The Extradomain A from fibronectin (EDA) has an immunomodulatory role as fusion protein with viral and tumor antigens, but its effect when administered with bacteria has not been assessed. Here, we investigated the adjuvant effect of EDA in mice immunizations against Salmonella enterica subspecies enterica serovar Enteritidis (Salmonella Enteritidis). Since lipopolysaccharide (LPS) is a major virulence factor and the LPS O-polysaccharide (O-PS) is the immunodominant antigen in serological diagnostic tests, Salmonella mutants lacking O-PS (rough mutants) represent an interesting approach for developing new vaccines and diagnostic tests to differentiate infected and vaccinated animals (DIVA tests). Here, antigenic preparations (hot-saline extracts and formalin-inactivated bacterins) from two Salmonella Enteritidis rough mutants, carrying either intact (SEΔwaaL) or deep-defective (SEΔgal) LPS-Core, were used in combination with EDA. Biotinylated bacterins, in particular SEΔwaaL bacterin, decorated with EDAvidin (EDA and streptavidin fusion protein) improved the protection conferred by hot-saline or bacterins alone and prevented significantly the virulent infection at least to the levels of live attenuated rough mutants. These findings demonstrate the adjuvant effect of EDAvidin when administered with biotinylated bacterins from Salmonella Enteritidis lacking O-PS and the usefulness of BEDA-SEΔwaaL as non-live vaccine in the mouse model.

Although Helicobacter Pylori (HP) was detected in some cases of chronic laryngitis, the results were not confirmed by polymerase chain reaction (PCR). By this time, it has not been found in laryngeal lesions by in house PCR, the most sensitive method for detecting the genome tracks. Regarding the previous results and also few numbers of studies about the presence of HP in benign laryngeal lesions, specifically by PCR, we aimed to investigate the presence of HP in benign laryngeal lesions by in-house PCR.

Caspase 2 was initially identified as a neuronally expressed developmentally down-regulated gene (HUGO gene nomenclature CASP2) and has been shown to be required for neuronal death induced by several stimuli, including NGF (nerve growth factor) deprivation and Aβ (β-amyloid). In non-neuronal cells the PIDDosome, composed of caspase 2 and two death adaptor proteins, PIDD (p53-inducible protein with a death domain) and RAIDD {RIP (receptor-interacting protein)-associated ICH-1 [ICE (interleukin-1β-converting enzyme)/CED-3 (cell-death determining 3) homologue 1] protein with a death domain}, has been proposed as the caspase 2 activation complex, although the absolute requirement for the PIDDosome is not clear. To investigate the requirement for the PIDDosome in caspase-2-dependent neuronal death, we have examined the necessity for each component in induction of active caspase 2 and in execution of caspase-2-dependent neuronal death. We find that both NGF deprivation and Aβ treatment of neurons induce active caspase 2 and that induction of this activity depends on expression of RAIDD, but is independent of PIDD expression. We show that treatment of wild-type or PIDD-null neurons with Aβ or NGF deprivation induces formation of a complex of caspase 2 and RAIDD. We also show that caspase-2-dependent execution of neurons requires RAIDD, not PIDD. Caspase 2 activity can be induced in neurons from PIDD-null mice, and NGF deprivation or Aβ use caspase 2 and RAIDD to execute death of these neurons.

Viruses are known to be the most abundant organisms on earth, yet little is known about their collective origin and evolutionary history. With exceptionally high rates of genetic mutation and mosaicism, it is not currently possible to resolve deep evolutionary histories of the known major virus groups. Metagenomics offers a potential means of establishing a more comprehensive view of viral evolution as vast amounts of new sequence data becomes available for comparative analysis.

Health utility (HU) measures are used as overall measures of quality of life and to determine quality adjusted life years (QALYs) in economic analyses. We compared baseline values of three HUs including Short Form 6 Dimensions (SF-6D), and Health Utilities Index, Mark II and Mark III (HUI2 and HUI3) and the feeling thermometer (FT) among type 2 diabetes participants in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial. We assessed relationships between HU and FT values and patient demographics and clinical variables.

D-phenylglycine aminotransferase (D-PhgAT) of Pseudomonas stutzeri ST-201 catalyzes the reversible stereo-inverting transamination potentially useful in the application for synthesis of D-phenylglycine and D-4-hydroxyphenylglycine using L-glutamate as a low cost amino donor substrate in one single step. The enzyme is a relatively hydrophobic homodimeric intracellular protein difficult to express in the soluble functionally active form. Over-expression of the dpgA gene in E. coli resulted in the majority of the D-PhgAT aggregated into insoluble inclusion bodies that failed to be re-natured. Expression in Pichia pastoris was explored as an alternative route for high level production of the D-PhgAT.

Gestational diabetes mellitus (GDM) affects 3% to 7% of pregnant women in the United States, and Asian, black, American Indian, and Hispanic women are at increased risk. Florida, the fourth most populous US state, has a high level of racial/ethnic diversity, providing the opportunity to examine variations in the contribution of maternal body mass index (BMI) status to GDM risk. The objective of this study was to estimate the race/ethnicity-specific percentage of GDM attributable to overweight and obesity in Florida.

We have developed a microarray to study the expression of L-chain V genes (V(L) genes) in healthy and SLE patient peripheral κ- and λ-sorted B cells. In all repertoires tested, one V(L) gene accounts for over 10% of all gene V(L) expression, consistent with positive selection acting on L-chains. While a few V(L) genes were highly expressed in all individuals, most V(L) genes were expressed at different levels. Some V(L) genes (5 out of a total of 78) were not detected. We attribute their absence from the repertoire to negative selection. Positive selection and negative selection were also found in SLE repertoires, but expression of V(L) genes was different; the differences point to less regulation of V(L) gene repertoires in SLE. Our data shows that V(L) gene expression is variable and supports a model where the L-chain repertoire is generated by both positive and negative selection on L-chains.

It is well known that GluK2-containing kainate receptors play essential roles in seizure and cerebral ischemia-induced neuronal death, while GluK1-containing kainate receptors could increase tonic inhibition of post-synaptic pyramidal neurons. This research investigated whether GluK1 could inhibit activation of c-Jun N-terminal kinase 3 (JNK3) signaling pathway mediated by the GluK2 in cerebral ischemia-reperfusion. The results show that GluK1 activation by (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) at 1nmol per rat could inhibit the assembly of GluK2·Postsynaptic density 95·mixed lineage kinase 3 signaling module, activation of JNK3 and its downstream signal molecules. However, the inhibition of ATPA could be prevented by GluK1 antagonist NS3763, GluK1 antisense, and GABA(A) receptor antagonist bicuculline. In addition, ATPA played a neuroprotective role against cerebral ischemia. In sum, the findings indicate that activation of GluK1 by ATPA at specific dosages may promote GABA release, which then suppresses post-synaptic GluK2-JNK3 signaling-mediated cerebral ischemic injury via GABA(A)R.

To determine the cause of an unprecedented outbreak of encephalitis among horses in New South Wales, Australia, in 2011, we performed genomic sequencing of viruses isolated from affected horses and mosquitoes. Results showed that most of the cases were caused by a variant West Nile virus (WNV) strain, WNV(NSW2011), that is most closely related to WNV Kunjin (WNV(KUN)), the indigenous WNV strain in Australia. Studies in mouse models for WNV pathogenesis showed that WNV(NSW2011) is substantially more neuroinvasive than the prototype WNV(KUN) strain. In WNV(NSW2011), this apparent increase in virulence over that of the prototype strain correlated with at least 2 known markers of WNV virulence that are not found in WNV(KUN). Additional studies are needed to determine the relationship of the WNV(NSW2011) strain to currently and previously circulating WNV(KUN) strains and to confirm the cause of the increased virulence of this emerging WNV strain.

The evolutionarily ancient arm of the E2f family of transcription factors consisting of the two atypical members E2f7 and E2f8 is essential for murine embryonic development. However, the critical tissues, cellular processes, and molecular pathways regulated by these two factors remain unknown. Using a series of fetal and placental lineage-specific cre mice, we show that E2F7/E2F8 functions in extraembryonic trophoblast lineages are both necessary and sufficient to carry fetuses to term. Expression profiling and biochemical approaches exposed the canonical E2F3a activator as a key family member that antagonizes E2F7/E2F8 functions. Remarkably, the concomitant loss of E2f3a normalized placental gene expression programs, corrected placental defects, and fostered the survival of E2f7/E2f8-deficient embryos to birth. In summary, we identified a placental transcriptional network tightly coordinated by activation and repression through two distinct arms of the E2F family that is essential for extraembryonic cell proliferation, placental development, and fetal viability.

Balancing progenitor cell self-renewal and differentiation is essential for brain development and is regulated by the activity of chromatin remodeling complexes. Nevertheless, linking chromatin changes to specific pathways that control cortical histogenesis remains a challenge. Here we identify a genetic interaction between the chromatin remodeler Snf2l and Foxg1, a key regulator of neurogenesis. Snf2l mutant mice exhibit forebrain hypercellularity arising from increased Foxg1 expression, increased progenitor cell expansion, and delayed differentiation. We demonstrate that Snf2l binds to the Foxg1 locus at midneurogenesis and that the phenotype is rescued by reducing Foxg1 dosage, thus revealing that Snf2l and Foxg1 function antagonistically to regulate brain size.

New therapies for children with high risk neuroblastoma are needed, and haploidentical stem cell transplantation with NK post-graft injections is a potential option. To develop this strategy, we compared and correlated two methods of NK cytotoxicity assay. The aim of this work is to optimize in vitro NK cytotoxicity assays, investigate the effect of interleukin stimulation on NK cells and use of antiGD2 antibodies against tumor target cells and finally establish an in vitro model for haploidentical stem cell transplantation.

It is well established that there is individual variability in pain and temperature sensitivity. Functional brain imaging studies have found that interindividual heat pain variability correlates with brain activity in sensory and pain modulation areas. Thus, it is possible that these individual differences are associated with variability in gray matter thickness of cortical regions involved in thermoreception and pain. To test this, we investigated the relationship between thermal thresholds and cortical thickness in 80 healthy subjects. Subjects underwent a psychophysical session to determine their cool detection (CD), warm detection (WD), cold pain (CP), and heat pain (HP) threshold. A high-resolution structural magnetic resonance imaging scan was acquired for each subject. We correlated each threshold measure to cortical thickness of regions associated with thermoreception and pain. The mean (± SD) thresholds were 30.7 °C (± 0.8) for CD, 33.8 °C (± 0.7) for WD, 11.7 °C (± 9.7) for CP, and 45.3 °C (± 2.8) for HP. The brain gray matter analysis revealed a strong correlation between greater thermal and pain sensitivity and cortical thickening of the primary somatosensory cortex. Additionally, greater sensitivity to cool stimuli correlated with cortical thickening in the paracentral lobule, and greater WD correlated with cortical thinning in the anterior midcingulate cortex. We also found that greater HP sensitivity correlated with thickening in the posterior midcingulate cortex and the orbitofrontal cortex. These cortical gray matter correlates of thermal and pain sensitivity provide a neural basis for individual differences in thermal sensitivity.

Fibrillar α-synuclein (AS) is the major component of Lewy bodies, the pathological hallmark of Parkinson's disease. Mouse AS (mAS) aggregates much faster than human AS (hAS), although mAS differs from hAS at only seven positions in its primary sequence. Currently, little is known about the site-specific structural differences between mAS and hAS fibrils. Here, we applied state-of-the-art solid-state nuclear magnetic resonance (ssNMR) methods to structurally characterize mAS fibrils. The assignment strategy employed a set of high-resolution 2D and 3D ssNMR spectra recorded on uniformly [(13)C, (15)N], [1-(13)C]glucose, and [2-(13)C]glucose labeled mAS fibrils. An almost complete resonance assignment (96% of backbone amide (15)N and 93% of all (13)C nuclei) was obtained for residues from Gly41 to Val95, which form the core of mAS fibrils. Six β-strands were identified to be within the fibril core of mAS based on a secondary chemical shift and NHHC analysis. Intermolecular (13)C:(15)N labeled restraints obtained from mixed 1:1 (13)C/(15)N-labeled mAS fibrils reveal a parallel, in-register supramolecular β-sheet arrangement. The results were compared in detail to recent structural studies on hAS fibrils and indicate the presence of a structurally conserved motif comprising residues Glu61-Lys80.

Microcin C (McC) is heptapeptide adenylate antibiotic produced by Escherichia coli strains carrying the mccABCDEF gene cluster encoding enzymes, in addition to the heptapeptide structural gene mccA, necessary for McC biosynthesis and self-immunity of the producing cell. The heptapeptide facilitates McC transport into susceptible cells, where it is processed releasing a non-hydrolyzable aminoacyl adenylate that inhibits an essential aminoacyl-tRNA synthetase. The self-immunity gene mccF encodes a specialized serine peptidase that cleaves an amide bond connecting the peptidyl or aminoacyl moieties of, respectively, intact and processed McC with the nucleotidyl moiety. Most mccF orthologs from organisms other than E. coli are not linked to the McC biosynthesis gene cluster. Here, we show that a protein product of one such gene, MccF from Bacillus anthracis (BaMccF), is able to cleave intact and processed McC, and we present a series of structures of this protein. Structural analysis of apo-BaMccF and its adenosine monophosphate complex reveals specific features of MccF-like peptidases that allow them to interact with substrates containing nucleotidyl moieties. Sequence analyses and phylogenetic reconstructions suggest that several distinct subfamilies form the MccF clade of the large S66 family of bacterial serine peptidases. We show that various representatives of the MccF clade can specifically detoxify non-hydrolyzable aminoacyl adenylates differing in their aminoacyl moieties. We hypothesize that bacterial mccF genes serve as a source of bacterial antibiotic resistance.

Epidemiological studies have repeatedly investigated the association between depression and metabolic syndrome (MetS). However, the results have been inconsistent. This meta-analysis aimed to summarize the current evidence from cross-sectional and prospective cohort studies that evaluated this association.

Recent genome-wide association studies (GWAS) have found a single nucleotide polymorphism (SNP, rs2274223 A>G) in PLCE1 to be associated with risk of gastric adenocarcinoma. In the present study, we validated this finding and also explored the risk associated with another unreported potentially functional SNP (rs11187870 G>C) of PLCE1 in a hospital-based case-control study of 1059 patients with pathologically confirmed gastric adenocarcinoma and 1240 frequency-matched healthy controls.

Leber Congenital Amaurosis (LCA) and Early Childhood Onset Severe Retinal Dystrophy are clinically and genetically heterogeneous retinal disorders characterised by visual impairment and nystagmus from birth or early infancy. We investigated the prevalence of sequence variants in AIPL1 in a large cohort of such patients (n = 392) and probed the likelihood of disease-causation of the identified variants, subsequently undertaking a detailed assessment of the phenotype of patients with disease-causing mutations. Genomic DNA samples were screened for known variants in the AIPL1 gene using a microarray LCA chip, with 153 of these cases then being directly sequenced. The assessment of disease-causation of identified AIPL1 variants included segregation testing, assessing evolutionary conservation and in silico predictions of pathogenicity. The chip identified AIPL1 variants in 12 patients. Sequencing of AIPL1 in 153 patients and 96 controls found a total of 46 variants, with 29 being novel. In silico analysis suggested that only 6 of these variants are likely to be disease-causing, indicating a previously unrecognized high degree of polymorphism. Seven patients were identified with biallelic changes in AIPL1 likely to be disease-causing. In the youngest subject, electroretinography revealed reduced cone photoreceptor function, but rod responses were within normal limits, with no measurable ERG in other patients. An increasing degree and extent of peripheral retinal pigmentation and degree of maculopathy was noted with increasing age in our series. AIPL1 is significantly polymorphic in both controls and patients, thereby complicating the establishment of disease-causation of identified variants. Despite the associated phenotype being characterised by early-onset severe visual loss in our patient series, there was some evidence of a degree of retinal structural and functional preservation, which was most marked in the youngest patient in our cohort. This data suggests that there are patients who have a reasonable window of opportunity for gene therapy in childhood.

Tularemia or vaccination with the live vaccine strain (LVS) of Francisella tularensis confers long-lived cell-mediated immunity. We hypothesized that this immunity depends on polyfunctional memory T cells, i.e., CD4(+) and/or CD8(+) T cells with the capability to simultaneously express several functional markers. Multiparametric flow cytometry, measurement of secreted cytokines, and analysis of lymphocyte proliferation were used to characterize in vitro recall responses of peripheral blood mononuclear cells (PBMC) to killed F. tularensis antigens from the LVS or Schu S4 strains. PBMC responses were compared between individuals who had contracted tularemia, had been vaccinated, or had not been exposed to F. tularensis (naïve). Significant differences were detected between either of the immune donor groups and naïve individuals for secreted levels of IL-5, IL-6, IL-10, IL-12, IL-13, IFN-γ, MCP-1, and MIP-1β. Expression of IFN-γ, MIP-1β, and CD107a by CD4(+)CD45RO(+) or CD8(+)CD45RO(+) T cells correlated to antigen concentrations. In particular, IFN-γ and MIP-1β strongly discriminated between immune and naïve individuals. Only one cytokine, IL-6, discriminated between the two groups of immune individuals. Notably, IL-2- or TNF-α-secretion was low. Our results identify functional signatures of T cells that may serve as correlates of immunity and protection against F. tularensis.