Transcriptomic mapping uncovers Purkinje neuron plasticity driving learning.

Cellular diversification is critical for specialized functions of the brain including learning and memory1. Single-cell RNA sequencing facilitates transcriptomic profiling of distinct major types of neuron2-4, but the divergence of transcriptomic profiles within a neuronal population and their link to function remain poorly understood. Here we isolate nuclei tagged5 in specific cell types followed by single-nucleus RNA sequencing to profile Purkinje neurons and map their responses to motor activity and learning. We find that two major subpopulations of Purkinje neurons, identified by expression of the genes Aldoc and Plcb4, bear distinct transcriptomic features. Plcb4+, but not Aldoc+, Purkinje neurons exhibit robust plasticity of gene expression in mice subjected to sensorimotor and learning experience. In vivo calcium imaging and optogenetic perturbation reveal that Plcb4+ Purkinje neurons have a crucial role in associative learning. Integrating single-nucleus RNA sequencing datasets with weighted gene co-expression network analysis uncovers a learning gene module that includes components of FGFR2 signalling in Plcb4+ Purkinje neurons. Knockout of Fgfr2 in Plcb4+ Purkinje neurons in mice using CRISPR disrupts motor learning. Our findings define how diversification of Purkinje neurons is linked to their responses in motor learning and provide a foundation for understanding their differential vulnerability to neurological disorders.

Developmental programming and lineage branching of early human telencephalon.

The dorsal and ventral human telencephalons contain different neuronal subtypes, including glutamatergic, GABAergic, and cholinergic neurons, and how these neurons are generated during early development is not well understood. Using scRNA-seq and stringent validations, we reveal here a developmental roadmap for human telencephalic neurons. Both dorsal and ventral telencephalic radial glial cells (RGs) differentiate into neurons via dividing intermediate progenitor cells (IPCs_div) and early postmitotic neuroblasts (eNBs). The transcription factor ASCL1 plays a key role in promoting fate transition from RGs to IPCs_div in both regions. RGs from the regionalized neuroectoderm show heterogeneity, with restricted glutamatergic, GABAergic, and cholinergic differentiation potencies. During neurogenesis, IPCs_div gradually exit the cell cycle and branch into sister eNBs to generate distinct neuronal subtypes. Our findings highlight a general RGs-IPCs_div-eNBs developmental scheme for human telencephalic progenitors and support that the major neuronal fates of human telencephalon are predetermined during dorsoventral regionalization with neuronal diversity being further shaped during neurogenesis and neural circuit integration.

Identification of a tumour immune barrier in the HCC microenvironment that determines the efficacy of immunotherapy.

BACKGROUND & AIMS: The tumour microenvironment (TME) is a crucial mediator of cancer progression and therapeutic outcome. The TME subtype correlates with patient response to immunotherapy in multiple cancers. Most previous studies have focused on the role of different cellular components in the TME associated with immunotherapy efficacy. However, the specific structure of the TME and its role in immunotherapy efficacy remain largely unknown. METHODS: We combined spatial transcriptomics with single-cell RNA-sequencing and multiplexed immunofluorescence to identify the specific spatial structures in the TME that determine the efficacy of immunotherapy in patients with hepatocellular carcinoma (HCC) receiving anti-PD-1 treatment. RESULTS: We identified a tumour immune barrier (TIB) structure, a spatial niche composed of SPP1+ macrophages and cancer-associated fibroblasts (CAFs) located near the tumour boundary, which is associated with the efficacy of immune checkpoint blockade. Furthermore, we dissected ligand‒receptor networks among malignant cells, SPP1+ macrophages, and CAFs; that is, the hypoxic microenvironment promotes SPP1 expression, and SPP1+ macrophages interact with CAFs to stimulate extracellular matrix remodelling and promote TIB structure formation, thereby limiting immune infiltration in the tumour core. Preclinically, the blockade of SPP1 or macrophage-specific deletion of Spp1 in mice led to enhanced efficacy of anti-PD-1 treatment in mouse liver cancer, accompanied by reduced CAF infiltration and increased cytotoxic T-cell infiltration. CONCLUSIONS: We identified that the TIB structure formed by the interaction of SPP1+ macrophages and CAFs is related to immunotherapy efficacy. Therefore, disruption of the TIB structure by blocking SPP1 may be considered a relevant therapeutic approach to enhance the therapeutic effect of immune checkpoint blockade in HCC. IMPACT AND IMPLICATIONS: Only a limited number of patients with hepatocellular carcinoma (HCC) benefit from tumour immunotherapy, which significantly hinders its application. Herein, we used multiomics to identify the spatial structure of the tumour immune barrier (TIB), which is formed by the interaction of SPP1+ macrophages and cancer-associated fibroblasts in the HCC microenvironment. This structure constrains immunotherapy efficacy by limiting immune cell infiltration into malignant regions. Preclinically, we revealed that blocking SPP1 or macrophage-specific deletion of Spp1 in mice could destroy the TIB structure and sensitize HCC cells to immunotherapy. These results provide the first key steps towards finding more effective therapies for HCC and have implications for physicians, scientists, and drug developers in the field of HCC.

Enhanced Cross-Reactive and Polyfunctional Effector-Memory T Cell Responses by ICVAX-a Human PD1-Based Bivalent HIV-1 Gag-p41 Mosaic DNA Vaccine.

Vaccine-induced protective T cell immunity is necessary for HIV-1 functional cure. We previously reported that rhesus PD1-Gag-based DNA vaccination sustained simian-human immunodeficiency virus (SHIV) suppression by inducing effector-memory CD8+ T cells. Here, we investigated a human PD1-Gag-based DNA vaccine, namely, ICVAX, for clinical translation. PD1-based dendritic cell targeting and mosaic antigenic designs were combined to generate the ICVAX by fusing the human soluble PD1 domain with a bivalent HIV-1 Gag-p41 mosaic antigen. The mosaic antigen was cross-reactive with patients infected with B, CRF07/08_BC, and CRF01_AE variants. In mice, ICVAX elicited stronger, broader, and more polyfunctional T cell responses than mosaic Gag-p41 alone, and suppressed EcoHIV infection more efficiently. In macaques, ICVAX elicited polyfunctional effector-memory T cell responses that targeted multiple nonoverlapping epitopes of the Gag-p41 antigen. Furthermore, ICVAX manufactured following good manufacturing practices proved potent immunogenicity in macaques after biannual homologous vaccination, warranting clinical evaluation of ICVAX as an immunotherapy against HIV-1. IMPORTANCE This study presents that ICVAX, a PD1-based DNA vaccine against HIV-1, could induce broad and polyfunctional T cell responses against different HIV-1 subtypes. ICVAX encodes a recombinant antigen consisting of the human soluble PD1 domain fused with two mosaic Gag-p41 antigens. The mosaic antigens cover more than 500 HIV-1 strains circulating in China including the subtypes B/B', CRF01_AE, and CRF07/08_BC. In mice, ICVAX elicited stronger, broader, and more polyfunctional T cell responses, with better EcoHIV suppression than the nontargeting mosaic Gag-p41 DNA vaccine. Moreover, both lab-generated and GMP-grade ICVAX also elicited strong polyfunctional effector-memory T cell responses in rhesus macaques with good immunogenicity against multiple nonoverlapping epitopes of the Gag-p41 antigen. This study therefore highlights the great potential to translate the PD1-based DNA vaccine approach into clinical use, and opens up new avenues for alternative HIV-1 vaccine design for HIV-1 preventive and functional cure.

Sustained viremia suppression by SHIVSF162P3CN-recalled effector-memory CD8+ T cells after PD1-based vaccination.

HIV-1 functional cure requires sustained viral suppression without antiretroviral therapy. While effector-memory CD8+ T lymphocytes are essential for viremia control, few vaccines elicit such cellular immunity that could be potently recalled upon viral infection. Here, we investigated a program death-1 (PD1)-based vaccine by fusion of simian immunodeficiency virus capsid antigen to soluble PD1. Homologous vaccinations suppressed setpoint viremia to undetectable levels in vaccinated macaques following a high-dose intravenous challenge by the pathogenic SHIVSF162P3CN. Poly-functional effector-memory CD8+ T cells were not only induced after vaccination, but were also recalled upon viral challenge for viremia control as determined by CD8 depletion. Vaccine-induced effector memory CD8+ subsets displayed high cytotoxicity-related genes by single-cell analysis. Vaccinees with sustained viremia suppression for over two years responded to boost vaccination without viral rebound. These results demonstrated that PD1-based vaccine-induced effector-memory CD8+ T cells were recalled by AIDS virus infection, providing a potential immunotherapy for functional cure.

Inhibition of TMEM16A improves cisplatin-induced acute kidney injury via preventing DRP1-mediated mitochondrial fission.

Acute kidney injury (AKI) is associated with high morbidity and mortality. Our previous study has demonstrated that TMEM16A, a Ca2+-activated chloride channel, contributes to renal fibrosis progression in chronic kidney disease. However, whether TMEM16A is involved in AKI is still unknown. In this study, we established cisplatin AKI mice model and found that TMEM16A expression was upregulated in the injured kidney. In vivo knockdown of TMEM16A effectively prevented cisplatin-induced tubular cell apoptosis, inflammation and kidney function loss. Western blot and transmission electron microscopy (TEM) revealed that TMEM16A knockdown inhibited Drp1 translocation from the cytoplasm to mitochondria and prevented mitochondrial fission in tubular cells. Consistently, in cultured HK2 cells, knockdown or inhibition of TMEM16A by shRNA or its specific inhibitor suppressed cisplatin-induced mitochondrial fission and its associated energy dysfunction, ROS accumulation, and cell apoptosis via inhibiting Drp1 activation. Further investigation showed that genetic knockdown or pharmacological inhibition of TMEM16A inhibited cisplatin-induced Drp1 Ser-616 site phosphorylation through ERK1/2 signaling pathway, whereas overexpression of TMEM16A promoted this effect. Treatment with Drp1 or ERK1/2 inhibitor could efficiently prevent cisplatin-induced mitochondrial fission. Collectively, our data suggest that TMEM16A inhibition alleviated cisplatin-induced AKI by preventing tubular cell mitochondrial fission through the ERK1/2 / Drp1 pathway. Inhibition of TMEM16A may be a novel therapeutic strategy for AKI.

Role of (pro)renin receptor in cyclosporin A-induced nephropathy.

Calcineurin inhibitors such as cyclosporin A (CsA) have been widely used to improve graft survival following solid-organ transplantation. However, the clinical use of CsA is often limited by its nephrotoxicity. The present study tested the hypothesis that activation of the (pro)renin receptor (PRR) contributes to CsA-induced nephropathy by activating the renin-angiotensin system (RAS). Renal injury in male Sprague-Dawley rats was induced by a low-salt diet combined with CsA as evidenced by elevated plasma creatinine and blood urea nitrogen levels, decreased creatinine clearance and induced renal inflammation, apoptosis and interstitial fibrosis, and elevated urinary N-acetyl-ß-d-glucosaminidase activity and urinary kidney injury molecule-1 content. Each index of renal injury was attenuated following 2 wk of treatment with the PRR decoy inhibitor PRO20. Although CsA-treated rats with kidney injury displayed increased renal soluble (s)PRR abundance, plasma sPRR, renin activity, angiotensin II, and heightened urinary total prorenin/renin content, RAS activation was attenuated by PRO20. Exposure of cultured human renal proximal tubular HK-2 cells to CsA induced expression of fibronectin and sPRR production, but the fibrotic response was attenuated by PRO20 and siRNA-mediated PRR knockdown. These findings support the hypothesis that activation of PRR contributes to CsA-induced nephropathy by activating the RAS in rats. Of importance, we provide strong proof of concept that targeting PRR offers a novel therapeutic strategy to limit nephrotoxic effects of immunosuppressant drugs.NEW & NOTEWORTHY The present study reports, for the first time, that activation of the (pro)renin receptor drives the renin-angiotensin system to induce renal injury during cyclosporin A administration. More importantly, our study has identified that antagonism with PRO20 offers a novel intervention in the management of side effects of cyclosporin A.

Cross-talk of four types of RNA modification writers defines tumor microenvironment and pharmacogenomic landscape in colorectal cancer.

BACKGROUND: The four major RNA adenosine modifications, i.e., m6A, m1A, alternative polyadenylation, and adenosine-to-inosine RNA editing, are mediated mostly by the "writer" enzymes and constitute critical mechanisms of epigenetic regulation in immune response and tumorigenesis. However, the cross-talk and potential roles of these "writers" in the tumor microenvironment (TME), drug sensitivity, and immunotherapy remain unknown. METHODS: We systematically characterized mRNA expression and genetic alterations of 26 RNA modification "writers" in colorectal cancer (CRC), and evaluated their expression pattern in 1697 CRC samples from 8 datasets. We used an unsupervised clustering method to assign the samples into two patterns of expression of RNA modification "writers". Subsequently, we constructed the RNA modification "writer" Score (WM_Score) model based on differentially expressed genes (DEGs) responsible for the RNA modification patterns to quantify the RNA modification-related subtypes of individual tumors. Furthermore, we performed association analysis for WM_Score and characteristics of TME, consensus molecular subtypes (CMSs), clinical features, transcriptional and post-transcriptional regulation, drug response, and the efficacy of immunotherapy. RESULTS: We demonstrated that multi-layer alterations of RNA modification "writer" are associated with patient survival and TME cell-infiltrating characteristics. We identified two distinct RNA modification patterns, characterized by a high and a low WM_Score. The WM_Score-high group was associated with worse patient overall survival and with the infiltration of inhibitory immune cells, such as M2 macrophages, EMT activation, and metastasis, while the WM_Score-low group was associated with a survival advantage, apoptosis, and cell cycle signaling pathways. WM_Score correlated highly with the regulation of transcription and post-transcriptional events contributing to the development of CRC. In response to anti-cancer drugs, WM_Score highly negatively correlated (drug sensitive) with drugs which targeted oncogenic related pathways, such as MAPK, EGFR, and mTOR signaling pathways, positively correlated (drug resistance) with drugs which targeted in apoptosis and cell cycle. Importantly, the WM_Score was associated with the therapeutic efficacy of PD-L1 blockade, suggesting that the development of potential drugs targeting these "writers" to aid the clinical benefits of immunotherapy. CONCLUSIONS: Our study is the first to provide a comprehensive analysis of four RNA modifications in CRC. We revealed the potential function of these writers in TME, transcriptional and post-transcriptional events, and identified their therapeutic liability in targeted therapy and immunotherapy. This work highlights the cross-talk and potential clinical utility of RNA modification "writers" in cancer therapy.

Severe fever with thrombocytopenia syndrome: a systematic review and meta-analysis of epidemiology, clinical signs, routine laboratory diagnosis, risk factors, and outcomes.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with the high case-fatality rate, and lack of vaccines. We aimed to systematically analysed the epidemiological characteristics, clinical signs, routine laboratory diagnosis, risk factors, and outcomes. METHODS: Documents on SFTS were collected by searching the Chinese National Knowledge Infrastructure, Wan Fang Data, PubMed, Embase, and Web of Science databases from 2011 to 2018. Meta-analysis was performed by using Review Manager and Stata software. RESULTS: Twenty-five articles involving 4143 cases were included. Diarrhea (odds ratio (OR) =1.60, 95% confidence interval (CI): 1.06 to 2.42, P = 0.02), and vomiting (OR = 1.56, 95% CI: 1.01 to 2.39, P = 0.04) on admission were associated with the fatal outcomes of SFTS. Compared to patients with mild symptoms, patients with severe symptoms had significantly elevated levels of lactic acid dehydrogenase (standard mean difference (SMD) =1.27, 95% CI: 0.59 to 1.94), alanine aminotransferase (SMD = 0.55, 95% CI: 0.24 to 0.85), aspirate aminotransferase (SMD = 1.01, 95% CI: 0.69 to 1.32), and creatine kinase (SMD = 1.04, 95% CI: 0.74 to 1.33) but had reduced platelet counts (SMD = -0.87, 95% CI: - 1.16 to - 0.58) and albumin levels (SMD = -1.00, 95% CI: - 1.32 to - 0.68). The risk factors for poor prognosis included age (mean difference (MD) =6.88, 95% CI: 5.41 to 8.35) and farming (OR = 2.01, 95% CI: 1.06 to 3.80). For the risk factors of contracting SFTS, the incidence of SFTS related to tick bites was 24% [95% CI: 0.18 to 0.31]. The pooled case-fatality rate of SFTS patients was 18% [95% CI: 0.16 to 0.21]. CONCLUSIONS: China is the country with the highest incidence of SFTS. May to July was the peak of the epidemic, and farmers were a high-risk group. The risk factor for SFTS included age (poor prognosis) and tick bites (contracting SFTS). Patients with severe diarrhea and vomiting symptoms on admission should be noted. Clinicians could use routine laboratory parameters and clinical symptoms as references for clinically suspected cases, classification of SFTS, and timely treatment, especially in basic hospitals.

Smooth muscle-specific TMEM16A expression protects against angiotensin II-induced cerebrovascular remodeling via suppressing extracellular matrix deposition.

Cerebrovascular remodeling is the leading factor for stroke and characterized by increased extracellular matrix deposition, migration and proliferation of vascular smooth muscle cells, and inhibition of their apoptosis. TMEM16A is an important component of Ca2+-activated Cl- channels. Previously, we showed that downregulation of TMEM16A in the basilar artery was negatively correlated with cerebrovascular remodeling during hypertension. However, it is unclear whether TMEM16A participates in angiotensin II (Ang II)-induced vascular remodeling in mice that have TMEM16A gene modification. In this study, we generated a transgenic mouse that overexpresses TMEM16A specifically in vascular smooth muscle cells. We observed that vascular remodeling in the basilar artery during Ang II-induced hypertension was significantly suppressed upon vascular smooth muscle-specific overexpression of TMEM16A relative to control mice. Specifically, we observed a large reduction in the deposition of fibronectin and collagen I. The expression of matrix metalloproteinases (MMP-2, MMP-9, and MMP-14), and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were upregulated in the basilar artery during Ang II-induced hypertension, but this was suppressed upon overexpression of TMEM16A in blood vessels. Furthermore, TMEM16A overexpression alleviated the overactivity of the canonical TGF-ß1/Smad3, and non-canonical TGF-ß1/ERK and JNK pathways in the basilar artery during Ang II-induced hypertension. These in vivo results were similar to the results derived in vitro with basilar artery smooth muscle cells stimulated by Ang II. Moreover, we observed that the inhibitory effect of TMEM16A on MMPs was mediated by decreasing the activation of WNK1, which is a Cl--sensitive serine/threonine kinase. In conclusion, this study demonstrates that TMEM16A protects against cerebrovascular remodeling during hypertension by suppressing extracellular matrix deposition. We also showed that TMEM16A exerts this effect by reducing the expression of MMPs via inhibiting WNK1, and decreasing the subsequent activities of TGF-ß1/Smad3, ERK, and JNK. Accordingly, our results suggest that TMEM16A may serve as a novel therapeutic target for vascular remodeling.

Surging HIV-1 CRF07_BC epidemic among recently infected men who have sex with men in Fujian, China.

A rapidly increasing number of HIV-1 infections have been identified among men who have sex with men (MSM) in Fujian province of China since 2010. We aimed to investigate the causative factors underlying this surging epidemic. Using immunoassays for HIV-1 diagnosis and phylogenetic analysis for viral genotyping, we found that the number of MSM infections doubled from 171 in 2011 to 340 in 2013 with a significantly increased prevalent rate from 4.1% to 5.2%. Majority of these increased infections took place in Fuzhou, Xiamen, and Quanzhou, three large cities in Fujian, mainly among youth, unemployed, business, and well-educated MSMs. Phylogenetic analysis revealed three major HIV-1 genotypes including CRF01_AE, CRF07_BC, and B/B' yet the surging MSM infections were primarily associated with the rapid sexual spread of CRF07_BC in addition to CRF01_AE. In particular, there was a significant proportional expansion of CRF07_BC infections among recently infected MSMs from 19% in 2012 to 41.9% in 2013. This increase was accompanied by emergence of complex patterns of viral recombination including multiple hybrid variants derived from CRF01_AE and CRF07_BC. Full-genome analysis indicated that CRF07_BC in Fujian was likely originated from similar strains previously found among IDUs in Yunnan province but with unique recombination break points. Our findings indicated that HIV-1 CRF07_BC has adapted for rapid sexual transmission, resulting in the surging HIV-1 epidemic and the emergence of new recombinant strains among MSMs in Fujian. Our findings have implications to vaccine and passive immunization trials in Fujian with emphasis on the induction of cross-subtype protective immunity.

ClC-3 promotes angiotensin II-induced reactive oxygen species production in endothelial cells by facilitating Nox2 NADPH oxidase complex formation.

Recent evidence suggests that ClC-3, a member of the ClC family of Cl- channels or Cl-/H+ antiporters, plays a critical role in NADPH oxidase-derived reactive oxygen species (ROS) generation. However, the underling mechanisms remain unclear. In this study we investigated the effects and mechanisms of ClC-3 on NADPH oxidase activation and ROS generation in endothelial cells. Treatment with angiotensin II (Ang II, 1 µmol/L) significantly elevated ClC-3 expression in cultured human umbilical vein endothelial cells (HUVECs). Furthermore, Ang II treatment increased ROS production and NADPH oxidase activity, an effect that could be significantly inhibited by knockdown of ClC-3, and further enhanced by overexpression of ClC-3. SA-ß-galactosidase staining showed that ClC-3 silencing abolished Ang II-induced HUVEC senescence, whereas ClC-3 overexpression caused the opposite effects. We further showed that Ang II treatment increased the translocation of p47phox and p67phox from the cytosol to membrane, accompanied by elevated Nox2 and p22phox expression, which was significantly attenuated by knockdown of ClC-3 and potentiated by overexpression of ClC-3. Moreover, overexpression of ClC-3 increased Ang II-induced phosphorylation of p47phox and p38 MAPK in HUVECs. Pretreatment with a p38 inhibitor SB203580 abolished ClC-3 overexpression-induced increase in p47phox phosphorylation, as well as NADPH oxidase activity and ROS generation. Our results demonstrate that ClC-3 acts as a positive regulator of Ang II-induced NADPH oxidase activation and ROS production in endothelial cells, possibly via promoting both Nox2/p22phox expression and p38 MAPK-dependent p47phox/p67phox membrane translocation, then increasing Nox2 NADPH oxidase complex formation.

Endophilin A2 protects H2O2-induced apoptosis by blockade of Bax translocation in rat basilar artery smooth muscle cells.

BACKGROUND: Apoptosis plays a central role in maintaining the normal cell number and tissue homeostasis. Endophilins are a family of evolutionarily conserved proteins that have the critical role in endocytosis. Here, we determined whether endophilin A2 (EndoII) contributes to hydrogen peroxide (H2O2)-induced apoptosis in rat basilar artery smooth muscle cells (BASMCs) and the underlying mechanisms. METHODS AND RESULTS: By using small interference RNA (siRNA) and EndoII overexpression strategy, we found that EndoII siRNA knockdown reduced cell viability and promoted H2O2-induced cell apoptosis, evidenced by loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase-9, 3 and poly (ADP-ribose) polymerase (PARP). In contrast, EndoII overexpression showed opposite effects and inhibited H2O2-induced BASMCs apoptosis. Further studies revealed that there was a direct interaction between EndoII and Bax. Upon H2O2-induced apoptosis, the association of EndoII with Bax were significantly decreased, while the interaction of Bax/tBid were increased, accompanied by a translocation of Bax from cytosol to mitochondria. Knockdown of EndoII did not affect the expression of Bax, but further promoted the binding of Bax with tBid and favored the accumulation of Bax to mitochondria as well as Bax activation; whereas EndoII overexpression produced the opposite effects. In addition, EndoII siRNA aggravated, but EndoII overexpression alleviated, the reduction of Bcl-2 expression in H2O2-treated cells. CONCLUSIONS: These data suggested a role of EndoII in protecting BASMCs apoptosis induced by H2O2, possibly by inhibiting the addressing of Bax to mitochondria. Targeting on EndoII may be a new strategy to treat apoptosis-associated diseases.

Pwp1 is required for the differentiation potential of mouse embryonic stem cells through regulating Stat3 signaling.

Leukemia inhibitory factor/Stat3 signaling is critical for maintaining the self-renewal and differentiation potential of mouse embryonic stem cells (mESCs). However, the upstream effectors of this pathway have not been clearly defined. Here, we show that periodic tryptophan protein 1 (Pwp1), a WD-40 repeat-containing protein associated with histone H4 modification, is required for the exit of mESCs from the pluripotent state into all lineages. Knockdown (KD) of Pwp1 does not affect mESC proliferation, self-renewal, or apoptosis. However, KD of Pwp1 impairs the differentiation potential of mESCs both in vitro and in vivo. PWP1 chromatin immunoprecipitation-seq results revealed that the PWP1-occupied regions were marked with significant levels of H4K20me3. Moreover, Pwp1 binds to sites in the upstream region of Stat3. KD of Pwp1 decreases the level of H4K20me3 in the upstream region of Stat3 gene and upregulates the expression of Stat3. Furthermore, Pwp1 KD mESCs recover their differentiation potential through suppressing the expression of Stat3 or inhibiting the tyrosine phosphorylation of STAT3. Together, our results suggest that Pwp1 plays important roles in the differentiation potential of mESCs.

A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18.

Viral RNA elements that facilitate mRNA export are useful tools for identifying cellular RNA export factors. Here we show that hepatitis B virus post-transcriptional element (PRE) is one such element, and using PRE several new cellular mRNA export factors were identified. We found that PRE drastically enhances the cytoplasmic accumulation of cDNA transcripts independent of any viral protein. Systematic deletion analysis revealed the existence of a 116 nt functional Sub-Element of PRE (SEP1). The RNP that forms on the SEP1 RNA was affinity purified, in which TREX components as well as several other proteins were identified. TREX components and the SEP1-associating protein ZC3H18 are required for SEP1-mediated mRNA export. Significantly, ZC3H18 directly binds to the SEP1 RNA, interacts with TREX and is required for stable association of TREX with the SEP1-containing mRNA. Requirements for SEP1-mediated mRNA export are similar to those for splicing-dependent mRNA export. Consistent with these similarities, several SEP1-interacting proteins, including ZC3H18, ARS2, Acinus and Brr2, are required for efficient nuclear export of polyA RNAs. Together, our data indicate that SEP1 enhances mRNA export by recruiting TREX via ZC3H18. The new mRNA export factors that we identified might be involved in cap- and splicing-dependent TREX recruitment to cellular mRNAs.

ClC-3 deficiency prevents atherosclerotic lesion development in ApoE-/- mice.

BACKGROUND: Recent evidence suggested that ClC-3, encoding Cl(-) channel or Cl(-)/H(+) antiporter, plays a critical role in regulation of a variety of physiological functions. However, remarkably little is known about whether ClC-3 is involved in atherosclerosis. This study aims to establish the involvement and direct role of ClC-3 in atherogenesis and underlying mechanisms by using ClC-3 and ApoE double null mice. METHODS AND RESULTS: After a 16-week western-type high-fat diet, the ClC-3(+/+)ApoE(-/-) mice developed widespread atherosclerotic lesions in aorta. However, the lesion size was significantly reduced in aorta of ClC-3(-/-)ApoE(-/-) mice. Compared with the ClC-3(+/+) controls, there was significantly decreased ox-LDL binding and uptake in isolated peritoneal macrophages from ClC-3(-/-) mice. Moreover, the expression of scavenger receptor SR-A, but not CD36, was significantly decreased in both ClC-3(-/-) peritoneal macrophages and aortic lesions from ClC-3(-/-)ApoE(-/-) mice. These findings were further confirmed in ox-LDL-treated RAW264.7 macrophages, which showed that silence of ClC-3 inhibited SR-A expression, ox-LDL accumulation and foam cell formation, whereas overexpression of ClC-3 produced the opposite effects. In addition, ClC-3 siRNA significantly inhibited, whereas ClC-3 overexpression increased, the phosphorylation of JNK/p38 MAPK in ox-LDL-treated RAW264.7 foam cells. Pretreatment with JNK or p38 inhibitor abolished ClC-3-induced increase in SR-A expression and ox-LDL uptake. Finally, the increased JNK/p38 phosphorylation and SR-A expression induced by ClC-3 could be mimicked by reduction of [Cl(-)]i by low Cl(-) solution. CONCLUSIONS: Our findings demonstrated that ClC-3 deficiency inhibits atherosclerotic lesion development, possibly via suppression of JNK/p38 MAPK dependent SR-A expression and foam cell formation.

Application of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for the detection of SFTSV-specific human IgG and IgM antibodies by indirect ELISA.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease that was first reported in China in 2011. It is caused by SFTS virus (SFTSV) which is a member of the Phlebovirus genus in the Bunyaviridae family. SFTSV has been classified as a BSL3 pathogen. There is a need to develop safe and affordable serodiagnostic methods for proper clinical management of infected patients. METHODS: The full length nucleocapsid (N) gene of SFTSV Yamaguchi strain was amplified by RT-PCR and cloned to an expression vector pQE30. The recombinant (r) SFTSV-N protein was expressed by using Escherichia coli (E. coli) expression system and purified under native conditions. rSFTSV-N protein based indirect IgG and IgM enzyme linked immunosorbent assay (ELISA) systems were established to detect specific human IgG and IgM antibodies, respectively. One hundred fifteen serum samples from clinically suspected-SFTS patients were used to evaluate the newly established systems and the results were compared with the total antibody detecting sandwich ELISA system. RESULTS: The native form of recombinant (r) SFTSV-N protein was expressed and purified. Application of the rSFTSV-N protein based indirect IgG ELISA to the 115 serum samples showed results that perfectly matched those of the total antibody sandwich ELISA with a sensitivity and specificity of 100 %. The rSFTSV-N protein based indirect IgM ELISA missed 8 positive samples that were detected by the total antibody sandwich ELISA. The sensitivity and specificity of rSFTSV-N-IgM capture ELISA were 90.59 and 100 %, respectively. CONCLUSIONS: The rSFTSV-N protein is highly immunoreactive and a good target for use as an assay antigen in laboratory diagnosis. Its preparation is simpler in comparison with that used for the total antibody sandwich system. Our rSFTSV-N protein-based IgG and IgM ELISA systems have the advantage of distinguishing two types of antibodies and require small volume of serum sample only. They are safe to use for diagnosis of SFTS virus infection and especially fit in large-scale epidemiological investigations.

[Discovery and whole genome sequences analysis of a dengue virus type 1 strain isolated in Henan province].

OBJECTIVE: To diagnose imported dengue fever case from Henan province, and to sequence and analyze the characteristics of whole genome sequence, and to explore the possible viral origin source. METHODS: A suspected dengue fever case was reported in Yuzhou city, Henan province. The patient returned from foshan, Guangdong province on September 19, 2014, after the epidemiological investigation and serum specimen collected, which dengue fever case was diagnosed in the laboratory, then it was inoculated on Vero cells. Whole genome sequence was amplified by several pairs primers and characterized using biologic software. RESULTS: The imported case was diagnosed as dengue virus 1 serotype infection. Dengue 1 strain was isolated using Vero cells successfully. Whole genome was 10,670 nt, which belonged to dengue virus 1 serotype V genotype and didn't found any recombination event. The phylogenetic analysis demonstrated that the strain was closed to Indian starins isolated in 2008-2011, and the homology of nucleotide sequence was between 98.2%-99.4%. CONCLUSION: It was the first time to discover imported dengue 1 serotype case in Henan province. However, according to the patient has been to Guangdong province before onset, it inferred that the Indian strain had been imported to Guangdong province before this case in Henan province.

Detection of new bunyavirus RNA by reverse transcription-loop-mediated isothermal amplification.

Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging and epidemic infectious disease in central and northeast China. It is caused by New Bunyavirus and carries an average 12% case fatality rate. Early and rapid detection is critical for prevention and control of New Bunyavirus infection, since no vaccine or antiviral drugs are currently available, and prevention requires careful attention to control of the suspected tick vector. In this study, a simple and sensitive reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid detection of New Bunyavirus. The detection limit of the RT-LAMP assay was approximately 10(3) 50% tissue culture infective doses/ml of New Bunyavirus in culture supernatants, and no cross-reactive amplification of other viruses known to cause similar clinical manifestations was observed. The assay was further evaluated using 138 specimens from clinically suspected SFTS and 40 laboratory-proven hantavirus infection with fever and renal syndrome patients, and the assay exhibited 97% agreement compared to real-time RT-PCR and conventional RT-PCR. Using real-time RT-PCR as the diagnostic gold standard, RT-LAMP was 99% sensitive and 100% specific. The RT-LAMP assay could become a useful alternative in clinical diagnosis of SFTS caused by New Bunyavirus, especially in resource-limited hospitals or rural clinics of China.

Mucosal priming with a replicating-vaccinia virus-based vaccine elicits protective immunity to simian immunodeficiency virus challenge in rhesus monkeys.

Mucosal surfaces are not targeted by most human immunodeficiency virus type 1 (HIV-1) vaccines, despite being major routes for HIV-1 transmission. Here we report a novel vaccination regimen consisting of a mucosal prime with a modified replicating vaccinia virus Tiantan strain (MVTT(SIVgpe)) and an intramuscular boost with a nonreplicating adenovirus strain (Ad5(SIVgpe)). This regimen elicited robust cellular immune responses with enhanced magnitudes, sustainability, and polyfunctionality, as well as higher titers of neutralizing antibodies against the simian immunodeficiency virus SIV(mac1A11) in rhesus monkeys. The reductions in peak and set-point viral loads were significant in most animals, with one other animal being protected fully from high-dose intrarectal inoculation of SIV(mac239). Furthermore, the animals vaccinated with this regimen were healthy, while ~75% of control animals developed simian AIDS. The protective effects correlated with the vaccine-elicited SIV-specific CD8(+) T cell responses against Gag and Pol. Our study provides a novel strategy for developing an HIV-1 vaccine by using the combination of a replicating vector and mucosal priming.