Many Gram-negative bacteria utilize specialized secretion systems to inject proteins (effectors) directly into host cells. Little is known regarding how bacteria ensure that only small subsets of the thousands of proteins they encode are recognized as substrates of the secretion systems, limiting their identification through bioinformatic analyses. Many of these proteins require chaperones to direct their secretion. Here, using the newly described protein interaction platform assay, we demonstrate that type 3 secretion system class IB chaperones from one bacterium directly bind their own effectors as well as those from other species. In addition, we observe that expression of class IB homologs from seven species, including pathogens and endosymbionts, mediate the translocation of effectors from Shigella directly into host cells, demonstrating that class IB chaperones are often functionally interchangeable. Notably, class IB chaperones bind numerous effectors. However, as previously proposed, they are not promiscuous; rather they recognize a defined sequence that we designate the conserved chaperone-binding domain (CCBD) sequence [(LMIF)(1)XXX(IV)(5)XX(IV)(8)X(N)(10)]. This sequence is the first defined amino acid sequence to be identified for any interspecies bacterial secretion system, i.e., a system that delivers proteins directly into eukaryotic cells. This sequence provides a new means to identify substrates of type III secretion systems. Indeed, using a pattern search algorithm for the CCBD sequence, we have identified the first two probable effectors from an endosymbiont, Sodalis glossinidius.

About 1 million expressed sequence tag (EST) sequences comprising 125.3 Mb nucleotides were accreted from 51 cDNA libraries constructed from a variety of tissues and organs under a range of conditions, including abiotic stresses and pathogen challenges in common wheat (Triticum aestivum). Expressed sequence tags were assembled with stringent parameters after processing with inbuild scripts, resulting in 37,138 contigs and 215,199 singlets. In the assembled sequences, 10.6% presented no matches with existing sequences in public databases. Functional characterization of wheat unigenes by gene ontology annotation, mining transcription factors, full-length cDNA, and miRNA targeting sites were carried out. A bioinformatics strategy was developed to discover single-nucleotide polymorphisms (SNPs) within our large EST resource and reported the SNPs between and within (homoeologous) cultivars. Digital gene expression was performed to find the tissue-specific gene expression, and correspondence analysis was executed to identify common and specific gene expression by selecting four biotic stress-related libraries. The assembly and associated information cater a framework for future investigation in functional genomics.

UDP-galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyzes the reversible conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). As in prokaryotic UGMs, the flavin needs to be reduced for the enzyme to be active. Here we present the first eukaryotic UGM structures from Aspergillus fumigatus (AfUGM). The structures are of UGM alone, with the substrate UDP-Galp and with the inhibitor UDP. Additionally, we report the structures of AfUGM bound to substrate with oxidized and reduced flavin. These structures provide insight into substrate recognition and structural changes observed upon substrate binding involving the mobile loops and the critical arginine residues Arg-182 and Arg-327. Comparison with prokaryotic UGM reveals that despite low sequence identity with known prokaryotic UGMs the overall fold is largely conserved. Structural differences between prokaryotic UGM and AfUGM result from inserts in AfUGM. A notable difference from prokaryotic UGMs is that AfUGM contains a third flexible loop (loop III) above the si-face of the isoalloxazine ring that changes position depending on the redox state of the flavin cofactor. This loop flipping has not been observed in prokaryotic UGMs. In addition we have determined the crystals structures and steady-state kinetic constants of the reaction catalyzed by mutants R182K, R327K, R182A, and R327A. These results support our hypothesis that Arg-182 and Arg-327 play important roles in stabilizing the position of the diphosphates of the nucleotide sugar and help to facilitate the positioning of the galactose moiety for catalysis.

Unbiased proteomic screens provide a powerful tool for defining protein-protein interaction networks. Previous studies employed multidimensional protein identification technology to identify the Sox2-interactome in embryonic stem cells (ESC) undergoing differentiation in response to a small increase in the expression of epitope-tagged Sox2. Thus far the Sox2-interactome in ESC has not been determined. To identify the Sox2-interactome in ESC, we engineered ESC for inducible expression of different combinations of epitope-tagged Sox2 along with Oct4, Klf4, and c-Myc. Epitope-tagged Sox2 was used to circumvent the lack of suitable Sox2 antibodies needed to perform an unbiased proteomic screen of Sox2-associated proteins. Although i-OS-ESC differentiate when both Oct4 and Sox2 are elevated, i-OSKM-ESC do not differentiate even when the levels of the four transcription factors are coordinately elevated ∼2-3-fold. Our findings with i-OS-ESC and i-OSKM-ESC provide new insights into the reasons why ESC undergo differentiation when Sox2 and Oct4 are elevated in ESC. Importantly, the use of i-OSKM-ESC enabled us to identify the Sox2-interactome in undifferentiated ESC. Using multidimensional protein identification technology, we identified >70 proteins that associate with Sox2 in ESC. We extended these findings by testing the function of the Sox2-assoicated protein Smarcd1 and demonstrate that knockdown of Smarcd1 disrupts the self-renewal of ESC and induces their differentiation. Together, our work provides the first description of the Sox2-interactome in ESC and indicates that Sox2 along with other master regulators is part of a highly integrated protein-protein interaction landscape in ESC.

We have previously shown that human mesenchymal stem cells (hMSCs) can reduce toxin-induced neurodegeneration in a well characterized rodent model of Parkinson's disease. However, the precise mechanisms, optimal cell concentration required for neuroprotection, and detailed cell tracking need to be defined. We exploited a near-infrared imaging platform to perform noninvasive tracing following transplantation of tagged hMSCs in live parkinsonian rats.

Superparamagnetic iron oxide nanoparticles are attractive materials that have been widely used in medicine for drug delivery, diagnostic imaging, and therapeutic applications. In our study, superparamagnetic iron oxide nanoparticles and the anticancer drug, doxorubicin hydrochloride, were encapsulated into poly (D, L-lactic-co-glycolic acid) poly (ethylene glycol) (PLGA-PEG) nanoparticles for local treatment. The magnetic properties conferred by superparamagnetic iron oxide nanoparticles could help to maintain the nanoparticles in the joint with an external magnet.

The immature (or reticulated) platelet fraction (IPF) is rich in nucleic acids, especially RNA, and can be used as a predictive factor for platelet recovery in platelet immunomediated consumption or in postchemotherapy myelosuppression. Our aim was to determine if transfusions with IPF-rich solutions, during autologous peripheral blood stem cell transplantation, reduce the occurrence of bleeding and hemorrhagic complications.

Effective implementation of evidence-based interventions in "real-world" settings can be challenging. Interventions based on externally valid trial findings can be even more difficult to apply in resource-limited settings, given marked differences-in provider experience, patient population, and health systems-between those settings and the typical clinical trial environment. Under the auspices of the Integrated Management of Physician-Delivered Alcohol Care for Tuberculosis Patients (IMPACT) study, a randomized, controlled effectiveness trial, and as an integrated component of tuberculosis treatment in Tomsk, Russia, we adapted two proven alcohol interventions to the delivery of care to 200 patients with alcohol use disorders. Tuberculosis providers performed screening for alcohol use disorders and also delivered naltrexone (with medical management) or a brief counseling intervention either independently or in combination as a seamless part of routine care. We report the innovations and challenges to intervention design, training, and delivery of both pharmacologic and behavioral alcohol interventions within programmatic tuberculosis treatment services. We also discuss the implications of these lessons learned within the context of meeting the challenge of providing evidence-based care in resource-limited settings.

We have shown previously that Rab6, a small, trans-Golgi-localized GTPase, acts upstream of the conserved oligomeric Golgi complex (COG) and ZW10/RINT1 retrograde tether complexes to maintain Golgi homeostasis. In this article, we present evidence from the unbiased and high-resolution approach of electron microscopy and electron tomography that Rab6 is essential to the trans-Golgi trafficking of two morphological classes of coated vesicles; the larger corresponds to clathrin-coated vesicles and the smaller to coat protein I (COPI)-coated vesicles. On the basis of the site of coated vesicle accumulation, cisternal dilation and the normal kinetics of cargo transport from the endoplasmic reticulum (ER) to Golgi followed by delayed Golgi to cell surface transport, we suggest that Golgi function in cargo transport is preferentially inhibited at the trans-Golgi/trans-Golgi network (TGN). The >50% increase in Golgi cisternae number in Rab6-depleted HeLa cells that we observed may well be coupled to the trans-Golgi accumulation of COPI-coated vesicles; depletion of the individual Rab6 effector, myosin IIA, produced an accumulation of uncoated vesicles with if anything a decrease in cisternal number. These results are the first evidence for a Rab6-dependent protein machine affecting Golgi-proximal, coated vesicle accumulation and probably transport at the trans-Golgi and the first example of concomitant cisternal proliferation and increased Golgi stack organization under inhibited transport conditions.

Assessment of cardiac function is key in the management of intensive care unit (ICU) patients and frequently relies on the use of standard transthoracic echocardiography (TTE). A commercially available new generation ultrasound system with two-dimensional imaging capability, which has roughly the size of a mobile phone, is adequately suited to extend the physical examination. The primary endpoint of this study was to evaluate the additional value of this new miniaturized device used as an ultrasonic stethoscope (US) for the determination of left ventricular (LV) systolic function, when compared to conventional clinical assessment by experienced intensivists. The secondary endpoint was to validate the US against TTE for the semi-quantitative assessment of left ventricular ejection fraction (LVEF) in ICU patients.

In humans, inflammation of either the urinary bladder or the distal colon often results in sensory cross-sensitization between these organs. Limited information is known about the mechanisms underlying this clinical syndrome. Studies with animal models have demonstrated that activation of primary afferent pathways may have a role in mediating viscero-visceral cross-organ sensitization.

In experimentally induced puromycine aminonucleoside nephrosis (PAN) animal models, nephrotic syndrome with minimal change disease and focal and segmental sclerosis-like nephritis similar to that in human is demonstrated; however, the real mechanism of PAN is not yet elucidated. Platelet derived endothelial cell growth factor (PD-ECGF), an endothelial mitogen protein, is believed to take part in microvessel formation and in stimulation of angiogenesis and its expression has not been totally demonstrated in PAN rats yet. In this study, we aimed to examine PD-ECGF expression in acute and chronic PAN induced in rats and find out the association between its expression and the stages of angiogenesis in kidney.

The structural and functional annotation of genomes is now heavily based on data obtained using automated pipeline systems. The key for an accurate structural annotation consists of blending similarities between closely related genomes with biochemical evidence of the genome interpretation. In this work we applied high-throughput proteogenomics to Ruegeria pomeroyi, a member of the Roseobacter clade, an abundant group of marine bacteria, as a seed for the annotation of the whole clade.

Individuals with a history of childhood maltreatment present increased rates of metabolic disturbances, but the underlying mechanisms for such phenomena are poorly understood. This study examined whether the secretion of adipokines, adipocyte-derived inflammation markers closely associated with metabolic disorders, is altered in individuals with a history of childhood maltreatment. The serum levels of inflammatory markers adiponectin and resistin were measured from 147 general population participants who had a history of adverse mental symptoms, and who also reported their experiences of childhood maltreatment. Participants with experiences of childhood maltreatment (n=30) had lowered levels of serum adiponectin (p=0.007) and resistin (p=0.028). The differences in adiponectin levels persisted in multivariate modeling with adjustments for age, gender, and body mass index (OR for each 1 standard deviation decrease in the serum adiponectin level 2.65, 95% CI 1.31-5.35, p=0.007). Additional adjustments for marital status or a diagnosis of major depressive disorder, or the exclusion of individuals using NSAIDs, oral corticosteroids, or antidepressants did not alter the results. The association between resistin levels and childhood maltreatment did not remain independent in the same models. Our findings suggest that in individuals with previously reported adverse mental symptoms, a history of childhood maltreatment is independently associated with lowered levels of the anti-inflammatory marker adiponectin. This may lead to a lowered anti-inflammatory buffer capacity, which can, in turn, increase the susceptibility to physical and psychological states characterized by pronounced pro-inflammation.

The potato genome sequence derived from the Solanum tuberosum Group Phureja clone DM1-3 516 R44 provides unparalleled insight into the genome composition and organisation of this important crop. A key class of genes that comprises the vast majority of plant resistance (R) genes contains a nucleotide-binding and leucine-rich repeat domain, and is collectively known as NB-LRRs.

We sought to assess the potential of human cord blood-derived mesenchymal stem cells (CB-MSCs) to derive insulin-producing, glucose-responsive cells. We show here that differentiation protocols based on stepwise culture conditions initially described for human embryonic stem cells (hESCs) lead to differentiation of cord blood-derived precursors towards a pancreatic endocrine phenotype, as assessed by marker expression and in vitro glucose-regulated insulin secretion. Transplantation of these cells in immune-deficient animals shows human C-peptide production in response to a glucose challenge. These data suggest that human cord blood may be a promising source for regenerative medicine approaches for the treatment of diabetes mellitus.

Receptor subunit composition is believed to play a major role in the synaptic trafficking of AMPA receptors (AMPARs), and thus in activity-dependent synaptic plasticity. To isolate a physiological role of GluA1-containing AMPARs in area CA3 of the hippocampus, pair recordings were performed in organotypic hippocampal slices taken from genetically modified mice lacking the GluA1 subunit. We report here that long-term potentiation (LTP) is impaired not only at active but also at silent synapses when the GluA1 subunit is absent. The GluA1 knockout mice also exhibited reduced AMPAR-mediated evoked currents between pairs of CA3 pyramidal neurons under baseline conditions suggesting a significant role for GluA1-containing AMPARs in regulating basal synaptic transmission. In two independent measures, however, long-term depression (LTD) was unaffected in tissue from these mice. These data provide a further demonstration of the fundamental role that GluA1-containing AMPARs play in activity-dependent increases in synaptic strength but do not support a GluA1-dependent mechanism for reductions in synaptic strength.

The study of the folding of single domains, in the context of their multidomain environment, is important because more than 70% of eukaryotic proteins are composed of multiple domains. The structures of the tandem immunoglobulin (Ig) domain pairs A164-A165 and A168-A169, from the A-band of the giant muscle protein titin, reveal that they form tightly associated domain arrangements, connected by a continuous β-strand. We investigate the thermodynamic and kinetic properties of these tandem domain pairs. While A164-A165 apparently behaves as a single cooperative unit at equilibrium, unfolding without the accumulation of a large population of intermediates, domains in A168-A169 behave independently. Although A169 appears to be stabilized in the tandem protein, we show that this is due to nonspecific stabilization by extension. We elucidate the folding and unfolding pathways of both tandem pairs and show that cooperativity in A164-A165 is a manifestation of the relative refolding and unfolding rate constants of each individual domain. We infer that the differences between the two tandem pairs result from a different pattern of interactions at the domain/domain interface.

A hallmark of the bacterial twin-arginine translocation (Tat) pathway is its ability to export folded proteins. Here, we discovered that overexpressed Tat substrate proteins form two distinct, long-lived translocation intermediates that are readily detected by immunolabeling methods. Formation of the early translocation intermediate Ti-1, which exposes the N- and C-termini to the cytoplasm, did not require an intact Tat translocase, a functional Tat signal peptide, or a correctly folded substrate. In contrast, formation of the later translocation intermediate, Ti-2, which exhibits a bitopic topology with the N-terminus in the cytoplasm and C-terminus in the periplasm, was much more particular, requiring an intact translocase, a functional signal peptide, and a correctly folded substrate protein. The ability to directly detect Ti-2 intermediates was subsequently exploited for a new protein engineering technology called MAD-TRAP (membrane-anchored display for Tat-based recognition of associating proteins). Through the use of just two rounds of mutagenesis and screening with MAD-TRAP, the intracellular folding and antigen-binding activity of a human single-chain antibody fragment were simultaneously improved. This approach has several advantages for library screening, including the unique involvement of the Tat folding quality control mechanism that ensures only native-like proteins are displayed, thus eliminating poorly folded sequences from the screening process.

Polymorphisms in PTPN22 are associated with many autoimmune diseases; while rs2476601 is supposed to play a major role, other experimental data suggest that rs2488457 may be even more important. Results in myasthenia gravis are controversial. In 356 Italian myasthenic patients and 439 controls genotyped for both polymorphisms, we found that rs2476601 was not associated with myasthenia, presence of autoantibodies, thymus pathology, sex or onset age unlike previous studies on other European populations (confirmed by the present meta-analysis). On the other hand, while rs2488457 was not associated with myasthenia or thymus pathology, we found a correlation of rs2488457 with low autoantibody titers and a trend of association with a less severe disease. Both polymorphisms were in tight linkage disequilibrium in controls, not in patients. Our results suggest that SNPs in this gene different from rs2476601, and/or epigenetic interactions, could play a greater role.