Peptidylarginine deiminase type 4 (PAD4/PADI4) post-translationally converts peptidylarginine to citrulline. Recent studies suggest that PADI4 represses expression of p53-regulated genes via citrullination of histones at gene promoters.

Autophagy, a major degradative pathway of the lysosomal system, has been implicated in various neurodegenerative diseases. During autophagic process, organelles and proteins are encapsulated in double-membrane vacuoles called autophagosomes, which finally fuse with lysosomes to form autolysosomes where incorporated materials are degraded. Despite extensive investigations in identifying the molecular components that participate in autophagy, little is known about routes and dynamics of autophagosomes/autolysosomes in the neurites of live cells. Hence, in the present study, we aim to investigate the biophysical characteristics of neuritic transport of autolysosomes in PC12 cells. Our study demonstrated that monomeric red fluorescence protein-light chain 3 (mRFP-LC3)-labeled autolysosomes were motile and moved along PC12 neurites in both anterograde and retrograde directions with a bias towards the nucleus during starvation. By using image processing, quantitative analysis was made to show the dynamic biophysical characteristics of these vesicles. The average velocity of anterograde and retrograde transport was 0.33±0.04μm/s and 0.39±0.05μm/s, respectively. Disruption of microtubules by nocodazole completely abolished their movements, suggesting the neuritic transport of autolysosomes depends on microtubules. The directional transport of autolysosomes was also affected by blockage of motor protein activity. Altogether, our study documents many aspects of the highly dynamic movement of autolysosome in PC12 neurites. Autolysosomes transported in a bi-directional manner along microtubules by dynein and kinesin motor proteins. These findings provide valuable insight into understanding the mechanism and control of autophagy in neurites under physiological and pathological conditions.

Tumor suppressors ZNRF3 and RNF43 inhibit Wnt signaling through promoting degradation of Wnt coreceptors Frizzled (FZD) and LRP6, and this activity is counteracted by stem cell growth factor R-spondin. The mechanism by which ZNRF3 and RNF43 recognize Wnt receptors remains unclear. Here we uncover an unexpected role of Dishevelled (DVL), a positive Wnt regulator, in promoting Wnt receptor degradation. DVL knockout cells have significantly increased cell surface levels of FZD and LRP6. DVL is required for ZNRF3/RNF43-mediated ubiquitination and degradation of FZD. Physical interaction with DVL is essential for the Wnt inhibitory activity of ZNRF3/RNF43. Binding of FZD through the DEP domain of DVL is required for DVL-mediated downregulation of FZD. Fusion of the DEP domain to ZNRF3/RNF43 overcomes their DVL dependency to downregulate FZD. Our study reveals DVL as a dual function adaptor to recruit negative regulators ZNRF3/RNF43 to Wnt receptors to ensure proper control of pathway activity.

Dunaliella salina is a unicellular green alga with exceptional halotolerance. Although the D. salina cells are capable to proliferate in hypersaline medium, the intracellular salt concentrations are maintained at a low level. Thus the extracellular but not intracellular Dunaliella proteins are expected to be highly halotolerant. In this research, we compared the salt-dependence of the activity and stability of Hsp90s from the halotolerant alga D. salina (dsHsp90) and the mesophilic alga Chlamydomonas reinhardtii (crHsp90). We found that the ATPase activity of crHsp90 could be enhanced about six-fold by 2M NaCl, while the activity of dsHsp90 showed a much weaker dependence on salinity. When denatured by urea, both crHsp90 and dsHsp90 exhibited an apparent three-state unfolding with the population of an unfolding intermediate. High salinity significantly decreased the Gibbs free energy change of crHsp90 but not dsHsp90 for the transition from the native state to the intermediate. The little dependence of dsHsp90 activity and folding on salinity suggests that dsHsp90 is halotolerant though it is an intracellular protein. We propose that the halotolerance of intracellular Dunaliella proteins might play a role in fighting against the transient intracellular salt fluctuations during hyperosmotic or hypoosmotic shock.

The aim of this study was to examine whether albumin reduced mortality when employed for the resuscitation of adult patients with severe sepsis and septic shock compared with crystalloid by meta-analysis.

Fibroblasts, far from being merely bystander cells, are known to play a specific role in inflammation resolution after an acute injury. As the endogenous "braking signal," resolvins possess potent anti-inflammatory and pro-resolution actions. We demonstrated that the expression of COX-2 protein was significantly peaked initially at 6 hours but then also at 48 hours after LPS stimulation in lung fibroblasts. PGE2 levels also peaked at 6 hours, and PGD2 levels were increased and peaked at 48 hours. However, no significant change in the protein expression of COX-1 was observed after treatment with LPS in lung fibroblasts. Exogenous resolvin D1 inhibited the first peak of COX-2 expression as well as the production of PGE2 induced by LPS. In contrast, exogenous resolvin D1 increased the second peak of COX-2 expression as well as the production of PGD2 induced by LPS. In addition, resolvin D1 inhibited COX-2 expression at 6 hours, which was partly through PI3K/AKT and ERK2 signalling pathways.

T-helper-17 (TH17) cells have critical roles in mucosal defence and in autoimmune disease pathogenesis. They are most abundant in the small intestine lamina propria, where their presence requires colonization of mice with microbiota. Segmented filamentous bacteria (SFB) are sufficient to induce TH17 cells and to promote TH17-dependent autoimmune disease in animal models. However, the specificity of TH17 cells, the mechanism of their induction by distinct bacteria, and the means by which they foster tissue-specific inflammation remain unknown. Here we show that the T-cell antigen receptor (TCR) repertoire of intestinal TH17 cells in SFB-colonized mice has minimal overlap with that of other intestinal CD4(+) T cells and that most TH17 cells, but not other T cells, recognize antigens encoded by SFB. T cells with antigen receptors specific for SFB-encoded peptides differentiated into RORγt-expressing TH17 cells, even if SFB-colonized mice also harboured a strong TH1 cell inducer, Listeria monocytogenes, in their intestine. The match of T-cell effector function with antigen specificity is thus determined by the type of bacteria that produce the antigen. These findings have significant implications for understanding how commensal microbiota contribute to organ-specific autoimmunity and for developing novel mucosal vaccines.

Prokaryotic gene expression is environment-dependent and temperature plays an important role in shaping the gene expression profile. Revealing the regulation mechanisms of gene expression pertaining to temperature has attracted tremendous efforts in recent years particularly owning to the yielding of transcriptome and proteome data by high-throughput techniques. However, most of the previous works concentrated on the characterization of the gene expression profile of individual organism and little effort has been made to disclose the commonality among organisms, especially for the gene sequence features. In this report, we collected the transcriptome and proteome data measured under heat stress condition from recently published literature and studied the sequence determinants for the expression level of heat-responsive genes on multiple layers. Our results showed that there indeed exist commonness and consistent patterns of the sequence features among organisms for the differentially expressed genes under heat stress condition. Some features are attributed to the requirement of thermostability while some are dominated by gene function. The revealed sequence determinants of bacterial gene expression level under heat stress complement the knowledge about the regulation factors of prokaryotic gene expression responding to the change of environmental conditions. Furthermore, comparisons to thermophilic adaption have been performed to reveal the similarity and dissimilarity of the sequence determinants for the response to heat stress and for the adaption to high habitat temperature, which elucidates the complex landscape of gene expression related to the same physical factor of temperature.

It is currently controversial whether remote ischemic preconditioning (RIPC) reduces the incidence of acute kidney injury (AKI) in patients undergoing cardiovascular interventions. The main objective of this meta-analysis was to investigate whether RIPC provides renal protection for patients undergoing cardiac or vascular surgery. We searched the PubMed database (1966-Oct 2015), Embase database (1966-Oct 2015), Google Scholar, Cochrane Library, ClinicalTrials Database and Open Grey. Then we conducted a meta-analysis of the randomized controlled trials that met the inclusion criteria of our study. The interventions included use of an inflatable tourniquet around the limbs or cross-clamping of the iliac arteries before surgery (RIPC groups) and general cardiovascular intervention (control groups). The main outcomes examined included the incidence of AKI; changes in acute kidney injury biomarkers; and use of renal replacement therapy. Other outcomes examined included in-hospital mortality and the lengths of hospital stay and intensive care unit (ICU) stay. Finally, we screened 26 eligible studies containing 6699 patients who underwent cardiac or vascular interventions with RIPC (n = 3343) or without RIPC (n = 3356). The AKI incidence was decreased in the RIPC group as was the length of ICU stay. There were no differences in the changes in AKI biomarkers, use of renal replacement therapy or in-hospital mortality between the two groups. Remote ischemic preconditioning may decrease the occurrence of AKI in cardiovascular surgery patients. Since studies included have a significant heterogeneity, meta-analyses using a stricter inclusion criteria are needed to clarify the renoprotection effect of RIPC.

To examine the comparative efficacy and safety of different antiplatelet regimens in patients with prior non-cardioembolic ischaemic stroke or transient ischaemic attack.

Many bacterial pathogens secrete virulence (effector) proteins that interfere with immune signaling in their host. SpvD is a Salmonella enterica effector protein that we previously demonstrated to negatively regulate the NF-κB signaling pathway and promote virulence of S. enterica serovar Typhimurium in mice. To shed light on the mechanistic basis for these observations, we determined the crystal structure of SpvD and show that it adopts a papain-like fold with a characteristic cysteine-histidine-aspartate catalytic triad comprising Cys-73, His-162, and Asp-182. SpvD possessed an in vitro deconjugative activity on aminoluciferin-linked peptide and protein substrates in vitro A C73A mutation abolished SpvD activity, demonstrating that an intact catalytic triad is required for its function. Taken together, these results strongly suggest that SpvD is a cysteine protease. The amino acid sequence of SpvD is highly conserved across different S. enterica serovars, but residue 161, located close to the catalytic triad, is variable, with serovar Typhimurium SpvD having an arginine and serovar Enteritidis a glycine at this position. This variation affected hydrolytic activity of the enzyme on artificial substrates and can be explained by substrate accessibility to the active site. Interestingly, the SpvDG161 variant more potently inhibited NF-κB-mediated immune responses in cells in vitro and increased virulence of serovar Typhimurium in mice. In summary, our results explain the biochemical basis for the effect of virulence protein SpvD and demonstrate that a single amino acid polymorphism can affect the overall virulence of a bacterial pathogen in its host.

Damage to vascular endothelial cells (VECs) is a critical hallmark of hemorrhagic diseases caused by dengue virus (DENV). However, the precise molecular event involved in DENV binding and infection of VECs has yet to be clarified. In this study, vimentin (55 kDa) was identified to be involved in DENV-2 adsorption into VECs. This protein is located on the surface of VECs and interacts with DENV-2 envelope protein domain III (EDIII). The expression level of the superficial vimentin on VECs was not affected by viral infection or siRNA interference, indicating that the protein exists in a particular mode. Furthermore, the rod domain of the vimentin protein mainly functions in DENV-2 adsorption into VECs. Molecular docking results predicted several residues in vimentin rod and DENV EDIII; these residues may be responsible for cell-virus interactions. We propose that the superficial vimentin could be a novel molecule involved in DENV binding and infection of VECs. DENV EDIII directly interacts with the rod domain of vimentin on the VEC surface and thus mediates the infection.

Sirt3, a mitochondrial NAD+-dependent histone deacetylase, is the only member proven to promote longevity in mammalian Sirtuin family. The processed short form of Sirt3 has been demonstrated to target many mediators of energy metabolism and mitochondrial stress adaptive program. Autophagy serves as a dynamic recycling mechanism and provides energy or metabolic substrates. Among the mechanisms triggered by cardiac stress, opinions vary as to whether autophagy is a protective or detrimental response. Here, by inducing the Sirt3-knockout mice to myocardial hypertrophy with chronic angiotensin II infusion for four weeks, we determined the role of Sirt3 in myocardial hypertrophy and autophagy. In this study, the Sirt3-knockout mice developed deteriorated cardiac function and impaired autophagy compared to wild-type mice. What's more, the overexpression of Sirt3 by lentivirus transfection attenuated cardiomyocytes hypertrophy by promoting autophagy. We further demonstrated that Sirt3 could bind to FoxO1 and activate its deacetylation. Sequentially, deacetylated FoxO1 translocates to the nucleus where it facilitates downstream E3 ubiquitin ligases such as Muscle RING Finger 1 (MuRF1) and muscle atrophy F-box (MAFbx, Atrogin1). Altogether, these results revealed that Sirt3 activation is essential to improve autophagy flux by reducing the acetylation modification on FoxO1, which in turn alleviates myocardial hypertrophy.

Recent studies have shown an association between peripheral arterial disease (PAD) and increased risk of mortality in hemodialysis (HD) patients; however, the estimates vary widely and are inconsistent. It is necessary to elucidate the degree of mortality risk for PAD patients in HD population.

Enteroaggregative Escherichia coli (EAEC) is an emerging cause of acute and persistent diarrhea worldwide. The pathogenesis of different EAEC stains is complicated, however, the early essential step begins with attachment of EAEC to intestinal mucosa via aggregative adherence fimbriae (AAFs). Currently, five different variants have been identified, which all share a degree of similarity in the gene organization of their operons and sequences. Here, we report the solution structure of Agg5A from the AAF/V variant. While preserving the major structural features shared by all AAF members, only Agg5A possesses an inserted helix at the beginning of the donor strand, which together with altered surface electrostatics, renders the protein unable to interact with fibronectin. Hence, here we characterize the first AAF variant with a binding mode that varies from previously described AAFs.

Cellular DNA damage response (DDR) triggered by infection of DNA viruses mediate cell cycle checkpoint activation, DNA repair, or apoptosis induction. In the present study, infection of porcine circovirus type 2 (PCV2), which serves as a major etiological agent of PCV2-associated diseases (PCVAD), was found to elicit a DNA damage response (DDR) as observed by the phosphorylation of H2AX and RPA32 following infection. The response requires active viral replication, and all the ATM (ataxia telangiectasia-mutated kinase), ATR (ATM- and Rad3-related kinase), and DNA-PK (DNA-dependent protein kinase) are the transducers of the DDR signaling events in the PCV2-infected cells as demonstrated by the phosphorylation of ATM, ATR, and DNA-PK signalings as well as reductions in their activations after treatment with specific kinase inhibitors. Inhibitions of ATM, ATR, and DNA-PK activations block viral replication and prevent apoptotic responses as observed by decreases in cleaved poly-ADP ribose polymerase (PARP) and caspase-3 as well as fragmented DNA following PCV2 infection. These results reveal that PCV2 is able to exploit the cellular DNA damage response machinery for its own efficient replication and for apoptosis induction, further extending our understanding for the molecular mechanism of PCV2 infection.

Objective. This study aimed to screen potential crucial lncRNAs and genes involved in aging. Methods. The data of 9 peripheral white adipocytes, respectively, taken from male C57BL/6J mice (6 months, 14 months, and 18 months of age) in GSE25905 were used in this study. Differentially time series expressed lncRNA genes (DE-lncRNAs) and mRNA genes (DEGs) were identified. After cluster analysis of lncRNAs expression pattern, target genes of DE-lncRNAs were predicted from the DEGs, and functional analysis for target genes was conducted. Results. A total of 8301 time series-related DEGs and 43 time series-related DE-lncRNAs were identified. Among them, 41 DE-lncRNAs targeted 1880 DEGs. The DEGs positively regulated by DE-lncRNAs were mainly related to the development of blood vessel and the pathways of cholesterol biosynthesis and elastic fibre formation. Furthermore, the DEGs negatively regulated by DE-lncRNAs were correlated with protein metabolism. Conclusion. These DE-lncRNAs and DEGs are potentially involved in the process of aging.

MicroRNAs (miRNAs) are small, non-coding RNAs capable of postranscriptionally regulating gene expression. Accurate expression profiling is crucial for understanding the biological roles of miRNAs, and exploring them as biomarkers of diseases.

Xenobiotic-mediated induction of cytochrome P450 (CYP) drug metabolizing enzymes (DMEs) is frequently encountered in drug discovery and can influence disposition, pharmacokinetic, and toxicity profiles. The CYP1A subfamily of DMEs plays a central role in the biotransformation of several drugs and environmental chemicals. Autoinduction of drugs through CYP3A enzymes is a common mechanism for their enhanced clearance. However, autoinduction via CYP1A is encountered less frequently. In this report, an experimental compound, A-998679 [3-(5-pyridin-3-yl-1,2,4-oxadiazol-3-yl) benzonitrile], was shown to enhance its own clearance via induction of Cyp1a1 and Cyp1a2. Rats were dosed for 5 days with 30, 100, and 200 mg/kg/day A-998679. During the dosing period, the compound's plasma AUC decreased at 30 mg/kg (95%) and 100 mg/kg (80%). Gene expression analysis and immunohistochemistry of the livers showed a large increase in the mRNA and protein levels of Cyp1a, which was involved in the biotransformation of A-998679. Induction of CYP1A was confirmed in primary rat, human, and dog hepatocytes. The compound also weakly inhibited CYP1A2 in human liver microsomes. A-998679 activated the aryl hydrocarbon receptor (AhR) in a luciferase gene reporter assay in HepG2 cells, upregulated expression of genes associated with AhR activation in rat liver and enhanced nuclear migration of AhR in HepG2 cells. Collectively these results demonstrate that A-998679 is an AhR activator that induces Cyp1a1 and Cyp1a2 expression, resulting in an autoinduction phenomenon. The unique properties of A-998679, along with its novel structure distinct from classical polycyclic aromatic hydrocarbons (PAHs), may warrant its further evaluation as a tool compound for use in studies involving AhR biology and CYP1A-related mechanisms of drug metabolism and toxicity.

The pleckstrin homology (PH) domain is a versatile fold that mediates a variety of protein-protein and protein-phosphatidylinositol lipid interactions. The Ran-binding protein 2 (RanBP2) contains four interspersed Ran GTPase-binding domains (RBD(n = 1-4)) with close structural homology to the PH domain of Bruton's tyrosine kinase. The RBD2, kinesin-binding domain (KBD) and RBD3 comprise a tripartite domain (R2KR3) of RanBP2 that causes the unfolding, microtubule binding and biphasic activation of kinesin-1, a crucial anterograde motor of mitochondrial motility. However, the interplay between Ran GTPase and R2KR3 of RanBP2 in kinesin-1 activation and mitochondrial motility is elusive. We use structure-function, biochemical, kinetic and cell-based assays with time-lapse live-cell microscopy of over 260,000 mitochondrial-motility-related events to find mutually exclusive subdomains in RBD2 and RBD3 towards Ran GTPase binding, kinesin-1 activation and mitochondrial motility regulation. The RBD2 and RBD3 exhibit Ran-GTP-independent, subdomain and stereochemical-dependent discrimination on the biphasic kinetics of kinesin-1 activation or regulation of mitochondrial motility. Further, KBD alone and R2KR3 stimulate and suppress, respectively, multiple biophysical parameters of mitochondrial motility. The regulation of the bidirectional transport of mitochondria by either KBD or R2KR3 is highly coordinated, because their kinetic effects are accompanied always by changes in mitochondrial motile events of either transport polarity. These studies uncover novel roles in Ran GTPase-independent subdomains of RBD2 and RBD3, and KBD of RanBP2, that confer antagonizing and multi-modal mechanisms of kinesin-1 activation and regulation of mitochondrial motility. These findings open new venues towards the pharmacological harnessing of cooperative and competitive mechanisms regulating kinesins, RanBP2 or mitochondrial motility in disparate human disorders.