Tolls and Toll-like receptors (TLRs), as innate immune-recognition receptors that recognize molecular patterns associated with microbial pathogens, play a critical role in antimicrobial immune responses. Here, we report on single nucleotide polymorphisms (SNPs) of Litopenaeus vannamei Toll3 (LvToll3). Multiple sequence alignment of the L. vannamei Toll3 Leucine rich repeat C-terminal domain (LvToll3-LRR-CT) with other L. vannamei Tolls LRR-CT domains showed 39.23% - 43.96% homology at the nucleic acid level and 20.31% - 30.00% identity at the amino acid level. Analysis of different shrimp tissues by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) revealed that LvToll3-LRR-CT had genetic polymorphisms at both the genomic deoxyribonucleic acid (gDNA) and complementary deoxyribonucleic acid (cDNA) levels. Further, high-throughput sequencing analysis confirmed the presence of 8 non-synonymous SNP (nsSNP) and 1 nsSNPs with frequency greater than 1% at the gDNA level, while 13 nsSNPs and 2 nsSNPs with frequency greater than 1% at the cDNA level. In silico analysis revealed that the α-helix secondary structure and tertiary structure of LvToll3 changed when 3 SNPs (C2039T, T2041C, T2228C) were mutated. Interestingly, 2 novel bands on PCR-DGGE, which were identified as 2 nsSNPs (C2140A, T2186A) were observed following challenge with Streptococcus iniae but not with Vibrio parahaemolyticus or White spot syndrome virus (WSSV). Moreover, the secondary and tertiary structures of LvToll3 changed when the nsSNP T2186A was mutated. The present findings therefore provide novel insight into the molecular basis of shrimp innate immune response to pathogens through the generation of specific SNPs.

Protein extracts obtained from cells or tissues often require removal of interfering substances for the preparation of high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples. TCA/acetone precipitation and phenol extraction, a common method of protein extraction, is thought to minimize protein degradation and activity of proteases as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation method relies on the complete pulverization and repeated rinsing of tissue powder to remove the interfering substances, which is laborious and time-consuming. In addition, by prolonged incubation in TCA/acetone, the precipitated proteins are more difficult to re-dissolve. We have described a modified method of TCA/acetone precipitation of plant proteins for proteomic analysis. Proteins of cells or tissues were extracted using SDS-containing buffer, precipitated with equal volume of 20% TCA/acetone, and washed with acetone. Compared to classical TCA/acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. The modified TCA/acetone precipitation method is simple, fast, and suitable for proteomic analysis of various plant tissues in proteomic analysis.

Prasugrel as a second generation P2Y12 adenosine diphosphate receptor antagonist which in the cerebral aneurysms with Endovascular treatment have become more emphasized.

The CRISPR-Cas9-based multiplexed gene editing (MGE) provides a powerful method to modify multiple genomic regions simultaneously controlling different agronomic traits in crops. We applied the MGE construct built by combining the tandemly arrayed tRNA-gRNA units to generate heritable mutations in the TaGW2, TaLpx-1, and TaMLO genes of hexaploid wheat. The knockout mutations generated by this construct in all three homoeologous copies of one of the target genes, TaGW2, resulted in a substantial increase in seed size and thousand grain weight. We showed that the non-modified gRNA targets in the early generation plants can be edited by CRISPR-Cas9 in the following generations. Our results demonstrate that transgenerational gene editing activity can serve as the source of novel variation in the progeny of CRISPR-Cas9-expressing plants and suggest that the Cas9-inducible trait transfer for crop improvement can be achieved by crossing the plants expressing the gene editing constructs with the lines of interest.

A growing number of studies suggest that synovitis plays an important role in the pathogenesis and progression of osteoarthritis (OA). As a negative mediator of the nuclear factor-kappa B (NF-κB) signaling pathway, the zinc finger protein A20 has significant anti-inflammatory properties. In this study, the differential expression of A20 was investigated at the mRNA and protein levels in human normal OA fibroblast-like synoviocytes (FLSs) and normal FLSs pretreated with TNF-α. We then measured the activation of the NF-κB pathway and expression of pro-inflammatory cytokines in the above three groups by western blotting, a human cytokine array and ELISA. We found that TNF-α activated the NF-κB pathway, increased the expression of the pro-inflammatory cytokines IL-6 and IL-8, and A20 expression in human normal FLSs. However, the role of A20 in FLSs was unclear. To clarify this, we investigated the effect of A20 overexpression in human normal FLSs. The results indicate that A20 inhibits the NF-κB signaling pathway activation and OA-associated pro-inflammatory cytokines release. The results of this study indicate that A20 has anti-inflammatory effects in FLSs, which makes it a potential target for OA synovitis treatment.

Polycystic ovary syndrome (PCOS) is linked to hyperinsulinemia and insulin resistance with dysfunctional glucose metabolism. Pilot studies suggests that acupuncture treatment with combined manual and low-frequency electrical stimulation (electroacupuncture (EA)) of the needles decrease circulating glycated haemoglobulin (HbA1c) and homeostatic model assessment-insulin resistance. Therefore, we here aim to investigate if acupuncture treatment or metformin together with lifestyle or lifestyle management alone improves insulin sensitivity and related symptoms in overweight/obese women with PCOS.

The HOTAIR transcript is transcribed from the antisense strand within the HOXC gene cluster, and it is thought to play a role in regulating the inductive capacity of dermal papilla cells during the reconstruction of hair-follicle. In the current investigation, we firstly isolated and characterized a lncRNA-HOTAIR transcript from the secondary hair follicle of cashmere goat. Also, we analyzed its transcriptional pattern and methylation level of HOTAIR gene promoter in secondary hair follicle of cashmere goat during anagen and telogen stages. Nucleotide composition analysis indicated that the contents of Adenine (A) and Thymine (T) are higher than that of Guanine (G) and Cytosine (C) in lncRNA-HOTAIR transcript of cashmere goat with the highest frequency distribution of AG nucleotide pair (8.06%). The regulatory network analysis showed a directly or indirectly complex regulatory relationships between lncRNA-HOTAIR of cashmere goat and its potential target molecules: miRNAs, mRNAs and proteins. Also, we showed that lncRNA-HOTAIR was properly transcribed at both anagen and telogen stages of secondary hair follicle of cashmere goat with the anagen being significantly higher than telogen in its expression, which suggest that lncRNA-HOTAIR transcript might be involved in the reconstruction of secondary hair follicle with the formation and growth of cashmere fiber. Taken together with methylation analysis of HOTAIR gene promoter, our data suggest that the promoter methylation of HOTAIR gene most likely is involved in its transcriptional suppression in secondary hair follicle of cashmere goat.

Identification of seed development regulatory genes is the key for the genetic improvement in rice grain quality. NF-Ys are the important transcription factors, but their roles in rice grain quality control and the underlying molecular mechanism remain largely unknown. Here, we report the functional characterization a rice NF-Y heterotrimer complex NF-YB1-YC12-bHLH144, which is formed by the binding of NF-YB1 to NF-YC12 and then bHLH144 in a sequential order. Knock-out of each of the complex genes resulted in alteration of grain qualities in all the mutants as well as reduced grain size in crnf-yb1 and crnf-yc12. RNA-seq analysis identified 1496 genes that were commonly regulated by NF-YB1 and NF-YC12, including the key granule-bound starch synthase gene Wx. NF-YC12 and bHLH144 maintain NF-YB1 stability from the degradation mediated by ubiquitin/26S proteasome, while NF-YB1 directly binds to the 'G-box' domain of Wx promoter and activates Wx transcription, hence to regulate rice grain quality. Finally, we revealed a novel grain quality regulatory pathway controlled by NF-YB1-YC12-bHLH144 complex, which has great potential for rice genetic improvement.

Accumulating evidence indicates that miRNAs can be promising diagnostic and/or prognostic markers for various cancers. In this study, we identified a novel miRNA, miR-3607-3p, and its targets in non-small cell lung cancer (NSCLC). The expression of miR-3607-3p was measured and its correlation with patient prognosis was determined. Ectopic expression in NSCLC cells, xenografts, and metastasis models was used to evaluate the effects of miR-3607-3p on proliferation and migration of NSCLC. Luciferase assay and western blotting were performed to validate the potential targets of miR-3607-3p after preliminary screening by microarray analysis and computer-aided algorithms. We demonstrated that miR-3607-3p was downregulated in NSCLC tissues and that miR-3607-3p might act as an independent predictor for overall survival in NSCLC. Moreover, serum miR-3607-3p may be a novel and stable marker for NSCLC. We found that overexpression of miR-3607-3p inhibited cell proliferation, colony formation, migration and invasion, and hampered the cell cycle of NSCLC cell lines in vitro. Our results suggested that miR-3607-3p directly targets TGFBR1 and CCNE2. In accordance with in vitro studies, we confirmed that miR-3607-3p functions as a potent suppressor miRNA of NSCLC. We showed that miR-3607-3p agomir could reduce tumor growth and inhibit TGFBR1 and CCNE2 protein expression. Taken together, our findings indicate that miR-3607-3p can inhibit NSCLC cell growth and metastasis by targeting TGFBR1 and CCNE2 protein expression, and provide new evidence of miR-3607-3p as a potential non-invasive biomarker and therapeutic target for NSCLC.

Prognostic and predictive markers are needed to predict the clinical outcomes of patients with advanced colorectal cancer (CRC) who receive standard first-line treatments. We performed a prospective cohort study in advanced CRC patients to identify a miRNA signature that could predict the benefit of receiving first-line chemotherapy for these patients. Twenty-one paired tumours and adjacent normal tissues were collected from advanced CRC patients and analysed by miRNA microarrays. Between tumour and normal tissues, 33 miRNAs were differentially expressed and was confirmed by qRT-PCR from another group of 67 patients from a prospective cohort study. A two-miRNA-based signature was obtained using the LASSO Cox regression model based on the association between the expression of each miRNA and the PFS of individual patients. Internal and external validation cohorts, including 40 and 44 patients with advanced CRC, respectively, were performed to prove the prognostic and predictive value of this signature. A signature was built based on two miRNAs, miR-125b-2-3p and miR-933. CRC patients were classified into low- and high-risk groups for disease progression based on this tool. The patients with low risk scores generally had better PFS than those with high risk scores. In the training set, the median PFS in the low- and high-risk groups were 12.00 and 7.40 months, respectively. In the internal validation set, the median PFS in the low- and high-risk groups were 9.90 and 5.10 months, respectively. In the external validation set, the median PFS in the low- and high-risk groups were 9.90 and 6.40 months, respectively. Furthermore, we detected miR-125b-2-3p associated with CRC cell sensitivity to first-line chemotherapy. Our two-miRNA-based signature was a reliable prognostic and predictive tool for tumour progression in patients with advanced CRC, and might be able to predict the benefit of receiving standard first-line chemotherapy in CRC.

Gefitinib, a tyrosine kinase inhibitor of epidermal growth factor receptor, has been used as the first choice of treatment for advanced non-small-cell lung cancer. However, during the course of treatment, cancer cells often develop resistance to gefitinib without fully understood mechanisms. In this study, we aimed to elucidate an important role of long intergenic non-coding RNA 00665 in developing resistance to gefitinib in non-small-cell lung cancer. We showed that long intergenic non-coding RNA 00665 expression was significantly upregulated in lung cancer tissues and cells with acquired gefitinib resistance. Long intergenic non-coding RNA 00665 knockdown restored gefitinib sensitivity both in vitro and in vivo by suppressing cell proliferation and inducing apoptosis. Moreover, knockdown of long intergenic non-coding RNA 00665 markedly reduced activation of EGFR and its downstream event protein kinase B (AKT). Moreover, LINC00665 could interact with EZH2 and regulate the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. Thus, our study suggests that long intergenic non-coding RNA 00665 is important for non-small-cell lung cancer to develop drug resistance and might be a potential biomarker for drug resistance and a therapeutic target for non-small-cell lung cancer.

Sexual development in Flammulina velutipes is controlled by two different mating type loci (HD and PR). The HD locus contains homeodomain (Hd) genes on two separate HD subloci: HD-a and HD-b. While the functionality of the HD-b sublocus has been largely confirmed, the status and content of the HD-a sublocus has remained unclear.

How the human brain differs from those of non-human primates is largely unknown and the complex drivers underlying such differences at the genomic level remain unclear. In this study, we selected 243 brain-related genes, based on Gene Ontology, and identified 184,113 DNaseI hypersensitive sites (DHSs) within their regulatory regions. To performed comprehensive evolutionary analyses, we set strict filtering criteria for alignment quality and filtered 39,132 DHSs for inclusion in the investigation and found that 2,397 (~6%) exhibited evidence of accelerated evolution (aceDHSs), which was a much higher proportion that DHSs genome-wide. Target genes predicted to be regulated by brain-aceDHSs were functionally enriched for brain development and exhibited differential expression between human and chimpanzee. Alignments indicated 61 potential human-specific transcription factor binding sites in brain-aceDHSs, including for CTCF, FOXH1, and FOXQ1. Furthermore, based on GWAS, Hi-C, and eQTL data, 16 GWAS SNPs, and 82 eQTL SNPs were in brain-aceDHSs that regulate genes related to brain development or disease. Among these brain-aceDHSs, we confirmed that one enhanced the expression of GPR133, using CRISPR-Cas9 and western blotting. The GPR133 gene is associated with glioblastoma, indicating that SNPs within DHSs could be related to brain disorders. These findings suggest that brain-related gene regulatory regions are under adaptive evolution and contribute to the differential expression profiles among primates, providing new insights into the genetic basis of brain phenotypes or disorders between humans and other primates.

Angiogenic factor with G-patch and FHA domain 1 (AGGF1), as a newly identified human angiogenic factor, is overexpressed in some types of malignant tumors and closely associated with patient's prognosis. However, the mechanisms involved in the regulation of AGGF1 in gastric cancer (GC) still remain unclear.

Immunoglobulin A nephropathy (IgAN) is characterized by mesangial IgA and IgG co-deposition. As the clinical course of IgAN is highly variable, a lot of patients will eventually develop to end-stage renal disease (ESRD) within years. Hirudin, a potent and specific thrombin inhibitor, has been reported to treat IgAN with hematuria, but the mechanism is unclear. Our study aims to explore the potential of hirudin and the underlying mechanism in the treatment of IgAN. The establishment of IgAN model was set up in rats through oral and intravenous immunization with bovine gamma-globulin (BGG). Results suggested that hirudin could reduce the increased level of proteinuria, serum creatinine and urea nitrogen in IgAN models. Besides that, hirudin ameliorated the elevated number of apoptotic bodies and expressions of apoptosis-related proteins (caspase-3 and caspase-9) in IgAN model. The fibrosis indexes (transforming growth factor β-1 (TGF-β1), Collagen-IV (CoI-IV) and Fibronectin-1) of kidney were remarkably suppressed in IgAN rats treated with hirudin compared with IgAN rats with no further treatment. IgAN rats exhibited remarkably increased inflammatory factors (IL-1β, IL-6, and IL-18), while hirudin treatment significantly alleviated these alterations. Moreover, the reduced levels of CD4+CD25+Foxp3+ Treg and CD4+IFN-γ+ Th1/CD4+IL-4+ Th2 could be reversed by hirudin in IgAN model. Furthermore, in the process of IgAN, hirudin could inactivate various pathways (IκBα, NF-κB, TNF-α, and VCAM-1) compared with IgAN model group. Taken together, our study indicated that hirudin could ameliorate IgAN through suppressing fibrosis and inflammatory response. These findings provide a new therapeutic method to treat IgAN.

Cellulolytic enzymes produced by the filamentous fungus Trichoderma reesei are commonly used in biomass conversion. The high cost of cellulase is still a significant challenge to commercial biofuel production. Improving cellulase production in T. reesei for application in the cellulosic biorefinery setting is an urgent priority.

Neutrophil extracellular traps (NETs) are one of the most powerful and specific tools for neutrophils to clean up extracellular microbes, but the mechanisms of NETosis under infection are scarcely studied. In this study, by examining the neutrophils from human peripheral blood and mouse abdomen, we demonstrated that PRAK dysfunction resulted in a significantly reduced NET formation and elevated apoptotic cells. Furthermore, PRAK dysfunction could lead to impaired NET-mediated antibacterial activity and shorten the survival of mice with CLP-induced sepsis. Mechanism studies revealed that attenuated NET formation in PRAK dysfunctional neutrophils correlated with overproduction of reactive oxygen species (ROS), which triggered apoptosis through excessive autophagy. The imbalance of NET formation and apoptosis was further regulated by treatment with lower ROS in hypoxia. Here, we propose a novel candidate, PRAK, which can sense the oxidative stress and regulate the releasing of ROS, may be the master molecular switch to regulate the NETosis-apoptosis axis of neutrophils.

Most of the previous studies combined all types of intramedullary ependymomas without providing accurate pathological subtypes. In addition, it was very difficult to evaluate the factors associated with postoperative outcomes of patients with different pathological subtypes of intramedullary Grade II ependymomas by traditional meta-analysis. This study evaluated the factors related with postoperative outcomes of patients with intramedullary Grade II ependymomas.

The expression of programmed cell death ligand 1 (PD-L1) is a biomarker for immunotherapy, but approved detection method is absent in diffuse large B-cell lymphoma (DLBCL). Here, we performed three methods including immunohistochemistry (IHC) (clone SP263 and SP142), RNAscope, and fluorescence in situ hybridization (FISH) to evaluate PD-L1 status on a cohort of DLBCL including 94 of DLBCL-NOS, 25 of primary mediastinal large B-cell lymphoma (PMBCL) and 7 of double-hit lymphoma (DHL). SP263 with 25% for immune cell (IC) or combined cell and SP142 with 10% for tumor cell (TC), 20% for both of IC and combined cell were proved to have corresponding survival prognostic. Combined+ showed comparable prognostic value with TC+ and IC+ . SP263 and SP142 showed strong concordance (k = 0.788) with combined+ rates of 33.3% (42/126) and 34.9% (44/126), respectively. In DLBCL-NOS, TC+ by SP263 preferred to non-GCB and immunoblastic variant DLBCL-NOS (P = 0.029 and P = 0.004). Combined+ (SP263 and SP142) were associated with more than one extranodal site involved (P = 0.006, P = 0.042), higher ECOG PS scores (P = 0.001, P < 0.001), high IPI risk (P = 0.012, P = 0.005), and poor treatment response (P = 0.095, P = 0.002). IC+ by SP263 and SP142 were both independent risk factors (P = 0.027, P = 0.037). 9p24.1 locus amplification and gain were identified in 4.3% and 7.6% DLBCL-NOS and indicated shorter overall survival (P = 0.004). Positive rate of PD-L1 by RNAscope was 36.5%, while no clinical significance shown. PD-L1 positive rates were all higher in PMBCL and DHL than in DLBCL-NOS by SP263, SP142, RNAscope, and FISH (P = 0.001, P < 0.001, P = 0.005 and P < 0.001, respectively). In conclusion, combined PD-L1 expression by IHC was potentially reliable and convenient as a predicting biomarker. SP263 staining was easier to evaluate and recognized more PD-L1-stained cells, but SP142 presented a better prognostic indicator. FISH and RNAscope could be used as supplementary assays. PMBCL itself was a sensitive cohort for immunotherapy.

Laryngeal carcinoma is one of the most common malignant tumors of the head, neck, and respiratory tract. The aim of the present study is to explore the biological function of SRY-related HMG-box 18 (SOX18) in laryngeal carcinoma cells and study the molecular mechanism involved. Initial findings indicate that the expression of SOX18 was increased in laryngeal carcinoma cell lines and tissues. The effect of SOX18 on laryngeal carcinoma cell proliferation, cell cycle, apoptosis, invasion, and migration was also identified. The results indicated that down-regulation of SOX18 significantly inhibited cell proliferation, migration, and invasion, and induced cell-cycle arrest in G0/G1 phase and apoptosis of laryngeal carcinoma cells. However, overexpression of SOX18 promoted cell proliferation, invasion, and migration, and inhibited cell apoptosis. The expression of cyclin D1, active-caspase-3, N-cadherin, MTA1, MMP-2, and MMP-7 was also regulated by the overexpression of siSOX18 or SOX18. In addition, it was found that SOX18 could also accelerate the phosphorylation of JAK2/STAT3 signaling in laryngeal carcinoma cells. Furthermore, our study indicated that SOX18 could stimulate cell proliferation, migration, and invasion of laryngeal carcinoma cells via regulation of JAK2/STAT3 signaling, which could provide a new strategy for laryngeal carcinoma diagnosis and molecular therapies.