Primary biliary cholangitis (PBC) is an autoimmune liver disease with a strong hereditary component. Here, we report a genome-wide association study that included 1,122 PBC cases and 4,036 controls of Han Chinese descent, with subsequent replication in a separate cohort of 907 PBC cases and 2,127 controls. Our results show genome-wide association of 14 PBC risk loci including previously identified 6p21 (HLA-DRA and DPB1), 17q12 (ORMDL3), 3q13.33 (CD80), 2q32.3 (STAT1/STAT4), 3q25.33 (IL12A), 4q24 (NF-κB) and 22q13.1 (RPL3/SYNGR1). We also identified variants in IL21, IL21R, CD28/CTLA4/ICOS, CD58, ARID3A and IL16 as novel PBC risk loci. These new findings and histochemical studies showing enhanced expression of IL21 and IL21R in PBC livers (particularly in the hepatic portal tracks) support a disease mechanism in which the deregulation of the IL21 signalling pathway, in addition to CD4 T-cell activation and T-cell co-stimulation are critical components in the development of PBC.

Cerebral cavernous malformations (CCMs) are a cause of stroke and seizure for which no effective medical therapies yet exist. CCMs arise from the loss of an adaptor complex that negatively regulates MEKK3-KLF2/4 signalling in brain endothelial cells, but upstream activators of this disease pathway have yet to be identified. Here we identify endothelial Toll-like receptor 4 (TLR4) and the gut microbiome as critical stimulants of CCM formation. Activation of TLR4 by Gram-negative bacteria or lipopolysaccharide accelerates CCM formation, and genetic or pharmacologic blockade of TLR4 signalling prevents CCM formation in mice. Polymorphisms that increase expression of the TLR4 gene or the gene encoding its co-receptor CD14 are associated with higher CCM lesion burden in humans. Germ-free mice are protected from CCM formation, and a single course of antibiotics permanently alters CCM susceptibility in mice. These studies identify unexpected roles for the microbiome and innate immune signalling in the pathogenesis of a cerebrovascular disease, as well as strategies for its treatment.

Two-pore domain potassium (K2P) channels generate leak currents that are responsible for the maintenance of the resting membrane potential, and they are thus potential drug targets for treating diseases. Here, we identify N-(4-cholorphenyl)-N-(2-(3,4-dihydrosioquinolin-2(1H)-yl)-2-oxoethyl)methanesulfonamide (TKDC) as an inhibitor of the TREK subfamily, including TREK-1, TREK-2 and TRAAK channels. Using TKDC as a chemical probe, a study combining computations, mutagenesis and electrophysiology reveals a K2P allosteric ligand-binding site located in the extracellular cap of the channels. Molecular dynamics simulations suggest that ligand-induced allosteric conformational transitions lead to blockage of the ion conductive pathway. Using virtual screening approach, we identify other inhibitors targeting the extracellular allosteric ligand-binding site of these channels. Overall, our results suggest that the allosteric site at the extracellular cap of the K2P channels might be a promising drug target for these membrane proteins.TREKs are members of the two-pore domain potassium (K2P) channels, being important clinical targets. Here the authors identify inhibitors of K2P that bind to an allosteric site located in their extracellular cap, suggesting that it might be a promising drug target for these channels.

Nodulation competition is a key factor that limits symbiotic nitrogen fixation between rhizobia and their host legumes. Soybean root exudates (SREs) are thought to act as signals that influence Bradyrhizobium ability to colonize roots and to survive in the rhizosphere, and thus they act as a key determinant of nodulation competitiveness. In order to find the competitiveness-related genes in B. diazoefficiens, the transcriptome of two SREs treated B. diazoefficiens with completely different nodulation abilities (B. diazoefficiens 4534 and B. diazoefficiens 4222) were sequenced and compared. In SREs treated strain 4534 (SREs-4534), 253 unigenes were up-regulated and 204 unigenes were down-regulated. In SREs treated strain 4534 (SREs-4222), the numbers of up- and down-regulated unigenes were 108 and 185, respectively. There were considerable differences between the SREs-4534 and SREs-4222 gene expression profiles. Some differentially expressed genes are associated with a two-component system (i.g., nodW, phyR-σEcfG), bacterial chemotaxis (i.g., cheA, unigene04832), ABC transport proteins (i.g., unigene02212), IAA (indole-3-acetic acid) metabolism (i.g., nthA, nthB), and metabolic fitness (i.g., put.), which may explain the higher nodulation competitiveness of B. diazoefficiens in the rhizosphere. Our results provide a comprehensive transcriptomic resource for SREs treated B. diazoefficiens and will facilitate further studies on competitiveness-related genes in B. diazoefficiens.

The aim of this study was to analyze the correlation between high-intensity zone (HIZ) on magnetic resonance imaging (MRI) of a lumbar disc and positive pain response assessed by discography for the diagnosis and treatment of discogenic low back pain (LBP). Thirty-seven patients aged 21 to 59 years with chronic LBP but without any neurological symptoms or lumbar disc herniation who underwent MRI and subsequent discography were included in this study. During discography, concordant pain was regarded as positive, whereas discordant pain and no pain were regarded as negative. X-ray and computed tomography (CT) after discography with positive pain response were analyzed to correlate with HIZ on MRI. A total of 98 discs underwent discography in 37 patients; 21 discs presented positive pain response, including 10 with HIZ (47.6%). Seventy-seven discs presented negative pain response, including 29 with HIZ (37.6%). The high grade of annular disruption group shows a high proportion of HIZ on MRI. A positive correlation between HIZ and degree of annular disruption was observed. However, no correlation between HIZ and positive pain response was established on discography. The findings confirm that the presence of HIZ on MRI is only a suggestive and screening iconography indication for the diagnosis of discogenic LBP and cannot replace the gold standard of the discography. MRI should be closely integrated with those of discography, and thus, they play a crucial role in selecting operative segments of multilevel lumbar degenerative disk disease.

A relationship between hyperthyroidism and insulin secretion in type 2 diabetes mellitus (T2DM) has been reported. Therefore, this study explored the use of first-phase insulin secretion in the differential diagnosis of thyroid diabetes (TDM) and T2DM.

Myogenic differentiation, which occurs during muscle development, is a highly ordered process that can be regulated by E2F transcription factors. Available data show that E2F3b, but not E2F3a, is upregulated and required for myogenic differentiation. However, the regulation of E2F3b expression in myogenic differentiation is not well understood. To investigate whether E2Fb expression is controlled by miRNAs, we used bioinformatics to combine the database of microRNAs downregulated during myogenesis and those predicted to target E2F3. This identified miR-17 and miR-20a as miRNAs potentially involved in E2F3 regulation. We found that miR-17-92 controls the expression of E2F3b in C2C12 cells during myogenic differentiation. Moreover, we confirmed that miR-20a regulates the expression of E2F3b proteins in vivo using a muscle regeneration model.

Damage to the small intestine secondary to heat stroke is a major factor in heat stroke-related morbidity and mortality. However, the underlying mechanisms by which heat stroke causes small intestinal lesions and dysfunction remain unclear. To explore the pathogenesis of small intestinal tissue and epithelial cell injury, the SW480 cell heat stress model and the mice heat stroke model were established to mimic heat stroke. Morphologic changes in intestinal tissue and increased TUNEL-positive index were induced by heat stress in vivo. Heat stress activated the lysosomal-mitochondrial apoptotic pathway in SW480 cells, increasing intracellular reactive oxygen species and causing lysosomal membrane permeabilization with subsequent release of cathepsin B to the cytosol, mitochondrial depolarization, and cytochrome C release to cytosol. An increase in the Bax/Bcl2 ratio, caspase-9 and caspase-3 were observed. N-Acetyl-L-Cysteine was shown to inhibit ROS generation, suppress permeabilization of lysosomal membranes, decrease levels of cathepsin B and cytochrome C in the cytosol, and inhibit Bax/Bcl2 ratio, caspase-9 and caspase-3 activity both in vitro and in vivo. Mitochondrial damage was alleviated when the models were pre-treated with CA-074 Me both in vitro and in vivo, decreasing cathepsin B and cytochrome C levels in the cytosol, Bax/Bcl2 ratio, caspase-9 and caspase-3 activity. In our models, heat stress-induced apoptosis of small intestinal tissue and epithelial cells through accumulation of ROS and activation of the lysosomal-mitochondrial apoptotic pathway involved the release of cathepsin B. These findings may offer potentially pharmaceutical targets and strategies to repair intestinal injury caused by heat stroke.

Tumor infiltrated type II (M2) macrophages promote tumorigenesis by suppressing immune clearance, promoting proliferation, and stimulating angiogenesis. Interestingly, macrophages were also found to enrich in small foci of altered hepatocytes containing liver tumor-initiating cells (TICs). However, whether and how TICs specifically recruit macrophages and the function of these macrophages in tumor initiation remain unknown due to technical difficulties. In this study, by generating genetically defined liver TICs, we demonstrate that TICs actively recruit M2 macrophages from as early as the single-cell stage. Elimination of TIC-associated macrophages (TICAMs) abolishes tumorigenesis in a manner dependent on the immune system. Mechanistically, activation of the Hippo pathway effector Yes-associated protein (YAP) underlies macrophage recruitment by TICs. These results demonstrate for the first time that macrophages play a decisive role in the survival of single TICs in vivo and provide a proof of principle for TIC elimination by targeting YAP or M2 macrophages.

CRISPR-Cpf1 is a newly identified CRISPR-Cas system, and Cpf1 was recently engineered as a molecular tool for targeted genome editing in mammalian cells. To test whether the engineered CRISPR-Cpf1 system could induce the production of rice mutants, we selected two genome targets in the OsPDS and OsBEL genes. Our results show that both targets could be efficiently mutated in transgenic rice plants using CRISPR-Cpf1. We found that pre-crRNAs with a full-length direct repeat sequence exhibited considerably increased efficiencies compared with mature crRNAs. In addition, the specificity and transmission of the mutation were investigated, and the behaviours of crRNA-Cpf1-induced plant targeted genome mutagenesis were assessed. Taken together, our results indicate that CRISPR-Cpf1 expression via stable transformation can efficiently generate specific and heritable targeted mutations in rice and thereby constitutes a novel and important approach to specific and precise plant genome editing.

Chronic allograft damage, defined by interstitial fibrosis and tubular atrophy (IF/TA), is a leading cause of allograft failure. Few effective therapeutic options are available to prevent the progression of IF/TA. We applied a meta-analysis approach on IF/TA molecular datasets in Gene Expression Omnibus to identify a robust 85-gene signature, which was used for computational drug repurposing analysis. Among the top ranked compounds predicted to be therapeutic for IF/TA were azathioprine, a drug to prevent acute rejection in renal transplantation, and kaempferol and esculetin, two drugs not previously described to have efficacy for IF/TA. We experimentally validated the anti-fibrosis effects of kaempferol and esculetin using renal tubular cells in vitro and in vivo in a mouse Unilateral Ureteric Obstruction (UUO) model. Kaempferol significantly attenuated TGF-β1-mediated profibrotic pathways in vitro and in vivo, while esculetin significantly inhibited Wnt/β-catenin pathway in vitro and in vivo. Histology confirmed significantly abrogated fibrosis by kaempferol and esculetin in vivo. We developed an integrative computational framework to identify kaempferol and esculetin as putatively novel therapies for IF/TA and provided experimental evidence for their therapeutic activities in vitro and in vivo using preclinical models. The findings suggest that both drugs might serve as therapeutic options for IF/TA.

Skeletal muscle development is a complex biological process regulated by numerous genes and non-coding RNAs, such as microRNAs (miRNAs). In the current study, we made use of the deep sequencing data from Jianzhou Da'er goat longissimus dorsi sampled on days 45, 60, and 105 of gestation, as well as day three after birth to identify miRNAs that regulate goat skeletal myogenesis, and examine their temporal expression profiles. A total of 410 known goat miRNAs, 752 miRNA homologs and 88 novel miRNAs were identified across four stages. Besides three myomiRs, the abundance of 17 miRNAs, including chi-miR-424, chi-miR-542-3p and chi-miR-136-5p was more than 10,000 reads per million mapped reads (RPM), on average. Furthermore, 50 miRNAs with more than 100 RPM clustered at the imprinted DLK1-DIO3 locus on chromosome 21 and showed similar expression patterns, indicating that these miRNAs played important roles in skeletal myogenesis of goats. Based on pairwise comparisons, 221 differentially expressed (DE), known miRNAs were identified across four stages. GO and KEGG analyses of the genes targeted by the DE miRNAs revealed the significantly enriched processes and pathways to be consistent with temporal changes of skeletal muscle development across all sampled stages. However, follow-up experimental studies were required to explore functions of these miRNAs and targets underlying skeletal myogenesis.

Hypertension is a major risk factor for cardiovascular and cerebrovascular disease. Prenatal exposure to lipopolysaccharide (LPS) leads to hypertension in a rat offspring. However, the mechanism is still unclear. This study unraveled epigenetic mechanism for this and explored the protective effects of ascorbic acid against hypertension on prenatal inflammation-induced offspring. Prenatal LPS exposure resulted in an increase of intrarenal oxidative stress and enhanced angiotensin-converting enzyme 1 (ACE1) gene expression at the mRNA and protein levels in 6- and 12-week-old offspring, correlating with the augmentation of histone H3 acetylation (H3AC) on the ACE1 promoter. However, the prenatal ascorbic acid treatment decreased the LPS-induced expression of ACE1, protected against intrarenal oxidative stress, and reversed the altered histone modification on the ACE1 promoter, showing the protective effect in offspring of prenatal LPS stimulation. Our study demonstrates that ascorbic acid is able to prevent hypertension in offspring from prenatal inflammation exposure. Thus, ascorbic acid can be a new approach towards the prevention of fetal programming hypertension.

Glioma has become a significant global health problem with substantial morbidity and mortality, underscoring the importance of elucidating its underlying molecular mechanisms. Recent studies have identified mir-218 as an anti-oncogene; however, the specific functions of mir-218-1 and mir-218-2 remain unknown, especially the latter. The objective of this study was to further investigate the role of mir-218-2 in glioma. Our results indicated that mir-218-2 is highly overexpressed in glioma. Furthermore, we showed that mir-218-2 is positively correlated with the growth, invasion, migration, and drug susceptibility (to β-lapachone) of glioma cells. In vitro, the overexpression of mir-218-2 promoted glioma cell proliferation, invasion, and migration. In addition, the overexpression of mir-218-2 in vivo was found to increase glioma tumor growth. Accordingly, the inhibition of mir-218-2 resulted in the opposite effects. Cell division cycle 27 (CDC27), the downstream target of mir-218-2, is involved in the regulation of glioma cells. Our results indicate that the overexpression of CDC27 counteracted the function of mir-218-2 in glioma cells. These novel findings provide new insight in the application of mir-218-2 as a potential glioma treatment.

Fms-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase that normally functions in hematopoietic cell survival, proliferation and differentiation. Constitutively activating mutations of FLT3 map predominately to the juxtamembrane domain (internal tandem duplications; ITD) or the activation loop (AL) of the kinase domain and are detected in about 1/3 of de novo acute myeloid leukemia (AML) patients. Small molecule tyrosine kinase inhibitors (TKI) effectively target FLT3/ITD mutations, but some activating mutations, particularly those on the AL, are relatively resistant to many FLT3 TKI. We reproduced many of the AL or other non-ITD activating mutations and tested 13 FLT3 TKI for their activity against these and wild-type FLT3. All 13 TKI tested inhibited BaF3/ITD cell proliferation in a concentration-dependent manner as reported, but most TKI exhibited a wide range of differential activity against AL and other point mutants. Western blotting results examining inhibition of FLT3 autophosphorylation and signaling pathways indicate that many AL mutations reduce TKI binding. Most FLT3 TKI effectively target wild-type FLT3 signaling. As a demonstration of this differential activity, treatment of BaF3 D835Y cells transplanted in BALB/c mice with sorafenib showed no effect in vivo against this mutant whereas lestaurtinib proved effective at reducing disease burden. Thus, while FLT3 TKI have been selected based on their ability to inhibit FLT3/ITD, the selection of appropriate TKI for AML patients with FLT3 AL and other activating point mutations requires personalized consideration.

Staphylococcus phages of the Myoviridae family have a wide host range and potential applications in phage therapy. In this report, safety assessments of these phages were conducted based on their complete genome sequences. The complete genomes of Staphylococcus phages of the Myoviridae family were analyzed, and the Open Reading Frame (ORFs) were compared with a pool of virulence and antibiotic resistance genes using the BLAST algorithm. In addition, the lifestyle of the phages (virulent or temperate) was also confirmed using PHACTS. The results showed that all phages were lytic and did not contain resistance or virulence genes based on bioinformatic analyses, excluding the possibility that they could be vectors for the dissemination of these undesirable genes. These findings suggest that the phages are safe at the genome level. The SceD-like transglycosylase, which is a biomarker for vancomycin-intermediate strains, was widely distributed in the phage genomes. Approximately 70% of the ORFs encoded in the phage genomes have unknown functions; therefore, their roles in the antibiotic resistance and virulence of Staphylococcus aureus are still unknown and require consideration before use in phage therapy.

Ribosomal protein S3 (RPS3) is a 40S ribosomal protein of the S3P family essential for implementing protein translation. RPS3 has recently been found to interact with the p65 subunit of the NF-κB complex and promote p65 DNA-binding activity. Persistent activation of the NF-κB pathway is evident in allergic asthma. We hypothesized that gene silencing of lung RPS3 can ameliorate allergic airway inflammation.

Arterial stiffness contributes to heart failure and is decreased by angiotensin receptor blockers (ARBs). This cross-sectional study aimed to assess associations of lung function and ARB with arterial stiffness in patients with chronic heart failure. 354 outpatients (168 males; 186 females; 68.2 ± 7.2 years old) with chronic heart failure were evaluated. Lung function parameters, including forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), and FEV1 to FVC ratio (FEV1/FVC), were assessed. The cardio-ankle vascular index (CAVI) was used to estimate arterial stiffness. Unadjusted correlation analyses revealed a positive association of CAVI with ARB but not ACEI, and a negative correlation with FEV1 (r = -0.2987, p < 0.0001). Multiple stepwise regression analyses showed that ARB and FEV1 (p < 0.0001) were independent predicting factors for CAVI. These findings suggest that reduced pulmonary function is associated with increased CAVI. Pulmonary function protection could be used to improve the prognosis in heart failure, but additional studies are necessary.

miRNAs play a key role in the carcinogenesis of many cancers, including bladder cancer. In the current study, the role of miR-5195-3p, a quite recently discovered and poorly studied miRNA, in the proliferation and invasion of human bladder cancer cells was investigated. Our data displayed that, compared with healthy volunteers (control) and SU-HUC-1 normal human bladder epithelial cells, miR-5195-3p was sharply downregulated in bladder cancer patients and five human bladder cancer cell lines. The oligo miR-5195-3p mimic or miR-5195-3p antagomir was subsequently transfected into both T24 and BIU-87 bladder cancer cell lines. The miR-5195-3p mimic robustly increased the miR-5195-3p expression level and distinctly reduced the proliferation and invasion of T24 and BIU-87 cells. In contrast, the miR-5195-3p antagomir had an opposite effect on miR-5195-3p expression, cell proliferation, and invasion. Our data from bioinformatic and luciferase reporter gene assays identified that miR-5195-3p targeted the mRNA 3'-UTR of Krüppel-like factor 5 (KLF5), which is a proven proto-oncogene in bladder cancer. miR-5195-3p sharply reduced KLF5 expression and suppressed the expression or activation of its several downstream genes that are kinases improving cell survival or promoting cell cycle regulators, including ERK1/2, VEGFA, and cyclin D1. In conclusion, miR-5195-3p suppressed proliferation and invasion of human bladder cancer cells via suppression of KLF5.

Acute myeloid leukemia (AML) is a disease of the hematopoietic progenitor cells associated with heterogeneous clonal proliferation. Vascular endothelial growth factor (VEGF) and its receptors play important roles in the regulation of angiogenesis during physiological and pathological processes. It is thought that AML cells have an autocrine VEGF pathway that contributes to the development and progression of AML. In addition, growing evidence has suggested that numerous microRNAs are involved in AML. The present study aimed to investigate the relationship between VEGF dysregulation and microRNA profiles in AML cells and patients. VEGF-overexpressing and VEGF-knockdown leukemia cells were constructed and changes in the patterns of microRNA expression were analyzed using a microRNA array. Subsequently, mononuclear cells from the blood of patients with AML showing high or low expression levels of VEGF were obtained and were used to assess the patterns of microRNA expression by reverse transcription-quantitative polymerase chain reaction. The results of the present study suggested that downregulation of VEGF markedly altered the profile of microRNAs in AML cells, while upregulation of VEGF did not. Examination of clinical samples from patients with AML showed that several microRNAs were closely associated with the expression level of VEGF, including miR-20a, miR-93, miR-16-5p, miR-17-5p, miR-124-5p and miR-17-3p. These results suggested that VEGF may be a pivotal protein that can both receive and initiate signals in leukemia cells.