Targeted functional investigations guided by integrative proteome network analysis revealed significant perturbations of renal tubular cell functions induced by high glucose

Recently, several studies employed various proteomic approaches to define diabetes‐induced changes in renal proteins. However, functional significance of those datasets in diabetic nephropathy remained unclear. We thus performed integrative proteome network analysis of such datasets followed by various targeted functional studies in distal renal tubular cells treated with high glucose (HG) (25 mM) compared to normal glucose (NG) (5.5 mM) and NG + mannitol (M) (5.5 + 19.5 mM). The data showed that at 96 h when cell proliferation/death, tight junction protein and β‐/F‐actin expression and organization, and transepithelial resistance remained unchanged, only HG caused increased levels of HSP90, HSP70, and HSP60, and increased accumulation of intracellular protein aggregates. In addition, HG also induced overproduction of intracellular ROS, decreased catalase level, increased level of oxidatively modified proteins, increased intracellular ATP level, and defective transepithelial Ca²⁺ transport. However, both HG and M increased the levels of ubiquitinated proteins. Taken together, this study demonstrated significant perturbations of distal renal tubular cells induced by HG based on targeted functional studies guided by integrative proteome network analysis. These data may, at least in part, lead to better understanding of the pathogenic mechanisms of diabetic nephropathy.     

Epigenetic changes and transposon reactivation in Thai rice hybrids

Inter- or intraspecific hybridization is the first step in transferring exogenous traits to the germplasm of a recipient crop. One of the complicating factors is the occurrence of epigenetic modifications of the hybrids, which in turn can change their gene expression and phenotype. In this study we present an analysis of epigenome changes in rice hybrids that were obtained by crossing rice cultivars, most of them of indica type and Thai origin. Comparing amplified fragment length polymorphism (AFLP) fingerprints of twenty-four cultivars, we calculated Nei’s indexes for measuring genetic relationships. Epigenetic changes in their hybrids were established using methylation-sensitive AFLP fingerprinting and transposon display of the rice transposable elements (TEs) Stowaway Os-1 and Mashu, leading to the question whether the relationship between parental genomes is a predictor of epigenome changes, TE reactivation and changes in TE methylation. Our study now reveals that the genetic relationship between the parents and DNA methylation changes in their hybrids is not significantly correlated. Moreover, genetic distance correlates only weakly with Mashu reactivation, whereas it does not correlate with Stowaway Os-1 reactivation. Our observations also suggest that epigenome changes in the hybrids are localized events affecting specific chromosomal regions and transposons rather than affecting the genomic methylation landscape as a whole. The weak correlation between genetic distance and Mashu methylation and reactivation points at only limited influence of genetic background on the epigenetic status of the transposon. Our study further demonstrates that hybridizations between and among specific japonica and indica cultivars induce both genomic DNA methylation and reactivation/methylation change in the Stowaway Os-1 and Mashu transposons. The observed epigenetic changes seem to affect the transposons in a clear manner, partly driven by stochastic processes, which may account for a broader phenotypic plasticity of the hybrids. A better understanding of the epigenome changes leading to such transposon activation can lead to the development of novel tools for more variability in future rice breeding.     

Discovery of Opisthorchis lobatus (Trematoda: Opisthorchiidae): a new record of small liver flukes in the Greater Mekong Sub-region

Metacercariae, morphologically similar to those of small liver flukes, were found to parasitize red-tailed snakehead fish, Channa limbata, collected from the city of Vientiane, Lao People's Democratic Republic. Adult worms that were recovered from experimentally-infected hamsters showed characteristics distinctly different from Opisthorchis viverrini, but closely similar to Opisthorchis lobatus, which was first reported in poultry (Anas sp.) from Pakistan. The present study aimed to redescribe O. lobatus based on the adult worms recovered from experimentally-infected hamsters. Additionally, it aimed to document the genetic relationships among O. lobatus and other opisthorchiid liver flukes using the mitochondrial cytochrome c oxidase subunit 1 (COI) gene and the internal transcribed spacer 2 (ITS2) region. DNA alignment of the O. lobatus and O. viverrini COI partial sequences (330 bp) showed 3.03% fixed differences (2.72% of amino acids changed) while the ITS2 region (350 bp) indicated a 0.86% difference for nucleotides. Species boundaries between the 2 parasites were determined by neighbor-joining analysis using the molecular sequence data. The phenogram confirmed that O. lobatus was distinctly different from O. viverrini, representing the first reported instance of O. lobatus in the Greater Mekong Sub-region (GMS) and the first record of C. limbata as the second intermediate host of a small liver fluke. Questions regarding human infection and the extent of the geographic distribution of these species should be investigated further.     

Glycoform-Selective Prion Formation in Sporadic and Familial Forms of Prion Disease

The four glycoforms of the cellular prion protein (PrP^C) variably glycosylated at the two N-linked glycosylation sites are converted into their pathological forms (PrP^Sc) in most cases of sporadic prion diseases. However, a prominent molecular characteristic of PrP^Sc in the recently identified variably protease-sensitive prionopathy (VPSPr) is the absence of a diglycosylated form, also notable in familial Creutzfeldt-Jakob disease (fCJD), which is linked to mutations in PrP either from Val to Ile at residue 180 (fCJD^V180I) or from Thr to Ala at residue 183 (fCJD^T183A). Here we report that fCJD^V180I, but not fCJD^T183A, exhibits a proteinase K (PK)-resistant PrP (PrP^res) that is markedly similar to that observed in VPSPr, which exhibits a five-step ladder-like electrophoretic profile, a molecular hallmark of VPSPr. Remarkably, the absence of the diglycosylated PrP^res species in both fCJD^V180I and VPSPr is likewise attributable to the absence of PrP^res glycosylated at the first N-linked glycosylation site at residue 181, as in fCJD^T183A. In contrast to fCJD^T183A, both VPSPr and fCJD^V180I exhibit glycosylation at residue 181 on di- and monoglycosylated (mono181) PrP prior to PK-treatment. Furthermore, PrP^V180I with a typical glycoform profile from cultured cells generates detectable PrP^res that also contains the diglycosylated PrP in addition to mono- and unglycosylated forms upon PK-treatment. Taken together, our current in vivo and in vitro studies indicate that sporadic VPSPr and familial CJD^V180I share a unique glycoform-selective prion formation pathway in which the conversion of diglycosylated and mono181 PrP^C to PrP^Sc is inhibited, probably by a dominant-negative effect, or by other co-factors.     

Spontaneous reconstitution of discoidal HDL from sphingomyelin-containing model membranes by apolipoprotein A-I

Nascent HDL is known to be formed by the interaction of apolipoprotein A-I (apoA-I) with transmembrane ABCA1, but the molecular mechanism by which nascent HDL forms is less well understood. Here, we studied how reconstituted high density lipoprotein (rHDL) forms spontaneously on the interaction of apoA-I with model membranes. The formation of rHDL from pure phosphatidylcholine (PC) large unilamellar vesicles (LUVs) proceeded very slowly at 37.0 degrees C, but sphingomyelin (SM) -rich PC/SM LUVs, which are in a gel/liquid-disordered phase (L(d) phase) at this temperature, were rapidly microsolubilized to form rHDL by apoA-I. The addition of cholesterol decreased the rate at which rHDL formed and induced the selective extraction of lipids by apoA-I, which preferably extracted lipids of L(d) phase rather than lipids of liquid-ordered phase. In addition, apoA-I extracted lipids from the outer and inner leaflets of LUVs simultaneously. These results suggest that the heterogeneous interface of the mixed membranes facilitates the insertion of apoA-I and induces L(d) phase-selective but leaflet-nonselective lipid extraction to form rHDL; they are compatible with recent cell works on apoA-I-dependent HDL generation.     

Inactivation of <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis><Emphasis Type="Italic">bsaQ</Emphasis> results in decreased invasion efficiency and delayed escape of bacteria from endocytic vesicles

Burkholderia pseudomallei, an infectious Gram-negative bacterium, is the causative pathogen of melioidosis. In the present study, a B. pseudomallei strain with mutation in the bsaQ gene, encoding a structural component of the type III secretion system (T3SS), was constructed. This bsaQ mutation caused a marked decrease in secretion of BopE effector and BipD translocator proteins into culture supernatant. The B. pseudomallei bsaQ mutant also exhibited decreased efficiencies of plaque formation, invasion into non-phagocytic cells and multinucleated giant cell (MNGC) development in a J774A.1 macrophage cell line. Co-localization of the bacteria and lysosome-associated membrane glycoprotein-1 (LAMP-1) containing vesicles suggested that defects in MNGC formation may result from the delayed ability of this B. pseudomallei mutant to escape from the vacuoles of macrophages. Veerachat Muangsombut and Supaporn Suparak contributed equally to this work.     

Panton-Valentine leukocidin is not the primary determinant of outcome for Staphylococcus aureus skin infections: evaluation from the CANVAS studies

The impact of Panton-Valentine leukocidin (PVL) on the severity of complicated skin and skin structure infections (cSSSI) caused by Staphylococcus aureus is controversial. We evaluated potential associations between clinical outcome and PVL presence in both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolates from patients enrolled in two large, multinational phase three clinical trials assessing ceftaroline fosamil for the treatment of cSSSI (the CANVAS 1 and 2 programs). Isolates from all microbiologically evaluable patients with monomicrobial MRSA or MSSA infections (n?=?473) were genotyped by PCR for pvl and underwent pulsed-field gel electrophoresis (PFGE). Genes encoding pvl were present in 266/473 (56.2%) isolates. Infections caused by pvl-positive S. aureus were associated with younger patient age, North American acquisition, and presence of major abscesses (P<0.001 for each). Cure rates of patients infected with pvl-positive and pvl-negative S. aureus were similar overall (93.6% versus 92.8%; P?=?0.72), and within MRSA-infected (94.5% vs. 93.1%; P?=?0.67) and MSSA-infected patients (92.2% vs. 92.7%; P?=?1.00). This finding persisted after adjustment for multiple patient characteristics. Outcomes were also similar when USA300 PVL+ and non-USA300 PVL+ infections were compared. The results of this contemporary, international study suggest that pvl presence was not the primary determinant of outcome in patients with cSSSI due to either MRSA or MSSA.     

Oral Streptococci Utilize a Siglec-Like Domain of Serine-Rich Repeat Adhesins to Preferentially Target Platelet Sialoglycans in Human Blood

Damaged cardiac valves attract blood-borne bacteria, and infective endocarditis is often caused by viridans group streptococci. While such bacteria use multiple adhesins to maintain their normal oral commensal state, recognition of platelet sialoglycans provides an intermediary for binding to damaged valvular endocardium. We use a customized sialoglycan microarray to explore the varied binding properties of phylogenetically related serine-rich repeat adhesins, the GspB, Hsa, and SrpA homologs from Streptococcus gordonii and Streptococcus sanguinis species, which belong to a highly conserved family of glycoproteins that contribute to virulence for a broad range of Gram-positive pathogens. Binding profiles of recombinant soluble homologs containing novel sialic acid-recognizing Siglec-like domains correlate well with binding of corresponding whole bacteria to arrays. These bacteria show multiple modes of glycan, protein, or divalent cation-dependent binding to synthetic glycoconjugates and isolated glycoproteins in vitro. However, endogenous asialoglycan-recognizing clearance receptors are known to ensure that only fully sialylated glycans dominate in the endovascular system, wherein we find these particular streptococci become primarily dependent on their Siglec-like adhesins for glycan-mediated recognition events. Remarkably, despite an excess of alternate sialoglycan ligands in cellular and soluble blood components, these adhesins selectively target intact bacteria to sialylated ligands on platelets, within human whole blood. These preferred interactions are inhibited by corresponding recombinant soluble adhesins, which also preferentially recognize platelets. Our data indicate that circulating platelets may act as inadvertent Trojan horse carriers of oral streptococci to the site of damaged endocardium, and provide an explanation why it is that among innumerable microbes that gain occasional access to the bloodstream, certain viridans group streptococci have a selective advantage in colonizing damaged cardiac valves and cause infective endocarditis.