Ischemic stroke often leads to lifelong disability or death in stroke patients. It is one of the most common causes of death and disability worldwide, so it is a global health problem. The objective of this protocol is to provide the methods for using overview and network meta-analysis to identify the more effective intervention for infarct volume and neurobehavioral score in animal models of ischemic stroke.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a single-stranded, positive-sense RNA virus. PHEV mainly causes two types of clinical manifestations representing vomiting and wasting and encephalomyelitis in piglets. However, our recent findings provide strong evidence that PHEV can also cause respiratory disease in older pigs. Genomic analysis of new PHEV strains identified in our former study further classifies PHEV into three genotypes. Detection and differentiation of these new mutants are critical in monitoring PHEV evolution in the field. In the present study, we report the development of a triplex real-time RT-PCR assay for detection and differentiation of three PHEV genotypes, 1, 2, and 3. Three sets of primers and probes were designed; one set of primers and probe targeting the conserved regions of the 3' end nucleocapsid for detection of all three genotypes and another two sets of primers and probes targeting the regions of NS2 with different patterns of deletions for detection of both genotypes 1 and 3, or genotype 3 only. Genotype 1 was positive when two probe dyes showed signals, genotype 2 was positive when only one probe dye showed a signal, and genotype 3 was positive when all three probes showed signals. The detection limit of the developed triplex real-time RT-PCR was as low as 8 or 9 DNA copies for three sets of primers and probes. The specificity test showed no cross reaction with other porcine viruses. Positive field-samples were correctly typed by this new assay, which was further confirmed by DNA sequencing. The triplex real-time RT-PCR provides a rapid and sensitive method to detect and differentiate all three US genotypes of PHEV from clinical samples.

To explore the non-linear relationship between platelet count (PLT) and hepatocellular carcinoma (HCC) overall survival (OS).

Sickle cell disease (SCD) results from a hemoglobin (Hb) mutation βGlu6 → βVal6 that changes normal Hb (HbA) into sickle Hb (HbS). Under hypoxia, HbS polymerizes into rigid fibers, causing red blood cells (RBCs) to sickle; leading to numerous adverse pathological effects. The RBC sickling is made worse by the low oxygen (O2) affinity of HbS, due to elevated intra-RBC concentrations of the natural Hb effector, 2,3-diphosphoglycerate. This has prompted the development of Hb modifiers, such as aromatic aldehydes, with the intent of increasing Hb affinity for O2 with subsequent prevention of RBC sickling. One such molecule, Voxelotor was recently approved by U.S. FDA to treat SCD. Here we report results of a novel aromatic aldehyde, VZHE-039, that mimics both the O2-dependent and O2-independent antisickling properties of fetal hemoglobin. The latter mechanism of action-as elucidated through crystallographic and biological studies-is likely due to disruption of key intermolecular contacts necessary for stable HbS polymer formation. This dual antisickling mechanism, in addition to VZHE-039 metabolic stability, has translated into significantly enhanced and sustained pharmacologic activities. Finally, VZHE-039 showed no significant inhibition of several CYPs, demonstrated efficient RBC partitioning and high membrane permeability, and is not an efflux transporter (P-gp) substrate.

Fluorofenidone (AKF-PD) is a novel pyridone agent that reduces the deposition of extracellular matrix (ECM) in various models of renal fibrosis. However, there are no reports on the effect of AKF-PD in preventing fibrosis in the folic acid nephropathy model. Besides, the mechanisms of action of AKF-PD in preventing renal fibrosis are not fully understood. In the study, we observed that AKF-PD reduced folate-induced kidney injury, ameliorated the deterioration of renal function, and suppressed the deposition of ECM by decreasing the expression of collagen I, collagen III, transforming growth factor-β (TGF-β), fibronectin (FN), and alpha smooth muscle actin (α-SMA) in the folic acid nephropathy model. Additionally, AKF-PD suppressed the activation of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome to reduce the production of caspase-1 and IL-1β, and alleviated mitochondrial oxidative damage by promoting mitochondrial energy metabolism and reducing the expression of NADPH oxidase 4 (NOX4). The results of in vitro experiments demonstrated that AKF-PD suppressed NLRP3 inflammasome activation in activated peritoneal-derived macrophages (PDMs) and renal tubular epithelial cells (RTECs). AKF-PD increased the intracellular ATP content and decreased the expression of NOX4, while preventing the excessive production of mitochondrial reactive oxygen species (mtROS) in activated PDMs. In conclusion, this study demonstrated that AKF-PD inhibited renal fibrosis by suppressing the mtROS-NLRP3 pathway in the folic acid nephropathy model. These findings provide new evidence in support of the clinical use of AKF-PD in the treatment of diseases related to renal fibrosis.

In this study, high-throughput sequencing methods were used to analyze the composition and diversity of the microbial communities of three different traditional fermented blueberry beverages (Jiaosu A, Jiaosu B, and Jiaosu C) produced in three different regions. Lactic acid bacteria and yeast counts, total soluble solids, total titration acid, total phenols, total flavonoids, total anthocyanin, superoxide dismutase, and antioxidant activity were analyzed in all samples. The results showed that at the phylum level, the bacteria in all samples were predominantly Firmicutes and Proteobacteria, while the majority of fungus belonged to Ascomycota. At the genus level, Lactobacillus, Gluconobacter, and Acetobacter were the dominant bacteria, and Dekkera and Issatchenkia were the dominant fungi. Our data show that the lactic acid bacteria counts in Jiaosu A were the lowest of the three products, in the range of 4.31-10.9 log CFU/mL, while yeast counts ranged from 6.71 to 7.35 log CFU/mL. The antioxidant activities of Jiaosu C were greater than those of Jiaosu A and Jiaosu B, and Spearman correlation analysis showed that the relative abundance of Lactobacillus and Dekkera was significantly positively correlated with total phenolics, total anthocyanin, total flavonoids, and antioxidant index.

The identification and functional characterization of natural variants in plants are essential for understanding phenotypic adaptation. Here we identify a molecular variation in At2g47310 that contributes to the natural variation in flowering time in Arabidopsis thaliana accessions. This gene, which we term SISTER of FCA (SSF), functions in an antagonistic manner to its close homolog FCA. Genome-wide association analysis screens two major haplotypes of SSF associated with the natural variation in FLC expression, and a single polymorphism, SSF-N414D, is identified as a main contributor. The SSF414N protein variant interacts more strongly with CUL1, a component of the E3 ubiquitination complex, than the SSF414D form, mediating differences in SSF protein degradation and FLC expression. FCA and SSF appear to have arisen through gene duplication after dicot-monocot divergence, with the SSF-N414D polymorphism emerging relatively recently within A. thaliana. This work provides a good example for deciphering the functional importance of natural polymorphisms in different organisms.

The two-component response regulator Rrp2 is a key activator controlling the production of numerous virulence factors of Borrelia burgdorferi, the Lyme disease pathogen. Previously it was shown that the cognate histidine kinase HK2 is not required for Rrp2 activation in vitro, nor for mammalian infection upon needle inoculation, raising the question whether HK2 has any role in the enzootic cycle of B. burgdorferi. In this study, we demonstrated that HK2 is not required for spirochetal survival in the tick vector. When fed on naive mice, the hk2 mutant had reduced infectivity through the route of tick bite, suggesting that the spirochetes lacking HK2 had a disadvantage in the enzootic cycle. Furthermore, overexpression of hk2 reduced the level of Rrp2 phosphorylation, suggesting that HK2 can function as a phosphatase to dephosphorylate Rrp2. Strains overexpressing hk2 impaired the expression of RpoN regulon whose activation is dependent on Rrp2 phosphorylation and activation, and had reduced infectivity in mice. Taken together, these results demonstrate that although HK2 does not play an essential role in Rrp2 activation, it is important for the optimal fitness of B. burgdorferi in the enzootic cycle.

Insights into the interaction between phages and their bacterial hosts are crucial for the development of phage therapy. However, only one study has investigated global gene expression of Clostridioides (formerly Clostridium) difficile carrying prophage, and transcriptional reprogramming during lytic infection has not been studied. Here, we presented the isolation, propagation, and characterization of a newly discovered 35,109-bp phage, JD032, and investigated the global transcriptomes of both JD032 and C. difficile ribotype 078 (RT078) strain TW11 during JD032 infection. Transcriptome sequencing (RNA-seq) revealed the progressive replacement of bacterial host mRNA with phage transcripts. The expressed genes of JD032 were clustered into early, middle, and late temporal categories that were functionally similar. Specifically, a gene (JD032_orf016) involved in the lysis-lysogeny decision was identified as an early expression gene. Only 17.7% (668/3,781) of the host genes were differentially expressed, and more genes were downregulated than upregulated. The expression of genes involved in host macromolecular synthesis (DNA/RNA/proteins) was altered by JD032 at the level of transcription. In particular, the expression of the ropA operon was downregulated. Most noteworthy is that the gene expression of some antiphage systems, including CRISPR-Cas, restriction-modification, and toxin-antitoxin systems, was suppressed by JD032 during infection. In addition, bacterial sporulation, adhesion, and virulence factor genes were significantly downregulated. This study provides the first description of the interaction between anaerobic spore-forming bacteria and phages during lytic infection and highlights new aspects of C. difficile phage-host interactions.IMPORTANCE C. difficile is one of the most clinically significant intestinal pathogens. Although phages have been shown to effectively control C. difficile infection, the host responses to phage predation have not been fully studied. In this study, we reported the isolation and characterization of a new phage, JD032, and analyzed the global transcriptomic changes in the hypervirulent RT078 C. difficile strain, TW11, during phage JD032 infection. We found that bacterial host mRNA was progressively replaced with phage transcripts, three temporal categories of JD032 gene expression, the extensive interplay between phage-bacterium, antiphage-like responses of the host and phage evasion, and decreased expression of sporulation- and virulence-related genes of the host after phage infection. These findings confirmed the complexity of interactions between C. difficile and phages and suggest that phages undergoing a lytic cycle may also cause different phenotypes in hosts, similar to prophages, which may inspire phage therapy for the control of C. difficile.

Inhibition of transforming growth factor β (TGF-β) type 1 receptor (ALK5) provides a feasible approach for the treatment of fibrotic diseases and malignant tumors. In this study, we designed and synthesized a new series of 4-(pyridin-4-oxy)-3-(3,3-difluorocyclobutyl)-pyrazole derivatives, and evaluated biologically as TGF-β type 1 receptor inhibitors. The most potent compound 15r inhibited the ALK5 enzyme and NIH3T3 cell viability with IC50 values of 44 and 42.5 nM, respectively. Compound 15r also displayed better oral plasma exposure and excellent bioavailability than LY-3200882, and in vivo inhibited 65.7% of the tumor growth in a CT26 xenograft mouse model.

Gamma-aminobutyric acid (GABA) is an important metabolite which functions in plant growth, development, and stress responses. However, its role in plant defense and how it is regulated are largely unknown. Here, we report a detailed analysis of GABA induction during the resistance response to Pseudomonas syringae in Arabidopsis thaliana. While searching for the mechanism underlying the pathogen-responsive mitogen-activated protein kinase (MPK)3/MPK6 signaling cascade in plant immunity, we found that activation of MPK3/MPK6 greatly induced GABA biosynthesis, which is dependent on the glutamate decarboxylase genes GAD1 and GAD4. Inoculation with Pseudomonas syringae pv tomato DC3000 (Pst) and Pst-avrRpt2 expressing the avrRpt2 effector gene induced GAD1 and GAD4 gene expression and increased the levels of GABA. Genetic evidence revealed that GAD1, GAD2, and GAD4 play important roles in both GABA biosynthesis and plant resistance in response to Pst-avrRpt2 infection. The gad1/2/4 triple and gad1/2/4/5 quadruple mutants, in which the GABA levels were extremely low, were more susceptible to both Pst and Pst-avrRpt2. Functional loss of MPK3/MPK6, or their upstream MKK4/MKK5, or their downstream substrate WRKY33 suppressed the induction of GAD1 and GAD4 expression after Pst-avrRpt2 treatment. Our findings shed light on both the regulation and role of GABA in the plant immunity to a bacterial pathogen.

Plants have evolved genetic and physiological mechanisms to mitigate the adverse effects of high temperature. CARBOXYL TERMINUS OF THE HSC70-INTERACTING PROTEINS (CHIP) is a conserved chaperone-dependent ubiquitin E3 ligase that targets misfolded proteins. Here, we report functional analysis of the SlCHIP gene from tomato (Solanum lycopersicum) in heat tolerance. SlCHIP encodes a CHIP protein with three tandem tetracopeptide repeat (TPR) motifs and a C-terminal U box domain. Phylogenetic analysis of CHIP homologs from animals, spore-bearing and seed plants revealed a tree topology similar to the evolutionary tree of the organisms. Expression of SlCHIP was induced under high temperature and was also responsive to plant stress hormones. Silencing of SlCHIP in tomato reduced heat tolerance based on increased heat stress symptoms, reduced photosynthetic activity, elevated electrolyte leakage and accumulation of insoluble protein aggregates. The accumulated protein aggregates in SlCHIP-silenced plants were still highly ubiquitinated, suggesting involvement of other E3 ligases in ubiquitination. SlCHIP restored the heat tolerance of Arabidopsis chip mutant to the wild type levels. These results indicate that tomato SlCHIP plays a critical role in heat stress responses most likely by targeting degradation of misfolded proteins that are generated during heat stress.

The aim of this work was to characterize biogenic amines (BAs) in different parts of Lycium barbarum L. using HPLC with dansyl chloride derivatization, and jointly, to provide referential data for further exploration and utilization of Lycium barbarum L. The linear correlation coefficients for all BAs were above 0.9989. The limits of detection and quantification were 0.015-0.075 and 0.05-0.25 μg/mL, respectively. The relative standard deviations for the intra-day and inter-day precision were 0.66-2.69% and 0.91-4.38%. The described method has good repeatability and intermediate precision for the quantitative determination of BAs in different parts of Lycium barbarum L. Satisfactory recovery for all amines was obtained (79.3-110.3%). The result showed that there were four kinds of BAs. The highest putrescine content (20.9 ± 3.2 mg/kg) was found in the flower. The highest histamine content (102.7 ± 5.8 mg/kg) was detected in the bark, and the highest spermidine (13.3 ± 1.6 mg/kg) and spermine (23.7 ± 2.0 mg/kg) contents were detected in the young leaves. The high histamine (HIS) content in the bark may be one of the reasons why all of the parts of Lycium barbarum L., except the bark, are used for medicine or food in China. Meanwhile, the issue of the high concentration of HIS should be considered when exploiting or utilizing the bark of Lycium barbarum L.

Vascular smooth muscle cells (VSMCs) are involved in the pathogenesis of many human cardiovascular diseases. They modulate their phenotype from "contractile" to "synthetic" in response to changes in local environmental cues. How glutamine regulates the differentiation of VSMCs and the underlying mechanisms remain largely unknown.

MicroRNAs (miRNAs) are thought to be involved in the development of cisplatin (DDP) resistance in gastric cancer (GC). Using RNA sequencing analysis (RNA-seq), we found that miR-95-3p is associated with DDP resistance in GC. We discovered that miR-95-3p is highly expressed in DDP-resistant GC tissues and cell lines (SGC7901/DDP and AGS/DDP). Furthermore, results from the BrdU and MTT assays indicated that miR-95-3p promotes GC cell proliferation. Additionally, data from transwell chamber assay, wound healing test and in vivo experiments illustrated that miR-95-3p can effectively promote invasion, migration and tumorigenic capacity, respectively, of DDP-resistant GC cells. Subsequently, results from dual luciferase assay and qRT-PCR collectively indicated that EMP1 is a target of miR-95-3p with inhibitory function through suppression of the EMT process and drug-resistance proteins. Furthermore, PI3K/AKT was identified as a downstream pathway of miR-95-3p, which promotes DDP resistance in GC. In summary, miR-95-3p helped develop DDP-resistance through down-regulation of EMP1 and increasing phosphorylation of the PI3K/Akt pathway in GC.

Trans-regulating elements such as noncoding RNAs are crucial in modifying cells, and has shown broad application in synthetic biology, metabolic engineering and RNA therapies. Although effective, titration of the regulatory levels of such elements is less explored. Encouraged by the need of fine-tuning cellular functions, we studied key parameters of the antisense RNA design including oligonucleotide length, targeting region and relative dosage to achieve differentiated inhibition. We determined a 30-nucleotide configuration that renders efficient and robust inhibition. We found that by targeting the core RBS region proportionally, quantifiable inhibition levels can be rationally obtained. A mathematic model was established accordingly with refined energy terms and successfully validated by depicting the inhibition levels for genomic targets. Additionally, we applied this fine-tuning approach for 4-hydroxycoumarin biosynthesis by simultaneous and quantifiable knockdown of multiple targets, resulting in a 3.58-fold increase in titer of the engineered strain comparing to that of the non-regulated. We believe the developed tool is broadly compatible and provides an extra layer of control in modifying living systems.

Type 2 diabetes mellitus (T2DM) is recognized as a chronic, low-grade inflammatory disease characterized by insulin resistance and pancreatic β cell dysfunction; however, the underlying molecular mechanism remains unclear. Here, we report a key β cell-macrophage crosstalk pathway mediated by the miRNA-29-TNF-receptor-associated factor 3 (TRAF3) axis. β cell-specific transgenic miR-29a/b/c mice are predisposed to develop glucose intolerance and insulin resistance when fed a high-fat diet (HFD). The metabolic effect of β cell miR-29 is largely mediated through macrophages because either depletion of macrophages or reconstitution with miR-29-signaling defective bone marrow improves metabolic parameters in the transgenic mice. Mechanistically, our data show that miR-29 promotes the recruitment and activation of circulating monocytes and macrophages and, hence, inflammation, via miR-29 exosomes in a TRAF3-dependent manner. Our results demonstrate the ability of β cells to modulate the systemic inflammatory tone and glucose homeostasis via miR-29 in response to nutrient overload.

Cervical cancer is one most common cancer types among females over the world. While its underlying mechanisms remain unclear. Circ-CCDC66 has been revealed to participate in multiple biological functions, and contribute to various diseases' progression. In the current study, we aimed to demonstrate the role of circ-CCDC66 in cervical cancer progression.

The etiology between homocysteine and polycystic ovary syndrome (PCOS) is unclear. In humans, the level of homocysteine is mainly affected by two enzymes: methylene tetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR). While the activity of these two enzymes is mainly affected by three missense mutations, namely C677T (MTHFR), A1298C (MTHFR), and A66G (MTRR). This study aims to examine the association between the three missense mutations and PCOS and investigate whether the three missense mutations exerted their effect on PCOS by affecting the homocysteine level.

Scales are symbolic characteristic of Lepidoptera; however, nothing is known about the contribution of cuticular proteins (CPs) to the complex patterning of lepidopteran scales. This is because scales are resistant to solubilization, thus hindering molecular studies. Here we succeeded in dissolving developing wing scales from Bombyx mori, allowing analysis of their protein composition. We identified a distinctive class of histidine rich (His-rich) CPs (6%-45%) from developing lepidopteran scales by LC-MS/MS. Functional studies using RNAi revealed CPs with different histidine content play distinct and critical roles in constructing the microstructure of the scale surface. Moreover, we successfully synthesized films in vitro by crosslinking a 45% His-rich CP (BmorCPR152) with laccase2 using N-acetyl- dopamine or N-β-alanyl-dopamine as the substrate. This molecular study of scales provides fundamental information about how such a fine microstructure is constructed and insights into the potential application of CPs as new biomaterials.