Transcript Levels Of Target Genes Research Articles

DeepCORE: An interpretable multi-view deep neural network model to detect co-operative regulatory elements

Gene transcription is an essential process involved in all aspects of cellular functions with significant impact on biological traits and diseases. This process is tightly regulated by multiple elements that co-operate to jointly modulate the transcription levels of target genes. To decipher the complicated regulatory network, we present a novel multi-view attention-based deep neural network that models the relationship between genetic, epigenetic, and transcriptional patterns and identifies co-operative regulatory elements (COREs). We applied this new method, named DeepCORE, to predict transcriptomes in various tissues and cell lines, which outperformed the state-of-the-art algorithms. Furthermore, DeepCORE contains an interpreter that extracts the attention values embedded in the deep neural network, maps the attended regions to putative regulatory elements, and infers COREs based on correlated attentions. The identified COREs are significantly enriched with known promoters and enhancers. Novel regulatory elements discovered by DeepCORE showed epigenetic signatures consistent with the status of histone modification marks.

Screening and stability verification of reference genes in Botrytis cinerea ZX2 fermentation

Botrytis cinerea, an airborne plant pathogen, holds the potential to synthesize sesquiterpenes, which have been used for the industrial production of abscisic acid. Previously, through our genetic technology, we obtained strain ZX2, whose main product 1´,4´-trans-ABA-diol is physiologically active in plants. In this study, 50 L of fed fermentation was carried out with ZX2 strain to study the stability of expression of TUA, TUB, ATC, EF-1, GAPDH, UCE and GTP genes. Four kinds of software (GeNorm, NormFinder, BestKeeper and Delta Ct) were used to analyze the expression stability of candidate genes, and finally the best reference gene was screened by RefFinder. Based on the results, the ACT was the most stable gene. It was used to normalize the expression levels of two genes related to 1´,4´-trans-ABA-diol production (hmgr and bcaba3) when fed-batch fermentation. Guide the selection of appropriate internal reference genes during the fermentation process to accurately quantify the relative transcription levels of target genes in B.cinerea ZX2.

TGFβ signaling pathway is altered by HLA-B27 expression, resulting in pathogenic consequences relevant for spondyloarthritis

BackgroundAssociation of HLA-B27 with spondyloarthritis (SpA) has been known for 50 years, but still remains unexplained. We recently showed that HLA-B27 expressed in wing imaginal disc from HLA-B27/human-β2 microglobulin (hβ2m) transgenic Drosophila deregulated bone morphogenetic protein (BMP) pathway by interacting physically with type I BMP receptor (BMPR1) Saxophone (Sax), leading to crossveinless phenotype.MethodsGenetic interaction was studied between activin/transforming growth factor β (TGFβ) pathway and HLA-B27/hβ2m in transgenic Drosophila wings. The HLA-B27-bound peptidome was characterized in wing imaginal discs. In mesenteric lymph node (mLN) T cells from HLA-B27/hβ2m rat (B27 rat), physical interaction between HLA-B27 and activin receptor-like kinase-2 (ALK2), ALK3 and ALK5 BMPR1s, phosphorylation of small mothers against decapentaplegic (SMADs) and proteins of the non-canonical BMP/TGFβ pathways induced by its ligands, and the transcript level of target genes of the TGFβ pathway, were evaluated.ResultsIn HLA-B27/hβ2m transgenic Drosophila, inappropriate signalling through the activin/TGFβ pathway, involving Baboon (Babo), the type I activin/TGFβ receptor, contributed to the crossveinless phenotype, in addition to deregulated BMP pathway. We identified peptides bound to HLA-B27 with the canonical binding motif in HLA-B27/hβ2m transgenic Drosophila wing imaginal disc. We demonstrated specific physical interaction, between HLA-B27/hβ2m and mammalian orthologs of Sax and Babo, i.e. ALK2 and ALK5 (i.e. TGFβ receptor I), in the mLN cells from B27 rat. The magnitude of phosphorylation of SMAD2/3 in response to TGFβ1 was increased in T cells from B27 rats, showing evidence for deregulated TGFβ pathway. Accordingly, expression of several target genes of the pathway was increased in T cells from B27 rats, in basal conditions and/or after TGFβ exposure, including Foxp3, Rorc, Runx1 and Maf. Interestingly, Tgfb1 expression was reduced in naive T cells from B27 rats, even premorbid, an observation consistent with a pro-inflammatory pattern.ConclusionsThis study shows that HLA-B27 alters the TGFβ pathways in Drosophila and B27 rat. Given the importance of this pathway in CD4 + T cells differentiation and regulation, its disturbance could contribute to the abnormal expansion of pro-inflammatory T helper 17 cells and altered regulatory T cell phenotype observed in B27 rats.

Effects of different concentrations of nicotinamide on hematopoietic stem cells cultured in vitro.

In vitro expansion to increase numbers of hematopoietic stem cells (HSCs) in cord blood could improve clinical efficacy of this vital resource. Nicotinamide (NAM) can promote HSC expansion ex vivo, but its effect on hematopoietic stem and progenitor cells (HSPCs, CD34+CD38) and functional subtypes of HSCs - short-term repopulating HSCs (ST-HSCs, CD34+CD38CD45RACD49f+) and long-term repopulating HSCs (LT-HSCs, CD34+CD38CD45RACD49f+CD90+) is not yet known. As a sirtuin 1 (SIRT1) inhibitor, NAM participates in regulating cell adhesion, polarity, migration, proliferation, and differentiation. However, SIRT1 exhibits dual effects by promoting or inhibiting differentiation in different tissues or cells. We propose that the concentration of NAM may influence proliferation, differentiation, and SIRT1 signaling of HSCs. To evaluate the effects and underlying mechanisms of action of different concentrations of NAM on HSC proliferation and differentiation. CD34+ cells were purified from umbilical cord blood using MacsCD34 beads, and cultured for 10-12 d in a serum-free medium supplemented with cytokines, with different concentrations of NAM added according to experimental requirements. Flow cytometry was used to detect phenotype, cell cycle distribution, and apoptosis of the cultured cells. Real-time polymerase chain reaction was used to detect the transcription levels of target genes encoding stemness-related factors, chemokines, components of hypoxia pathways, and antioxidant enzymes. Dichloro-dihydro-fluorescein diacetate probes were used to evaluate intracellular production of reactive oxygen species (ROS). Determination of the effect of different culture conditions on the balance of cytokine by cytometric bead array. Compared with the control group, the proportion and expansion folds of HSPCs (CD34+CD38) incubated with 5 mmol/L or 10 mmol/L NAM were significantly increased (all P < 0.05). The ST-HSCs ratio and fold expansion of the 5 mmol/L NAM group were significantly higher than those of the control and 10 mmol/L NAM groups (all P < 0.001), whereas the LT-HSCs ratio and fold expansion of the 10 mmol/L NAM group were significantly higher than those of the other two groups (all P < 0.05). When the NAM concentration was > 10 mmol/L, cell viability significantly decreased. In addition, compared with the 5 mmol/L NAM group, the proportion of apoptotic cells in the 10 mmol/L NAM group increased and the proportion of cells in S and G2 phase decreased. Compared with the 5 mmol/L NAM group, the HSCs incubated with 10 mmol/L NAM exhibited significantly inhibited SIRT1 expression, increased intracellular ROS content, and downregulated expression of genes encoding antioxidant enzymes (superoxide dismutase 1, peroxiredoxin 1). Low concentrations (5 mmol/L) of NAM can better regulate the balance between proliferation and differentiation, thereby promoting expansion of HSCs. These findings allow adjustment of NAM concentrations according to expansion needs.

Investigating the role of aae-miR-34-5p in the regulation of juvenile hormone biosynthesis genes in the mosquito Aedes aegypti

Juvenile hormone (JH) controls the development and reproduction of insects. Therefore, a tight regulation of the expression of JH biosynthetic enzymes is critical. microRNAs (miRNAs) play significant roles in the post-transcriptional regulation of gene expression by interacting with complementary sequences in target genes. Previously, we reported that several miRNAs were differentially expressed during three developmental stages of Aedes aegypti mosquitoes with different JH levels (no JH, high JH, and low JH). One of these miRNAs was aae-miR-34-5p. In this study, we identified the presence of potential target sequences of aae-miR-34-5p in the transcripts of some genes encoding JH biosynthetic enzymes. We analysed the developmental expression patterns of aae-miR-34-5p and the predicted target genes involved in JH biogenesis. Increases in miRNA abundance were followed, with a delay, by decreases in transcript levels of target genes. Application of an inhibitor and a mimic of aae-miR-34-5p led respectively to increased and decreased levels of thiolase transcripts, which is one of the early genes of JH biosynthesis. Female adult mosquitoes injected with an aae-miR-34-5p inhibitor exhibited significantly increased transcript levels of three genes encoding JH biosynthetic enzymes, acetoacetyl-CoA thiolase (thiolase), farnesyl diphosphate phosphatase, and farnesal dehydrogenase. Overall, our results suggest a potential role of miRNAs in JH production by directly targeting genes involved in its biosynthesis.

Discovery and validation of PURA as a transcription target of 20(S)-protopanaxadiol: Implications for the treatment of cognitive dysfunction

Background20(S)-protopanaxadiol (PPD), a ginsenoside metabolite, has prominent benefits for the central nervous system, especially in improving learning and memory. However, its transcriptional targets in brain tissue remain unknown. MethodsIn this study, we first used mass spectrometry-based drug affinity responsive target stability (DARTS) to identify the potential proteins of ginsenosides and intersected them with the transcription factor library. Second, the transcription factor PURA was confirmed as a target of PPD by biolayer interferometry (BLI) and molecular docking. Next, the effect of PPD on the transcriptional levels of target genes of PURA in brain tissues was determined by qRT-PCR. Finally, bioinformatics analysis was used to analyze the potential biological features of these target proteins. ResultsThe results showed three overlapping transcription factors between the proteomics of DARTS and transcription factor library. BLI analysis further showed that PPD had a higher direct interaction with PURA than parent ginsenosides. Subsequently, BLI kinetic analysis, molecular docking, and mutations in key amino acids of PURA indicated that PPD specifically bound to PURA. The results of qRT-PCR showed that PPD could increase the transcription levels of PURA target genes in brain. Finally, bioinformatics analysis showed that these target proteins were involved in learning and memory function. ConclusionThe above-mentioned findings indicate that PURA is a transcription target of PPD in brain, and PPD upregulate the transcription levels of target genes related to cognitive dysfunction by binding PURA, which could provide a chemical and biological basis for the study of treating cognitive impairment by targeting PURA.

Genome-wide analysis reveals Hsf1 maintains high transcript abundance of target genes controlled by strong constitutive promoter in Saccharomyces cerevisiae

BackgroundIn synthetic biology, the strength of promoter elements is the basis for precise regulation of target gene transcription levels, which in turn increases the yield of the target product. However, the results of many researches proved that excessive transcription levels of target genes actually reduced the yield of the target product. This phenomenon has been found in studies using different microorganisms as chassis cells, thus, it becomes a bottleneck problem to improve the yield of the target product.ResultsIn this study, promoters PGK1p and TDH3p with different strengths were used to regulate the transcription level of alcohol acetyl transferase encoding gene ATF1. The results demonstrated that the strong promoter TDH3p decreased the production of ethyl acetate. The results of Real-time PCR proved that the transcription level of ATF1 decreased rapidly under the control of TDH3p, and the unfolded protein reaction was activated, which may be the reason for the abnormal production caused by the strong promoter. RNA-sequencing analysis showed that the overexpression of differential gene HSP30 increased the transcriptional abundance of ATF1 gene and production of ethyl acetate. Interestingly, deletion of the heat shock protein family (e.g., Hsp26, Hsp78, Hsp82) decreased the production of ethyl acetate, suggesting that the Hsp family was also involved in the regulation of ATF1 gene transcription. Furthermore, the results proved that the Hsf1, an upstream transcription factor of Hsps, had a positive effect on alleviating the unfolded protein response and that overexpression of Hsf1 reprogramed the pattern of ATF1 gene transcript levels. The combined overexpression of Hsf1 and Hsps further increased the production of ethyl acetate. In addition, kinase Rim15 may be involved in this regulatory pathway. Finally, the regulation effect of Hsf1 on recombinant strains constructed by other promoters was verified, which confirmed the universality of the strategy.ConclusionsOur results elucidated the mechanism by which Rim15–Hsf1–Hsps pathway reconstructed the repression of high transcription level stress and increased the production of target products, thereby providing new insights and application strategies for the construction of recombinant strains in synthetic biology.

The Role of Sigma‐1‐Receptor in Endoreticulum Stress and Alzheimer's Disease.

AbstractBackgroundOne evident hallmark of Alzheimer's Disease (AD) is the irregular accumulation of proteins such as Aβ and pTau. This accumulation could alleviate ER stress and trigger an integrated signaling cascade called the unfolded protein response (UPR). UPR reduces the number of misfolded proteins and further inhibits abnormal protein accumulation. Targeting ER stress may therefore be an effective treatment for AD. One protein of interest that plays a role in modulating ER stress is the Sigma‐1 receptor (Sig1R). Sig1R agonists have been shown to be neuroprotective and anti‐amnesic in AD mouse models. However, the mechanism of action remains largely unknown.MethodIn our study, we use primary MEF cells derived from Wild‐type and Knock‐out Sig1R mouse models to examine the role of Sig1R in ER stress. We treated cells with an acute ER stressor, DTT, and measured transcript levels of target genes under the UPR branches. Furthermore, we used Chromosomal Immunoprecipitation (ChIP) to identify regulators of SIG1R gene during stress.ResultWe observed that the loss of Sig1R compromised the PERK pathway. KO MEFs had a slow recovery from stress suggesting Sig1R plays a role in re‐establishing homeostasis following stress. Following DTT treatment, mRNA and protein levels of Sig1R increase significantly over the course of recovery. ChIP uncovered three CHOP, a major player in the UPR, binding sites within the promoter of SIG1R, and each site had a distinct temporal binding pattern over the course of cell recovery. This increase in binding correlates with an increase in Sig1R expression. This showed that Sig1R is a chaperone downstream of CHOP, and that the up‐regulation of Sig1R might be crucial in re‐establishing homeostasis. Finally, our imaging data showed an increase in localization of Sig1R with extracellular vesicles. We further hypothesized that the high expression of Sig‐1R may have a role in the removal of misfolded proteins, perhaps Aβ. Therefore, we have ongoing experiments looking at the effect of Aβ on our models.ConclusionOur study has the potential to unravel the implications of Sig1R in AD by showing that it is transcriptionally and translationally regulated in response to stress induced by misfolded proteins.

Susceptibility of Neohelice granulata (Decapoda, Varunidae) to white spot syndrome virus (WSSV)

White spot syndrome virus (WSSV) continues to be the most severe viral pathogen to the shrimp industry worldwide. Pacific white shrimp Litopenaeus vannamei is particularly affected by WSSV, and this virus has been detected in a wide range of wild crustaceans, including penaeid and non-penaeid shrimp and crayfish, as well as crabs. Crabs have been considered as a dangerous threat to shrimp farms because they are generally believed to be highly tolerant to WSSV and to remain infected for long periods without signs of disease. The burrowing crab Neohelice (= Chasmagnathus) granulata can be commonly found in the surroundings of the shrimp farms in southern Brazil. Here, we investigated the susceptibility of N. granulata to WSSV infection in comparison to L. vannamei. WSSV infectability and host susceptibility were investigated by viral challenge (intramuscular injection) in both species. Viral load in challenged animals was quantified by qPCR in both hemolymph and gills. Furthermore, the transcript levels of sixteen target genes related to the molecular defense status were assessed. The results revealed that N. granulata experimentally infected by WSSV, as well as those naturally infected, showed lethargy, lack of appetite, and later gross signs of the disease. Moreover, N. granulata showed to be less susceptible to WSSV when compared to L. vannamei. While no death was observed in crabs before a post-challenge interval of 120 h, shrimp death was seen as early as 48 h post-infection. Comparative viral load was also assessed by qPCR in gills of captured wild crabs and farmed shrimp naturally infected by WSSV. Transcript levels of target genes were also investigated after WS challenge between 24 and 48 hpi in shrimp and between 96 and 120 hpi in crab. Differences in gene transcripts were particularly noteworthy with the increase of viral copies at 106 copies µl−1. These results indicated that WSSV infection modulated genes related of important cellular responses, such as apoptosis, chaperone function, and ion storage, in both shrimp and crab.Graphical Abstract

Impact of nickel mining in New Caledonia on marbled eels Anguilla marmorata

New Caledonia is particularly affected by nickel open pit mining activities because of the presence of ultramafic soils rich in metals. The particles dispersed by atmospheric transport and soil erosion during the excavation of nickel end up by deposition or leaching in rivers where they may be bioaccumulated by organisms living downstream the mines. Despite alarming freshwater metals concentrations, no study investigated the level of their bioaccumulation in eels, and if high bioaccumulation levels occur, the potential consequences on their health. The aim of this study was to determine how eels Anguilla marmorata are impacted in situ by metals issued from mining activity by measuring: morphometric parameters; metal concentrations in tissues and organs and transcription levels of target genes encoding proteins involved in several metabolic key functions. Among organs, liver was found to be the most affected by mining with average nickel concentrations of 5.14 mg/kg versus 1.63 mg/kg for eels away from mines leading to dysregulation of numerous genes involved in oxidative stress, DNA repair, apoptosis, reproduction and both lipid and mitochondrial metabolisms. This study should allow us to define in an integrated way if metals released by mining activities influence metals bioaccumulation in eels and induce biological effects.

Polydatin Glycosides Improve Monocrotaline-Induced Pulmonary Hypertension Injury by Inhibiting Endothelial-To-Mesenchymal Transition.

Objective: To study the effect of polydatin on the injury of pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT). Methods: SD rats were induced to develop PAH injury by a single subcutaneous injection of MCT (60 mg/kg). From the second day, rats in the administration group were orally given sildenafil (20 mg/kg) and polydatin (30 or 60 mg/kg) for 3 weeks. At the end of the experiment, right ventricular hypertrophy (RVH) index of SD rats was calculated, pathological damage was assessed by HE staining, transcription levels of target genes were detected by RT-PCR and Elisa, and expression levels of Endothelial-to-mesenchymal transition (EndMT) related proteins were detected by immunohistochemistry (IHC) and immunofluorescence (IF). Finally, molecular docking analysis was used to verify the interaction of polydatin on the main targets. Results: Polydatin could significantly restore the body function, reduce MCT-induced PAH injury, reduce serum biochemical indices; polydatin could effectively inhibit EndMT process by decreasing the expression of N-cadherin, β-catenin and vimentin; polydatin could down-regulate TAGLN expression and increase PECAM1 expression to reduce pulmonary vascular remodeling. The interaction between polydatin and EndMT target was confirmed by molecular docking operation. Conclusion: Pharmacological experiments combined with Combining molecular docking was first used to clarify that polydatin can reduce the pulmonary endothelial dysfunction and pulmonary vascular remodeling induced by MCT by inhibiting EndMT. The results of the study provide new ideas for the further treatment of PAH injury.

Bacterially expressed dsRNA induces Varroa destructor gene knockdown by honey bee-mediated oral administration

The ectoparasite Varroa destructor causes serious losses of Apis mellifera colonies and negatively impacts the beekeeping industry around the world. New control methods have been proposed based on the RNA interference technique. Previous reports showed that parasitized honey bees fed with double-stranded RNA (dsRNA) synthesized in vitro reduce the transcription levels of target genes in Varroa mites. An efficient and inexpensive alternative to produce dsRNA is the use of bacteria capable of achieving high levels of in vivo synthesis. In the present study, dsRNA synthetized in vivo was used to induce gene silencing in V. destructor and evaluate their effect on the survival of both honey bees and the parasitic Varroa mites. The results evidenced that dsRNA fed to the bees engendered gene silencing in mites, inhibiting expression levels of target genes by 50%. Indeed, a reduction of 50% in Varroa survival was observed when bacterially expressed dsRNAs were administered to mite-parasitized bees. Worker bees that were fed with Varroa-targeted dsRNA by oral route showed no survival differences compared to control bees, fed with sucrose or dsRNA-GFP solutions. Our results demonstrated that specific dsRNA over-expressed in bacteria is capable of reducing mite survival by bee-mediated oral administration. This study provides an efficient and low-cost method for dsRNA production to control parasites and honey bee diseases.

Super-enhancer mediated regulation of adult β-globin gene expression: the role of eRNA and Integrator.

Super-enhancers (SEs) mediate high transcription levels of target genes. Previous studies have shown that SEs recruit transcription complexes and generate enhancer RNAs (eRNAs). We characterized transcription at the human and murine β-globin locus control region (LCR) SE. We found that the human LCR is capable of recruiting transcription complexes independently from linked globin genes in transgenic mice. Furthermore, LCR hypersensitive site 2 (HS2) initiates the formation of bidirectional transcripts in transgenic mice and in the endogenous β-globin gene locus in murine erythroleukemia (MEL) cells. HS2 3′eRNA is relatively unstable and remains in close proximity to the globin gene locus. Reducing the abundance of HS2 3′eRNA leads to a reduction in β-globin gene transcription and compromises RNA polymerase II (Pol II) recruitment at the promoter. The Integrator complex has been shown to terminate eRNA transcription. We demonstrate that Integrator interacts downstream of LCR HS2. Inducible ablation of Integrator function in MEL or differentiating primary human CD34+ cells causes a decrease in expression of the adult β-globin gene and accumulation of Pol II and eRNA at the LCR. The data suggest that transcription complexes are assembled at the LCR and transferred to the globin genes by mechanisms that involve Integrator mediated release of Pol II and eRNA from the LCR.

Effects of vascular normalizing agents on immune marker expression in T cells, dendritic cells, and melanoma cells

Tertiary lymphoid structures (TLS) are lymph node-like structures that form at sites of inflammation, and their presence in cancer patients is predictive of a better clinical outcome. One significant obstacle to TLS formation is reduced immune cell infiltration into the tumor microenvironment (TME), resulting from aberrant vasculature within the TME. Recent studies have shown that low doses of vasculature normalizing (VN) agents may override this defect, leading to improved tissue perfusion and increased immune cell entry into the TME. While the effect of VN agents on vascular endothelial cells has been well documented, their effects on immune cell and tumor cell phenotype remain understudied. We hypothesized that treating immune cells with VN agents would induce a pro-inflammatory phenotype in T cells and dendritic cells (DCs), while treating tumor cells would reduce their immunosuppressive phenotype and promote production of chemokines that recruit immune cells and foster TLS formation. To test this, a mouse melanoma cell line, primary murine T cells, and DCs were treated overnight with VN agents. The next day, treated and control cells were harvested for analyses to measure transcript levels of target genes as well as levels of surface markers. Overall, VN agents were observed to have differential but predominantly immune-supportive effects on immune and tumor cell phenotypes. These findings will guide future experiments which may result in an effective clinical treatment.

RNA silencing of hormonal biosynthetic genes impairs larval growth and development in cotton bollworm, Helicoverpa armigera

The cotton bollworm, Helicoverpa armigera, is a highly polyphagous pest, causing enormous losses to various economically important crops. The identification and in vitro functional validation of target genes of a pest is a prerequisite to combat pest via host-mediated RNA interference (RNAi). In the present study, six hormonal biosynthesis genes of H. armigera were chosen and evaluated by feeding insect larvae with dsRNAs corresponding to each target gene, viz., juvenile hormone acid methyltransferase (HaJHAMT), prothoracicotropic hormone (HaPTTH), pheromone biosynthesis-activating peptide (HaPBAP), molt regulating transcription factor (HaHR3), activated protein 4 (HaAP-4) and eclosion hormone precursor (HaEHP). The loss of function phenotypes for these hormonal genes were observed by releasing second instar larvae on to artificial diet containing target gene-specific dsRNAs. Ingestion of dsRNAs resulted in mortality ranging from 60% to 90%, reduced larval weight, phenotypic deformities and delayed pupation. The quantitative real-time PCR (qRT-PCR) analysis showed that the target gene transcript levels were decreased drastically (31% to 77%) as compared to control or unrelated control (GFP-dsRNA), and correlated well with the mortality and developmental defects of larvae. Also, a comparison of the silencing efficacy of un-diced long HaPTTH -dsRNAwith RNase III diced HaPTTH-dsRNA (siRNAs) revealed that long dsRNAs were more efficient in silencing the target gene. These results indicated that the hormonal biosynthesis genes have varied sensitivity towards RNAi and could be the vital targets for insect resistance in crop plants like cotton which are infested by H. armigera.

The sug operon involves in resistance to quaternary ammonium compounds in Listeria monocytogenes EGD-e.

In recent years, an increasing number of Listeria monocytogenes strains with resistance to quaternary ammonium compounds (QACs) have been reported. However, the genetic basis for QACs resistance in L. monocytogenes remains poorly understood. In the present study, we have characterized the operon lmo0852/lmo0853/lmo0854 (designated sugR/sugE1/sugE2) that contributes to QACs' resistance in L. monocytogenes EGD-e. We constructed the gene deletion mutants and the complemented strains, determined minimum inhibitory concentrations (MICs) of these strains against antimicrobial agents, assessed the transcription levels of target genes by RT-qPCR, and measured the promoter activity by using β-galactosidase assays. We also investigated the interaction between the promoter DNA and the putative regulatory protein by electrophoretic mobility shift assay (EMSA). The sug operon consists of a putative TetR family regulator encoded by sugR and two small multidrug resistance (SMR) efflux pumps encoded by sugE1 and sugE2. Our results showed that either SugE1 or SugE2 is sufficient for QACs' resistance, indicating their function overlapping in QACs' resistance. Interestingly, lacking one sugE gene could lead to a significant increase in transcription of the other sugE gene in the presence of benzalkonium chloride (BC). Additionally, SugR negatively regulates the transcription of the sug locus by binding to the operon promoter. Given that QACs are commonly used in food industry, the findings from this study will help us to have a better understanding of the adaptations of L. monocytogenes to this type of disinfectant. Key points •Either SugE1 or SugE2 was sufficient for QACs resistance. •The functions of two sug genes overlap and compensate each other in QACs resistance. •SugR is the transcriptional suppressor of sugE1 and sugE2. •SugR regulates the sug locus by binding to the operon promoter.

Selection of lethal genes for ingestion RNA interference against western flower thrips, Frankliniella occidentalis, via leaf disc-mediated dsRNA delivery

The western flower thrips, Frankliniella occidentalis, is a major pest that damages a wide variety of crops and vegetables. Following extensive use of insecticides, it has developed high levels of resistance to almost all groups of insecticides due to its high reproduction rate and short generation time. Therefore, an alternative pest control strategy, such as RNA interference (RNAi)-based control, is essential. To establish an ingestion RNAi-based control, a total of 57 genes involved in various biological processes were selected, and their double-stranded RNAs (dsRNA) were delivered to an insecticide-susceptible strain of F. occidentalis via the leaf disc-feeding method using a bioassay chamber optimized by 3D printing. The mortality of dsRNA-ingested thrips was examined every 24 h until 120 h post-treatment. Of the 57 genes screened, dsRNAs of the Toll-like receptor 6, apolipophorin, coatomer protein subunit epsilon and sorting and assembly machinery component were most lethal when ingested by thrips. The dsRNA-fed thrips showed substantially reduced transcription levels of target genes, demonstrating that the observed mortality was likely due to RNAi. When these genes were tested for ingestion RNAi against an insecticide-resistant strain of F. occidentalis, bioassay results were similar. In conclusion, this study provides the first evidence that ingestion RNAi can be lethal to F. occidentalis, a mesophyll sucking pest, and further suggests that transgenic plants expressing hairpin RNA of these essential genes can be employed to control insecticide-resistant thrips.

Predictive models of eukaryotic transcriptional regulation reveals changes in transcription factor roles and promoter usage between metabolic conditions.

Transcription factors (TF) are central to transcriptional regulation, but they are often studied in relative isolation and without close control of the metabolic state of the cell. Here, we describe genome-wide binding (by ChIP-exo) of 15 yeast TFs in four chemostat conditions that cover a range of metabolic states. We integrate this data with transcriptomics and six additional recently mapped TFs to identify predictive models describing how TFs control gene expression in different metabolic conditions. Contributions by TFs to gene regulation are predicted to be mostly activating, additive and well approximated by assuming linear effects from TF binding signal. Notably, using TF binding peaks from peak finding algorithms gave distinctly worse predictions than simply summing the low-noise and high-resolution TF ChIP-exo reads on promoters. Finally, we discover indications of a novel functional role for three TFs; Gcn4, Ert1 and Sut1 during nitrogen limited aerobic fermentation. In only this condition, the three TFs have correlated binding to a large number of genes (enriched for glycolytic and translation processes) and a negative correlation to target gene transcript levels.

Expression Level of Caspase Genes in Colorectal Cancer

Background:Caspases proteins are protease enzymes involved in the initiation and execution of apoptosis process. Regulation of apoptosis process plays an important role in the normal biological events and development. In addition to developmental abnormalities, dysregulated apoptosis system may lead to tumorigenesis, autoimmunity, and other serious health problems. Aberrant regulation of apoptosis may also be the paramount cause of chemoresistance during cancer therapy. It is aimed through this study to evaluate the transcript levels of Caspase 3, 8, and 9 in tumoral tissues from patients with colorectal cancer (CRC) and compare it with normal marginal tissues.Methods:Fifty tumor tissues and their matched marginal tissues, as control group, were obtained from CRC patients. Total mRNA of all tissue samples was extracted and cDNA was synthesized. Using SYBR Green PCR master mix and Real-time gene expression technique, the transcript level of target genes was quantified.Results:Experiments indicated that mRNA expressions of caspase 9 and 3 were downregulated in tumoral tissues from CRC patients in comparison to marginal tissues. In contrast, tumoral tissues expressed mRNA of caspase 8 higher than normal marginal tissues. Modified transcript levels of caspase 3, 8, and 9 were correlated with the clinical manifestations of the patients.Conclusions:Alteration in the mRNA level of caspase genes may be involved in the development of CRC.

Development and use of a switchgrass (Panicum virgatum L.) transformation pipeline by the BioEnergy Science Center to evaluate plants for reduced cell wall recalcitrance

BackgroundThe mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based biofuel production. One BESC goal was to decrease poplar and switchgrass biomass recalcitrance to biofuel conversion while not affecting plant growth. A transformation pipeline (TP), to express transgenes or transgene fragments (constructs) in these feedstocks with the goal of understanding and decreasing recalcitrance, was considered essential for this goal. Centralized data storage for access by BESC members and later the public also was essential.ResultsA BESC committee was established to codify procedures to evaluate and accept genes into the TP. A laboratory information management system (LIMS) was organized to catalog constructs, plant lines and results from their analyses. One hundred twenty-eight constructs were accepted into the TP for expression in switchgrass in the first 5 years of BESC. Here we provide information on 53 of these constructs and the BESC TP process. Eleven of the constructs could not be cloned into an expression vector for transformation. Of the remaining constructs, 22 modified expression of the gene target. Transgenic lines representing some constructs displayed decreased recalcitrance in the field and publications describing these results are tabulated here. Transcript levels of target genes and detailed wall analyses from transgenic lines expressing six additional tabulated constructs aimed toward modifying expression of genes associated with wall structure (xyloglucan and lignin components) are provided. Altered expression of xyloglucan endotransglucosylase/hydrolases did not modify lignin content in transgenic plants. Simultaneous silencing of two hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferases was necessary to decrease G and S lignin monomer and total lignin contents, but this reduced plant growth.ConclusionsA TP to produce plants with decreased recalcitrance and a LIMS for data compilation from these plants were created. While many genes accepted into the TP resulted in transgenic switchgrass without modified lignin or biomass content, a group of genes with potential to improve lignocellulosic biofuel yields was identified. Results from transgenic lines targeting xyloglucan and lignin structure provide examples of the types of information available on switchgrass lines produced within BESC. This report supplies useful information when developing coordinated, large-scale, multi-institutional reverse genetic pipelines to improve crop traits.