Magnetic separation is an efficient method for target enrichment and elimination of inhibitors in the molecular detection systems for foodborne pathogens. In this study, we prepared magnetic capture probes by modifying oligonucleotides complementary to target sequences on the surface of amino-modified silica-coated magnetic nanoparticles and optimized the conditions and parameters of probe synthesis and hybridization. We innovatively put the complexes of magnetic capture probes and target sequences into qPCR without any need for denaturation and purification steps. This strategy can reduce manual steps and save time. We used the magnetic capture probes to separate invA mRNA from Salmonella in artificially contaminated milk samples. The detection sensitivity was 104 CFU/ml, which could be increased to 10 CFU/ml after a 12 h enrichment step. The developed method is robust enough to detect live bacteria in a complex environmental matrix.

Previous studies have demonstrated that long non‑coding RNA (lncRNA) is involved in vascular remodeling. The metastasis‑associated lung adenocarcinoma transcript 1 (MALAT1) lncRNA is associated with the proliferation and migration of vascular smooth muscle and endothelial cells; however, its biological role in pulmonary artery hypertension (PAH) is currently unclear. The aim of the present study was to investigate the post‑transcriptional regulation of MALAT1 in human pulmonary artery smooth muscle cells (HPASMCs). The results revealed that MALAT1 expression levels were significantly upregulated in the pulmonary arteries (PAs) and HPASMCs obtained from patients with PAH compared with adjacent normal PA tissues and HPASMCs. Knockdown of MALAT1 suppressed the viability and proliferation of HPASMCs and prevented cells entering the G0/G1 cell cycle phase. MALAT1 overexpression exerted the opposite effects. Bioinformatics analysis predicted complementary binding of hsa‑microRNA (miR)‑124‑3p.1 with the 3'‑untranslated region of MALAT1. Luciferase reporter assays and RNA immunoprecipitation experiments demonstrated molecular binding between MALAT1 and hsa‑miR‑124‑3p.1. This resulted in the formation of an RNA‑induced silencing complex. In addition, Kruppel‑like factor 5 (KLF5) was confirmed to be a target gene of MALAT1/hsa‑miR‑124‑3p.1. MALAT1 silencing did not inhibit the proliferation and migration of HPASMCs following knockdown of hsa‑miR‑124‑3p.1. In addition, MALAT1 knockdown was demonstrated to attenuate the expression of KLF5. Following MALAT1 knockdown, the expression level of KLF5 was rescued by inhibition of hsa‑miR‑124‑3p.1 expression. The results of the current study indicate that the MALAT1/hsa‑miR‑124‑3p.1/KLF5 axis may serve a key role in HPASMCs. In addition, the results contribute to what is known regarding the role of MALAT1 in PAH development and provide a novel theoretical basis for the development of new therapeutic interventions for patients with PAH.

Protein interactions with the microRNA (miRNA)-mediated gene silencing protein Argonaute 2 (AGO2) control miRNA expression. miRNA biogenesis starts with the production of precursor transcripts and culminates with the loading of mature miRNA onto AGO2 by DICER1. Here we reveal an additional component to the regulatory mechanism for miRNA biogenesis involving the adaptor protein, growth factor receptor-bound protein 2 (GRB2). The N-terminal SH3 domain of GRB2 is recruited to the PAZ domain of AGO2 forming a ternary complex containing GRB2, AGO2 and DICER1. Using small-RNA sequencing we identified two groups of miRNAs which are regulated by the binding of GRB2. First, mature and precursor transcripts of mir-17~92 and mir-221 miRNAs are enhanced. Second, mature, but not precursor, let-7 family miRNAs are diminished suggesting that GRB2 directly affects loading of these miRNAs. Notably, the resulting loss of let-7 augments expression of oncogenic targets such as RAS. Thus, a new role for GRB2 is established with implications for cancer pathogenesis through regulation of miRNA biogenesis and oncogene expression.

Over the past 10 years, genomes of cultivated rice cultivars and their wild counterparts have been sequenced although most efforts are focused on genome assembly and annotation of two major cultivated rice (Oryza sativa L.) subspecies, 93-11 (indica) and Nipponbare (japonica). To integrate information from genome assemblies and annotations for better analysis and application, we now introduce a comparative rice genome database, the Rice Genome Knowledgebase (RGKbase, http://rgkbase.big.ac.cn/RGKbase/). RGKbase is built to have three major components: (i) integrated data curation for rice genomics and molecular biology, which includes genome sequence assemblies, transcriptomic and epigenomic data, genetic variations, quantitative trait loci (QTLs) and the relevant literature; (ii) User-friendly viewers, such as Gbrowse, GeneBrowse and Circos, for genome annotations and evolutionary dynamics and (iii) Bioinformatic tools for compositional and synteny analyses, gene family classifications, gene ontology terms and pathways and gene co-expression networks. RGKbase current includes data from five rice cultivars and species: Nipponbare (japonica), 93-11 (indica), PA64s (indica), the African rice (Oryza glaberrima) and a wild rice species (Oryza brachyantha). We are also constantly introducing new datasets from variety of public efforts, such as two recent releases-sequence data from ∼1000 rice varieties, which are mapped into the reference genome, yielding ample high-quality single-nucleotide polymorphisms and insertions-deletions.

Human noroviruses (HuNoVs) are major contributors to acute nonbacterial gastroenteritis outbreaks. Many aspects of HuNoVs are poorly understood due to both the current inability to culture HuNoVs, and the lack of efficient small animal models. Surrogates for HuNoVs, such as recombinant viral like particles (VLPs) expressed in eukaryotic system or P particles expressed in prokaryotic system, have been used for studies in immunology and interaction between the virus and its receptors. However, it is difficult to use VLPs or P particles to collect or isolate potential ligands binding to these recombinant capsid proteins. In this study, a new strategy was used to collect HuNoVs binding ligands through the use of ice nucleation protein (INP) to display recombinant capsid proteins of HuNoVs on bacterial surfaces. The viral protein-ligand complex could be easily separated by a low speed centrifugation step. This system was also used to explore interaction between recombinant capsid proteins of HuNoVs and their receptors. In this system, the VP1 capsid encoding gene (ORF2) and the protruding domain (P domain) encoding gene (3' terminal fragment of ORF2) of HuNoVs GI.1 and GII.4 were fused with 5' terminal fragment of INP encoding gene (inaQn). The results demonstrated that the recombinant VP1 and P domains of HuNoVs were expressed and anchored on the surface of Escherichia coli BL21 cells after the bacteria were transformed with the corresponding plasmids. Both cell surface displayed VP1 and P domains could be recognized by HuNoVs specific antibodies and interact with the viral histo-blood group antigens receptors. In both cases, displayed P domains had better binding abilities than VP1. This new strategy of using displayed HuNoVs capsid proteins on the bacterial surface could be utilized to separate HuNoVs binding components from complex samples, to investigate interaction between the virus and its receptors, as well as to develop an oral vaccine for HuNoVs.

Genome-wide analysis of glioblastoma (GBM) reveals pervasive aberrations in apoptotic signaling pathways that collectively contribute to therapeutic resistance. Inhibitors of apoptosis proteins (IAP) exert critical control on the terminal segment of apoptosis leading to apoptosis evasion. In this study, we uncover a unique role for BIRC3, as an IAP that is critical in GBM in response to therapy. Using the TCGA dataset of 524 unique samples, we identify BIRC3 is the only IAP whose differential expression is associated with long-term survival in GBM patients. Using patient tissue samples we further show that BIRC3 expression increases with recurrence. When extrapolated to a preclinical model of a human GBM cell line, we find an increase in BIRC3 expression in response to irradiation (RT) and temozolomide (TMZ) treatment. More importantly, we mechanistically implicate STAT3 and PI3K signaling pathways as drivers of RT-induced up-regulation of BIRC3 expression. Lastly, we demonstrate that both in-vivo and in-vitro BIRC3 up-regulation results in apoptosis evasion and therapeutic resistance in GBM. Collectively, our study identifies a novel translational and targetable role for BIRC3 expression as a predictor of aggressiveness and therapeutic resistance to TMZ and RT mediated by STAT3 and PI3K signaling in GBM.

We explore cell heterogeneity during spontaneous and transcription-factor-driven commitment for network inference in hematopoiesis. Since individual genes display discrete OFF states or a distribution of ON levels, we compute and combine pairwise gene associations from binary and continuous components of gene expression in single cells. Ddit3 emerges as a regulatory node with positive linkage to erythroid regulators and negative association with myeloid determinants. Ddit3 loss impairs erythroid colony output from multipotent cells, while forcing Ddit3 in granulo-monocytic progenitors (GMPs) enhances self-renewal and impedes differentiation. Network analysis of Ddit3-transduced GMPs reveals uncoupling of myeloid networks and strengthening of erythroid linkages. RNA sequencing suggests that Ddit3 acts through development or stabilization of a precursor upstream of GMPs with inherent Meg-E potential. The enrichment of Gata2 target genes in Ddit3-dependent transcriptional responses suggests that Ddit3 functions in an erythroid transcriptional network nucleated by Gata2.

The purpose of this study was to investigate whether cinnabar causes renal inflammation and fibrosis in rats. Rats were dosed orally with cinnabar (1 g/kg/day) for 8 weeks or 12 weeks. The control rats were treated with solvent (5% carboxymethylcellulose solution) over the same time periods, respectively. Renal mercury (RHg), urinary mercury (UHg), serum creatinine (SCr), urine kidney injury molecule 1 (KIM-1), renal pathology, and renal mediators were examined. At both 8 weeks and 12 weeks, RHg, UHg, and urine KIM-1 were significantly higher in the cinnabar group than in the control group, although SCr was unchanged. Kidney lesions in the cinnabar-treated rats occurred mainly in the tubules and interstitium, including vacuolization, protein casts, infiltration of inflammatory cells, and slight increase in interstitial collagen. In addition, mild mesangial proliferation was observed in glomeruli. Moreover, the expression of inflammatory and fibrogenic mediators was upregulated in the cinnabar group. In conclusion, cinnabar may cause kidney damage due to the accumulation of mercury, and renal inflammation and slight fibrogenesis may occur in rats. In the clinic, the potential risk of renal injury due to the prolonged consumption of cinnabar should be considered even though the agent is relatively nontoxic.

ETV6-RUNX1 is associated with childhood acute B-lymphoblastic leukemia (cALL) functioning as a first-hit mutation that initiates a clinically silent pre-leukemia in utero. Because lineage commitment hierarchies differ between embryo and adult, and the impact of oncogenes is cell-context dependent, we hypothesized that the childhood affiliation of ETV6-RUNX1 cALL reflects its origins in a progenitor unique to embryonic life. We characterize the first emerging B cells in first-trimester human embryos, identifying a developmentally restricted CD19-IL-7R+ progenitor compartment, which transitions from a myeloid to lymphoid program during ontogeny. This developmental series is recapitulated in differentiating human pluripotent stem cells (hPSCs), thereby providing a model for the initiation of cALL. Genome-engineered hPSCs expressing ETV6-RUNX1 from the endogenous ETV6 locus show expansion of the CD19-IL-7R+ compartment, show a partial block in B lineage commitment, and produce proB cells with aberrant myeloid gene expression signatures and potential: features (collectively) consistent with a pre-leukemic state.

T cell receptor (TCR) stimulation of naive CD8+ T cells initiates reprogramming of cis-regulatory landscapes that specify effector and memory cytotoxic T lymphocyte (CTL) differentiation. We mapped regions of hyper-accessible chromatin in naive cells during TCR stimulation and discovered that the transcription factor (TF) Runx3 promoted accessibility to memory CTL-specific cis-regulatory regions before the first cell division and was essential for memory CTL differentiation. Runx3 was specifically required for accessibility to regions highly enriched with IRF, bZIP and Prdm1-like TF motifs, upregulation of TFs Irf4 and Blimp1, and activation of fundamental CTL attributes in early effector and memory precursor cells. Runx3 ensured that nascent CTLs differentiated into memory CTLs by preventing high expression of the TF T-bet, slowing effector cell proliferation, and repressing terminal CTL differentiation. Runx3 overexpression enhanced memory CTL differentiation during iterative infections. Thus, Runx3 governs chromatin accessibility during TCR stimulation and enforces the memory CTL developmental program.

Tissue-resident memory CD8+ T (TRM) cells are found at common sites of pathogen exposure, where they elicit rapid and robust protective immune responses. However, the molecular signals that control TRM cell differentiation and homeostasis are not fully understood. Here we show that mouse TRM precursor cells represent a unique CD8+ T cell subset that is distinct from the precursors of circulating memory cell populations at the levels of gene expression and chromatin accessibility. Using computational and pooled in vivo RNA interference screens, we identify the transcription factor Runx3 as a key regulator of TRM cell differentiation and homeostasis. Runx3 was required to establish TRM cell populations in diverse tissue environments, and supported the expression of crucial tissue-residency genes while suppressing genes associated with tissue egress and recirculation. Furthermore, we show that human and mouse tumour-infiltrating lymphocytes share a core tissue-residency gene-expression signature with TRM cells that is associated with Runx3 activity. In a mouse model of adoptive T cell therapy for melanoma, Runx3-deficient CD8+ tumour-infiltrating lymphocytes failed to accumulate in tumours, resulting in greater rates of tumour growth and mortality. Conversely, overexpression of Runx3 enhanced tumour-specific CD8+ T cell abundance, delayed tumour growth, and prolonged survival. In addition to establishing Runx3 as a central regulator of TRM cell differentiation, these results provide insight into the signals that promote T cell residency in non-lymphoid sites, which could be used to enhance vaccine efficacy or adoptive cell therapy treatments that target cancer.

We previously have studied the insertion and deletion polymorphism by sequencing no more than one hundred introns in a mixed human population and found that the minimal introns tended to maintain length at an optimal size. Here we analyzed re-sequenced 179 individual genomes (from African, European, and Asian populations) from the data released by the 1000 Genome Project to study the size dynamics of minimal introns.

Histo-blood group antigens (HBGAs) are considered as receptors/co-receptors for human norovirus (HuNoV). It has been reported that binding of HuNoV-derived virus-like particles (VLPs) to HBGA-like molecules-expressing bacteria increased the stability of VLPs to heat-denaturation (HD). In this study, we tested for HBGA-like-binding-conveyed protection against HD on viral replication using Tulane virus (TV) and Escherichia coli O86:H2 (O86:H2), with E. coli K-12 (K-12) used as a control. Expression of HBGA type B was confirmed by ELISA in O86:H2 but not in K-12. Binding of TV was confirmed by ELISA in O86:H2 (P/N = 2.23) but not in K-12 (P/N = 1.90). Pre-incubation of TV with free HBGA could completely inhibit its ability to bind to O86:H2 (p = 0.004), while producing no significant change in its ability to bind K-12 (p = 0.635). We utilized a bacterial-capture-RT-qPCR procedure to confirm that both bacterial strains were capable of binding TV, and that O86:H2 exhibited fivefold greater binding capacity than K-12. Pre-incubation of TV with free HBGA would partially inhibit the binding of TV to O86:H2 (p = 0.047). In contrast, not only did pre-incubation of TV with free HBGA not inhibit the binding of TV to K-12, binding was slightly enhanced (p = 0.13). The viral infectivity assay allowed us to conduct a direct evaluation of the ability of HBGA-like-bound bacteria to confer HD protection to TV. Prior to inoculate to LLC-MK2 cells, TV was incubated with each bacterial strain at ratios of 1:0, 1:1 and 100:1, then both partially and fully HD. The viral amplification was quantitated by RT-qPCR 48 h later. The binding of bacteria to TV reduced viral replication in a dose-dependent matter. We found that neither bound O86:H2 nor K-12 conferred protection of TV against partial or full HD conditions. Partial HD reduction of viral replication was not significantly impacted by the binding of either bacterial strain, with infectivity losses of 99.03, 99.42, 96.32, 96.10, and 98.88% for TV w/o bacteria, TV w/O86:H2 (1:1), TV w/O86:H2 (100:1), TV w/K-12 (1:1), and TV w/K-12 (100:1), respectively. Full HD reduction of viral replication was not impacted by the binding of either bacterial strain, as full loss of infectivity was observed in all cases.

Autophagy is a catabolic process essential for preserving cellular homeostasis, and the epithelial-to-mesenchymal transition (EMT) is involved during tissue development and cancer progression. In arsenite-treated human hepatic epithelial (L-02) cells, arsenite reduced the autophagic flux, which caused accumulation of p62, an adaptor and receptor of autophagy. Further, in arsenite-transformed L-02 cells, the levels of E-cadherin were attenuated, but the levels of vimentin, which is expressed in mesenchymal cells, and Snail, a transcription regulator of the EMT, were up-regulated. Thus, after chronic exposure of L-02 cells to arsenite, the impaired autophagic flux induced the accumulation of p62, which up-regulated the expression of Snail, a protein involved in arsenite-induced EMT of these cells. Knockdown of p62 by siRNA reversed the arsenite-induced EMT and decreased the capacities of arsenite-transformed L-02 cells for colony formation and invasion and migration. Therefore, in arsenite-induced transformation of L-02 cells, the accumulation of p62, by impairing autophagic flux, mediates the EMT via Snail. These results provide a previously unknown mechanism underlying arsenic toxicity and carcinogenicity.

Pseudomonas syringae pv. actinidiae (Psa) is a phytopathogen that causes canker in kiwifruit. Few conventional control methods are effective against this bacterium. Therefore, alternative approaches, such as phage therapy are warranted. In this study, a lytic bacteriophage (PN09) of Psa was isolated from surface water collected from a river in Hangzhou, China in 2019. Morphologically, PN09 was classified into the Myoviridae family, and could lyse all 29 Psa biovar 3 strains. The optimal temperature and pH ranges for PN09 activity were determined as 25 to 35 ∘C and 6.0 to 9.0, respectively. The complete genome of PN09 was found to be composed of a linear 99,229 bp double-stranded DNA genome with a GC content of 48.16%. The PN09 endolysin (LysPN09) was expressed in vitro and characterized. LysPN09 was predicted to belong to the Muraidase superfamily domain and showed lytic activity against the outer-membrane-permeabilized Psa strains. The lytic activity of LysPN09 was optimal over temperature and pH ranges of 25 to 40 ∘C and 6.0 to 8.0, respectively. When recombinant endolysin LysPN09 was combined with EDTA, Psa strains were effectively damaged. All these characteristics demonstrate that the phage PN09 and its endolysin, LysPN09, are potential candidates for biocontrol of Psa in the kiwifruit industry.

Some new trials have reported the effectiveness of chronic kidney disease on recurrence of atrial fibrillation following catheter ablation. Limited by small number of studies and insufficient outcomes, previous meta-analyses also failed to draw a consistent conclusion on this topic. We thus conducted a new meta-analysis to systematically analyze the effect of chronic kidney disease on recurrence of atrial fibrillation following catheter ablation.

Skeletal muscle satellite cells cultured on soft surfaces (12 kPa) show improved differentiation than cells cultured on stiff surfaces (approximately 100 kPa). To better understand the reasons for this, we performed an RNA-Seq analysis for a single satellite cell clone (C1F) derived from the H2kb-tsA58 immortomouse, which differentiates into myotubes under tightly regulated conditions (withdrawal of ɣ-interferon, 37 °C). The largest change in overall gene expression occurred at day 1, as cells switched from proliferation to differentiation. Surprisingly, further analysis showed that proliferating C1F cells express Pax3 and not Pax7, confirmed by immunostaining, yet their subsequent differentiation into myotubes is normal, and enhanced on softer surfaces, as evidenced by significantly higher expression levels of myogenic regulatory factors, sarcomeric genes, enhanced fusion and improved myofibrillogenesis. Levels of mRNA encoding extracellular matrix structural constituents and related genes were consistently upregulated on hard surfaces, suggesting that a consequence of differentiating satellite cells on hard surfaces is that they attempt to manipulate their niche prior to differentiating. This comprehensive RNA-Seq dataset will be a useful resource for understanding Pax3 expressing cells.

Human Noroviruses (HuNoVs) are the main cause of non-bacterial gastroenteritis. Contaminated produce is a main vehicle for dissemination of HuNoVs. In this study, we used an ice nucleation protein mediated surface display system to present the protruding domain of GII.4 HuNoV capsid protein on bacterial surface and used it as a new strategy to explore interaction between HuNoV protein and receptor candidates from romaine lettuce. The surface-displayed HuNoV proteins were confirmed on the surface of the transformed bacteria by an immunofluorescence assay. The distribution patterns of the surface-displayed HuNoV proteins in romaine lettuce were identified through a confocal immunofluorescence assay. The surface-displayed HuNoV proteins could be found in the stomata, and the surfaces of vein and leaf of romaine lettuce. The surface-displayed HuNoV proteins could be captured by an ELISA assay utilizing extract from leaf (LE) or vein (VE). The binding of the surface-displayed HuNoV proteins to LE or VE could be competitively blocked by histo-blood group antigens from human saliva. In addition, the binding of the surface-displayed HuNoV proteins to LE or VE could also be attenuated by heat denaturation of lettuce proteins, and abolished by oxidation of lettuce carbohydrates. The results indicated that histo-blood group antigen-like molecules in LE or VE were involved in the binding of the surface-displayed HuNoV proteins to romaine lettuce. All data demonstrated that the surface-displayed HuNoV proteins could be utilized in a new and simple system for investigation of the interaction between the HuNoVs and their candidate ligands.

Tumor hypoxia is an established facilitator of survival adaptation and mesenchymal transformation in glioblastoma (GBM). The underlying mechanisms that direct hypoxia-mediated survival in GBM habitats are unclear. We previously identified BIRC3 as a mediator of therapeutic resistance in GBM to standard temozolomide (TMZ) chemotherapy and radiotherapy (RT). Here we report that BIRC3 is a biomarker of the hypoxia-mediated adaptive mesenchymal phenotype of GBM. Specifically, in the TCGA dataset elevated BIRC3 gene expression was identified as a superior and selective biomarker of mesenchymal GBM versus neural, proneural and classical subtypes. Further, BIRC3 protein was highly expressed in the tumor cell niches compared to the perivascular niche across multiple regions in GBM patient tissue microarrays. Tumor hypoxia was found to mechanistically induce BIRC3 expression through HIF1-alpha signaling in GBM cells. Moreover, in human GBM xenografts robust BIRC3 expression was noted within hypoxic regions of the tumor. Importantly, selective inhibition of BIRC3 reversed therapeutic resistance of GBM cells to RT in hypoxic microenvironments through enhanced activation of caspases. Collectively, we have uncovered a novel role for BIRC3 as a targetable biomarker and mediator of hypoxia-driven habitats in GBM.

Norovirus is recognized as one of the leading causes of acute gastroenteritis outbreaks. Genotype GII.9 was first detected in Norfolk, VA, USA, in 1997. However, the complete genome sequence of this genotype has not yet been determined. In this study, a complete genome sequence of GII.9[P7] norovirus, SCD1878_GII.9[P7], from a patient was determined using high-throughput sequencing and rapid amplification of cDNA ends (RACE) technology. The complete genome sequence of SCD1878_GII.9[P7] is 7544 nucleotides (nt) in length with a 3' poly(A) tail and contains three open reading frames. Sequence comparisons indicated that SCD1878_GII.9[P7] shares 92.1%-92.3% nucleotide sequence identity with GII.P7 (AB258331 and AB039777) and 96.7%-97.4% identity with GII.9 (AY038599 and DQ379715). The results suggested that SCD1878_GII.9[P7] is a member of P genotype GII.P7 and G genotype GII.9. This viral sequence fills a gap at the whole-genome level for the GII.9 genotype.