Incomplete transcripts dominate the Mycobacterium tuberculosis transcriptome.

Mycobacterium tuberculosis (Mtb) is a bacterial pathogen that causes tuberculosis (TB), an infectious disease that is responsible for major health and economic costs worldwide1. Mtb encounters diverse environments during its life cycle and responds to these changes largely by reprogramming its transcriptional output2. However, the mechanisms of Mtb transcription and how they are regulated remain poorly understood. Here we use a sequencing method that simultaneously determines both termini of individual RNA molecules in bacterial cells3 to profile the Mtb transcriptome at high resolution. Unexpectedly, we find that most Mtb transcripts are incomplete, with their 5' ends aligned at transcription start sites and 3' ends located 200-500 nucleotides downstream. We show that these short RNAs are mainly associated with paused RNA polymerases (RNAPs) rather than being products of premature termination. We further show that the high propensity of Mtb RNAP to pause early in transcription relies on the binding of the σ-factor. Finally, we show that a translating ribosome promotes transcription elongation, revealing a potential role for transcription-translation coupling in controlling Mtb gene expression. In sum, our findings depict a mycobacterial transcriptome that prominently features incomplete transcripts resulting from RNAP pausing. We propose that the pausing phase constitutes an important transcriptional checkpoint in Mtb that allows the bacterium to adapt to environmental changes and could be exploited for TB therapeutics.

mRNA recognition and packaging by the human transcription-export complex.

Newly made mRNAs are processed and packaged into mature ribonucleoprotein complexes (mRNPs) and are recognized by the essential transcription-export complex (TREX) for nuclear export1,2. However, the mechanisms of mRNP recognition and three-dimensional mRNP organization are poorly understood3. Here we report cryo-electron microscopy and tomography structures of reconstituted and endogenous human mRNPs bound to the 2-MDa TREX complex. We show that mRNPs are recognized through multivalent interactions between the TREX subunit ALYREF and mRNP-bound exon junction complexes. Exon junction complexes can multimerize through ALYREF, which suggests a mechanism for mRNP organization. Endogenous mRNPs form compact globules that are coated by multiple TREX complexes. These results reveal how TREX may simultaneously recognize, compact and protect mRNAs to promote their packaging for nuclear export. The organization of mRNP globules provides a framework to understand how mRNP architecture facilitates mRNA biogenesis and export.

MYC multimers shield stalled replication forks from RNA polymerase.

Oncoproteins of the MYC family drive the development of numerous human tumours1. In unperturbed cells, MYC proteins bind to nearly all active promoters and control transcription by RNA polymerase II2,3. MYC proteins can also coordinate transcription with DNA replication4,5 and promote the repair of transcription-associated DNA damage6, but how they exert these mechanistically diverse functions is unknown. Here we show that MYC dissociates from many of its binding sites in active promoters and forms multimeric, often sphere-like structures in response to perturbation of transcription elongation, mRNA splicing or inhibition of the proteasome. Multimerization is accompanied by a global change in the MYC interactome towards proteins involved in transcription termination and RNA processing. MYC multimers accumulate on chromatin immediately adjacent to stalled replication forks and surround FANCD2, ATR and BRCA1 proteins, which are located at stalled forks7,8. MYC multimerization is triggered in a HUWE16 and ubiquitylation-dependent manner. At active promoters, MYC multimers block antisense transcription and stabilize FANCD2 association with chromatin. This limits DNA double strand break formation during S-phase, suggesting that the multimerization of MYC enables tumour cells to proliferate under stressful conditions.

Baseline and post-treatment biomarkers of resistance to anti-PD-1 therapy in acral and mucosal melanoma: an observational study.

BACKGROUND: Immunotherapies targeting programmed cell death-1 (PD-1) and its ligands have improved clinical outcomes for advanced melanoma. However, many tumors exhibit primary resistance or acquire secondary resistance after an initial positive response. The mechanisms of resistance are not well understood, and no validated predictive biomarkers are available. This exploratory study aimed to characterize baseline differences and molecular changes arising during treatment in acral and mucosal melanomas that exhibited primary or secondary resistance to anti-PD-1 monotherapy. METHODS: This was an observational retrospective study of 124 patients who had been treated for metastatic acral or mucosal melanoma with anti-PD-1 monotherapy. Tumor samples were collected at baseline (all patients) and post-treatment (resistant tumors only) and were assayed by immunohistochemistry, whole-exome sequencing, and RNA sequencing. RESULTS: At baseline, more non-progressor than resistant tumors exhibited expression of PD-L1, immune cell infiltration, and high tumor mutational burden (TMB); baseline PD-L1 expression was also more common in secondary-resistant than in primary-resistant tumors as well as in late versus early secondary-resistant tumors. Non-progressor tumors also had higher median baseline expression of an 18-gene T cell-inflamed gene expression profile (TcellinfGEP). Among resistant tumors, the proportion of PD-L1-positive melanomas and the expression of the TcellinfGEP mRNA signature increased during treatment, while the expression of mRNA signatures related to WNT and INFA1 signaling decreased. There was evidence for greater changes from baseline in secondary-resistant versus primary-resistant tumors for some markers, including expression of RAS-related and WNT-related mRNA signatures and density of CD11c+ and FOXP3+ T cells. Greater changes in CD11c+ cell density were observed in early compared with late secondary-resistant tumors. CONCLUSIONS: Our findings suggest that TcellinfGEP and PD-L1 expression, TMB, immune cell infiltration, and RAS and WNT signaling warrant further investigation as potential mechanisms and/or biomarkers of anti-PD-1 therapy resistance in acral and mucosal melanomas. Confirmation of these findings in larger populations is needed.

Real-time visualization of mRNA synthesis during memory formation in live mice.

Memories are thought to be encoded in populations of neurons called memory trace or engram cells. However, little is known about the dynamics of these cells because of the difficulty in real-time monitoring of them over long periods of time in vivo. To overcome this limitation, we present a genetically encoded RNA indicator (GERI) mouse for intravital chronic imaging of endogenous Arc messenger RNA (mRNA)-a popular marker for memory trace cells. We used our GERI to identify Arc-positive neurons in real time without the delay associated with reporter protein expression in conventional approaches. We found that the Arc-positive neuronal populations rapidly turned over within 2 d in the hippocampal CA1 region, whereas ∼4% of neurons in the retrosplenial cortex consistently expressed Arc following contextual fear conditioning and repeated memory retrievals. Dual imaging of GERI and a calcium indicator in CA1 of mice navigating a virtual reality environment revealed that only the population of neurons expressing Arc during both encoding and retrieval exhibited relatively high calcium activity in a context-specific manner. This in vivo RNA-imaging approach opens the possibility of unraveling the dynamics of the neuronal population underlying various learning and memory processes.

Synonymous mutations in representative yeast genes are mostly strongly non-neutral.

Synonymous mutations in protein-coding genes do not alter protein sequences and are thus generally presumed to be neutral or nearly neutral1-5. Here, to experimentally verify this presumption, we constructed 8,341 yeast mutants each carrying a synonymous, nonsynonymous or nonsense mutation in one of 21 endogenous genes with diverse functions and expression levels and measured their fitness relative to the wild type in a rich medium. Three-quarters of synonymous mutations resulted in a significant reduction in fitness, and the distribution of fitness effects was overall similar-albeit nonidentical-between synonymous and nonsynonymous mutations. Both synonymous and nonsynonymous mutations frequently disturbed the level of mRNA expression of the mutated gene, and the extent of the disturbance partially predicted the fitness effect. Investigations in additional environments revealed greater across-environment fitness variations for nonsynonymous mutants than for synonymous mutants despite their similar fitness distributions in each environment, suggesting that a smaller proportion of nonsynonymous mutants than synonymous mutants are always non-deleterious in a changing environment to permit fixation, potentially explaining the common observation of substantially lower nonsynonymous than synonymous substitution rates. The strong non-neutrality of most synonymous mutations, if it holds true for other genes and in other organisms, would require re-examination of numerous biological conclusions about mutation, selection, effective population size, divergence time and disease mechanisms that rely on the assumption that synoymous mutations are neutral.

Concomitant Expression of IL-6 and TGF-ß Cytokines and their Receptors in Peripheral Blood of Patients with Multiple Sclerosis: The Effects of INFß Drugs.

BACKGROUND: Concomitant signals from IL-6 and TGF-ß have a central role in the Th17 cells development and differentiation, and these cells are the main promoters of demyelinating inflammation in the central nervous system (CNS) resulting in multiple sclerosis (MS). OBJECTIVES: To evaluate the simultaneous IL-6 and TGF-ß gene and their receptor protein expression in patients with Relapsing-Remitting (RR)-MS. MATERIALS AND METHODS: IL-6 and TGF-ß mRNA and their receptor expression on the surface of CD4+T cells were evaluated using real-time PCR (RT-PCR) and flow cytometry, respectively. RESULTS: The IL-6 mRNA expression in patients with RRMS was significantly higher than in the controls (p= 0.019). When patients who did not receive any other treatment were compared with the controls, the significant difference was substantial (p=0.006). The TGF-ß mRNA expression in patients was lower than in the controls (p = 0.03). However, in patients receiving IFNß, it increased compared with the other patients (p= 0.036). There was no difference in cytokine receptor expression between patients and the control group. CONCLUSION: Our data conclude an increase and decrease in mRNA expression levels of IL-6 and TGF-ß in patients with RRMS, respectively. Moreover, there were no significant differences in receptor expression of either cytokines. Based on our data the balance of TGF and IL-6 appears to have a positive impact on the disease control.

Effect of 17ß-estradiol on the daily pattern of ACE2, ADAM17, TMPRSS2 and estradiol receptor transcription in the lungs and colon of male rats.

Covid-19 progression shows sex-dependent features. It is hypothesized that a better Covid-19 survival rate in females can be attributed to the presence of higher 17ß-estradiol (E2) levels in women than in men. Virus SARS-CoV-2 is enabled to enter the cell with the use of angiotensin converting enzyme 2 (ACE2). The expression of several renin-angiotensin system components has been shown to exert a rhythmic pattern, and a role of the circadian system in their regulation has been implicated. Therefore, the aim of the study is to elucidate possible interference between E2 signalling and the circadian system in the regulation of the expression of ACE2 mRNA and functionally related molecules. E2 was administered at a dosage of 40 µg/kg/day for 7 days to male Wistar rats, and sampling of the lungs and colon was performed during a 24-h cycle. The daily pattern of expression of molecules facilitating SARS-CoV-2 entry into the cell, clock genes and E2 receptors was analysed. As a consequence of E2 administration, a rhythm in ACE2 and TMPRSS2 mRNA expression was observed in the lungs but not in the colon. ADAM17 mRNA expression showed a pronounced rhythmic pattern in both tissues that was not influenced by E2 treatment. ESR1 mRNA expression exerted a rhythmic pattern, which was diminished by E2 treatment. The influence of E2 administration on ESR2 and GPER1 mRNA expression was greater in the lungs than in the colon as a significant rhythm in ESR2 and GPER1 mRNA expression appeared only in the lungs after E2 treatment. E2 administration also increased the amplitude of bmal1 expression in the lungs, which implicates altered functioning of peripheral oscillators in response to E2 treatment. The daily pattern of components of the SARS-CoV-2 entrance pathway and their responsiveness to E2 should be considered in the timing of pharmacological therapy for Covid-19.

Novel Benzoxazoles Containing 4-Amino-Butanamide Moiety Inhibited LPS-Induced Inflammation by Modulating IL-6 or IL-1ß mRNA Expression.

LPS induces inflammatory cytokines, including IL-1ß, IL-6, and TNF-α, and causes an inflammatory response. The development of small molecules that have suppressive effect on those inflammatory cytokines is a desirable strategy for the treatment of inflammatory diseases. We synthesized 12 novel compounds with 4-amino-N-(4-(benzo[d]oxazol-2-ylamino)phenyl)butanamide moiety and evaluated their biological activities. Among them, 4 compounds (compound 5d, 5c, 5f, 5m and synthetic intermediate 4d) showed potent inhibition activities on IL-1ß and IL-6 mRNA expression in vitro. Further, in vivo activity was evaluated with two compounds (5f and 4d) and mRNA levels of IL-1ß, IL-6, and TNF-α were significantly decreased without hepatotoxicity. From the in vivo and in vitro test results, we confirmed that our synthesized compounds are effective for suppression of representative inflammatory cytokines.

The Four mRNA Bases Have Quite Different (Un)folding Free Energies, Applications to RNA Splicing and Translation Initiation with BindOligoNet.

Expression of mRNA is often regulated by the binding of a small RNA (miRNA, snoRNA, siRNA). While the pairing contribution to the net free energy is well parameterized and can be computed in O(N) time, the cost of removing pre-existing mRNA secondary structure has not received sufficient attention. Conventional methods for computing the unfolding free energy of a target mRNA are costly, scaling like the cube of the number of target bases O(N3). Here we introduce a model to describe the unfolding costs of the binding site, which features surprisingly big differences in the free energy parameters for the four bases. The model is implemented in our O(N) algorithm, BindOligoNet. Donor splice site prediction is more accurate when using our calculation of spliceosomal U1-snRNA to mRNA net binding free energy. Our base-dependent free energies also correlate with efficient ribosome docking near the start codon.

DDX3X mRNA Expression in T Cells Is Associated with the Severity and Mortality of Septic Patients.

PURPOSE: DDX3X acts as the critical checkpoint of death in stressed cells. The purpose of this study was to evaluate the mRNA expression level of DDX3X in T cells in peripheral blood of patients with sepsis and to explore its correlation with the prognosis of sepsis. METHODS: Seventy-nine patients with traumatic sepsis were enrolled in this prospective cohort study. Blood samples were collected within 24 hours after the diagnosis of sepsis or septic shock, and the mRNA expression level of DDX3X in T cells was detected by PCR. RESULTS: The level of DDX3X mRNA in T cells was significantly increased in septic patients as well as in septic shock patients. The level of DDX3X mRNA was negatively correlated with T cell count and positively correlated with acute physiological and chronic health assessment (APACHE) score and sequential organ failure assessment (SOFA) score (P < 0.01). The area under the curve (AUC) of the receiver operating characteristic (ROC) curve was 0.79 (95% confidence interval (CI), 0.68-0.90). A Cox proportional hazard model identified an association between an increased DDX3X mRNA level (≥1.575) and the risk of 28-day mortality (hazard ratio = 9.540, 95% CI, 2.452-37.108). CONCLUSIONS: High level of DDX3X mRNA in T cells in sepsis is associated with the severity of sepsis and the mortality of patients with sepsis.

IRF6 rs2235371 as a risk factor for non-syndromic cleft palate only among the Deutero-Malay race in Indonesia and its effect on the IRF6 mRNA expression level.

BACKGROUND: During the early embryological development of the face, complex orofacial failure results in a non-syndromic cleft lip and palate (NS CLP). The interferon regulatory factor 6 gene (IRF6) rs2235371 is a non-synonymous polymorphism that is one of the strong candidate genes associated with NS CLP. OBJECTIVES: The purpose of this study was to determine IRF6 rs2235371 as a risk factor for NS CLP and its phenotypes, including complete unilateral cleft lip and palate (CUCLP), bilateral cleft lip and palate (BCLP), cleft lip only (CL), and cleft palate only (CP), as well as to examine the effect of the polymorphism on the IRF6 mRNA expression levels among the Deutero-Malay race in Indonesia. MATERIAL AND METHODS: This study used a case-control design and enrolled 264 samples, including 158 NS CLP cases (42 NS CUCLP, 34 NS BCLP, 33 NS CL, and 49 NS CP) and 106 control subjects. DNA was extracted from venous blood, and then subjected to polymerase chain reaction (PCR) and sequencing. The odds ratio (OR) was used to determine the risk factor for NS CLP and its phenotypes. The Livak, KruskalWallis and Mann-Whitney U tests were used to determine mRNA expression levels in the oral epithelium, followed by real-time quantitative PCR (RT-qPCR). RESULTS: Among all of the NS CLP cases, in the NS CP phenotype, OR for the A mutant allele and the GA genotype was 2,492 (p = 0.017) and 2,114 (p = 0.048), respectively. The IRF6 mRNA expression level of the GA genotype was higher in the NS CP subjects as compared to the GG genotype (p = 0.031). CONCLUSIONS: The IRF6 rs2235371 polymorphism is associated with the NS CP phenotype in DeuteroMalay patients from Indonesia and it affects the IRF6 mRNA expression level.

Turmeric Extract (Curcuma longa) Mediates Anti-Oxidative Effects by Reduction of Nitric Oxide, iNOS Protein-, and mRNA-Synthesis in BV2 Microglial Cells.

Plant-derived products have been used since the beginnings of human history to treat various pathological conditions. Practical experience as well as a growing body of research suggests the benefits of the use of turmeric (Curcuma longa) and some of its active components in the reduction of oxidative stress, a mechanism leading to neurodegeneration. In this current study, we investigated the effects of a preparation of Curcuma longa, and its constituents curcumin, tetrahydrocurcumin, and curcumenol, in one of the molecular pathways leading to oxidative stress, which is the release of NO, a free radical involved in stress conditions, using the BV2 microglial cell line. The concentration-dependent reduction of NO is linked to reduced amounts of iNOS protein- and mRNA-synthesis and is possibly mediated by the phosphorylation of mitogen-activated protein kinases (MAPK) such as p42/44 or p38 MAPK. Therefore, the use of turmeric extract is a promising therapeutic option for diseases linked to the dysregulation of oxidative stress, with fewer side-effects in comparison to the currently used pharmacotherapeutics.

Fewer LAG-3+ T Cells in Relapsing-Remitting Multiple Sclerosis and Type 1 Diabetes.

The coinhibitory receptor lymphocyte activation gene 3 (LAG-3) is an immune checkpoint molecule that negatively regulates T cell activation, proliferation, and homeostasis. Blockade or deletion of LAG-3 in autoimmune-prone backgrounds or induced-disease models has been shown to exacerbate disease. We observed significantly fewer LAG-3+ CD4 and CD8 T cells from subjects with relapsing-remitting multiple sclerosis (RRMS) and type 1 diabetes. Low LAG-3 protein expression was linked to alterations in mRNA expression and not cell surface cleavage. Functional studies inhibiting LAG-3 suggest that in subjects with RRMS, LAG-3 retains its ability to suppress T cell proliferation. However, LAG-3 expression was associated with the expression of markers of apoptosis, indicating a role for low LAG-3 in T cell resistance to cell death. In T cells from subjects with RRMS, we observed a global dysregulation of LAG-3 expression stemming from decreased transcription and persisting after T cell stimulation. These findings further support the potential clinical benefits of a LAG-3 agonist in the treatment of human autoimmunity.

Differential expression analysis of microRNAs and mRNAs in the mouse hippocampus of post-stroke depression (PSD) based on transcriptome sequencing.

To clarify the differential expressions of microRNAs and mRNAs in a PSD model, this study employed PSD mice for model construction by injecting vasoconstrictor ET-1 (angioendothelin-1) into the medial prefrontal cortex (mPFC) of mice. The animals underwent elevated plus maze test, open field test, tail suspension test, and forced swimming test subsequently. Transcriptome sequencing was performed to analyze the differentially expressed mRNAs and microRNAs. The results showed that open arm entries and time of PSD mice were markedly decreased. Times of the entry to center for mice in the model group were apparently decreased. The climbing time of mice in the model group was greatly decreased. The behavior of PSD mice indicated a marked change, and several indicators of the behavioral tests were significantly lower than those of the control group. Transcriptome sequencing analysis demonstrated that expressions of 1 206 genes and 21 microRNAs were markedly upregulated in model group, whereas expressions of 2 113 genes and 32 microRNAs were markedly downregulated. GO analysis revealed that the differentially expressed genes were mainly involved in regulatory pathways of single-multicellular organism process, developmental process, cell periphery, plasma membrane, and neuron projection. Meanwhile, KEGG analysis results indicated that the differentially expressed genes mostly participated in signaling pathways of neuroactive ligand-receptor interaction, calcium signaling pathway, and cytokine-cytokine receptor interaction. In conclusion, differentially expressed microRNAs and mRNAs were screened, which offers a theoretical foundation for further investigation of molecular mechanisms and novel insight for the early identification, prevention, and treatment of PSD.

Clinical significance of FAT1 gene mutation and mRNA expression in patients with head and neck squamous cell carcinoma.

The FAT1 gene functions as a tumor suppressor or promoter and remains incompletely understood. We examined the clinical significance of FAT1 in head and neck squamous cell carcinoma (HNSCC) using four publicly available HNSCC cohorts and one HNSCC cohort enrolled at a tertiary medical center. We developed FAT1 signatures reflecting FAT1 mutations and mRNA expression using one cohort. Patients with HNSCC were classified into FAT1-associated low risk (FAT1-LR; n = 195) and FAT1-associated high risk (FAT1-HR; n = 371) subgroups. The five-year overall survival and recurrence-free survival rates were significantly lower in the FAT1-HR subgroup than in the FAT1-LR subgroup (P = 0.01 and 0.003, respectively). The clinical significance of FAT1 was validated using four independent cohorts. Cox proportional hazards models showed that the FAT1 signature was an independent prognostic factor for HNSCC patients. In addition, FAT1 signature was associated with the response to radiotherapy, advanced stage, and human papilloma virus (HPV) status in HNSCC patients. In conclusion, the FAT1 gene signature was associated with prognosis of HNSCC and may help to provide personalized treatments for HNSCC patients.

Human and mouse muscle transcriptomic analyses identify insulin receptor mRNA downregulation in hyperinsulinemia-associated insulin resistance.

Hyperinsulinemia is commonly viewed as a compensatory response to insulin resistance, yet studies have demonstrated that chronically elevated insulin may also drive insulin resistance. The molecular mechanisms underpinning this potentially cyclic process remain poorly defined, especially on a transcriptome-wide level. Transcriptomic meta-analysis in >450 human samples demonstrated that fasting insulin reliably and negatively correlated with INSR mRNA in skeletal muscle. To establish causality and study the direct effects of prolonged exposure to excess insulin in muscle cells, we incubated C2C12 myotubes with elevated insulin for 16 h, followed by 6 h of serum starvation, and established that acute AKT and ERK signaling were attenuated in this model of in vitro hyperinsulinemia. Global RNA-sequencing of cells both before and after nutrient withdrawal highlighted genes in the insulin receptor (INSR) signaling, FOXO signaling, and glucose metabolism pathways indicative of 'hyperinsulinemia' and 'starvation' programs. Consistently, we observed that hyperinsulinemia led to a substantial reduction in Insr gene expression, and subsequently a reduced surface INSR and total INSR protein, both in vitro and in vivo. Bioinformatic modeling combined with RNAi identified SIN3A as a negative regulator of Insr mRNA (and JUND, MAX, and MXI as positive regulators of Irs2 mRNA). Together, our analysis identifies mechanisms which may explain the cyclic processes underlying hyperinsulinemia-induced insulin resistance in muscle, a process directly relevant to the etiology and disease progression of type 2 diabetes.

Regulations of expressions of rat/human sulfotransferases by anticancer drug, nolatrexed, and micronutrients.

Cancer is related to the cellular proliferative state. Increase in cell-cycle regulatory function augments cellular folate pool. This pathway is therapeutically targeted. A number of drugs influences this metabolism, that is, folic acid, folinic acid, nolatrexed, and methotrexate. Our previous study showed methotrexate influences on rat/human sulfotransferases. Present study explains the effect of nolatrexed (widely used in different cancers) and some micronutrients on the expressions of rat/human sulfotransferases. Female Sprague-Dawley rats were treated with nolatrexed (01-100 mg/kg) and rats of both sexes were treated to folic acid (100, 200, or 400 mg/kg) for 2-weeks and their aryl sulfotransferase-IV (AST-IV; ß-napthol sulfation) and sulfotransferase (STa; DHEA sulfation) activities, protein expression (western blot) and mRNA expression (RT-PCR) were tested. In human-cultured hepatocarcinoma (HepG2) cells nolatrexed (1 nM-1.2 mM) or folinic acid (10 nM-10 µM) were applied for 10 days. Folic acid (0-10 µM) was treated to HepG2 cells. PPST (phenol catalyzing), MPST (dopamine and monoamine), DHEAST (dehydroepiandrosterone and DHEA), and EST (estradiol sulfating) protein expressions (western-blot) were tested in HepG2 cells. Present results suggest that nolatrexed significantly increased sulfotransferases expressions in rat (protein, STa, F = 4.87, P < 0.05/mRNA, AST-IV, F = 6.702, P < 0.014; Student's t test, P < 0.01-0.05) and HepG2 cells. Folic acid increased sulfotransferases activity/protein in gender-dependant manner. Both folic and folinic acid increased several human sulfotransferases isoforms with varied level of significance (least or no increase at highest dose) in HepG2 cells pointing its dose-dependent multiphasic responses. The clinical importance of this study may be furthered in the verification of sulfation metabolism of several exogenous/endogenous molecules, drug-drug interaction and their influences on cancer pathophysiological processes. Further studies are necessary.

A New Hybrid Protein Is a Novel Regulator for Experimental Colitis in Rats.

We newly developed a hybrid protein, tentatively named rMIKO-1, using gene technology. We herein investigated the effects of rMIKO-1 on activated macrophages and discussed its potential as a suppressor of experimental colitis. Fluorescent microscopy was used to observe the dynamic mobility of rMIKO-1 in macrophages. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, fluorescent immunochemical staining, flow cytometry, enzyme-linked immunosorbent assays, a polymerase chain reaction/quantitative polymerase chain reaction, and hematoxylin and eosin staining were conducted to assess the potential activity of rMIKO-1. A large amount of bleeding was observed in rats treated with 5% dextran sulfate sodium (DSS) alone on day 8 after treatment initiation, but not in those treated with 5% DSS plus rMIKO-1. In the in vitro assay, rMIKO-1 rapidly bound to macrophages, immediately entered cells by an unknown mechanism, and then migrated inside the nucleus. This result suggests that rMIKO-1 plays important immunological roles in the nucleus. Despite the activation of macrophages by lipopolysaccharide, the mRNA expression of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin-6, and interleukin-1ß, was significantly suppressed in macrophages preliminarily treated with rMIKO-1 for 1 h. Complexes of rMIKO-1 with nuclear factor-kappa B (NF-κB)/p65 and ß-actin formed in activated macrophages, which attenuated experimental colitis in rats. These results strongly suggest that rMIKO-1 negatively regulates excessively activated macrophages through the NF-κB/p65 signaling pathway. Therefore, rMIKO-1 is a novel suppressor of experimental colitis in rats through the negative regulation of activated macrophages.

Predicting functional circular RNA-based competitive endogenous RNA network in gastric carcinoma using novel bioinformatics analysis.

Gastric cancer (GC) remains one of the most prevalent types of malignancies worldwide, and also one of the most reported lethal tumor-related diseases. Circular RNAs (circRNAs) have been certified to be trapped in multiple aspects of GC pathogenesis. Yet, the mechanism of this regulation is mostly undefined. This research is designed to discover the vital circRNA-microRNA (miRNA)-messenger RNA (mRNA) regulatory network in GC. Expression profiles with diverse levels including circRNAs, miRNAs, and mRNAs were all determined using microarray public datasets from Gene Expression Ominous (GEO). The differential circRNAs expressions were recognized against the published robust rank aggregation algorithm. Besides, a circRNA-based competitive endogenous RNA (ceRNA) interaction network was visualized via Cytoscape software (version 3.8.0). Functional and pathway enrichment analysis associated with differentially expressed targeted mRNAs were conducted using Cytoscape and an online bioinformatics database. Furthermore, an interconnected protein-protein interaction association network which consisted of 51 mRNAs was predicted, and hub genes were screened using STRING and CytoHubba. Then, several hub genes were chosen to explore their expression associated with survival rate and clinical stage in GEPIA and Kaplan-Meier Plotter databases. Finally, a carefully designed circRNA-related ceRNA regulatory subnetwork including four circRNAs, six miRNAs, and eight key hub genes was structured using the online bioinformatics tool.