Functional blood vessel growth depends on generation of distinct but coordinated responses from endothelial cells. Bone morphogenetic proteins (BMP), part of the TGFβ superfamily, bind receptors to induce phosphorylation and nuclear translocation of SMAD transcription factors (R-SMAD1/5/8) and regulate vessel growth. However, SMAD1/5/8 signalling results in both pro- and anti-angiogenic outputs, highlighting a poor understanding of the complexities of BMP signalling in the vasculature. Here we show that BMP6 and BMP2 ligands are pro-angiogenic in vitro and in vivo, and that lateral vessel branching requires threshold levels of R-SMAD phosphorylation. Endothelial cell responsiveness to these pro-angiogenic BMP ligands is regulated by Notch status and Notch sets responsiveness by regulating a cell-intrinsic BMP inhibitor, SMAD6, which affects BMP responses upstream of target gene expression. Thus, we reveal a paradigm for Notch-dependent regulation of angiogenesis: Notch regulates SMAD6 expression to affect BMP responsiveness of endothelial cells and new vessel branch formation.

Evidence of the prognostic role of serine peptidase inhibitor Kazal type 1 (SPINK1) in prostate cancer (PCa) is controversial. The aim of this study was, therefore, to evaluate the association between SPINK1 and clinical outcomes in PCa. Searches were made of PubMed, Medline, Embase, and the China Biology Medicine disc (CBMdisc) up to January 2017. The Newcastle-Ottawa Scale was used to assess the risk of bias of included studies. RevMan software was used to perform meta-analysis, and the Grading of Recommendations Assessment, Development and Evaluation (GRADE) method was employed for assessing the quality of the evidence. Ten studies with 17,161 patients were included in the analysis. Random-effect models were adopted for all outcomes with significant heterogeneities. In patients treated with radical prostatectomy, SPINK1 was associated with biochemical recurrence (BCR) (hazard ratio [HR] =1.41, 95% confidence interval [CI]: 1.01-1.97; P=0.04), but not PCa-specific mortality (HR =0.93, 95% CI: 0.33-2.57; P=0.88), and overall survival (OS) (HR =0.89, 95% CI: 0.58-1.35; P=0.57). In metastatic PCa, SPINK1 was significantly associated with castration-resistant PCa-free survival (HR =3.87, 95% CI: 1.87-8.00; P=0.0003) and OS (HR =2.59, 95% CI: 1.16-5.78; P=0.02). However, the quality of the evidence was very low for all study outcome measures. In conclusion, although SPINK1 was not a predictor of PCa mortality or OS among patients who underwent radical prostatectomy, it may have prognostic value in metastatic PCa.

In clinical practice, the detection of biomarkers is mostly based on primary tumors for its convenience in acquisition. However, immune checkpoints may express differently between primary and metastatic tumor. Therefore, we aimed to compare the differential expressions of PD-1, PD-L1 and PD-L2 between the primary and metastatic sites of renal cell carcinoma (RCC).

Direct reprogramming has revolutionized the fields of stem cell biology and regenerative medicine. However, the common mechanisms governing how reprogramming cells undergo transcriptome and epigenome remodeling (i.e., regulatome remodeling) have not been investigated. Here, by characterizing early changes in the regulatome of three different types of direct reprogramming, we identify lineage-specific features as well as common regulatory transcription factors. Of particular interest, we discover that the neuronal factor Ascl1 possesses cross-lineage potential; together with Mef2c, it drives efficient cardiac reprogramming toward a mature and induced cardiomyocyte phenotype. Through ChIP-seq and RNA-seq, we find that MEF2C drives the shift in ASCL1 binding away from neuronal genes toward cardiac genes, guiding their co-operative epigenetic and transcription activities. Together, these findings demonstrate the existence of common regulators of different direct reprogramming and argue against the premise that transcription factors possess only lineage-specific capabilities for altering cell fate - the basic premise used to develop direct reprogramming approaches.

Unraveling the gene regulatory networks that govern development and function of the mammalian heart is critical for the rational design of therapeutic interventions in human heart disease. Using the Drosophila heart as a platform for identifying novel gene interactions leading to heart disease, we found that the Rho-GTPase Cdc42 cooperates with the cardiac transcription factor Tinman/Nkx2-5. Compound Cdc42, tinman heterozygous mutant flies exhibited impaired cardiac output and altered myofibrillar architecture, and adult heart-specific interference with Cdc42 function is sufficient to cause these same defects. We also identified K(+) channels, encoded by dSUR and slowpoke, as potential effectors of the Cdc42-Tinman interaction. To determine whether a Cdc42-Nkx2-5 interaction is conserved in the mammalian heart, we examined compound heterozygous mutant mice and found conduction system and cardiac output defects. In exploring the mechanism of Nkx2-5 interaction with Cdc42, we demonstrated that mouse Cdc42 was a target of, and negatively regulated by miR-1, which itself was negatively regulated by Nkx2-5 in the mouse heart and by Tinman in the fly heart. We conclude that Cdc42 plays a conserved role in regulating heart function and is an indirect target of Tinman/Nkx2-5 via miR-1.

During heart formation, a network of transcription factors and signaling pathways guide cardiac cell fate and differentiation, but the genetic mechanisms orchestrating heart assembly and lumen formation remain unclear. Here, we show that the small GTPase Cdc42 is essential for Drosophila melanogaster heart morphogenesis and lumen formation. Cdc42 genetically interacts with the cardiogenic transcription factor tinman; with dDAAM which belongs to the family of actin organizing formins; and with zipper, which encodes nonmuscle myosin II. Zipper is required for heart lumen formation, and its spatiotemporal activity at the prospective luminal surface is controlled by Cdc42. Heart-specific expression of activated Cdc42, or the regulatory formins dDAAM and Diaphanous caused mislocalization of Zipper and induced ectopic heart lumina, as characterized by luminal markers such as the extracellular matrix protein Slit. Placement of Slit at the lumen surface depends on Cdc42 and formin function. Thus, Cdc42 and formins play pivotal roles in heart lumen formation through the spatiotemporal regulation of the actomyosin network.

Truncating mutations in the giant sarcomeric protein Titin result in dilated cardiomyopathy and skeletal myopathy. The most severely affected dilated cardiomyopathy patients harbor Titin truncations in the C-terminal two-thirds of the protein, suggesting that mutation position might influence disease mechanism. Using CRISPR/Cas9 technology, we generated six zebrafish lines with Titin truncations in the N-terminal and C-terminal regions. Although all exons were constitutive, C-terminal mutations caused severe myopathy whereas N-terminal mutations demonstrated mild phenotypes. Surprisingly, neither mutation type acted as a dominant negative. Instead, we found a conserved internal promoter at the precise position where divergence in disease severity occurs, with the resulting protein product partially rescuing N-terminal truncations. In addition to its clinical implications, our work may shed light on a long-standing mystery regarding the architecture of the sarcomere.

Bone marrow mesenchymal stem cells (MSCs) are among the most common cell types to be used and studied for cardiac regeneration. Low survival rate and difficult retention of delivered MSCs in infarcted heart remain as major challenges in the field. Co-delivery of stem cell-derived exosomes (Exo) is expected to improve the recruitment and survival of transplanted MSCs.

To define the functional roles of Grk1 and Grk7 in zebrafish cones in vivo.

A polylactide composite fracture fixator loaded with vancomycin cationic liposome (PLA@VL) was prepared by reverse evaporation method. The method of cationic liposome encapsulating vancomycin could effectively improve antibacterial property and achieve drug sustained release effect, so as to reduce toxicity of antibiotics in vivo. Scanning electron microscope (SEM) was used to observe morphology and Fourier transform infrared spectroscopy (FTIR) was used to detect the composition of the internal fixator. In vitro drug release model, in vitro degradation model and body fluid osteogenesis model were designed in this study. On the other hand, the experiments of inhibition zone and MC3T3-E1 osteoblasts in mice were conducted to explore antibacterial property, cell activity and adhesion of the PLA@VL composite internal fixator. Alkaline phosphatase (ALP) staining method and alizarin red assay were used to detect the osteogenic induction ability of the composite internal fixator. Finally, mice fracture models were established to verify osteogenic and anti-infection abilities of the composite internal fixator in vivo. The results showed that MC3T3-E1 cells had better adhesion and proliferation abilities on the PLA@VL composite internal fixator than on the PLA fixator, which indicated that the PLA@VL composite internal fixator possessed excellent osteogenic and anti-infection abilities both in vivo and in vitro. Therefore, the above experiments showed that the fracture internal fixator combined with vancomycin cationic liposome had better biocompatibility, antibacterial ability and osteogenic ability, which provides a promising anti-infection material for the clinical field of fracture.

Gastric cardia adenocarcinoma (GCA) is one of two main gastric cancer subtypes and has its own epidemiological, pathogenic and clinical characteristics. Genetic polymorphisms locating in a microRNA (miRNA) gene enhancer could transcriptionally regulates miRNA expression via impacting binding of transcriptional factors. It is still unclear how miR-1262 and a potential regulatory rs12740674 polymorphism mapping to a strong enhancer region of miR-1262 contribute to GCA development. We genotyped miR-1262 rs12740674 in two independent case-control sets consisting of 1,024 GCA patients and 1,118 controls, and found that the rs12740674 CT or TT genotype carriers had a 0.69-fold decreased risk to develop GCA compared to the CC genotype carriers (95% confidence interval=0.57-0.84, P=2.1×10-4). In the genotype-phenotype correlation analyses of 21 pairs of GCA-normal tissues, the rs12740674 CT or TT genotype was associated with significantly increased levels of miR-1262. Cell proliferation, wound healing and transwell assays elucidated that miR-1262 is a novel GCA tumor suppressor. Consistently, a significantly down-regulated level of miR-1262 exists in GCA specimens compared to normal tissues. Furthermore, multiple lines of evidences indicated that oncogene ULK1 is the target gene of miR-1262 in GCA. Our findings demonstrate miR-1262 transcriptionally modulated by an enhancer genetic variant suppresses GCA via targeting oncogene ULK1. Our data highlight miR-1262 as a promising diagnostic marker and therapeutic target for GCA.

Celastrol plays a significant role in cerebral ischemia-reperfusion injury. Although previous studies have confirmed that celastrol post-treatment has a protective effect on ischemic stroke, the therapeutic effect of celastrol on ischemic stroke and the underlying molecular mechanism remain unclear. In the present study, focal transient cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) in mice and celastrol was administered immediately after reperfusion. We performed lncRNA and mRNA analysis in the ischemic hemisphere of adult mice with celastrol post-treatment through RNA-Sequencing (RNA-Seq). A total of 50 differentially expressed lncRNAs (DE lncRNAs) and 696 differentially expressed mRNAs (DE mRNAs) were identified between the sham and tMCAO group, and a total of 544 DE lncRNAs and 324 DE mRNAs were identified between the tMCAO and tMCAO + celastrol group. Bioinformatic analysis was done on the identified deregulated genes through gene ontology (GO) analysis, KEGG pathway analysis and network analysis. Pathway analysis indicated that inflammation-related signaling pathways played vital roles in the treatment of ischemic stroke by celastrol. Four DE lncRNAs and 5 DE mRNAs were selected for further validation by qRT-PCR in brain tissue, primary neurons, primary astrocytes, and BV2 cells. The results of qRT-PCR suggested that most of selected differentially expressed genes showed the same fold change patterns as those in RNA-Seq results. Our study suggests celastrol treatment can effectively reduce cerebral ischemia-reperfusion injury. The bioinformatics analysis of lnRNAs and mRNAs profiles in the ischemic hemisphere of adult mice provides a new perspective in the neuroprotective effects of celastrol, particularly with regards to ischemic stroke.

Direct lineage conversion offers a new strategy for tissue regeneration and disease modelling. Despite recent success in directly reprogramming fibroblasts into various cell types, the precise changes that occur as fibroblasts progressively convert to the target cell fates remain unclear. The inherent heterogeneity and asynchronous nature of the reprogramming process renders it difficult to study this process using bulk genomic techniques. Here we used single-cell RNA sequencing to overcome this limitation and analysed global transcriptome changes at early stages during the reprogramming of mouse fibroblasts into induced cardiomyocytes (iCMs). Using unsupervised dimensionality reduction and clustering algorithms, we identified molecularly distinct subpopulations of cells during reprogramming. We also constructed routes of iCM formation, and delineated the relationship between cell proliferation and iCM induction. Further analysis of global gene expression changes during reprogramming revealed unexpected downregulation of factors involved in mRNA processing and splicing. Detailed functional analysis of the top candidate splicing factor, Ptbp1, revealed that it is a critical barrier for the acquisition of cardiomyocyte-specific splicing patterns in fibroblasts. Concomitantly, Ptbp1 depletion promoted cardiac transcriptome acquisition and increased iCM reprogramming efficiency. Additional quantitative analysis of our dataset revealed a strong correlation between the expression of each reprogramming factor and the progress of individual cells through the reprogramming process, and led to the discovery of new surface markers for the enrichment of iCMs. In summary, our single-cell transcriptomics approaches enabled us to reconstruct the reprogramming trajectory and to uncover intermediate cell populations, gene pathways and regulators involved in iCM induction.

TFE3-translocation renal cell carcinoma (TFE3-tRCC) is a rare and heterogeneous subtype of kidney cancer with no standard treatment for advanced disease. We describe comprehensive molecular characteristics of 63 untreated primary TFE3-tRCCs based on whole-exome and RNA sequencing. TFE3-tRCC is highly heterogeneous, both clinicopathologically and genotypically. ASPSCR1-TFE3 fusion and several somatic copy number alterations, including the loss of 22q, are associated with aggressive features and poor outcomes. Apart from tumors with MED15-TFE3 fusion, most TFE3-tRCCs exhibit low PD-L1 expression and low T-cell infiltration. Unsupervised transcriptomic analysis reveals five molecular clusters with distinct angiogenesis, stroma, proliferation and KRAS down signatures, which show association with fusion patterns and prognosis. In line with the aggressive nature, the high angiogenesis/stroma/proliferation cluster exclusively consists of tumors with ASPSCR1-TFE3 fusion. Here, we describe the genomic and transcriptomic features of TFE3-tRCC and provide insights into precision medicine for this disease.

Hepatic cysts are fluid-filled lesions in the liver that are estimated to occur in 5% of the population. They may cause hepatomegaly and abdominal pain. Progression to secondary fibrosis, cirrhosis, or cholangiocarcinoma can lead to morbidity and mortality. Previous studies of patients and rodent models have associated hepatic cyst formation with increased proliferation and fluid secretion in cholangiocytes, which are partially due to impaired primary cilia. Congenital hepatic cysts are thought to originate from faulty bile duct development, but the underlying mechanisms are not fully understood. In a forward genetic screen, we identified a zebrafish mutant that developed hepatic cysts during larval stages. The cyst formation was not due to changes in biliary cell proliferation, bile secretion, or impairment of primary cilia. Instead, time-lapse live imaging data showed that the mutant biliary cells failed to form interconnecting bile ducts because of defects in motility and protrusive activity. Accordingly, immunostaining revealed a disorganized actin and microtubule cytoskeleton in the mutant biliary cells. By whole-genome sequencing, we determined that the cystic phenotype in the mutant was caused by a missense mutation in the furinb gene, which encodes a proprotein convertase. The mutation altered Furinb localization and caused endoplasmic reticulum (ER) stress. The cystic phenotype could be suppressed by treatment with the ER stress inhibitor 4-phenylbutyric acid and exacerbated by treatment with the ER stress inducer tunicamycin. The mutant liver also exhibited increased mammalian target of rapamycin (mTOR) signaling. Treatment with mTOR inhibitors halted cyst formation at least partially through reducing ER stress. Conclusion: Our study has established a vertebrate model for studying hepatic cystogenesis and illustrated the contribution of ER stress in the disease pathogenesis.

The serine/threonine kinase, PINK1, and the E3 ubiquitin ligase, Parkin, are known to facilitate LC3-dependent autophagosomal encasement and lysosomal clearance of dysfunctional mitochondria, and defects in this process contribute to a variety of cardiometabolic and neurological diseases. Although recent evidence indicates that dynamic actin remodeling plays an important role in PINK1/Parkin-mediated mitochondrial autophagy (mitophagy), the underlying signaling mechanisms remain unknown. Here, we identify the RhoGAP GRAF1 (Arhgap26) as a PINK1 substrate that regulates mitophagy. GRAF1 promotes the release of damaged mitochondria from F-actin anchors, regulates mitochondrial-associated Arp2/3-mediated actin remodeling and facilitates Parkin-LC3 interactions to enhance mitochondria capture by autophagosomes. Graf1 phosphorylation on PINK1-dependent sites is dysregulated in human heart failure, and cardiomyocyte-restricted Graf1 depletion in mice blunts mitochondrial clearance and attenuates compensatory metabolic adaptations to stress. Overall, we identify GRAF1 as an enzyme that coordinates cytoskeletal and metabolic remodeling to promote cardioprotection.

Cardiac regeneration requires coordinated participation of multiple cell types whereby their communications result in transient activation of proregenerative cell states. Although the molecular characteristics and lineage origins of these activated cell states and their contribution to cardiac regeneration have been studied, the extracellular signaling and the intrinsic genetic program underlying the activation of the transient functional cell states remain largely unexplored. In this study, we delineated the chromatin landscapes of the noncardiomyocytes (nonCMs) of the regenerating heart at the single-cell level and inferred the cis-regulatory architectures and trans-acting factors that control cell type-specific gene expression programs. Moreover, further motif analysis and cell-specific genetic manipulations suggest that the macrophage-derived inflammatory signal tumor necrosis factor-α, acting via its downstream transcription factor complex activator protein-1, functions cooperatively with discrete transcription regulators to activate respective nonCM cell types critical for cardiac regeneration. Thus, our study defines the regulatory architectures and intercellular communication principles in zebrafish heart regeneration.

Porcine contagious pleuropneumonia (PCP) is a highly contagious disease that is caused by Actinobacillus pleuropneumoniae (APP) and characterized by severe fibrinous necrotizing hemorrhagic pleuropneumonia, which is a severe threat to the swine industry. In addition to APP RTX-toxins I (ApxI), APP RTX-toxin II (ApxII), APP RTX-toxin III (ApxIII) and Outer membrane protein (OMP), there may be other useful antigens that can contribute to protection. In the development of an efficacious vaccine against APP, the immunogenicities of multicomponent recombinant subunit vaccines were evaluated.

Interleukin-21 (IL-21), which belongs to IL-2 γ chain receptor cytokine family, is as an important regulator of immune responses. In this study, we developed a novel strategy for immunizing mice with a DNA/vaccinia/protein vaccine in the presence or absence of mouse IL-21 (mIL-21) to evaluate whether mIL-21 could enhance immune responses. Our results demonstrated that co-immunization with mIL-21 did not increase significantly the capacity of vaccine induced antibodies to bind to HIV-1 GP140. An effect of mIL-21 in adjusting the efficacy of HIV-1 vaccine through enhancing Th1 type immune response was however observed. The frequencies of HIV-1-specific cytokine-producing CD4+ T and CD4+ TEM cells, especially multifunctional T cell responses, were significantly increased by co-administrating with mIL-21. A significant increase was also observed in the frequency of NK cells in mIL-21 adjuvant groups. Taken together, combination of mIL-21 with HIV-1 vaccines led to distinct enhancement of NK cells and T cell immune responses associated with immune protection.

Cloning of multiple genes in a single vector has greatly facilitated both basic and translational studies that require co-expression of multiple factors or multi-units of complex protein. Many strategies have been adopted, among which 2A "self-cleaving" peptides have garnered increased interest for their polycistronic nature, small size and high "cleavage" efficiency. However, broad application of 2 A peptides is limited by the lack of systematic comparison of different 2As alone or in combination. Here we characterized the effect of varying gene position and 2As on the expression of proteins encoded in bi-, tri-, or quad-cistronic constructs. Using direct cardiac reprogramming as an example, we further determined the effect of varied 2As on the efficiency of fluorescent cell labeling and cell fate conversion. We found that the expression of fluorophores decreased as it was moved towards the end of the construct while reprogramming was most efficient with the fluorophore at the second position. Moreover, quad-cistronic TPE2A constructs resulted in more efficient reprogramming than 3P2A or PTE2A constructs. We expect that the bi-, tri-, and quad-cistronic vectors constructed here and our results on protein expression ratios from different 2A constructs could serve to guide future utilization of 2A peptides in basic research and clinical applications.