Identification and preliminary validation of novel biomarkers of acute hepatic ischaemia/reperfusion injury using dual-platform proteomic/degradomic approaches

Hepatic ischaemia/reperfusion (I/R), a major cause of liver damage associated with multiple trauma, haemorrhagic and septic shock, and liver transplantation, contributes significantly to multiple organ failure. Development of novel sensitive biomarkers that detect early stages of liver damage is vital for effective management and treatment of ischaemic liver injury. By using high-throughput immunoblotting and cation–anion exchange chromatography/reversed-phase liquid chromatography-tandem mass-spectrometry, we identified several hepatic proteins, including argininosuccinate synthase (ASS) and estrogen sulfotransferase (EST-1), which were degraded in the liver and rapidly released into circulation during I/R injury. ASS accumulated in serum within 10 min, reached a steady state at 30 min, and persisted up until 3 h after reperfusion following 30 min of total hepatic ischaemia. EST-1 appeared rapidly in blood and attained maximum within 1 hour followed by a decline at 3 h of reperfusion. No ASS or EST-1 protein was detected in serum of control or sham operated rats. ASS and EST-1 exhibited greater sensitivity and specificity toward I/R liver injury as compared with alanine aminotransferase (ALT), an established marker of hepatocellular necrosis. In contrast, serum ASS and EST-1 were undetectable in rats with chronic alcoholic liver disease, while the levels of ALT protein were significantly increased. In addition, ASS, but not EST-1 or ALT accumulated in blood only 6 h after treatment with hepatotoxic combination of lipopolysaccharide and D-galactosamine. These data demonstrate the utility of ASS and EST-1 as novel sensitive and specific biomarkers of acute liver ischaemic injury for prospective clinical studies.     

Bioactives from probiotics for dermal health: functions and benefits

Probiotics have been extensively reviewed for decades, emphasizing on improving general gut health. Recently, more studies showed that probiotics may exert other health‐promoting effects beyond gut well‐being, attributed to the rise of the gut–brain axis correlations. Some of these new benefits include skin health such as improving atopic eczema, atopic dermatitis, healing of burn and scars, skin‐rejuvenating properties and improving skin innate immunity. Increasing evidence has also showed that bacterial compounds such as cell wall fragments, their metabolites and dead bacteria can elicit certain immune responses on the skin and improve skin barrier functions. This review aimed to underline the mechanisms or the exact compounds underlying the benefits of bacterial extract on the skin based on evidences from in vivo and in vitro studies. This review could be of help in screening of probiotic strains with potential dermal enhancing properties for topical applications.     

Novel Systems for Dynamically Assessing Insulin Action in Live Cells Reveals Heterogeneity in the Insulin Response

Regulated GLUT4 trafficking is a key action of insulin. Quantitative stepwise analysis of this process provides a powerful tool for pinpointing regulatory nodes that contribute to insulin regulation and insulin resistance. We describe a novel GLUT4 construct and workflow for the streamlined dissection of GLUT4 trafficking; from simple high throughput screens to high resolution analyses of individual vesicles. We reveal single cell heterogeneity in insulin action highlighting the utility of this approach – each cell displayed a unique and highly reproducible insulin response, implying that each cell is hard‐wired to produce a specific output in response to a given stimulus. These data highlight that the response of a cell population to insulin is underpinned by extensive heterogeneity at the single cell level. This heterogeneity is pre‐programmed within each cell and is not the result of intracellular stochastic events.     

Importin‐7 Mediates Nuclear Trafficking of DNA in Mammalian Cells

Eukaryotic cells have the ability to uptake and transport endogenous and exogenous DNA in their nuclei, however little is known about the specific pathways involved. Here we show that the nuclear transport receptor importin 7 (imp7) supports nuclear import of supercoiled plasmid DNA and human mitochondrial DNA in a Ran and energy‐dependent way. The imp7‐dependent pathway was specifically competed by excess DNA but not by excess of maltose‐binding protein fused with the classical nuclear localizing signal (NLS) or the M9 peptides. Transport of DNA molecules complexed with poly‐l ‐lysine was impaired in intact cells depleted of imp7, and DNA complexes remained localized in the cytoplasm. Poor DNA nuclear import in cells depleted of imp7 directly correlated with lower gene expression levels in these cells compared to controls. Inefficient nuclear import of transfected DNA induced greater upregulation of the interferon pathway, suggesting that rapid DNA nuclear import may prevent uncontrolled activation of the innate immune response. Our results provide evidence that imp7 is a non‐redundant component of an intrinsic pathway in mammalian cells for efficient accumulation of exogenous and endogenous DNA in the nucleus, which may be critical for the exchange of genetic information between mitochondria and nuclear genomes and to control activation of the innate immune response .     

A Sorting Nexin 17‐Binding Domain Within the LRP1 Cytoplasmic Tail Mediates Receptor Recycling Through the Basolateral Sorting Endosome

Sorting nexin 17 (SNX17) is an adaptor protein present in early endosomal antigen 1 (EEA1)‐positive sorting endosomes that promotes the efficient recycling of low‐density lipoprotein receptor‐related protein 1 (LRP1) to the plasma membrane through recognition of the first NPxY motif in the cytoplasmic tail of this receptor. The interaction of LRP1 with SNX17 also regulates the basolateral recycling of the receptor from the basolateral sorting endosome (BSE). In contrast, megalin, which is apically distributed in polarized epithelial cells and localizes poorly to EEA1‐positive sorting endosomes, does not interact with SNX17, despite containing three NPxY motifs, indicating that this motif is not sufficient for receptor recognition by SNX17. Here, we identified a cluster of 32 amino acids within the cytoplasmic domain of LRP1 that is both necessary and sufficient for SNX17 binding. To delineate the function of this SNX17‐binding domain, we generated chimeric proteins in which the SNX17‐binding domain was inserted into the cytoplasmic tail of megalin. This insertion mediated the binding of megalin to SNX17 and modified the cell surface expression and recycling of megalin in non‐polarized cells. However, the polarized localization of chimeric megalin was not modified in polarized Madin‐Darby canine kidney cells. These results provide evidence regarding the molecular and cellular mechanisms underlying the specificity of SNX17‐binding receptors and the restricted function of SNX17 in the BSE .      

Aquaporin Trafficking in Plant Cells: An Emerging Membrane‐Protein Model

Aquaporins (AQPs) are channel proteins that facilitate the transport of water and small solutes across biological membranes. In plants, AQPs exhibit a high multiplicity of isoforms in relation to a high diversity of sub‐cellular localizations, at the plasma membrane (PM) and in various intracellular compartments. Some members also exhibit a dual localization in distinct cell compartments, whereas others show polarized or domain‐specific expression at the PM or tonoplast, respectively. A diversity of mechanisms controlling the routing of newly synthesized AQPs towards their destination membranes and involving diacidic motifs, phosphorylation or tetramer assembly is being uncovered. Recent approaches using single particle tracking, fluorescence correlation spectroscopy and fluorescence recovery after photobleaching have, in combination with pharmacological interference, stressed the peculiarities of AQP sub‐cellular dynamics in environmentally challenging conditions. A role for clathrin and sterol‐rich domains in cell surface dynamics and endocytosis of PM AQPs was uncovered. These recent advances provide deep insights into the cellular mechanisms of water transport regulation in plants. They also point to AQPs as an emerging model for studying the sub‐cellular dynamics of plant membrane proteins .     

Spatial and temporal population genetic variation and structure of Nothotsuga longibracteata (Pinaceae), a relic conifer species endemic to subtropical China

Nothotsuga longibracteata, a relic and endangered conifer species endemic to subtropical China, was studied for examining the spatial-temporal population genetic variation and structure to understand the historical biogeographical processes underlying the present geographical distribution. Ten populations were sampled over the entire natural range of the species for spatial analysis, while three key populations with large population sizes and varied age structure were selected for temporal analyses using both nuclear microsatellites (nSSR) and chloroplast microsatellites (cpSSR). A recent bottleneck was detected in the natural populations of N. longibracteata. The spatial genetic analysis showed significant population genetic differentiation across its total geographical range. Notwithstanding, the temporal genetic analysis revealed that the level of genetic diversity between different age class subpopulations remained constant over time. Eleven refugia of the Last Glacial Maximum were identified, which deserve particular attention for conservation management.     

Cryo‐Immunoelectron Microscopy of Adherent Cells Improved by the Use of Electrospun Cell Culture Substrates

Electrospun nanofibres are an excellent cell culture substrate, enabling the fast and non‐disruptive harvest and transfer of adherent cells for microscopical and biochemical analyses. Metabolic activity and cellular structures are maintained during the only half a minute‐long harvest and transfer process. We show here that such samples can be optimally processed by means of cryofixation combined either with freeze‐substitution, sample rehydration and cryosection‐immunolabelling or with freeze‐fracture replica‐immunolabelling. Moreover, electrospun fibre substrates are equally suitable for complementary approaches, such as biochemistry, fluorescence microscopy and cytochemistry.     

A trs20 Mutation That Mimics an SEDT‐Causing Mutation Blocks Selective and Non‐Selective Autophagy: A Model for TRAPP III Organization

TRAPP is a multisubunit complex that functions in membrane traffic. Mutations in the mammalian TRAPP protein C2 are linked to the skeletal disorder spondyloepiphyseal dysplasia tarda (SEDT) that is thought to arise from an inability to secrete procollagen from the endoplasmic reticulum. Here, we show that C2 binds to the SNARE protein Syntaxin 5 and this interaction is weakened by an SEDT‐causing missense mutation (D47Y). Interestingly, the equivalent mutation (D46Y) in the yeast C2 homolog Trs20p does not block anterograde traffic but did affect endocytosis. The trs20D46Y mutation interfered with the interaction between Trs20p and Trs85p (TRAPP III‐specific subunit), Trs120p and Trs130p (TRAPP II‐specific subunits). Size exclusion chromatography suggested that this yeast mutation destabilized the TRAPP III complex that is involved in autophagy. We further show that this mutation blocks both the selective cytosol‐to‐vacuole (cvt) pathway as well as non‐selective autophagy. We demonstrate that the apparent molecular size of the TRAPP III complex is dependent upon membranes, and that the presence of TRAPP III is dependent upon Atg9p. Finally, we demonstrate that lipidated Bet3p is enriched in TRAPP III and that lipidation increases the efficiency of autophagy. Our study suggests that Trs20p acts as an adaptor for Trs85p and Trs120p and reveals complexities in TRAPP III assembly and function. The implications of C2D47Y in SEDT are discussed .