcriptional profile of pancreatic islets was highly specific in comparison to other organs of the same KO mice, a significant specific repression of Pttg1 was a common transcriptional alteration shared with other tissues of neuroectodermal origin. This observation, together with the remarkable pancreatic phenotypic similarities between RasGrf1 KO and Pttg1 KO mice suggested the possibility of proximal functional regulatory links between RasGrf1 and Pttg1 in pancreatic cell lineages expressing these proteins. [Conclusions]: The specific transcriptional profile and signaling behavior of RasgGrf1 KO pancreatic islets, together with the dominance of Pttg1 over RasGrf1 with regards to the generation of these phenotypes in mouse pancreas, suggest that RasGrf1 is an important upstream component of signal transduction pathways regulating Pttg1 expression and controlling beta cell development and physiological responses., Work supported by grants FIS PI13/02846 and RTICC RD12/0036/0001 from Instituto de Salud Carlos III (ISCIII), and grant SA181U13 from JCyL, Spain. We are grateful to Dr. Douglas Lowy (National Cancer Institute, Bethesda, MD) for providing plasmid pBK-CMV RasGrf1 and to Dr. Shlomo Melmed (Cedars-Sinai Medical Center, Los Angeles, CA) for providing reporter plasmid pGL3-Pttg1 and the single PTTG1 KO mouse strain used in these studies.

Oral cancers have been one of the leading causes of deaths particularly in the developing countries. Prime reason for this high mortality and morbidity is attributed to the delay in diagnosis and prompt treatment. Relentless research in the field of oncology has led to the advent of novel procedures for the early detection of oral cancers. Molecular biology is highly promising in this regard. It is a procedure that detects alterations at a molecular level much before they are seen under a microscope and much before clinical changes occur. Molecular studies serve as the basis by which we will eventually be able not only to augment clinical assessment and classification of oral lesions but also predict malignant potential of oral lesions, thus reducing the incidence and increasing the scope for early diagnosis and treatment of oral cancers. However, making such sophisticated tools available for the common man in developing countries is one of the most important challenges faced today.

Background Human genome is enriched with thousands of conserved non-coding elements (CNEs). Recently, a medium throughput strategy was employed to analyze the ability of human CNEs to drive tissue specific expression during mouse embryogenesis. These data led to the establishment of publicly available genome wide catalog of functionally defined human enhancers. Scattering of enhancers over larger regions in vertebrate genomes seriously impede attempts to pinpoint their precise target genes. Such associations are prerequisite to explore the significance of this in vivo characterized catalog of human enhancers in development, disease and evolution. Results This study is an attempt to systematically identify the target gene-bodies for functionally defined human CNE-enhancers. For the purpose we adopted the orthology/paralogy mapping approach and compared the CNE induced reporter expression with reported endogenous expression pattern of neighboring genes. This procedure pinpointed specific target gene-bodies for the total of 192 human CNE-enhancers. This enables us to gauge the maximum genomic search space for enhancer hunting: 4 Mb of genomic sequence around the gene of interest (2 Mb on either side). Furthermore, we used human-rodent comparison for a set of 159 orthologous enhancer pairs to infer that the central nervous system (CNS) specific gene expression is closely associated with the cooperative interaction among at least eight distinct transcription factors: SOX5, HFH, SOX17, HNF3β, c-FOS, Tal1beta-E47S, MEF and FREAC. Conclusions In conclusion, the systematic wiring of cis-acting sites and their target gene bodies is an important step to unravel the role of in vivo characterized catalog of human enhancers in development, physiology and medicine.

The human lung consists of multiple cell types derived from early embryonic compartments. The morphogenesis of the lung, as well as the injury repair of the adult lung, is tightly controlled by a network of signaling pathways with key transcriptional factors. Lung cancer is the third most cancer-related death in the world, which may be developed due to the failure of regulating the signaling pathways. Sox (sex-determining region Y (Sry) box-containing) family transcriptional factors have emerged as potent modulators in embryonic development, stem cells maintenance, tissue homeostasis, and cancerogenesis in multiple processes. Recent studies demonstrated that the members of the Sox gene family played important roles in the development and maintenance of lung and development of lung cancer. In this context, we summarize our current understanding of the role of Sox family transcriptional factors in the morphogenesis of lung, their oncogenic potential in lung cancer, and their potential impact in the diagnosis, prognosis, and targeted therapy of lung cancer.

This study aimed to determine the changes in the expression of genes for selected specific transcriptional factors that have both activating and repressing functions in in vitro ductal breast cancer cells, under the influence of paclitaxel, applying the microarray technique. The cells are treated with 60 ng/ml and 300 ng/ml doses of paclitaxel that correspond to those applied in breast cancer therapy. About 60 ng/ml doses of paclitaxel cause a statistically significant increase in expression of all the 16 analysed genes coding transcriptional factors, ranging from 1.84-fold (for PO4F2) to 4.65-fold (for LMO4) (p < 0.05) in comparison with the control cells, and enhanced the taxane mechanism of action. The 300 ng/ml doses of paclitaxel cause a cytotoxic effect in the cells. In this article, we argue that these changes in gene expression values may constitute prognostic and predictive factors in ductal breast cancer therapy.

A 1.8 kb 5'-flanking region of the large subunit of ADP-glucose pyrophosphorylase, isolated from watermelon (Citrullus vulgaris S.), has fruit-specific promoter activity in transgenic tomato plants. Two negative regulatory regions, from -986 to -959 and from -472 to -424, were identified in this promoter region by fine deletion analyses. Removal of both regions led to constitutive expression in epidermal cells. Gain-of-function experiments showed that these two regions were sufficient to inhibit RFP (red fluorescent protein) expression in transformed epidermal cells when fused to the cauliflower mosaic virus (CaMV) 35S minimal promoter. Gel mobility shift experiments demonstrated the presence of leaf nuclear factors that interact with these two elements. A TCCAAAA motif was identified in these two regions, as well as one in the reverse orientation, which was confirmed to be a novel specific cis-element. A quantitative beta-glucuronidase (GUS) activity assay of stable transgenic tomato plants showed that the activities of chimeric promoters harbouring only one of the two cis-elements, or both, were approximately 10-fold higher in fruits than in leaves. These data confirm that the TCCAAAA motif functions as a fruit-specific element by inhibiting gene expression in leaves.

We have previously identified two homologous groups of BnDREBs in Brassica napus, the trans-active BnDREBI and the trans-inactive BnDREBII, which provided an ideal system to study the trans-activation of DREB1/CBF. Deletion analysis indicated that the two additional regions in BnDREBI contributed little to the transcriptional activity. Domain swapping analysis indicated that all the domains contributed to the activity of BnDREBI, including the ERF/AP2 DNA binding domain. Through site-directed mutagenesis, we identified nine residues that were involved in the activity of BnDREBI, among which six residues are specific to BnDREBI, and three are common to DREB1A.

Background Xylose-based ethanol production by recombinant S. cerevisiae is of great interest to basic and applied bioenergy research. By expressing three different fungal pathways in two S. cerevisiae hosts respectively, we found that the xylose utilization efficiency by recombinant S. cerevisiae depends not only on the choice of xylose pathway but also on the choice of host, exhibiting an obvious host or context dependence. To investigate molecular mechanisms of this context dependence, we applied RNA-seq analysis in this study for a systematic characterization of the xylose utilization via different pathways in different S. cerevisiae hosts. Results Based on the RNA-seq analysis, the transcripts that were regulated during xylose utilization have been identified. Three transcription factors involved in regulation of amino acid metabolism, responses to oxidative stresses, and degradation of aggregated proteins, respectively, were found to participate in xylose metabolism regulation regardless of which pathway was expressed and which host the xylose pathway was expressed in. Nine transcription factors, involved in homeostasis, regulation of amino acid metabolism, and stress responses, were identified as the key modules responsible for the host-specific responses to the same xylose pathway. In addition, the transcriptional regulations of xylose utilization in different yeast hosts were compared to two reference regulation patterns, which indicated that diverse regulation strategies were adopted by different hosts for improved xylose utilization. Conclusions This study provides the first transcriptomic study of the host dependence of xylose utilization in S. cerevisiae. Both the conserved regulatory modules for xylose metabolism and the key modules responsible for host dependence were identified. As indicated by the functions of the conserved transcription factors involved in xylose metabolism regulation, the xylose utilization in recombinant S. cerevisiae may be affected by both carbohydrate metabolism regulation and stress responses. Based on the comparison of transcriptional regulation patterns, the metabolic optimizations of xylose utilization in different hosts went toward different directions, which may explain the host dependence observed in this study. The knowledge revealed by this study could provide valuable insights towards the improvement of metabolic engineering strategies for cellulosic ethanol production.

This study was performed in order to reveal the effect of Noopept (ethyl ester of N-phenylacetyl-Lprolylglycine, GVS-111) on the DNA-binding activity of transcriptional factors (TF) in HEK293 cells transiently transfected with luciferase reporter constructs containing sequences for CREB, NFAT, NF-B, p53, STAT1, GAS, VDR, HSF1, and HIF-1. Noopept (10 M) was shown to increase the DNA-binding activity of HIF-1 only, while lacking the ability to affect that of CREB, NFAT, NF-B, p53, STAT1, GAS, VDR, and HSF1. Noopept provoked an additional increase in the DNA-binding activity of HIF-1 when applied in conditions of CoCl2-induced HIF- 1 stabilization. The degree of this HIF-positive effect of Noopept was shown to be concentration-dependent. Piracetam (1 mM) failed to affect significantly any of the TF under study. The results of molecular docking showed that Noopept (L-isomer), as well as its metabolite, L-isomer of N-phenyl-acetylprolyl, unlike its pharmacologically ineffective D-isomer, is able to bind to the active site of prolyl hydroxylase 2. Taking into account the important role of the genes activated by HIF-1 in the formation of an adaptive response to hypoxia, data on the ability of Noopept to provoke a selective increase in the DNA-binding activity of HIF-1 explain the wide spectrum of neurochemical and pharmacological effects of Noopept revealed before. The obtained data allow one to propose the HIF-positive effect as the primary mechanism of the activity of this Pro-Gly-containing dipeptide.

Tendon injuries and tendinopathy are common disorders, but the underlying mechanisms are not well understood. The overall aim of this thesis was to better understand the mechanisms underlying tendon healing, pain, and inflammation. The aim of the first study was to assess biomarkers of tendon healing, including procollagen type I (PINP) and type III (PIIINP) in relation to patient outcome in 65 patients with Achilles tendon rupture (ATR). At two weeks post-ATR, PINP and PIIINP-levels were quantified using microdialysis followed by ELISA. At one-year post-ATR patient outcome was assessed using the validated Achilles tendon Total Rupture Score. We found that higher ratio of PINP and PIIINP to total protein were significantly associated with less pain but more fatigue in the affected limb. In the second study, we applied Intermittent Pneumatic Compression (IPC) therapy for two weeks to stimulate tendon healing. The patients received either adjuvant IPC treatment or treatment-as-usual in a plaster cast without IPC. We observed that IPC therapy significantly increased PINP levels in the injured tendon, suggesting enhanced healing response. In our third study, we investigated healing response and the role of mast cells (MCs) in-vivo using an ATR rat model. Three weeks postoperatively, we demonstrated an increased number of MCs and a higher proportion of degranulated MCs in the injured tendon compared to the control. We further established that MCs in the injured tendon were positive for the glutamate receptor NMDAR1. In our final study, we assessed the effect of glutamate stimulation on in-vitro-derived mouse bone marrow MCs. Mast cell degranulation was quantified through β-hexosaminidase release, immunofluorescence was used to quantify NMDARs at the protein level, and RT-qPCR/microarray was used to study the expression of NMDARs and associated genes. Glutamate induced a robust upregulation of glutamate receptors of both ionotropic and metabotropic type, both at the mRNA and at protein level. NMDAR1 co-localized with glutamate in the membrane of MCs, thereby confirming an interaction between glutamate and its receptor. Glutamate also induced expression of pro-inflammatory compounds such as IL-6 and CCL2 and transcription factors such as Egr2, Egr3 and FosB. Moreover, the NMDA-channel blocker MK-801 completely abrogated the response of MCs to glutamate, supporting a functional glutamate–glutamate receptor axis in MCs. Together, findings presented in this dissertation reveal possible mechanisms of tendon healing in relation to pain and function, and establish a novel principle for how immune cells can communicate with nerve cells after ATR. Senskador och tendinopati är vanliga problem, men de underliggande mekanismerna otillräckligt undersökta. Målet med denna avhandling är att öka kunskapen om mekanismer under läkningen av senor, och hur dessa relaterar till smärta och inflammation. Syftet med den första studien var att kvantifiera biomarkörer för läkning av Achillessena, genom analys av prokollagen typ I (PINP) och typ III (PIIINP) hos 65 patienter efter ruptur av Achillessenan. Två veckor efter achillesruptur kvantifierades PINP och PIIINP-nivåerna med mikrodialys följt av ELISA-analys. Ett år efter achillesruptur bedömdes patienternas upplevda besvär med användning av ett validerat formulär, Achilles Tendon Total Rupture Score. Ökad andel PINP och PIIINP av totala mängden protein vid två veckor var signifikant associerat med mindre smärta men ökad fatigue i skadat ben efter ett år. I nästa studie utvärderade vi effekten av intermittent pneumatisk kompression (IPC) under två veckor av senans läkning efter achillesruptur. Patienterna fick antingen tilläggsbehandling med IPC eller vanlig behandling i gipsskena utan IPC. Vi kunde visa att behandling med IPC signifikant ökade nivån av PINP i den skadade senan, vilket tyder på förbättrad läkning. I den tredje studien undersökte vi mastcellers roll under läkningen av Achillessena efter ruptur i en råttmodell. Tre veckor postoperativt visade vi ett ökat antal mastceller och en högre andel degranulerade av mastceller i den läkande senan jämfört med senan på den andra (den friska) sidan. Vi kunde också påvisa glutamatreceptorn NMDAR1 hos mastceller i den läkande senan. I den fjärde studien bedömde vi effekten av glutamatstimulering in-vitro, på mastceller från benmärg hos mus. Degranulering av mastceller kvantifierades genom frisättning av β-hexosaminidas. För att kvantifiera NMDAR på proteinnivå använde vi immunfluorescens, och för att studera uttrycket av NMDAR och associerade gener använde vi RT-qPCR/mikroarray. Vi kunde visa att glutamat inducerar uppreglering av glutamatreceptorer av både jonotropisk och metabotropisk typ i mastceller, både på mRNA- och proteinnivå. NMDAR1 samlokaliserade med glutamat i membranet på mastceller, vilket därmed bekräftar en interaktion mellan glutamat och dess receptor. Glutamat inducerade också uttryck av pro-inflammatoriska proteiner såsom IL-6 och CCL2, samt transkriptionsfaktorer såsom Egr2, Egr3 och FosB. Dessutom upphävde NMDA-kanalblockeraren MK-801 fullständigt effekten av glutamat på mastceller, vilket talar för en funktionell betydelse av interaktionen mellan glutamat och glutamatreceptorer i mastceller. Sammantaget visar de fynd som presenteras i denna avhandling möjliga mekanismer för läkning av Achillessena i relation till smärta och funktion och introducerar en ny princip för hur immunceller kan kommunicera med nervceller efter achillesruptur.

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