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Selecting suitable promoters to drive gene overexpression can provide significant insight into the development of engineered bacteria. In this study, we analyzed the transcriptome data of Burkholderia pyrrocinia JK-SH007 and identified 54 highly expressed genes. The promoter sequences were located using genome-wide data and scored using the prokaryotic promoter prediction software BPROM to further screen out 18 promoter sequences. We also developed a promoter trap system based on two reporter proteins adapted for promoter optimization in B. pyrrocinia JK-SH007: firefly luciferase encoded by the luciferase gene set (Luc) and trimethoprim (TP)-resistant dihydrofolate reductase (TPr). Ultimately, eight constitutive promoters were successfully inserted into the probe vector and transformed into B. pyrrocinia JK-SH007. The transformants were successfully grown on Tp antibiotic plates, and firefly luciferase expression was determined by measuring the relative light unit (RLU). Five of the promoters (P4, P9, P10, P14, and P19) showed 1.01–2.51-fold higher activity than the control promoter λ phage transcriptional promoter (PRPL). The promoter activity was further validated via qPCR analysis, indicating that promoters P14 and P19 showed stable high transcription levels at all time points. Then, GFP and RFP proteins were overexpressed in JK-SH007. In addition, promoters P14 and P19 were successfully used to drive gene expression in Burkholderia multivorans WS-FJ9 and Escherichia coli S17-1. The two constitutive promoters can be used not only in B. pyrrocinia JK-SH007 itself to gene overexpression but also to expand the scope of application. [ABSTRACT FROM AUTHOR]

Cancer is a complex disease with usually multiple disease mechanisms. Target combination is a better strategy than a single target in developing cancer therapies. However, target combinations are generally more difficult to be predicted. Current CRISPR-cas9 technology enables genome-wide screening for potential targets, but only a handful of genes have been screend as target combinations. Thus, an effective computational approach for selecting candidate target combinations is highly desirable. Selected target combinations also need to be translational between cell lines and cancer patients. We have therefore developed DSCN (double-targetselection guided byCRISPR screening andnetwork), a method that matches expression levels in patients and gene essentialities in cell lines through spectral-clustered protein-protein interaction (PPI) network. In DSCN, a sub-sampling approach is developed to model first-target knockdown and its impact on the PPI network, and it also facilitates the selection of a second target. Our analysis first demonstrated a high correlation of the DSCN sub-sampling-based gene knockdown model and its predicted differential gene expressions using observed gene expression in 22 pancreatic cell lines before and after MAP2K1 and MAP2K2 inhibition (R2 = 0.75). In DSCN algorithm, various scoring schemes were evaluated. The 'diffusion-path' method showed the most significant statistical power of differentialting known synthetic lethal (SL) versus non-SL gene pairs (P = 0.001) in pancreatic cancer. The superior performance of DSCN over existing network-based algorithms, such as OptiCon and VIPER, in the selection of target combinations is attributable to its ability to calculate combinations for any gene pairs, whereas other approaches focus on the combinations among optimized regulators in the network. DSCN's computational speed is also at least ten times fast than that of other methods. Finally, in applying DSCN to predict target combinations and drug combinations for individual samples (DSCNi), DSCNi showed high correlation between target combinations predicted and real synergistic combinations (P = 1e-5) in pancreatic cell lines. In summary, DSCN is a highly effective computational method for the selection of target combinations. Author summary: Cancer therapies require targets to function. Compared to a single target, a target combination is a better strategy for developing cancer therapies. However, predicting target combination is more complicated than predicting a single target. Current CRISPR technology enables whole-genome screening of potential targets. But most of the experiments have been conducted on a single target (gene) level. To facilitate the discovery of novel target (combinations), we developed DSCN (double-targetselection guided byCRISPR screening andnetwork) that utilize single target-level CRISPR screening data and expression profiles for predicting target combinations by connecting cell-line omics-data with tissue omics-data. DSCN showed great accuracy on different cancer types and superior performance compared to existing network-based prediction tools. We also introduced DSCNi derived from DSCN and designed specifically for predicting target combinations for single-tient patient. Our results showed synergistic target combinations predicted by DSCNi accurately reflected synergies on drug combination levels. Thus, DSCN and DSCNi have the potential to be further applied in the clinical personalized medicine practice. [ABSTRACT FROM AUTHOR]

Bacterial intestinal inflammation is a common disease of aquatic animal in high-density aquaculture. This study aims to construct a model of intestinal inflammation in northern snakehead, and analyze the intestinal transcriptome after Aeromonas hydrophila (A. hydrophila) infection by high-throughput sequencing technologies (RNA-seq), in order to obtain candidate genes and regulatory pathways that may participate in inflammatory response. Northern snakehead [(13.05 ± 2.39) g] were infected through anal for 24 h with different concentrations of A. hydrophila (1.0 × 10−4, 1.0 × 10−5, 1.0 × 10−6, 1.0 × 10−7 and 1.0 × 10−8 CFU/mL). Intestinal histology observation showed that infection with A. hydrophila caused obvious inflammatory responses in intestine, especially in hindgut. The results also indicated that intestinal permeability related parameters (D-LA and Na+-K+-ATP), immune-related parameters (C3, LZM, AKP, MPO, IL-1β and TNF-α) and immune-related gene expression (il-1β and tnf-α) were significantly changed compared to the CK group. In this process, high concentration of A. hydrophila infection caused the death of snakehead. On the basis of the above evaluation, we successfully constructed a model of intestinal inflammation induced by A. hydrophila in northern snakehead, and the optimal dosage of A. hydrophila was 1.0 × 10−6 CFU/mL. Subsequently, the constructed model was analyzed on the Illumina Genome Analyser II platform for intestinal transcriptome. Results showed that a total of 370,492,420 clean reads, 145,236 transcripts and 89,431 unigenes were obtained. Moreover, a total of 7566 differentially expressed genes (DEGs) were found, including 4486 upregulated genes and 3080 downregulated genes. Notably, the key pathways [Toll-like receptors (TLRs) and NOD-like receptors (NLRs)] and genes [ tlr1 , tlr2 , tlr5 , nf-κb , tnf-α , il-1β , il-10 , nod1 ] related to inflammation were discovered. These results revealed the response of northern snakehead to A. hydrophila at the transcriptional level, which provide insights for the regulatory mechanisms of intestinal inflammation in northern snakehead. • A model of intestinal inflammation in northern snakehead was constructed by Aeromonas hydrophila. • The optimal dosage of Aeromonas hydrophila for inducing intestinal inflammation in northern snakehead was 1.0 × 10−6 CFU/mL. • The key pathways and genes related to inflammation were discovered by high-throughput sequencing technologies. [ABSTRACT FROM AUTHOR]

Purpose: Abnormal expression of the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor may potentially increase the susceptibility to neuropsychiatric diseases. The purpose of this study was to investigate the functional sequence of the 3ʹUTR of the human GRIN1 gene, which encodes the GluN1 receptor to determine the effect on the expression of GluN1 receptor. Methods: We transferred seven recombinant pmirGLO recombinant vectors containing the 3ʹUTR truncated fragment of the GRIN1 gene into HEK-293, SK-N-SH, and U87 cell lines and compared the relative fluorescence intensity of adjacent length fragments. The TargetScan database was used to predict miRNAs. Then, miRNA mimics/inhibitors were co-transfected into the three cell lines with the 3ʹUTR of GRIN1 (pmirGLO - GRIN1), to investigate their influence on GRIN1 gene expression. Results: Compared with the pmirGLo-Basic vector, the relative fluorescence intensity of the complete GRIN1 gene 3ʹUTR recombinant sequence − 27 bp – +1284 bp (the next base of the stop codon is +1) was significantly decreased in all three cell lines. The relative fluorescence intensities were significantly different between − 27 bp – +294 bp and − 27 bp – +497 bp regions, and between − 27 bp – +708 bp and − 27 bp – +907 bp regions. According to the prediction of the TargetScan database and analysis, miR-212-5p, miR-324-3p and miR-326 may bind to +295 bp – +497 bp, while miR-491-5p may bind to +798 bp – +907 bp. After co-transfection of miRNA mimic/inhibitor or mimic/inhibitor NC with a recombinant vector in the 3ʹUTR region of GRIN1 gene, we found that has-miR-491-5p inhibited GRIN1 expression significantly in all three cell lines, while has-miR-326 inhibitor upregulated GRIN1 expression in HEK-293 and U87 cells. Conclusion: miR-491-5p may bind to the 3ʹUTR of the GRIN1 gene (+799 bp – +805 bp, the next base of the stop codon is +1) and down-regulate gene expression in HEK-293, SK-N-SH, and U87 cell lines, which implicates a potential role of miR-491-5p in central nervous system diseases. [ABSTRACT FROM AUTHOR]

Sex differences play an important role in depression, the basis of which is an excessive stress response. We aimed at revealing the neurobiological sex differences in the same study in acute- and chronically-stressed rats. Female Sprague-Dawley (SD) rats were randomly divided into 6 groups: chronic unpredictable mild stress (CUMS), acute foot shock (FS) and controls, animals in all 3 groups were sacrificed in proestrus or diestrus. Male SD rats were randomly divided into 3 groups: CUMS, FS and controls. Comparisons were made of behavioral changes in CUMS and control rats, plasma levels of corticosterone (CORT), testosterone (T) and estradiol (E2), and of the hypothalamic mRNA-expression of stress-related molecules, i.e. estrogen receptor α and β, androgen receptor, aromatase, mineralocorticoid receptor, glucocorticoid receptor, corticotropin-releasing hormone, arginine vasopressin and oxytocin. CUMS resulted in disordered estrus cycles, more behavioral and hypothalamic stress-related molecules changes and a stronger CORT response in female rats compared with male rats. Female rats also showed decreased E2 and T levels after FS and CUMS, while male FS rats showed increased E2 and male CUMS rats showed decreased T levels. Stress affects the behavioral, endocrine and the molecular response of the stress systems in the hypothalamus of SD rats in a clear sexual dimorphic way, which has parallels in human data on stress and depression. [ABSTRACT FROM AUTHOR]

Background and aims: Whereas the expression patterns and kinetic properties of the rice ( Oryza sativa) phosphate transporter gene OsPht1; 6 ( OsPT6) are well documented, little is known about the biological functions of this gene. The aim of this study was to investigate the roles of OsPT6 in inorganic phosphate (Pi) acquisition and mobilization, and examine its potential to enhance agricultural production. Methods: Here, we generated OsPT6 overexpression transgenic plants using Wuyujing 7, a widely cultivated variety of japonica rice, and then treated transgenic lines and wild type with different Pi supply in hydroponic and soil experiments to explore the functions of OsPT6 in rice. Results: The OsPT6-overexpressing rice lines grew better and accumulated more biomass than wild-type plants, and exhibited significant increases in P accumulation in various tissues, including reproductive tissues under both hydroponic and soil culture conditions. Phosphate-uptake experiment using radiolabeled Pi (P) showed that the rate of Pi uptake was 75 % and 73 % greater in transgenic plants grown under Pi-sufficient and -deficient conditions, respectively, than the wild-type controls, and that the shoot/root ratio of P was 104 % and 42 % greater, respectively. In addition, the grain yield per transgenic plant was much higher than that of the wild-type plants under field conditions. Conclusions: Taken together, our results demonstrate that OsPT6 plays a vital role in Pi acquisition and mobilization in rice and suggest that this gene may be used for genetic engineering rice plants that require less Pi fertilizer. [ABSTRACT FROM AUTHOR]

Identifying protein complexes from the large-scale protein interaction network is crucial to understand principles of cellular organization and predict protein functions, which is one of the most important issues in the post-genomic era. Generally, the traditional protein complex discovery algorithms are only based on the protein-protein interaction network (PPI network), and are not so accurate. In this paper, a novel algorithm IPCIPG is proposed based on the integration of the PPI network and the gene expression data. Different from other previous methods which use gene expression data to evaluate the reliability of PPIs, IPCIPG integrates the gene expression data into PPI network during the identification of protein complexes. IPCIPG uses the edge clustering coefficient (ECC) and the co-expression correlation between proteins (PCC) to calculate the weight of each node in the PPI network. And then the node with the highest weight is selected as seed, then, a dense sub-graph will be obtained by extending from the seed. The experiment results on the data of Saccharomyces cerevisiae show that IPCIPG can identify the protein complexes with specific biological meaning more effectively, precisely and comprehensively than the other algorithms HUNTER, HC-PIN, CMC, SPICI, MOCDE, and MCL. [ABSTRACT FROM AUTHOR]

Motivation Clustered regularly interspaced short palindromic repeats (CRISPR)-based genetic perturbation screen is a powerful tool to probe gene function. However, experimental noises, especially for the lowly expressed genes, need to be accounted for to maintain proper control of false positive rate. Methods We develop a statistical method, named CRISPR screen with Expression Data Analysis (CEDA), to integrate gene expression profiles and CRISPR screen data for identifying essential genes. CEDA stratifies genes based on expression level and adopts a three-component mixture model for the log-fold change of single-guide RNAs (sgRNAs). Empirical Bayesian prior and expectation–maximization algorithm are used for parameter estimation and false discovery rate inference. Results Taking advantage of gene expression data, CEDA identifies essential genes with higher expression. Compared to existing methods, CEDA shows comparable reliability but higher sensitivity in detecting essential genes with moderate sgRNA fold change. Therefore, using the same CRISPR data, CEDA generates an additional hit gene list. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

• −337 to −159 bp and −704 to −556 bp of GRIN1 contain transcription repression regions. • −556 to −337 bp of GRIN1 enhanced gene expression in neuronal cell lines. • The proximal promoter region of GRIN1 may related to neuropsychiatric diseases. Abnormal expression of ionotropic glutamate receptor NMDA type subunit 1, the key subunit of the NMDA receptor, may be related to many neuropsychiatric disorders. In this study, we explored the functional sequence of the 5′ regulatory region of the human GRIN1 gene and discussed the transcription factors that may regulate gene expression. Twelve recombinant pGL3 vectors with gradually truncated fragment lengths were constructed, transfected into HEK-293, U87, and SK-N-SH cell lines, and analyzed through the luciferase reporter gene assay. JASPAR database is used to predict transcription factors. In SK-N-SH and U87 cell lines, regions from −337 to −159 bp, −704 to −556 bp inhibited gene expression, while −556 to −337 bp upregulated gene expression. In HEK-293 and U87 cell lines, the expression of fragment −1703 to + 188 bp was significantly increased compared to adjacent fragments −1539 to + 188 bp and −1843 to + 188 bp. The protein expressions of fragments −2162 to + 188 bp and −2025 to + 188 bp, −1539 to + 188 bp and −1215 to + 188 bp, −1215 to + 188 bp and −1066 to + 188 bp were significantly different in HEK-293 and SK-N-SH cells. According to the predictions of the JASPAR database, the transcription factors REST, EGR1, and CREB1/HIC2 may bind the DNA sequences of GRIN1 gene from the −337 to −159, −556 to −337, and −704 to −556, respectively. In addition, zinc finger transcription factors may regulate the expression of other differentially expressed fragments. Abnormal transcription regulation in the proximal promoter region of GRIN1 (-704 to + 188 bp) may be involved in the course of neuropsychiatric diseases. [ABSTRACT FROM AUTHOR]

Abstract: Antizyme 1 (AZ1) is a member of the antizyme family that is involved in many biological processes. As a natural inhibitor, AZ1 controls the normal level of polyamines, which is indispensable to cellular function. Our prior research showed that the expression of the AZ1 gene in Longissimus doris of Landrace pigs was higher (P <0.05) than in Lantang pigs. The AZ1 gene might be involved in the development of muscle and potentially serves as an important target for muscle improvement in pigs. In this study, the molecular characterization, tissue expression, and sequence variation of porcine AZ1 gene were analyzed. A 4082bp sequence including the 5′-flanking region and gene sequence was obtained through RACE and sequencing. The genomic sequence of AZ1 gene consists of six exons and five introns. The mRNA of AZ1 gene contains three elements: 5′-untranslated regions (UTR, 79bp), CDS (684 or 207bp), and 3′-UTR (161 or 423bp for the two transcripts). Three termination signals of AATAAA were found in the longer 3′-UTR. The mRNA sequence of the AZ1 gene contained two ORFs with a frameshifting site at the 69th codon. The amino acid sequence from the porcine AZ1 gene was similar to other vertebrates and exhibited the highest similarity to cattle. The partial 5′-flanking region was 852bp with typical cis-regulatory elements such as TATA boxes and CAAT boxes. By DNA pooling and sequencing, nine and 12 single nucleotide polymorphisms (SNPs) were detected in the 5′-flanking region and introns, respectively. Except the SNP at −713, the other eight SNPs were found in putative cis-acting elements and might alter the binding of trans-acting factors. Expression patterns showed that in Lantang pig, the AZ1 gene is differentially expressed in various tissues and displayed higher expression in lung and skeletal muscle. [Copyright &y& Elsevier]

Abstract: Adenosine triphosphate–sensitive potassium (KATP) channels are suggested to be involved in pathogenesis of neuropathic pain, but remain underinvestigated in primary afferents and in the spinal cord. We examined alterations of KATP channels in rat spinal cord and tested whether and how they could contribute to neuropathic pain. The results showed that protein expression for KATP channel subunits SUR1, SUR2, and Kir6.1, but not Kir6.2, were significantly downregulated and associated with thermal hyperalgesia and mechanical allodynia after sciatic nerve injury. Spinal administration of a KATP channel opener cromakalim (CRO, 5, 10, and 20μg, respectively) prevented or suppressed, in a dose-dependent manner, the hyperalgesia and allodynia. Nerve injury also significantly increased expression and phosphorylation of connexin 43, an astroglial gap junction protein. Such an increase of phosphorylation of connexin 43 was inhibited by CRO treatment. Furthermore, preadministration of an astroglial gap junction decoupler carbenoxolone (10μg) completely reversed the inhibitory effects of CRO treatment on the hyperalgesia and allodynia and phosphorylation of NR1 and NR2B receptors and the subsequent activation of Ca2+-dependent signals Ca2+/calmodulin-dependent kinase II and cyclic adenosine monophosphate (cAMP) response element binding protein. These findings suggest that nerve injury–induced downregulation of the KATP channels in the spinal cord may interrupt the astroglial gap junctional function and contribute to neuropathic pain, thus the KATP channels opener can reduce neuropathic pain probably partly via regulating the astroglial gap junctions. This study may provide a new strategy for treating neuropathic pain using KATP channel openers in the clinic. [Copyright &y& Elsevier]

Objective: Screening of feature genes involved in cytokine release syndrome (CRS) from the coronavirus disease 19 (COVID-19). Methods: The data sets related to COVID-19 were retrieved using Gene Expression Omnibus (GEO) database, the differentially expressed genes (DEGs) related to CRS were analyzed with R software and Venn diagram, and the biological processes and signaling pathways involved in DEGs were analyzed with GO and KEGG enrichment. Core genes were screened using Betweenness and MCC algorithms. GSE164805 and GSE171110 dataset were used to verify the expression level of core genes. Immunoinfiltration analysis was performed by ssGSEA algorithm in the GSVA package. The DrugBank database was used to analyze the feature genes for potential therapeutic drugs. Results: This study obtained 6950 DEGs, of which 971 corresponded with CRS disease genes (common genes). GO and KEGG enrichment showed that multiple biological processes and signaling pathways associated with common genes were closely related to the inflammatory response. Furthermore, the analysis revealed that transcription factors that regulate these common genes are also involved in inflammatory response. Betweenness and MCC algorithms were used for common gene screening, yielding seven key genes. GSE164805 and GSE171110 dataset validation revealed significant differences between the COVID-19 and normal controls in four core genes (feature genes), namely IL6R, TLR4, TLR2, and IFNG. The upregulated IL6R, TLR4, and TLR2 genes were mainly involved in the Toll-like receptor signaling pathway of the inflammatory pathway, while the downregulated IFNG genes primarily participated in the necroptosis and JAK-STAT signaling pathways. Moreover, immune infiltration analysis indicated that higher expression of these genes was associated with immune cell infiltration that mediates inflammatory response. In addition, potential therapeutic drugs for these four feature genes were identified via the DrugBank database. Conclusion: IL6R, TLR4, TLR2, and IFNG may be potential pathogenic genes and therapeutic targets for the CRS associated with COVID-19. [ABSTRACT FROM AUTHOR]

Background: The serotonin neurotransmitter (5-HT) and its receptors have important roles in neuropsychiatric disorders such as schizophrenia. The aim of this study was to investigate the functional sequences of the 5′ regulation region of the human HTR1A gene to explore the effects on the expression of the 5-HT1A receptor. Methods: Fourteen recombinant pGL3-basic vectors containing deletion fragments of the HTR1A gene regulatory region were transfected with HEK-293 and SK-N-SH cells. The relative chemiluminescence intensities of different length fragments were analyzed. The JASPAR software was used for the prediction of transcription factors. Results: In the HEK-293 cells, the relative chemiluminescence intensity of the − 1649 bp to − 1550 bp (ATG + 1) fragment was significantly different. Two inhibitory activity regions were found in the − 1409 bp to − 1381 bp and − 1196 bp to − 1124 bp fragments, which might be bound to the GATA or SOX10 transcription factors as predicted by the JASPAR software. In addition, the fragments located from − 1124 bp to − 1064 bp and from − 908 bp to − 722 bp up-regulated protein expression. Only the sequence from − 1550 bp to − 1409 bp demonstrated a difference in luciferase expression in the both cell lines. According to the results of the 5'-UTR truncated vectors, there was a repression region at the distal end of the 5'-UTR, an enhancer region might be present at the proximal end of the transcription start site. Conclusions: Although the functional sequences of the HTR1A gene regulatory region were confirmed, the regulatory factors and functional components require further investigation. [ABSTRACT FROM AUTHOR]

Background: Abnormal RGS4 gene expression may cause neurotransmitter disorders, resulting in schizophrenia. The association between RGS4 and the risk of schizophrenia is controversial, and there has been little research on the SNPs in the promoter region of RGS4. Purpose: The present study was performed to detect the association between SNPs in the promoter region of the RGS4 gene and the risk of schizophrenia. Materials and Methods: In this study, the 1757-bp fragment (− 1119–+600, TSS+1) of RGS4 was amplified and sequenced in 198 schizophrenia patients and 264 healthy controls of the northern Chinese Han population. Allele, genotype and haplotype frequencies were analyzed by chi‐square test. Results: Four SNPs were detected in the region. LD analysis determined that rs7515900 was linked to rs10917671 (D' = 1, r2 = 1). Therefore, the data for rs10917671 were eliminated from further analysis. Genotype TT of rs12041948 (P = 0.009, OR = 1.829, and 95% CI = 0.038– 0.766) was significantly different between the two groups in the northern Chinese Han population. In males, genotype GG of rs6678136 (P = 0.009, OR = 2.292, and 95% CI = 1.256– 4.18) and CC of rs7515900 (P = 0.003, OR = 2.523, and 95% CI = 1.332– 4.778) were significantly different. Conclusion: The results of this study suggested that genotype TT of rs12041948 in the pooled male and female samples and GG of rs6678136 and CC of rs7515900 in the male samples could be risk factors for schizophrenia. The present study is the first to detect an association between SNPs in the promoter region of the RGS4 gene and the risk of schizophrenia in the northern Chinese Han population. Functional studies are required to confirm these findings. [ABSTRACT FROM AUTHOR]

Immunotherapy has been found to be efficient in a variety of cancers and, therefore, may be a promising strategy for breast cancer (BC), particularly due to the limited therapeutic options currently available for triple-negative BC (TNBC). However, heterogeneity of tumor mutation burden (TMB), immune gene expression and mismatch repair (MMR) gene activity across BC subtypes has not been well characterized. In the present study, a comprehensive analysis of TMB, expression levels of immune cell type marker genes, and expression levels of MMR-associated genes was performed. A total of 5 MMR-associated genes, including MLH1, MLH3, MSH2, MSH6 and PMS2, were analyzed. Patients that harbored at least two MMR genes with expression levels in the lower 10% percentile across the cohort were considered as potentially aberrant (lost expression). Hormone receptor (HR)-negative BC is associated with a higher TMB and immune gene expression compared with HR-positive BC [TMB, estrogen receptor (ER)-negative vs. ER-negative, 55 vs. 32, respectively; P=4.1×10−13; progesterone receptor (PR)-negative vs. PR-positive, 53 vs. 31, respectively; P<2.2×10−16]. By contrast, human epidermal growth factor receptor 2 (HER2)-negative BC tended to have a lower TMB and decreased immune gene expression compared with HER2-positive BC (TMB, HER2-negative vs. HER2-positive, 36 vs. 48, respectively; P=0.02). Furthermore, aberrant expression of MMR genes was found to be more common in HR-negative compared with HR-positive BC (P<0.001). Significantly higher expression levels of each immune marker gene of four major immune cell types were found in patients who were HR-negative compared with patients who were HR-positive (P<0.001). The findings of the present study suggest that HR-negative or HER2-positive BC exhibits elevated TMB and immunogenic activity, and immunotherapeutic options are recommended. [ABSTRACT FROM AUTHOR]

Background: Tumour immune microenvironment heterogeneity is prevalent in numerous cancers and can negatively impact immunotherapy response. Immune heterogeneity and evolution in gastroesophageal adenocarcinoma (GEA) have not been studied in the past. Methods: Together with a multi-region sampling of normal, primary and metastatic tissues, we performed whole exome sequencing, TCR sequencing as well as immune cell infiltration estimation through deconvolution of gene expression signals. Results: We discovered high TCR repertoire and immune cell infiltration heterogeneity among metastatic sites, while they were homogeneous among primary and normal samples. Metastatic sites shared high levels of abundant TCR clonotypes with blood, indicating immune surveillance via blood. Metastatic sites also had low levels of tumour-eliminating immune cells and were undergoing heavy immunomodulation compared to normal and primary tumour tissues. There was co-evolution of neo-antigen and TCR repertoire, but only in patients with late diverging mutational evolution. Co-evolution of TCR repertoire and immune cell infiltration was seen in all except one patient. Conclusions: Our findings revealed immune heterogeneity and co-evolution in GEA, which may inform immunotherapy decision-making. [ABSTRACT FROM AUTHOR]

Cell-free DNA (cfDNA) is short, extracellular, fragmented double-stranded DNA found in plasma. Plasma of patients with solid tumor has been found to show significantly increased quantities of cfDNA. Although currently poorly understood, the mechanism of cfDNA generation is speculated to be a product of genomic DNA fragmentation during cellular apoptosis and necrosis. Sequencing of cfDNA with tumor origin has identified tumor biomarkers, elucidating molecular pathology and assisting in accurate diagnosis. In this study, we performed whole-genome sequencing ofcfDNA samples with matching tumor and whole blood samples from five patients diagnosed with stage IV gastric or lung cancer. We analyzed the coverage spectrum of the human genome in our cfDNA samples. cfDNA exhibited no large regions with significant under-coverage, although we observed unbalanced coverage depth in cfDNA at transcription start sites and exon boundaries as a consequence of biased fragmentation due to ordered nucleosome positioning. We also analyzed the copy number variant status based on the whole-genome sequencing results and found high similarity between copy number profile constructed from tumor samples and cfDNA samples. Overall, we conclude that cfDNA comprises a good representation of the tumor genome in late stage gastric and lung cancer. [ABSTRACT FROM AUTHOR]

Simple Summary: Bladder cancer ranks fourth among the most prevalent cancers in men and is the most expensive cancer to treat on a per-patient basis. The muscle-invasive form of bladder cancer is one of the deadliest cancers, with a 5-year survival rate of only 6% in patients with distant metastasis. Effective therapeutic options remain few and far between. SNHG1 is a long non-coding RNA that is over-expressed in 95% of muscle-invasive bladder cancers. However, very little information is available about the role and mechanisms of SNHG1 in bladder tumor formation and progression. Here, we provide experimental evidence establishing that SNHG1 drives bladder cancer cell invasion and stem-cell-like behaviors through a specific signaling pathway. Our results reveal novel biomarkers predictive of the progression of muscle-invasive bladder cancer and potential new targets for therapeutic intervention. The stem-cell-like behavior of cancer cells plays a central role in tumor heterogeneity and invasion and correlates closely with drug resistance and unfavorable clinical outcomes. However, the molecular underpinnings of cancer cell stemness remain incompletely defined. Here, we show that SNHG1, a long non-coding RNA that is over-expressed in ~95% of human muscle-invasive bladder cancers (MIBCs), induces stem-cell-like sphere formation and the invasion of cultured bladder cancer cells by upregulating Rho GTPase, Rac1. We further show that SNHG1 binds to DNA methylation transferase 3A protein (DNMT3A), and tethers DNMT3A to the promoter of miR-129-2, thus hyper-methylating and repressing miR-129-2-5p transcription. The reduced binding of miR-129-2 to the 3′-UTR of Rac1 mRNA leads to the stabilization of Rac1 mRNA and increased levels of Rac1 protein, which then stimulates MIBC cell sphere formation and invasion. Analysis of the Human Protein Atlas shows that a high expression of Rac1 is strongly associated with poor survival in patients with MIBC. Our data strongly suggest that the SNHG1/DNMT3A/miR-129-2-5p/Rac1 effector pathway drives stem-cell-like and invasive behaviors in MIBC, a deadly form of bladder cancer. Targeting this pathway, alone or in combination with platinum-based therapy, may reduce chemoresistance and improve longer-term outcomes in MIBC patients. [ABSTRACT FROM AUTHOR]

Background: Cryptosporidium is an important zoonotic pathogen responsible for severe enteric diseases in humans and animals. However, the molecular mechanisms underlying host and Cryptosporidium interactions are still not clear. Methods: To study the roles of circRNAs in host cells during Cryptosporidium infection, the expression profiles of circRNAs in HCT-8 cells infected with C. parvum were investigated using a microarray assay, and the regulatory role of a significantly upregulated circRNA, ciRS-7, was investigated during C. parvum infection. Results: C. parvum infection caused notable alterations in the expression profiles of circRNAs in HCT-8 cells, and a total of 178 (including 128 up- and 50 downregulated) circRNAs were significantly differentially expressed following C. parvum infection. Among them, ciRS-7 was significantly upregulated and regulated the NF-κB signaling pathway by sponging miR-1270 during C. parvum infection. Furthermore, the ciRS-7/miR-1270/relA axis markedly affected the propagation of C. parvum in HCT-8 cells. Conclusions: Our results revealed that ciRS-7 would promote C. parvum propagation by regulating the miR-1270/relA axis and affecting the NF-κB pathway. To the best of our knowledge, this is the first study to investigate the role of circRNA during Cryptosporidium infection, and the findings provide a novel view for implementing control strategies against Cryptosporidium infection. [ABSTRACT FROM AUTHOR]

Background: The 5-hydroxytryptamine 1B receptor (5-HT1B) plays an essential role in the serotonin (5-HT) system and is widely involved in a variety of brain activities. HTR1B is the gene encoding 5-HT1B. Genome-wide association studies have shown that HTR1B polymorphisms are closely related to multiple mental and behavioral disorders; however, the functional mechanisms underlying these associations are unknown. This study investigated the effect of several HTR1B haplotypes on regulation of gene expression in vitro and the functional sequences in the 5′ regulatory region of HTR1B to determine their potential association with mental and behavioral disorders. Methods: Six haplotypes consisting of rs4140535, rs1778258, rs17273700, rs1228814, rs11568817, and rs130058 and several truncated fragments of the 5′ regulatory region of HTR1B were transfected into SK-N-SH and HEK-293 cells. The relative fluorescence intensities of the different haplotypes and truncated fragments were detected using a dual-luciferase reporter assay system. Results: Compared to the major haplotype T-G-T-C-T-A, the relative fluorescence intensities of haplotypes C-A-T-C-T-A, C-G-T-C-T-A, C-G-C-A-G-T, and C-G-T-A-T-A were significantly lower, and that of haplotype C-G-C-A-G-A was significantly higher. Furthermore, the effects of the rs4140535T allele, the rs17273700C-rs11568817G linkage combination, and the rs1228814A allele made their relative fluorescence intensities significantly higher than their counterparts at each locus. Conversely, the rs1778258A and rs130058T alleles decreased the relative fluorescence intensities. In addition, we found that regions from − 1587 to − 1371 bp (TSS, + 1), − 1149 to − 894 bp, − 39 to + 130 bp, + 130 to + 341 bp, and + 341 to + 505 bp upregulated gene expression. In contrast, regions − 603 to − 316 bp and + 130 to + 341 bp downregulated gene expression. Region + 341 to + 505 bp played a decisive role in gene transcription. Conclusions: HTR1B 5′ regulatory region polymorphisms have regulatory effects on gene expression and potential correlate with several pathology and physiology conditions. This study suggests that a crucial sequence for transcription is located in region + 341 ~ + 505 bp. Regions − 1587 to − 1371 bp, − 1149 to − 894 bp, − 603 to − 316 bp, − 39 to + 130 bp, and + 130 to + 341 bp contain functional sequences that can promote or suppress the HTR1B gene expression. [ABSTRACT FROM AUTHOR]

In the prediction of the synergy of drug combinations, systems pharmacology models expand the scope of experiment screening and overcome the limitations of current computational models posed by their lack of mechanical interpretation and integration of gene essentiality. We therefore investigated the synergy of drug combinations for cancer therapies utilizing records in NCI ALMANAC, and we employed logistic regression to test the statistical significance of gene and pathway features in that interaction. We trained our predictive models using 43 NCI-60 cell lines, 165 KEGG pathways, and 114 drug pairs. Scores of drug-combination synergies showed a stronger correlation with pathway than gene features in overall trend analysis and a significant association with both genes and pathways in genome-wide association analyses. However, we observed little overlap of significant gene expressions and essentialities and no significant evidence that associated target and non-target genes and their pathways. We were able to validate four drug-combination pathways between two drug combinations, Nelarabine-Exemestane and Docetaxel-Vermurafenib, and two signaling pathways, PI3K-AKT and AMPK, in 16 cell lines. In conclusion, pathways significantly outperformed genes in predicting drug-combination synergy, and because they have very different mechanisms, gene expression and essentiality should be considered in combination rather than individually to improve this prediction. [ABSTRACT FROM AUTHOR]

Background: The HTR1B gene encodes the 5-hydroxytryptamine (5-HT1B) receptor, which is involved in a variety of brain activities and mental disorders. The regulatory effects of non-coding regions on genomic DNA are one of many reasons for the cause of genetic-related diseases. Post-transcriptional regulation that depends on the function of 3′ regulatory regions plays a particularly important role. This study investigated the effects, on reporter gene expression, of several haplotypes of the HTR1B gene (rs6297, rs3827804, rs140792648, rs9361234, rs76194807, rs58138557, and rs13212041) and truncated fragments in order to analyze the function of the 3′ region of HTR1B. Results: We found that the haplotype, A-G-Del-C-T-Ins-A, enhanced the expression level compared to the main haplotype; A-G-Del-C-G-Ins-A; G-G-Del-C-G-Ins-G decreased the expression level. Two alleles, rs76194807T and rs6297G, exhibited different relative luciferase intensities compared to their counterparts at each locus. We also found that + 2440 ~ + 2769 bp and + 1953 ~ + 2311 bp regions both had negative effects on gene expression. Conclusions: The 3′ region of HTR1B has a regulatory effect on gene expression, which is likely closely associated with the interpretation of HTR1B-related disorders. In addition, the HTR1B 3′ region includes several effector binding sites that induce an inhibitory effect on gene expression. [ABSTRACT FROM AUTHOR]

Polysaccharide from Ma-chi-xian (Portulacae oleracea L., POLP) was prepared and the therapeutic effect on dextran sodium sulfate-induced colitis mice was investigated in this study. The results of clinical activity score and H&E staining confirmed the therapeutic effect of POLP. POLP could diminished the symptoms of colitis and improve colon histopathological structure of the colitis mice. The expression levels of four cytokines were determined. The concentrations of PGE2 and IL-6 were downregulated by POLP treatment. The COX-2 protein expression levels and the STAT3 phosphorylation levels were detected. The results showed that these two protein levels were all increased in colitis and decreased after POLP treatment, indicating that these two proteins were closely related with the protective effect of POLP. Because the synthesis of PGE2 is catalyzed by COX-2 and phosphorylation of STAT3 can induce the expression of COX-2, it was concluded that STAT3 was a key protein related to the POLP exerting its activity in colitis. [ABSTRACT FROM AUTHOR]

Postoperative pain is a common form of acute pain that, if not managed effectively, can become chronic pain. Evidence has shown that glia, especially microglia, mediate neuroinflammation, which plays a vital role in pain sensitization. Moreover, toll-like receptor 4 (TLR4), the tumor necrosis factor receptor (TNF-R), the interleukin-1 receptor (IL-1R), and the interleukin-6 receptor (IL-6R) have been considered key components in central pain sensitization and neuroinflammation. Therefore, we hypothesized that activation of the body’s endogenous “immune brakes” will inhibit these receptors and achieve inflammation tolerance as well as relieve postoperative pain. After searching for potential candidates to serve as this immune brake, we identified and focused on the suppressor of cytokine signaling 3 (SOCS3) gene. To regulate SOCS3 expression, we used paeoniflorin to induce heat shock protein 70 (HSP70)/TLR4 signaling. We found that paeoniflorin significantly induced SOCS3 expression both in vitro and in vivo and promoted the efflux of HSP70 from the cytoplasm to the extracellular environment. Furthermore, paeoniflorin markedly attenuated incision-induced mechanical allodynia, and this effect was abolished by small interfering RNAs targeting SOCS3. These findings demonstrated an effective and safe strategy to alleviate postoperative pain. [ABSTRACT FROM AUTHOR]

Polyploidy is common in Saccharomyces cerevisiae strains, but the physiological and phenotypic effects of ploidy changes have not been fully clarified. Here, isogenic diploid, triploid, and tetraploid S. cerevisiae strains were constructed from a haploid strain, CEN.PK2-1C. Stress tolerance and ethanol fermentation performance of the four euploid strains were compared. Each euploid strain had strengths and weaknesses in tolerance to certain stressors, and no single strain was tolerant of all stressors. The diploid had higher ethanol production than the other strains in normal fermentation medium, while the triploid strain showed the fastest fermentation rate in the presence of inhibitors found in lignocellulosic hydrolysate. Physiological determination revealed diverse physiological attributes, such as trehalose, ergosterol, glutathione, and anti-oxidative enzymes among the strains. Our analyses suggest that both ploidy parity and number of chromosome sets contribute to changes in physiological status. Using qRT-PCR, different expression patterns of genes involved in the regulation of cell morphology and the biosynthesis of key physiological attributes among strains were determined. Our data provide novel insights into the multiple effects of genome duplication on yeast cells and are a useful reference for breeding excellent strains used in specific industrial applications. [ABSTRACT FROM AUTHOR]

The limited cardiomyocyte proliferation is insufficient for repair of the myocardium. Therefore, activating cardiomyocyte proliferation might be a reasonable option for myocardial regeneration. Here, we investigated effect of retinoic acid (RA) on inducing adult cardiomyocyte proliferation and assessed efficacy of self-assembling peptide (SAP)-released RA in activating regeneration of the infarcted myocardium. Effect of RA on inducing cardiomyocyte proliferation was examined with the isolated cardiomyocytes. Expression of the cell cycle-associated genes and paracrine factors in the infarcted myocardium was examined at one week after treatment with SAP-carried RA. Cardiomyocyte proliferation, myocardial regeneration and improvement of cardiac function were assessed at four weeks after treatment. In the adult rat myocardium, expression of RA synthetase gene Raldh2 and RA concentration were decreased significantly. After treatment with RA, the proliferated cardiomyocytes were increased. The formulated SAP could sustainedly release RA. After treatment with SAP-carried RA, expression of the pro-proliferative genes in cell cycle and paracrine factors in the infarcted myocardium were up-regulated. Myocardial regeneration was enhanced, and cardiac function was improved significantly. These results demonstrate that RA can induce adult cardiomyocytes to proliferate effectively. The sustained release of RA with SAP is a promise strategy to enhance repair of the infarcted myocardium. [Display omitted] • Synthesis of retinoic acid in adult myocardium is decreased. • Retinoic acid can induce proliferation of adult cardiomyocytes. • Sustained release of retinoic acid is effective for repair of the infarcted myocardium. [ABSTRACT FROM AUTHOR]

Hypertrophic cardiomyopathy (HCM) is a leading cause of sudden death in children and young adults. Abnormalities in several signaling pathways are implicated in the pathogenesis of HCM, but the role of the RAS-RAF-MEK-ERK MAPK pathway has been controversial. Noonan syndrome (NS) is one of several autosomal-dominant conditions known as RASopathies, which are caused by mutations in different components of this pathway. Germline mutations in RAF1 (which encodes the serine-threonine kinase RAF1) account for approximately 3%-5% of cases of NS. Unlike other NS alleles, RAF1 mutations that confer increased kinase activity are highly associated with HCM. To explore the pathogenesis of such mutations, we generated knockin mice expressing the NS-associated Raf1(L613V) mutation. Like NS patients, mice heterozygous for this mutation (referred to herein as L613V/+ mice) had short stature, craniofacial dysmorphia, and hematologic abnormalities. Valvuloseptal development was normal, but L613V/+ mice exhibited eccentric cardiac hypertrophy and aberrant cardiac fetal gene expression, and decompensated following pressure overload. Agonist-evoked MEK-ERK activation was enhanced in multiple cell types, and postnatal MEK inhibition normalized the growth, facial, and cardiac defects in L613V/+ mice. These data show that different NS genes have intrinsically distinct pathological effects, demonstrate that enhanced MEK-ERK activity is critical for causing HCM and other RAF1-mutant NS phenotypes, and suggest a mutation-specific approach to the treatment of RASopathies. [ABSTRACT FROM AUTHOR]