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Background Extending service scope of primary care facilities (PCFs) has been widely concerned in China. However, no current data about association between service scope of PCFs with patient outcomes are available. This study aims to investigate association between service scope of PCFs and patient outcomes. Methods A multistage, stratified clustered sampling method was used to collect information about service scope of PCFs from rural Guizhou, China. Claim data of 299,633 inpatient cases covered by 64 PCFs were derived from local information system of New Rural Cooperation Medical Scheme. Service scope of PCFs was collected with self-administrated questionnaires. Primary outcomes were (1) level of inpatient institutions, (2) length of stay, (3) per capita total health cost, (4) per capita out-of-pocket cost, (5) reimbursement ratio, (6) 30-day readmission. A total of 64 PCFs were categorized into five groups per facility-level service scope scores. Generalized linear regression models, logistic regression model, and ordinal regression model were conducted to identify association between service scope of PCFs and patient outcomes. Results On average, the median service scope score of PCFs was 20, with wide variation across PCFs. After controlling for demographic and clinical characteristics, patients living in communities with PCFs of greatest service scope (Quintile V vs. I) tended to have smaller rates of admission by county-level hospitals (-6.2 % [-6.5 %, -5.9 %], city-level hospitals (-1.9 % [-2.0 %, -1.8 %]), and provincial hospitals (-2.1 % [-2.2 %, -2.0 %]), smaller rate of 30-day readmission (-0.5 % [-0.7 %, -0.2 %]), less total health cost (-201.8 [-257.9, -145.8]) and out-of-pocket cost (-210.2 [-237.2, -183.2]), and greater reimbursement ratio (2.3 % [1.9 %, 2.8 %]) than their counterparts from communities with PCFs of least service scope. Conclusions Service scope of PCFs varied a lot in rural Guizhou, China. Greater service scope was associated with a reduction in secondary and tertiary hospital admission, reduced total cost and out-of-pocket cost, and 30-day readmission and increased reimbursement ratio. These results raised concerns about access to care for patients discharged from hospitals, which suggests potential opportunities for cost savings and improvement of quality of care. However, further evidence is warranted to investigate whether extending service scope of PCFs is cost-effective and sustainable.

Background: Receptor signaling is central to vascular endothelial function and is dysregulated in vascular diseases such as atherosclerosis and pulmonary arterial hypertension (PAH). Signaling pathways involved in endothelial function include vascular endothelial growth factor receptors (VEGFRs) and G protein–coupled receptors, which classically activate distinct intracellular signaling pathways and responses. The mechanisms that regulate these signaling pathways have not been fully elucidated and it is unclear what nodes for cross talk exist between these diverse signaling pathways. For example, multifunctional β-arrestin (ARRB) adapter proteins are best known as regulators of G protein–coupled receptor signaling, but their role at other receptors and their physiological importance in the setting of vascular disease are unclear. Methods: We used a combination of human samples from PAH, human microvascular endothelial cells from lung, and Arrb knockout mice to determine the role of ARRB1 in endothelial VEGFR3 signaling. In addition, a number of biochemical analyses were performed to determine the interaction between ARRB1 and VEGFR3, signaling mediators downstream of VEGFR3, and the internalization of VEGFR3. Results: Expression of ARRB1 and VEGFR3 was reduced in human PAH, and the deletion of Arrb1 in mice exposed to hypoxia led to worse PAH with a loss of VEGFR3 signaling. Knockdown of ARRB1 inhibited VEGF-C–induced endothelial cell proliferation, migration, and tube formation, along with reduced VEGFR3, Akt, and endothelial nitric oxide synthase phosphorylation. This regulation was mediated by direct ARRB1 binding to the VEGFR3 kinase domain and resulted in decreased VEGFR3 internalization. Conclusions: Our results demonstrate a novel role for ARRB1 in VEGFR regulation and suggest a mechanism for cross talk between G protein–coupled receptors and VEGFRs in PAH. These findings also suggest that strategies to promote ARRB1-mediated VEGFR3 signaling could be useful in the treatment of pulmonary hypertension and other vascular disease.

Ulcerative colitis (UC) is an inflammatory bowel disease with complex pathogenesis, which is affected by genetic factors, intestinal immune status and intestinal microbial homeostasis. Intestinal epithelial barrier defect is crucial to the development of UC. Berberine, extracted from Chinese medicine, can identify bitter taste receptor on intestinal Tuft cells and activate IL-25-ILC2-IL-13 immune pathway to impair damaged intestinal tract by promoting differentiation of intestinal stem cells, which might be a potential approach for the treatment of UC.

Hyperhomocysteinemia (HHcy) is an established and potent independent risk factor for degenerative diseases, including cardiovascular disease (CVD), Alzheimer disease, type II diabetes mellitus, and chronic kidney disease. HHcy has been shown to inhibit proliferation and promote inflammatory responses in endothelial cells (EC), and impair endothelial function, a hallmark for vascular injury. However, metabolic processes and molecular mechanisms mediating HHcy-induced endothelial injury remains to be elucidated. This study examined the effects of HHcy on the expression of microRNA (miRNA) and mRNA in human aortic EC treated with a pathophysiologically relevant concentration of homocysteine (Hcy 500 μM). We performed a set of extensive bioinformatics analyses to identify HHcy-altered metabolic and molecular processes. The global functional implications and molecular network were determined by Gene Set Enrichment Analysis (GSEA) followed by Cytoscape analysis. We identified 244 significantly differentially expressed (SDE) mRNA, their relevant functional pathways, and 45 SDE miRNA. HHcy-altered SDE inversely correlated miRNA-mRNA pairs (45 induced/14 reduced mRNA) were discovered and applied to network construction using an experimentally verified database. We established a hypothetical model to describe the biochemical and molecular network with these specified miRNA/mRNA axes, finding: 1) HHcy causes metabolic reprogramming by increasing glucose uptake and oxidation, by glycogen debranching and NAD+/CoA synthesis, and by stimulating mitochondrial reactive oxygen species production via NNT/IDH2 suppression-induced NAD+/NADP-NADPH/NADP+ metabolism disruption; 2) HHcy activates inflammatory responses by activating inflammasome-pyroptosis mainly through ↓miR193b→↑CASP-9 signaling and by inducing IL-1β and adhesion molecules through the ↓miR29c→↑NEDD9 and the ↓miR1256→↑ICAM-1 axes, as well as GPCR and interferon α/β signaling; 3) HHcy promotes cell degradation by the activation of lysosome autophagy and ubiquitin proteasome systems; 4) HHcy causes cell cycle arrest at G1/S and S/G2 transitions, suppresses spindle checkpoint complex and cytokinetic abscission, and suppresses proliferation through ↓miRNA335/↑VASH1 and other axes. These findings are in accordance with our previous studies and add a wealth of heretofore-unexplored molecular and metabolic mechanisms underlying HHcy-induced endothelial injury. This is the first study to consider the effects of HHcy on both global mRNA and miRNA expression changes for mechanism identification. Molecular axes and biochemical processes identified in this study are useful not only for the understanding of mechanisms underlying HHcy-induced endothelial injury, but also for discovering therapeutic targets for CVD in general., Graphical abstract Image 1, Highlights • Identified multiple HHcy-altered metabolic and molecular processes potentially responsible for HHcy-induced endothelial injury via examining global mRNA/miRNA expression changes in Hcy-treated EC and performing comprehensive bioinformatic studies. • HHcy may activate glucose uptake signaling via the ↓miR148b→↑SLC2A axis. • HHcy may induce glucose oxidation signaling by switching pyruvate metabolism from lactate synthesis to mitochondrial oxidation via expression changes of ↑MPC1 & ↓LDHB. • HHcy may disrupt redox homeostasis mostly by suppressing NNT/IDH2-related mt-NADPH/mt-NAD+ signaling. • HHcy may increase FA β-oxidation, glutamine, TCA cycle and OXPHOS signaling. • HHcy may activate inflammatory signaling via the ↓miR29c→↑NEDD9 and the ↓miR1256→↑ICAM-1 axes. • HHcy may activate inflammasome/pyroptosis-related signaling by the ↓miR137→↑TLR3, the ↓miR574→↑TRAF5, and the ↓miR193b→↑CASP-9 axes, and induce IL1α/β and CASP-10/7. • HHcy may induce inflammation signaling via GPCR activation through the ↓miRNA335→↑CXCR4/↑GNA14 axes. • HHcy may activate molecular degradation process signaling through the ↓miRNA335→↑ASAH1/↑ABCB9 axes. • HHcy may suppress cell cycle and proliferation through the miR491→↓HMGA2→↓CCNA2/CCNB2, the ↓miR335→↑VASH1, the ↓miR181a→↑PHLDA1, the miR6045→↓CENPH, the miR22→↓PRR11/↓BRCA2, and the miR605/miR497/miR514a→CEP55 axes

Protein phosphorylation is a critical post-translational modification (PTM). Despite recent technological advances in reversed-phase liquid chromatography (RPLC)-mass spectrometry (MS)-based proteomics, comprehensive phosphoproteomic coverage in complex biological systems remains challenging, especially for hydrophilic phosphopeptides with enriched regions of serines, threonines, and tyrosines that often orchestrate critical biological functions. To address this issue, we developed a simple, easily implemented method to introduce a commonly used tandem mass tag (TMT) to increase peptide hydrophobicity, effectively enhancing RPLC-MS analysis of hydrophilic peptides. Different from conventional TMT labeling, this method capitalizes on using a nonprimary amine buffer and TMT labeling occurring before C18-based solid phase extraction. Through phosphoproteomic analyses of MCF7 cells, we have demonstrated that this method can greatly increase the number of identified hydrophilic phosphopeptides and improve MS detection signals. We applied this method to study the peptide QPSSSR, a very hydrophilic tryptic peptide located on the C-terminus of the G protein-coupled receptors (GPCR) CXCR3. Identification of QPSSSR has never been reported, and we were unable to detect it by traditional methods. We validated our TMT labeling strategy by comparative RPLC-MS analyses of both a hydrophilic QPSSSR peptide library as well as common phosphopeptides. We further confirmed the utility of this method by quantifying QPSSSR phosphorylation abundances in HEK 293 cells under different treatment conditions predicted to alter QPSSSR phosphorylation. We anticipate that this simple TMT labeling method can be broadly used not only for decoding GPCR phosphoproteome but also for effective RPLC-MS analysis of other highly hydrophilic analytes.

Heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) are common drug targets and canonically couple to specific G(α) protein subtypes and β-arrestin adaptor proteins. G protein–mediated signaling and β-arrestin–mediated signaling have been considered separable. We show here that GPCRs promote a direct interaction between G(αi) protein subtype family members and β-arrestins regardless of their canonical G(αi) protein subtype coupling. G(αi):β-arrestin complexes bound extracellular signal-regulated kinase (ERK), and their disruption impaired both ERK activation and cell migration, which is consistent with β-arrestins requiring a functional interaction with G(αi) for certain signaling events. These results introduce a GPCR signaling mechanism distinct from canonical G protein activation in which GPCRs cause the formation of G(αi):β-arrestin signaling complexes.

Endocytosis is a cellular process mostly responsible for membrane receptor internalization. Cell membrane receptors bind to their ligands and form a complex which can be internalized. We previously proposed that F-BAR protein initiates membrane curvature and mediates endocytosis via their binding partners. However, F-BAR protein partners involved in membrane receptor endocytosis and the regulatory mechanism remain unknown. In this study, we established a group of database mining strategies to explore mechanisms underlying receptor-related endocytosis. We identified 34 endocytic membrane receptors and 10 regulating proteins for vesicle formation in clathrin-dependent endocytosis (CDE), a major process of membrane receptor internalization. We found that F-BAR protein FCHSD2 (Carom) may facilitate endocytosis via 9 endocytic partners. Carom is highly expressed, along with highly expressed endocytic membrane receptors and partners, in endothelial cells and macrophages. We established 3 models of Carom-receptor complex and their intracellular trafficking based on protein-protein interaction and subcellular localization. We conclude that Carom may mediate receptor endocytosis and transport endocytic receptors to the cytoplasm for receptor signaling and lysosome/proteasome degradation, or to the nucleus for RNA processing, gene transcription and DNA repair.

Carom is a novel protein that regulates membrane curvature and transmits pathophysiological signaling. The tissue expression of Carom is unclear and its functional role and signaling are unknown. We employed a group of combined database mining strategies and established a working model of Carom signaling. We identified 26 Carom partners and established their expression profiles in human and mouse tissues. We classified three tiers of tissues for Carom/partner expression and found lymph node was the tier 1 tissue expressing Carom and most of its partners. Using GEO database, we discovered that four conditions (hypoxia, endometriosis, PPARgamma deletion and iPSC reprogramming) altered Carom/partner expression in endothelial cells. We identified 26 Carom partner signalings by Ingenuity pathway analysis. Ten of the 26 pathways and three genes (ITSN1, UBC and HSPA5) were reported to be regulated in the above four conditions. Paired induction of Carom/ITSN1 elevation was associated with pathological angiogenesis. Whereas, paired reduction of Carom/HSPA5 or UBC was associated with iPSC generation. These results provide an insight on identifying Carom complex model and predicting its functional implications.

Objective The preferred amino acids in the proteolytic sites have been considered to be similar between caspase-1 and caspase-9, which do not support their differential functions in inflammatory pyroptosis and apoptosis. We attempted to solve this problem. Methods We analyzed the flanking 20 amino acid residues in the cleavage sites in 34 caspase-1 and 11 capase-9 experimentally identified substrates. Results This study has made the following findings: first, we verified that caspase-1 and caspase-9 shared 100% aspartic acid in the P1 position. However, the structures in the cleavage sites of most caspase-1 substrates are different from that of caspase-9 substrates in the following three aspects, (a) the amino acid residues with the statistically high frequencies; (b) the hydrophobic amino acid occurrence frequencies; and (c) the charged amino acid occurrence frequencies; second, the amino acid pairs P1–P1′ are different; third, our identified cleavage site patterns are useful in the prediction for the 91.4% cleavage sites of 35 new caspase-1 substrates. Conclusion Since most caspase-1 substrates are involved in vascular function, inflammation and atherogenesis, our novel structural patterns for the caspases’ substrates are significant in developing new diagnostics and therapeutics.

A G2P[4]/NSP4[A] rotavirus strain TB-Chen was isolated from a 2-year-old patient hospitalized with acute gastroenteritis in Kunming, China. The strain TB-Chen was demonstrated having group A-specific antigenicity, a “short” (subgroup II) electropherotype. To investigate its overall genomic relatedness and to determine which group it belonged, the complete genome of strain TB-Chen was determined. Genomic comparison based on amino acid sequence identity and phylogenetic analysis revealed that all 11 gene segments of strain TB-Chen were highly identical (>91.80%) with the representative G2P[4]/NSP4[A] human strains DS-1, S2, NR1 and IS2, suggesting that this rotavirus strain was derived from human host. Besides, almost all the available representative rotavirus gene segments among group A were analyzed and identified within 15 G-types, 28 P-types, and 6 NSP4 genotypes. This is the first report of group A rotavirus genomic analyses in China and the findings have important implications for rotavirus vaccine development.

It has been demonstrated that the immunodominant region of the HCV core protein and the hepatitis B surface antigen (HBsAg) have high degree of reactivity. In order to construct a chimeric protein that carries HCV and HBV epitopes and possesses immunogenicity to both HCV and HBV, four epitopes derived from residues aa2-21 (epitope C1), aa22-40 (epitope C2) of the core protein, residues aa315-328 (epitope E) of E1 protein of HCV, and residues aa124-147 (epitope S) of HBsAg were chosen to be displayed in a conformation-specific manner on the outer surface of the Flock House virus capsid protein and expressed in E. coli cells. The reactivity of these epitopes with antisera from hepatitis C and hepatitis B patients and induction of immune response in guinea pigs were determined. The results showed that when displayed in this system, the chimeric protein carrying only epitope S could react with anti-HBsAg positive human sera, elicit an anti-HBsAg response in guinea pigs. The chimeric protein carrying epitopes C1, C2 and E could react with antibodies to different HCV genotypes, elicit an anti-HCV response in guinea pigs. The chimeric protein carrying epitopes C1, C2, E, and S could react with antibodies against HCV and HBV, elicit anti-HCV and anti-HBsAg responses in guinea pigs. The results suggested that these epitopes displayed in this form could be considered for development of epitope-based vaccines against HCV/HBV infections.

Transcription factor families are a small number of upstream master genes in "higher hierarchy" that control the expression of a large number of downstream genes. These transcription factors have been found to integrate the signaling pathways underlying the pathogenesis of cardiovascular diseases with or without autoimmune inflammatory mechanisms. In this chapter, we organize our analysis of recent progress in characterization of forkhead (Fox) transcription factor family members in vascular pathology and immune regulation into the following sections: (1) Introduction of the FOX transcription factor superfamily; (2) FOX transcription factors and endotheial cell pathology; (3) FOX transcription factors and vascular smooth muscle cells; and (4) FOX transcription factors, inflammation and immune system. Advances in these areas suggest that the FOX transcription factor family is important in regulating vascular development and the pathogenesis of autoimmune inflammatory cardiovascular diseases.

Eukaryotic cell membrane dynamics change in curvature during physiological and pathological processes. In the past ten years, a novel protein family, Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain proteins, has been identified to be the most important coordinators in membrane curvature regulation. The F-BAR domain family is a member of the Bin/Amphiphysin/Rvs (BAR) domain superfamily that is associated with dynamic changes in cell membrane. However, the molecular basis in membrane structure regulation and the biological functions of F-BAR protein are unclear. The pathophysiological role of F-BAR protein is unknown. This review summarizes the current understanding of structure and function in the BAR domain superfamily, classifies F-BAR family proteins into nine subfamilies based on domain structure, and characterizes F-BAR protein structure, domain interaction, and functional relevance. In general, F-BAR protein binds to cell membrane via F-BAR domain association with membrane phospholipids and initiates membrane curvature and scission via Src homology-3 (SH3) domain interaction with its partner proteins. This process causes membrane dynamic changes and leads to seven important cellular biological functions, which include endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation, via distinct signaling pathways determined by specific domain-binding partners. These cellular functions play important roles in many physiological and pathophysiological processes. We further summarize F-BAR protein expression and mutation changes observed in various diseases and developmental disorders. Considering the structure feature and functional implication of F-BAR proteins, we anticipate that F-BAR proteins modulate physiological and pathophysiological processes via transferring extracellular materials, regulating cell trafficking and mobility, presenting antigens, mediating extracellular matrix degradation, and transmitting signaling for cell proliferation.