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As one of the most important post-translational modifications, protein glycosylation plays vital role in various physiological processes. With multitudinous glycosyltransferases, N-glycans present structural diversity in linkages and branching styles. Structure-specific glycan profiling may provide more potential biological information than compositional profiling. In this work, N-glycans released from human serum samples were derivatized with reduction and methylamination prior to profiling using nanoLC-ESI-MS with PGC as stationary phase. In addition, α 2-3 neuraminidase was also applied for distinguishing the linkage types of sialic acid corresponding to different isomers. Relative abundances of 280 isomeric N-glycans were compared and 20 isomers showed significant difference between multiple myeloma cases and healthy controls. ROC was performed to assess the significantly altered isomeric glycans and 6 AUCs have exceeded 0.80, providing high diagnostic accuracy for MM. PCA is also employed to establish the differences among sample sets. Furthermore, these specific isomers have also been used for early detection of multiple myeloma, presenting important clinical application value. Isomer-specific biomarker discovery in multiple myeloma with dual-derivatized N-glycans.

This paper presents a lattice Boltzmann model to simulate the aqueous humor (AH) dynamics in the human eye by involving incompressible Navier-Stokes flow, heat convection and diffusion, and Darcy seepage flow. Verifying simulations indicate that the model is stable, convergent and robust. Further investigations were carried out, including the effects of heat convection and buoyancy, AH production rate, permeability of trabecular meshwork, viscosity of AH and anterior chamber angle on intraocular pressure (IOP). The heat convection and diffusion can significantly affect the flow patterns in the healthy eye, and the IOP can be controlled by increasing the anterior chamber angle or decreasing the secretion rate, the drainage resistance and viscosity of AH. However, the IOP is insensitive to the viscosity of AH, which may be one of the causes that the viscosity would not have been considered as a factor for controlling the IOP. It's interesting that all these factors have more significant influences on the IOP in pathologic eye than healthy one. The temperature difference and the eye-orientation have obvious influence on the cornea and iris wall shear stresses. The present model and simulation results are expected to provide an alternative tool and theoretical reference for the study of AH dynamics.

Sepsis is a life-threatening disease caused by infection. Inflammation is a key pathogenic process in sepsis. Paeonol, an active ingredient in moutan cortex (a Chinese herb), has many pharmacological activities, such as anti-inflammatory and antitumour actions. Previous studies have indicated that paeonol inhibits the expression of HMGB1 and the transcriptional activity of NF-κB. However, its underlying mechanism is still unknown. In this study, microarray assay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results confirmed that paeonol could significantly up-regulate the expression of miR-339-5p in RAW264.7 cells stimulated by LPS. Dual-luciferase assays indicated that miR-339-5p interacted with the 3′ untranslated region (3′-UTR) of HMGB1. Western blot, immunofluorescence and enzyme-linked immunosorbent assay (ELISA) analyses indicated that miR-339-5p mimic and siHMGB1 both negatively regulated the expression and secretion of inflammatory cytokines (e.g., HMGB1, IL-1β and TNF-α) in LPS-induced RAW264.7 cells. Studies have confirmed that IKK-β is targeted by miR-339-5p, and we further found that paeonol could inhibit IKK-β expression. Positive mutual feedback between HMGB1 and IKK-β was observed when we silenced HMGB1 or IKK-β. These results indicated that paeonol could attenuate the inflammation mediated by HMGB1 and IKK-β by upregulating miR-339-5p expression. In addition, we constructed CLP model mice by cecal ligation and puncture. Paeonol was used to intervene to investigate its anti-inflammatory effect in vivo. The results showed that paeonol could improve the survival rate of sepsis mice and protect the kidney of sepsis mice.

Hypertrophic scar (HS) is a dermal fibro-proliferative disorder result from abnormal wound healing after skin injury. MicroRNA-9-5p (miR-9-5p) has been reported to be upregulated and closely related to collagen proteins in human dermal fibroblasts. However, the correlation and possible mechanism between miR-9-5p and HS require further investigation. The expressions of miR-9-5p in HS tissues and HS fibroblasts were detected by quantitative real-time PCR (RT-qPCR). The expression level of peroxisome proliferator-activated receptor β (PPARβ) was measured by RT-qPCR assay. The protein levels of PPARβ, α-SMA, Vimentin, COL1A, cyclin D1, bcl-2, and bax were detected by western blot assay. The effect of miR-9-5p and PPARβ on HS fibroblasts proliferation and apoptosis were detected by cell counting kit-8 (CCK-8) and flow cytometry assays. The interaction between miR-9-5p and PPARβ was predicted by TargetScan, and then confirmed by dual-luciferase reporter assay. MiR-9-5p expression was downregulated in HS tissues and HS fibroblasts. MiR-9-5p inhibited the levels of extracellular matrix-associated genes (α-SMA, Vimentin, COL1A) in HS fibroblasts. MiR-9-5p repressed proliferation and induced apoptosis of HS fibroblasts. PPARβ is a target gene of miR-9-5p. The silencing of PPARβ expression hindered proliferation and expedited apoptosis of HS fibroblasts. MiR-9-5p suppressed proliferation and promoted apoptosis of HS fibroblasts by targeting PPARβ. In this paper, we firstly disclosed that miR-9-5p hampered extracellular matrix deposition and proliferation, and induced apoptosis by targeting PPARβ in HS fibroblasts. Our findings provided a new role of miR-9-5p/PPARβ in the occurrence and development of HS fibroblasts, promising a new target for HS., Summary: Our findings provided a new role of miR-9-5p/PPAR in the occurrence and development of HS fibroblasts, promising a new target for HS.

The abnormalities of blood pressure (BP) nocturnal decline have been found to be predictive for carotid plaque and lacunar infarction in patients with hypertension. In this study, BP dipping patterns in postmenopausal females with hypertension were investigated. The nocturnal decline of systolic BP (SBP) was evaluated using 24-hour ambulatory BP monitoring (ABPM). A total of 163 postmenopausal females were eventually included in our study. The prevalence of reverse-dipper BP pattern was 32.3% in females with menopause age in their 40s and 40% in their 50s. However, after multivariate logistic regression analysis, menopause age was shown to be an independent risk factor for BP reverse dipping (Odds ratio [OR] 1.148; 95%CI 1.020 - 1.292; P = 0.020). Moreover, menopause age was negatively correlated with the decline rate of nocturnal SBP (r = -0.159; P < 0.05) and diastolic BP (r = -0.161; P < 0.05). Our study suggested that the menopause age might serve as a risk factor for reverse-dipper BP pattern in postmenopausal females with hypertension.

Aims. The purpose of the study was to assess the effects of leisure-time physical activity on undetected prediabetes.Methods. Data from the National Health and Nutrition Examination Survey 2007–2012 were used in our analyses. Logistic regression was conducted to estimate the odds ratios (ORs) with 95% confidence intervals (CIs) of prediabetes associated with leisure-time physical activity.Results. A total of 8204 subjects were eligible for our analyses. For all subjects, high level of total leisure-time physical activity (OR = 0.78, 95% CI: 0.66, 0.94) and low level of vigorous leisure-time physical activity (OR = 0.72, 95% CI: 0.58, 0.90) were inversely associated with the risk of prediabetes in multivariate-adjusted model. For subjects under 45 years of age, high level of total leisure-time physical activity (OR = 0.78, 95% CI: 0.61, 0.99) and low (OR = 0.61, 95% CI: 0.45, 0.83) and high (OR = 0.72, 95% CI: 0.53, 1.00) level of vigorous leisure-time physical activity were associated with a decreased risk of prediabetes. In the 45 to 65 age group, only high level of total leisure-time physical activity (OR = 0.73, 95% CI: 0.57, 0.95) had protective effect on prediabetes.Conclusions. Leisure-time physical activity may be associated with a decreased risk of prediabetes.

The ability to generate spiral ganglion neurons (SGNs) from stem cells is a necessary prerequisite for development of cell-replacement therapies for sensorineural hearing loss. We present a protocol that directs human embryonic stem cells (hESCs) toward a purified population of otic neuronal progenitors (ONPs) and SGN-like cells. Between 82% and 95% of these cells express SGN molecular markers, they preferentially extend neurites to the cochlear nucleus rather than nonauditory nuclei, and they generate action potentials. The protocol follows an in vitro stepwise recapitulation of developmental events inherent to normal differentiation of hESCs into SGNs, resulting in efficient sequential generation of nonneuronal ectoderm, preplacodal ectoderm, early prosensory ONPs, late ONPs, and cells with cellular and molecular characteristics of human SGNs. We thus describe the sequential signaling pathways that generate the early and later lineage species in the human SGN lineage, thereby better describing key developmental processes. The results indicate that our protocol generates cells that closely replicate the phenotypic characteristics of human SGNs, advancing the process of guiding hESCs to states serving inner-ear cell-replacement therapies and possible next-generation hybrid auditory prostheses. © Stem Cells Translational Medicine 2017;6:923–936

A classic metabolic concept posits that insulin promotes energy storage and adipose expansion, while catecholamines stimulate release of adipose energy stores by hydrolysis of triglycerides through β-adrenergic receptor (βARs) and protein kinase A (PKA) signaling. Here, we have shown that a key hub in the insulin signaling pathway, activation of p70 ribosomal S6 kinase (S6K1) through mTORC1, is also triggered by PKA activation in both mouse and human adipocytes. Mice with mTORC1 impairment, either through adipocyte-specific deletion of Raptor or pharmacologic rapamycin treatment, were refractory to the well-known βAR-dependent increase of uncoupling protein UCP1 expression and expansion of beige/brite adipocytes (so-called browning) in white adipose tissue (WAT). Mechanistically, PKA directly phosphorylated mTOR and RAPTOR on unique serine residues, an effect that was independent of insulin/AKT signaling. Abrogation of the PKA site within RAPTOR disrupted βAR/mTORC1 activation of S6K1 without affecting mTORC1 activation by insulin. Conversely, a phosphomimetic RAPTOR augmented S6K1 activity. Together, these studies reveal a signaling pathway from βARs and PKA through mTORC1 that is required for adipose browning by catecholamines and provides potential therapeutic strategies to enhance energy expenditure and combat metabolic disease.

Objective The current study was designed to investigate the effects and underlying mechanisms of adipose tissue-derived stem cells (ADSCs) on hypertrophic scar (HS) fibrosis. Method Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot analysis were performed to detect the expression of collagen I (Col1), collagen III (Col3), and α-smooth muscle actin (α-SMA) after fibroblasts and cultured HS tissues were treated with ADSC medium. All data were analyzed by using SPSS17.0 software. Statistical analysis was performed by Student t tests. Results The in vitro study showed that ADSC medium decreased the expression of Col1, Col3, and α-SMA. In addition, the protein level of p-p38 was downregulated by ADSC medium treatment in a concentration dependent manner. Conclusion The current study demonstrated that ADSC could decrease collagen deposition and scar formation in in vitro experiments. The regulation of the p38/MAPK signaling pathway might play an important role in the process.

Uric acid (UA) is generalized as a byproduct the terminal steps of purine catabolism, which are catalyzed by xanthine oxidoreductase. Xanthine oxidase activity and uric acid synthesis are reported to be increased under tissue ischemia. Therefore, elevated uric acid may act as a prognostic marker of acute myocardial infarction (AMI). A few studies have showed that UA is associated with therapeutic outcomes in patients with acute myocardial infarction. The purpose of this meta-analysis is to evaluate the prognostic significance of the UA as a predictor of in-hospital mortality. We performed a systematic review and included studies that used both UA and in-hospital mortality from Embase and PubMed. Six studies have been included in this review with totally 5,686 patients. During the follow-up, high UA level was found to be associated with an increased risk of in-hospital mortality [risk ratios (RR) 2.10 (1.03-4.26), number needed to harm (NNH) 37], MACE [RR 3.44 (2.33-5.08), NNH 17]. High UA level has the potential to be an important prognostic marker for in-hospital mortality in individuals with AMI.

Recent genome-wide association studies (GWAS) have indicated an association of histone deacetylase 9 (HDAC9) genetic variant with large-vessel stroke and coronary artery disease, among the European population. However, whether HDAC9 gene is associated with an increased susceptibility to acute coronary syndrome (ACS) in Chinese Han population is not known. A total of 472 patients, including patients with ACS (N=309), and those with chest pain syndrome (controls,N=163) were enrolled. Genotyping for HDAC9 gene was performed using the ligation detection reaction assay. A series of statistical analyses were performed to investigate the correlation between HDAC9 gene SNPs and the susceptibility to ACS. The results revealed a significant association of rs2240419 with ACS risk in which the A allele (P= 0.047) and the A allele carriers (AA + AG) (P= 0.037) were more likely to be in ACS group as compared to those in the control group. None of two other SNPs, rs2389995 and rs2107595, were significantly associated with ACS risk (P> 0.05). Logistic regression analyses further revealed an increased risk for ACS in A allele carrier among rs2240419 genotypes, as compared to those with GG homozygotes (odds ratio: 1.869, 95% CI 1.143, 3.056,P= 0.013). A significant correlation between rs2240419 polymorphism of HDAC9 gene and the susceptibility to ACS in Chinese Han population was observed in this study.

Hepatitis C virus (HCV) alters the global behavior of the host cell to create an environment conducive to its own replication, but much remains unknown about how HCV proteins elicit these changes. Thus, a better understanding of the interface between the virus and host cell is required. Here we report the results of a large-scale yeast two-hybrid screen to identify protein–protein interactions between HCV genotype 2a (strain JFH1) and cellular factors. Our study identified 112 unique interactions between 7 HCV and 94 human proteins, over 40% of which have been linked to HCV infection by other studies. These interactions develop a more complete picture of HCV infection, providing insight into HCV manipulation of pathways, such as lipid and cholesterol metabolism, that were previously linked to HCV infection and implicating novel targets within microtubule-organizing centers, the complement system and cell cycle regulatory machinery. In an effort to understand the relationship between HCV and related viruses, we compared the HCV 2a interactome to those of other HCV genotypes and to the related dengue virus. Greater overlap was observed between HCV and dengue virus targets than between HCV genotypes, demonstrating the value of parallel screening approaches when comparing virus–host cell interactomes. Using siRNAs to inhibit expression of cellular proteins, we found that five of the ten shared targets tested (CUL7, PCM1, RILPL2, RNASET2, and TCF7L2) were required for replication of both HCV and dengue virus. These shared interactions provide insight into common features of the viral life cycles of the family Flaviviridae.

Traditional loudness models have been based on the average energy and the critical band analysis of steady-state sounds. However, most environmental sounds, including speech, are dynamic stimuli, in which the average level [e.g., the root-mean-square (rms) level] does not account for the large temporal fluctuations. The question addressed here was whether two stimuli of the same rms level but different peak levels would produce an equal loudness sensation. A modern adaptive procedure was used to replicate two classic experiments demonstrating that the sensation of "beats" in a two- or three-tone complex resulted in a louder sensation [E. Zwicker and H. Fastl, Psychoacoustics-Facts and Models (Springer-Verlag, Berlin, 1990)]. Two additional experiments were conducted to study exclusively the effects of the temporal envelope on the loudness sensation of dynamic stimuli. Loudness balance was performed by normal-hearing listeners between a white noise and a sinusoidally amplitude-modulated noise in one experiment, and by cochlear implant listeners between two harmonic stimuli of the same magnitude spectra, but different phase spectra, in the other experiment. The results from both experiments showed that, for two stimuli of the same rms level, the stimulus with greater temporal fluctuations sometimes produced a significantly louder sensation, depending on the temporal frequency and overall stimulus level. In normal-hearing listeners, the louder sensation was produced for the amplitude-modulated stimuli with modulation frequencies lower than 400 Hz, and gradually disappeared above 400 Hz, resulting in a low-pass filtering characteristic which bore some similarity to the temporal modulation transfer function. The extent to which loudness was greater was a nonmonotonic function of level in acoustic hearing and a monotonically increasingly function in electric hearing. These results suggest that the loudness sensation of a dynamic stimulus is not limited to a 100-ms temporal integration process, and may be determined jointly by a compression process in the cochlea and an expansion process in the brain. A level-dependent compression scheme that may better restore normal loudness of dynamic stimuli in hearing aids and cochlear implants is proposed.

In the present study, we characterized biologic and electrophysiologic consequences of A422T mutation in HERG K(+) channel and the role of pharmacologic or molecular chaperons by employing a heterogeneous expression system in HEK 293 cells. It was found that A422T mutation led to a marked decrease in whole-cell recording currents, and that a complexly glycosylated form protein band at 155 kDa was missing by Western blotting analysis compared to wild type (WT). And the mutant protein was mainly located in the cytoplasm as illustrated in immunocytochemical assay, indicating that the mutation underwent a trafficking defect. In addition, A422T mutation exerted remarkable dominant-negative suppression on WT, resulting in the alteration in the kinetic processes. Strikingly, trafficking-deficient A422T mutation was partially rescued by incubating the cells at a lower temperature, administration of pharmacologic chaperon, E4031 or overexpression of a chaperon molecule, Hsp90, but not Hsp70. In conclusion, missense A422T mutation in HERG K(+) channel results in its trafficking defect, which is rescurable by pharmacologic or molecular chaperones.

The goal of this study was to determine if neurofibromin is expressed in cultured human osteoblasts and chondrocytes that were isolated from ilia and ilial growth plate. Purity of the osteoblast and chondrocyte cultures was confirmed by alkaline phosphatase and toluidine blue staining, respectively. The reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect neurofibromin mRNA. Indirect immunofluorescence and Western blot studies were done to delineate the cellular distribution and expression levels of neurofibromin. These experiments show that neurofibromin is expressed at low levels in human osteoblasts and chondrocytes and is located mainly in the cytoplasm. Only the type II isoform of neurofibromin is detected in these cells. These findings suggest that the type II isoform of neurofibromin plays a physiological role in human osteoblasts and chondrocytes. Whether functional deficiency of neurofibromin is responsible for skeletal abnormalities remains to be established.

International audience; The ability of mammals to resist body fat accumulation is linked to their ability to expand the number and activity of "brown adipocytes" within white fat depots. Activation of β-adrenergic receptors (β-ARs) can induce a functional "brown-like" adipocyte phenotype. As cardiac natriuretic peptides (NPs) and β-AR agonists are similarly potent at stimulating lipolysis in human adipocytes, we investigated whether NPs could induce human and mouse adipocytes to acquire brown adipocyte features, including a capacity for thermogenic energy expenditure mediated by uncoupling protein 1 (UCP1). In human adipocytes, atrial NP (ANP) and ventricular NP (BNP) activated PPARγ coactivator-1α (PGC-1α) and UCP1 expression, induced mitochondriogenesis, and increased uncoupled and total respiration. At low concentrations, ANP and β-AR agonists additively enhanced expression of brown fat and mitochondrial markers in a p38 MAPK-dependent manner. Mice exposed to cold temperatures had increased levels of circulating NPs as well as higher expression of NP signaling receptor and lower expression of the NP clearance receptor (Nprc) in brown adipose tissue (BAT) and white adipose tissue (WAT). NPR-C(-/-) mice had markedly smaller WAT and BAT depots but higher expression of thermogenic genes such as Ucp1. Infusion of BNP into mice robustly increased Ucp1 and Pgc-1α expression in WAT and BAT, with corresponding elevation of respiration and energy expenditure. These results suggest that NPs promote "browning" of white adipocytes to increase energy expenditure, defining the heart as a central regulator of adipose tissue biology.

Dengue virus (DENV), an emerging mosquito-transmitted pathogen capable of causing severe disease in humans, interacts with host cell factors to create a more favorable environment for replication. However, few interactions between DENV and human proteins have been reported to date. To identify DENV-human protein interactions, we used high-throughput yeast two-hybrid assays to screen the 10 DENV proteins against a human liver activation domain library. From 45 DNA-binding domain clones containing either full-length viral genes or partially overlapping gene fragments, we identified 139 interactions between DENV and human proteins, the vast majority of which are novel. These interactions involved 105 human proteins, including six previously implicated in DENV infection and 45 linked to the replication of other viruses. Human proteins with functions related to the complement and coagulation cascade, the centrosome, and the cytoskeleton were enriched among the DENV interaction partners. To determine if the cellular proteins were required for DENV infection, we used small interfering RNAs to inhibit their expression. Six of 12 proteins targeted (CALR, DDX3X, ERC1, GOLGA2, TRIP11, and UBE2I) caused a significant decrease in the replication of a DENV replicon. We further showed that calreticulin colocalized with viral dsRNA and with the viral NS3 and NS5 proteins in DENV-infected cells, consistent with a direct role for calreticulin in DENV replication. Human proteins that interacted with DENV had significantly higher average degree and betweenness than expected by chance, which provides additional support for the hypothesis that viruses preferentially target cellular proteins that occupy central position in the human protein interaction network. This study provides a valuable starting point for additional investigations into the roles of human proteins in DENV infection.

Genome-wide yeast two-hybrid (Y2H) screens were conducted to elucidate the molecular functions of open reading frames (ORFs) encoded by murine γ-herpesvirus 68 (MHV-68). A library of 84 MHV-68 genes and gene fragments was generated in a Gateway entry plasmid and transferred to Y2H vectors. All possible pair-wise interactions between viral proteins were tested in the Y2H assay, resulting in the identification of 23 intra-viral protein-protein interactions (PPIs). Seventy percent of the interactions between viral proteins were confirmed by co-immunoprecipitation experiments. To systematically investigate virus-cellular protein interactions, the MHV-68 Y2H constructs were screened against a cellular cDNA library, yielding 243 viral-cellular PPIs involving 197 distinct cellar proteins. Network analyses indicated that cellular proteins targeted by MHV-68 had more partners in the cellular PPI network and were located closer to each other than expected by chance. Taking advantage of this observation, we scored the cellular proteins based on their network distances from other MHV-68-interacting proteins and segregated them into high (Y2H-HP) and low priority/not-scored (Y2H-LP/NS) groups. Significantly more genes from Y2H-HP altered MHV-68 replication when their expression was inhibited with siRNAs (53% of genes from Y2H-HP, 21% of genes from Y2H-LP/NS, and 16% of genes randomly chosen from the human PPI network; p, Author Summary Persistent infections by the herpesviruses Epstein Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) are associated with tumor formation. To better understand how these and other related viruses interact with their host cells to promote virus replication and cause disease, we studied murine gamma-herpesvirus 68 (MHV-68). MHV-68 belongs to the same group of herpesviruses as EBV and KSHV, but has the advantage of being able to replicate efficiently in cell culture. Our study used genome-wide screens to identify 23 protein-protein interactions between the 80 MHV-68 proteins. Several of these interactions are likely to be important for assembling new viruses. We also discovered 243 interactions between MHV-68 and cellular proteins. To help prioritize cellular proteins for follow up studies, we developed a new computational tool to analyze our data. Proteins with high priority scores were more likely to affect viral replication than low priority proteins. Among the cellular proteins that had the greatest effect on MHV-68 replication was PCBP1, which negatively regulated MHV-68 late gene expression. This study identified many novel cellular proteins involved in MHV-68 replication and established a method to identify important proteins from high-throughput virus-cellular protein-protein interaction data sets.