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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.

SummaryG-protein-coupled receptors (GPCRs) enable cells to sense and respond appropriately to hormonal and environmental signals, and are a target of ~30% of all FDA-approved medications. Canonically, each GPCR couples to distinct Gαproteins, such as Gαs, Gαi, Gαqor Gα12/13, as well as β-arrestins. These transducer proteins translate and integrate extracellular stimuli sensed by GPCRs into intracellular signals through what are broadly considered separable signalling pathways. However, the ability of Gαproteins to directly interact with β-arrestins to integrate signalling has not previously been appreciated. Here we show a novel interaction between Gαiprotein family members and β-arrestin. Gαi:β-arrestin complexes were formed by all GPCRs tested, regardless of their canonical G protein isoform coupling, and could bind both GPCRs as well as the extracellular signal-regulated kinase (ERK). This novel paradigm of Gαi:β-arrestin scaffolds enhances our understanding of GPCR signalling.

Hyperpolarized 129Xe magnetic resonance imaging (MRI) is capable of regional mapping of pulmonary gas-exchange and has found application in a wide range of pulmonary disorders in humans and animal model analogs. This study is the first application of 129Xe MRI to the monocrotaline rat model of pulmonary hypertension. Such models of preclinical pulmonary hypertension, a disease of the pulmonary vasculature that results in right heart failure and death, are usually assessed with invasive procedures such as right heart catheterization and histopathology. The work here adapted from protocols from clinical 129Xe MRI to enable preclinical imaging of rat models of pulmonary hypertension on a Bruker 7 T scanner. 129Xe spectroscopy and gas-exchange imaging showed reduced 129Xe uptake by red blood cells early in the progression of the disease, and at a later time point was accompanied by increased uptake by barrier tissues, edema, and ventilation defects—all of which are salient characteristics of the monocrotaline model. Imaging results were validated by H&E histology, which showed evidence of remodeling of arterioles. This proof-of-concept study has demonstrated that hyperpolarized 129Xe MRI has strong potential to be used to non-invasively monitor the progression of pulmonary hypertension in preclinical models and potentially to also assess response to therapy.

With lots of carbon nanoparticles (CNPs) applied in the industry, the possibilities of their environmental release have received much attention. As the CNPs may enter drinking water systems, and persist in water and wastewater treatment systems, their possible reaction with disinfectants should be studied. In this study, the formation of trihalomethanes (THMs) with 5 types of carbon nanotubes (CNTs), graphene oxide (GO) and reduced graphene oxide (rGO) was investigated. All CNPs could act as precursors of THMs in aqueous phase. Total concentrations of THMs formed with CNPs varied from 0.24 to 0.95 μM, much lower than that formed from chlorinated Suwannee River Natural Organic Matter (SRNOM) (approximately 9 μM). The kinetics of THMs formation with GO was 0.0814 M −1 s −1 , which is higher than most of the chlorinated humic acid obtained from different natural waters. The study indicates that during chlorination, C–Cl bond could be formed on the surface of CNPs. However, carbon atoms at the middle of two meta -positioned OH groups on the benzene ring are more active and may prefer to form THMs with chlorine oxidation. The influences of pH and reactant doses on the formation of THMs were also discussed.

Explosion caused by zirconium powder was revealed as one of main reasons in accidents happened in reprocessing of spent fuel in nuclear industry. It is urgent to study the explosion severity characteristic of zirconium dust cloud due to the great harm of its explosion. According to the equipment used in the actual post-treatment process in nuclear industry, the 20L cylindrical explosion equipment as a scale model was manufactured as the experimental device. The experimental results showed that Pmax and (dp/dt)max increased at first and then decreased with the increase of concentration. Small zirconium particles produced larger value of explosion severity parameters. Interestingly, initial temperature had no significant effect on Pmax of zirconium powder. However, the value of (dp/dt)max was strongly dependent on the initial temperature. Additionally, the oxidation degree of zirconium dust and temperature generated during explosion were studied by means of oxygen content and crystal form of explosion products. The study found that the particles develop toward spheroidization and its size became smaller, indicating that zirconium particles combustion is a heterogeneous shrinking core process. Under the condition of constant mass, increased number of ZrO2 particles leads to enlarged particle total surface area, increasing the amount of radioactive material released.

Deficient angiogenesis may contribute to worsen the prognosis of myocardial ischemia, peripheral arterial disease, ischemic stroke, etc. Dyslipidemic and inflammatory environments attenuate endothelial cell (EC) proliferation and angiogenesis, worsening the prognosis of ischemia. Under these dyslipidemic and inflammatory environments, EC-caspase-1 becomes activated and induces inflammatory cell death that is defined as pyroptosis. However, the underlying mechanism that correlates caspase-1 activation with angiogenic impairment and the prognosis of ischemia remains poorly defined. By using flow cytometric analysis, enzyme and receptor inhibitors, and hind limb ischemia model in caspase-1 knock-out (KO) mice, we examined our novel hypothesis, i.e. inhibition of caspase-1 in ECs under dyslipidemic and inflammatory environments attenuates EC pyroptosis, improves EC survival mediated by vascular endothelial growth factor receptor 2 (VEGFR-2), angiogenesis, and the prognosis of ischemia. We have made the following findings. Proatherogenic lipids induce higher caspase-1 activation in larger sizes of human aortic endothelial cells (HAECs) than in smaller sizes of HAECs. Proatherogenic lipids increase pyroptosis significantly more in smaller sizes of HAECs than in larger sizes of the cells. VEGFR-2 inhibition increases caspase-1 activation in HAECs induced by lysophosphatidylcholine treatment. Caspase-1 activation inhibits VEGFR-2 expression. Caspase-1 inhibition improves the tube formation of lysophosphatidylcholine-treated HAECs. Finally, caspase-1 depletion improves angiogenesis and blood flow in mouse hind limb ischemic tissues. Our results have demonstrated for the first time that inhibition of proatherogenic caspase-1 activation in ECs improves angiogenesis and the prognosis of ischemia.

Introduction: Angiotensin II Type 1 receptor (AT1R) is a member of the G protein-coupled receptors (GPCRs) family, playing an important role in several cardiovascular diseases. Depending on the stimulus, AT1R activates a cascade of signaling pathways including those mediated by G proteins and the multifunctional proteins β-arrestins. AT1R is also mechanosensor and respond to membrane stretch by activating ligand-independent β-arrestin-biased signaling. Objective: The aim of this study is to investigate the precise molecular mechanism for mechanoactivation of the AT1R. Methods and Results: Our previous work demonstrated that mechanical stretch induced by hypotonicity (osmotic-stretch, OSM) allosterically activates the AT1R to mediate β-arrestin signaling. Using Proximity Ligation Assays (PLA) and co-IP we now demonstrate that OSM uniquely promotes Gαi recruitment to AT1R to initiate β-arrestin signaling. In sharp contrast to the β-arrestin-biased ligand TRV120023, the Gαi inhibitor Pertussis Toxin (PTX) blocked the OSM-induced β-arrestin recruitment, EGFR transactivation and ERK signaling. To determine whether the two biased stimuli (OSM and TRV120023) can activate different β-arrestin conformations, we used β-arrestin FLAsH constructs and monitored BRET following AT1R activation by either ligand or stretch stimulation. A FlAsH constructs located in the N-domain domain and two located in the C-domain domain showed a distinct BRET pattern indicating that in response to OSM, β-arrestin assumes a distinct conformation from either the balanced ligand Angiontensin II and the TRV120023 (N=7; p Conclusion: The principal driver for differential signaling is thought to be different receptor conformations stabilized by each ligand. Our data suggest that in response to two β-arrestin-biased stimuli, AT1R likely adopt distinct conformations indicating remarkable conformational heterogeneity in activating intracellular responses.

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.

The novel magnetic PW-doped PEDOT (PW@PEDOT) imprinted photocatalyst with good reproducibility was prepared by the surface imprinting technique and microwave heating method. Due to the existence of PW@PEDOT and imprinted cavity in the imprinted layer, the as-prepared magnetic PW@PEDOT imprinted photocatalyst not only had higher photocatalytic activity, but also had the excellent specific recognition ability for selective photodegradation of TC. This paper proposed a new idea to prepare the imprinted photocatalysts.

A small number of upstream master genes in "higher hierarchy" controls the expression of a large number of downstream genes and integrates the signaling pathways underlying the pathogenesis of cardiovascular diseases with or without autoimmune inflammatory mechanisms. In this brief review, we organize our analysis of recent progress in characterization of forkhead (FOX) transcription factor family members in vascular pathology, diabetes and regulatory T cells into the following sections: (1) Overview of the FOX transcription factor superfamily; (2) Vascular pathology of mice deficient in FOX transcription factors; (3) Roles of FOX transcription factors in endothelial cell pathology; (4) Roles of FOX transcription factors in vascular smooth muscle cells; (5) Roles of FOX transcription factors in the pathogenesis of diabetes; and (6) Immune system phenotypes of mice deficient in FOX transcription factors. Advances in these areas suggest that the FOX transcription factor family plays important roles in vascular development and in the pathogenesis of autoimmune inflammatory cardiovascular diseases.

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.

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 endothelial 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.