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In the light of growing global epidemic of type 2 diabetes mellitus (T2DM), significant efforts are made to discover next-generation biomarkers for early detection of the disease. Multiple mechanisms including inflammatory response, abnormal insulin secretion and glucose metabolism contribute to the development of T2DM. Platelet activation, on the other hand, is known to be one of the underlying mechanisms of atherosclerosis, which is a common T2DM complication that frequently results in ischemic events at later stages of the disease. Available data suggest that platelets contain large amounts of microRNAs (miRNAs) that are found in circulating body fluids, including the blood. Since miRNAs have been illustrated to play an important role in metabolic homeostasis through regulation of multiple genes, they attracted substantial scientific interest as diagnostic and prognostic biomarkers in T2DM. Various miRNAs, as well as their target genes are implicated in the complex pathophysiology of T2DM. This article will first review the different miRNAs studied in the context of T2DM and platelet reactivity, and subsequently present original results from bioinformatic analyses of published reports, identifying a common gene ( PRKAR1A ) linked to glucose metabolism, blood coagulation and insulin signalling and targeted by miRNAs in T2DM. Moreover, miRNA–target gene interaction networks built upon Gene Ontology information from electronic databases were developed. According to our results, miR-30a-5p, miR-30d-5p and miR-30c-5p are the most widely regulated miRNAs across all specified ontologies, hence they are the most promising biomarkers of T2DM to be investigated in future clinical studies.

Summary The CRISPR/Cas9 system is an RNA‐guided sequence‐specific genome editing tool, which has been adopted for single or multiple gene editing in a wide range of organisms. When working with gene families with functional redundancy, knocking out multiple genes within the same family may be required to generate a phenotype. In this study, we tested the possibility of exploiting the known tolerance of Cas9 for mismatches between the single‐guide RNA (sgRNA) and target site to simultaneously introduce indels in multiple homologous genes in the marine diatom Phaeodactylum tricornutum. As a proof of concept, we designed two sgRNAs that could potentially target the same six light‐harvesting complex (LHC) genes belonging to the LHCF subgroup. Mutations in up to five genes were achieved simultaneously using a previously established CRISPR/Cas9 system for P. tricornutum. A visible colour change was observed in knockout mutants with multiple LHCF lesions. A combination of pigment, LHCF protein and growth analyses was used to further investigate the phenotypic differences between the multiple LHCF mutants and WT. Furthermore, we used the two same sgRNAs in combination with a variant of the existing Cas9 where four amino acids substitutions had been introduced that previously have been shown to increase Cas9 specificity. A significant reduction of off‐target editing events was observed, indicating that the altered Cas9 functioned as a high‐fidelity (HiFi) Cas9 nuclease.

Pax5 controls the identity and development of B cells by repressing lineage‐inappropriate genes and activating B‐cell‐specific genes. Here, we used genome‐wide approaches to identify Pax5 target genes in pro‐B and mature B cells. In these cell types, Pax5 bound to 40% of the cis ‐regulatory elements defined by mapping DNase I hypersensitive (DHS) sites, transcription start sites and histone modifications. Although Pax5 bound to 8000 target genes, it regulated only 4% of them in pro‐B and mature B cells by inducing enhancers at activated genes and eliminating DHS sites at repressed genes. Pax5‐regulated genes in pro‐B cells account for 23% of all expression changes occurring between common lymphoid progenitors and committed pro‐B cells, which identifies Pax5 as an important regulator of this developmental transition. Regulated Pax5 target genes minimally overlap in pro‐B and mature B cells, which reflects massive expression changes between these cell types. Hence, Pax5 controls B‐cell identity and function by regulating distinct target genes in early and late B lymphopoiesis. Genome‐wide sequencing approaches reveal that the transcription factor Pax5 controls the identity and function of B cells by regulating the expression of distinct target genes in pro‐B and mature B cells.

Cyclophosphamide (CP), an important alkylating agent which is used in the treatment therapy for chronic myeloid leukemia (CML). However, acquired drug resistance owing to the inactivation of its active metabolite aldophosphamide via tumoral-overexpressing aldehyde dehydrogenase (ALDH1A1) is one of the major issues with the CP therapy. However, the underlying mechanism of ALDH1A1 overexpression in cancer cells remains poorly defined. Therefore, the current study focused on analyzing the ALDH1A1-overexpressing microarray data for CP resistance and CP-sensitive CML cell lines. In this study, the microarray dataset was obtained from Gene Expression Omnibus GEO. The GEO2R tool was used to identify Differentially Expressing Genes (DEGs). Further, protein–protein interaction (PPI) network of DEGs were constructed using STRING database. Finally, Hub gene-miRNA-TFs interaction were constructed using miRNet tool. A total of 749 DEGs including 387 upregulated and 225 downregulated genes were identified from this pool of microarray data. The construction of DEGs network resulted in identification of three genes including ZEB2, EZH2, and MUC1 were found to be majorly responsible for ALDH1A1 overexpression. miRNA analysis identified that, hsa-mir-16-5p and hsa-mir-26a-5p as hub miRNA which are commonly interacting with maximum target genes. Additionally, drug-gene interaction analysis was performed to identify drugs which are responsible for ALDH1A1 expression. The entire study may provide a deeper understanding about ALDH1A1 regulatory genes responsible for its overexpression in CP resistance cancer. This understanding may be further explore for developing possible co-therapy to avoid the ALDH1A1-mediated CP resistance.

The present study aimed to investigate the molecular mechanisms underlying glioblastoma multiform (GBM) and its biomarkers. The differentially expressed genes (DEGs) were diagnosed using the limma software package. The ToppGene (ToppFun) was used to perform pathway and Gene Ontology (GO) enrichment analysis of the DEGs. Protein-protein interaction (PPI) networks, extracted modules, miRNA-target genes regulatory network and TF-target genes regulatory network were used to obtain insight into the actions of DEGs. Survival analysis for DEGs was carried out. A total of 590 DEGs, including 243 up regulated and 347 down regulated genes, were diagnosed between scrambled shRNA expression and Lin7A knock down. The up-regulated genes were enriched in ribosome, mitochondrial translation termination, translation, and peptide biosynthetic process. The down-regulated genes were enriched in focal adhesion, VEGFR3 signaling in lymphatic endothelium, extracellular matrix organization, and extracellular matrix. The current study screened the genes in the PPI network, extracted modules, miRNA-target genes regulatory network, and TF-target genes regulatory network with higher degrees as hub genes, which included NPM1, CUL4A, YIPF1, SHC1, AKT1, VLDLR, RPL14, P3H2, DTNA, FAM126B, RPL34, and MYL5. Survival analysis indicated that the high expression of RPL36A and MRPL35 were predicting longer survival of GBM, while high expression of AP1S1 and AKAP12 were predicting shorter survival of GBM. High expression of RPL36A and AP1S1 were associated with pathogenesis of GBM, while low expression of ALPL was associated with pathogenesis of GBM. In conclusion, the current study diagnosed DEGs between scrambled shRNA expression and Lin7A knock down samples, which could improve our understanding of the molecular mechanisms in the progression of GBM, and these crucial as well as new diagnostic markers might be used as therapeutic targets for GBM.

Alzheimer's disease (AD) is the most common form of dementia in the older population, however, the precise cause of the disease is unknown. The neuropathology is characterized by the presence of aggregates formed by amyloid-β (Aβ) peptide and phosphorylated tau; which is accompanied by progressive impairment of memory. Diverse signaling pathways are linked to AD, and among these the Wnt signaling pathway is becoming increasingly relevant, since it plays essential roles in the adult brain. Initially, Wnt signaling activation was proposed as a neuroprotective mechanism against Aβ toxicity. Later, it was reported that it participates in tau phosphorylation and processes of learning and memory. Interestingly, in the last years we demonstrated that Wnt signaling is fundamental in amyloid precursor protein (APP) processing and that Wnt dysfunction results in Aβ production and aggregation in vitro. Recent in vivo studies reported that loss of canonical Wnt signaling exacerbates amyloid deposition in a transgenic (Tg) mouse model of AD. Finally, we showed that inhibition of Wnt signaling in a Tg mouse previously at the appearance of AD signs, resulted in memory loss, tau phosphorylation and Aβ formation and aggregation; indicating that Wnt dysfunction accelerated the onset of AD. More importantly, Wnt signaling loss promoted cognitive impairment, tau phosphorylation and Aβ1-42 production in the hippocampus of wild-type (WT) mice, contributing to the development of an Alzheimer's-like neurophatology. Therefore, in this review we highlight the importance of Wnt/β-catenin signaling dysfunction in the onset of AD and propose that the loss of canonical Wnt signaling is a triggering factor of AD.

microRNAs (miRNAs) exert their functions by repressing the expression of their target genes, but most miRNA target genes are unknown, and the degree to which a miRNA differentially inhibits the expression of its targets is underappreciated. We selected human miR-1, miR-122, and miR-124 as representatives to investigate the reliability of miRNA target predictions and examine how miRNAs suppress their targets. We constructed miRNA target gene reporter libraries based on prediction programs TargetScan, miRanda, and PicTar, and performed large-scale reporter assays to directly evaluate whether and how strongly a predicted target gene is repressed by its miRNA. We then performed statistical analyses to examine parameters that contributed to the miRNA inhibition of target genes. We found that the three programs have approximately 72–85% success rates in predicting genuine targets and that the miRNA inhibition of different targets varies in extent. We also identified parameters that could predict the degrees of miRNA repression, and further showed that differential miR-124 repression might contribute to differential gene expression in vivo. Our studies systematically investigated hundreds of miRNA target genes, shed light on factors influencing miRNA functions, and suggested a new mechanism by which differential target repression by miRNAs regulates endogenous gene expression.

(LM) is an important food-borne pathogen, and the risk of its ingestion is a serious public health issue. The better its environmental adaptation mechanisms and pathogenicity are understood, the better the risk it poses can be countered. The regulatory role of the small non-coding RNA (sRNA) in the environmental adaptation and pathogenicity of LM is still unclear and this study investigated that role through its biological function. An LM- gene deletion strain and an LM- gene complementation strain were constructed using the homologous recombination technique. Then, the adaptation of these strains to temperature, alkalinity, acidity, salinity, ethanol and oxidative stressors, their biofilm-forming ability and their pathogenicity in mice were investigated to show the regulatory roles of sRNA in LM. The target gene of was also predicted, and the interaction between it and was verified by a two-plasmid co-expressing system based on and Western blot analysis. The adaptation of LM- to environmental stressors of pH 9, 5% NaCl and 8% NaCl, 3.8% ethanol and 5 mM H O was significantly reduced when compared to the parental (LM EGD-e) and complementation strains. Also, the biofilm formation, cell adhesion, invasion, intracellular proliferation and pathogenicity of LM- in mice were significantly reduced. The results of two-plasmid co-expression and Western blot showed that can interact with the mRNA of the predicted target gene. The sRNA may positively regulate the expression of the gene in LM. This study sheds light on its regulatory roles in environmental adaptation and pathogenicity, providing new insights into the molecular mechanism of sRNA mediation in LM .

MicroRNAs (miRNAs) can regulate the modulation of the phenotype of Schwann cells. Numerous novel miRNAs have been discovered and identified in rat sciatic nerve segments, including miR-3099. In the current study, miR-3099 expression levels following peripheral nerve injury were measured in the proximal stumps of rat sciatic nerves after surgical crush. Real-time reverse transcription-polymerase chain reaction was used to determine miR-3099 expression in the crushed nerve segment at 0, 1, 4, 7, and 14 days post sciatic nerve injury, which was consistent with Solexa sequencing outcomes. Expression of miR-3099 was up-regulated following peripheral nerve injury. EdU and transwell chamber assays were used to observe the effect of miR-3099 on Schwann cell proliferation and migration. The results showed that increased miR-3099 expression promoted the proliferation and migration of Schwann cells. However, reduced miR-3099 expression suppressed the proliferation and migration of Schwann cells. The potential target genes of miR-3099 were also investigated by bioinformatic tools and high-throughput outcomes. miR-3099 targets genes Aqp4, St8sia2, Tnfsf15, and Zbtb16 and affects the proliferation and migration of Schwann cells. This study examined the levels of miR-3099 at different time points following peripheral nerve injury. Our results confirmed that increased miR-3099 level induced by peripheral nerve injury can promote the proliferation and migration of Schwann cells.

microRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level by mRNA cleavage or translation inhibition. They play diverse roles in plant growth and development as well as abiotic stress responses. In response to abiotic stresses such as drought, salt, cold, heat, and nutrient limitations, the expression levels of some miRNAs change, resulting in a modulation of the expression patterns of miRNA target genes that are associated with stress adaptations. In rice, stress-responsive miRNAs have been identified and characterized, and conserved regulation of conserved miRNAs as well as new regulation by conserved miRNAs and rice-specific miRNAs is evident. The regulatory mechanisms controlling target gene expression by stressresponsive miRNAs include both the coherent and incoherent regulatory networks that are dynamic and complex. A better understanding of the regulation of miRNAs and targets during stress responses can contribute to rice breeding for improving yield, quality and tolerance to abiotic stresses. Here, we review current advances in the area of rice miRNAs and target RNAs associated with abiotic stresses and discuss how they relate to miRNA-mediated stresstolerance.