Ultrastructural features and synaptic connections of hilar ectopic granule cells in the rat dentate gyrus are different from those of granule cells in the granule cell layer.

Several investigators have shown the existence of dentate granule cells in ectopic locations within the hilus and molecular layer using both Golgi and retrograde tracing studies but the ultrastructural features and synaptic connections of ectopic granule cells were not previously examined. In the present study, the biocytin retrograde tracing technique was used to label ectopic granule cells following injections into stratum lucidum of CA3b of hippocampal slices obtained from epileptic rats. Electron microscopy was used to study hilar ectopic granule cells that were located 20-40 microm from the granule cell layer (GCL). They had ultrastructural features similar to those of granule cells in the GCL but showed differences, including nuclei that often displayed infoldings and thicker apical dendrites. At their origin, these dendrites were 6 microm in diameter and they tapered down to 2 microm at the border with the GCL. Both biocytin-labeled and unlabeled axon terminals formed exclusively asymmetric synapses with the somata and proximal dendrites of hilar ectopic granule cells. The mean number of axosomatic synapses for these cells was three times that for granule cells in the GCL. Together, these data indicate that hilar ectopic granule cells are postsynaptic to mossy fibers and have less inhibitory input on their somata and proximal dendrites than granule cells in the GCL. This finding is consistent with recent physiological results showing that hilar ectopic granule cells from epileptic rats are more hyperexcitable than granule cells in the GCL

Recovering probabilities for nucleotide trimming processes for T cell receptor TRA and TRG V-J junctions analyzed with IMGT tools

<p>Abstract</p> <p>Background</p> <p>Nucleotides are trimmed from the ends of variable (V), diversity (D) and joining (J) genes during immunoglobulin (IG) and T cell receptor (TR) rearrangements in B cells and T cells of the immune system. This trimming is followed by addition of nucleotides at random, forming the N regions (N for nucleotides) of the V-J and V-D-J junctions. These processes are crucial for creating diversity in the immune response since the number of trimmed nucleotides and the number of added nucleotides vary in each B or T cell. IMGT^®sequence analysis tools, IMGT/V-QUEST and IMGT/JunctionAnalysis, are able to provide detailed and accurate analysis of the final observed junction nucleotide sequences (tool "output"). However, as trimmed nucleotides can potentially be replaced by identical N region nucleotides during the process, the observed "output" represents a <it>biased </it>estimate of the "true trimming process."</p> <p>Results</p> <p>A probabilistic approach based on an analysis of the standardized tool "output" is proposed to infer the probability distribution of the "true trimmming process" and to provide plausible biological hypotheses explaining this process. We collated a benchmark dataset of TR alpha (TRA) and TR gamma (TRG) V-J rearranged sequences and junctions analysed with IMGT/V-QUEST and IMGT/JunctionAnalysis, the nucleotide sequence analysis tools from IMGT^®, the international ImMunoGeneTics information system^®, <url>http://imgt.cines.fr</url>. The standardized description of the tool output is based on the IMGT-ONTOLOGY axioms and concepts. We propose a simple first-order model that attempts to transform the observed "output" probability distribution into an estimate closer to the "true trimming process" probability distribution. We use this estimate to test the hypothesis that Poisson processes are involved in trimming. This hypothesis was not rejected at standard confidence levels for three of the four trimming processes: TRAV, TRAJ and TRGV.</p> <p>Conclusion</p> <p>By using trimming of rearranged TR genes as a benchmark, we show that a probabilistic approach, applied to IMGT^®standardized tool "outputs" opens the way to plausible hypotheses on the events involved in the "true trimming process" and eventually to an exact quantification of trimming itself. With increasing high-throughput of standardized immunogenetics data, similar probabilistic approaches will improve understanding of processes so far only characterized by the "output" of standardized tools.</p

Expression of core antigen of HCV genotype 3a and its evaluation as screening agent for HCV infection in Pakistan

<p>Abstract</p> <p>Background</p> <p>Pakistan is facing a threat from hepatitis C infection which is increasing at an alarming rate throughout the country. More specific and sensitive screening assays are needed to timely and correctly diagnose this infection.</p> <p>Methods</p> <p>After RNA extraction from specimen (HCV-3a), cDNA was synthesized that was used to amplify full length core gene of HCV 3a. After verification through PCR, DNA sequencing and BLAST, a properly oriented positive recombinant plasmid for core gene was digested with proper restriction enzymes to release the target gene which was then inserted downstream of GST encoding DNA in the same open reading frame at proper restriction sites in multiple cloning site of pGEX4t2 expression vector. Recombinant expression vector for each gene was transformed in <it>E. coli </it>BL21 (DE3) and induced with IPTG for recombinant fusion protein production that was then purified through affinity chromatography. Western blot and Enzyme Linked Immunosorbant Assay (ELISA) were used to detect immuno-reactivity of the recombinant protein.</p> <p>Results</p> <p>The HCV core antigen produced in prokaryotic expression system was reactive and used to develop a screening assay. After validating the positivity (100%) and negativity (100%) of in-house anti-HCV screening assay through a standardized panel of 200 HCV positive and 200 HCV negative sera, a group of 120 serum specimens of suspected HCV infection were subjected to comparative analysis of our method with commercially available assay. The comparison confirmed that our method is more specific than the commercially available assays for HCV strains circulating in this specific geographical region of the world and could thus be used for HCV screening in Pakistan.</p> <p>Conclusion</p> <p>In this study, we devised a screening assay after successful PCR amplification, isolation, sequencing, expression and purification of core antigen of HCV genotype 3a. Our developed screening assay is more sensitive, specific and reproducible than the commercially available screening assays in Pakistan.</p

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente

Two mechanisms of the enhanced antibody-dependent cellular cytotoxicity (ADCC) efficacy of non-fucosylated therapeutic antibodies in human blood

<p>Abstract</p> <p>Background</p> <p>Antibody-dependent cellular cytotoxicity (ADCC) has recently been identified as one of the critical mechanisms underlying the clinical efficacy of therapeutic antibodies, especially anticancer antibodies. Therapeutic antibodies fully lacking the core fucose of the Fc oligosaccharides have been found to exhibit much higher ADCC in humans than their fucosylated counterparts. However, data which show how fully non-fucosylated antibodies achieve such a high ADCC in human whole blood have not yet been disclosed. The precise mechanisms responsible for the high ADCC mediated by fully non-fucosylated therapeutic antibodies, even in the presence of human plasma, should be explained based on direct evidence of non-fucosylated antibody action in human blood.</p> <p>Methods</p> <p>Using a human <it>ex vivo </it>B-cell depletion assay with non-fucosylated and fucosylated anti-CD20 IgG1s rituximab, we monitored the binding of the therapeutic agents both to antigens on target cells (target side interaction) and to leukocyte receptors (FcγR) on effector cells (effector side interaction), comparing the intensities of ADCC in human blood.</p> <p>Results</p> <p>In the target side interaction, down-modulation of CD20 on B cells mediated by anti-CD20 was not observed. Simple competition for binding to the antigens on target B cells between fucosylated and non-fucosylated anti-CD20s was detected in human blood to cause inhibition of the enhanced ADCC of non-fucosylated anti-CD20 by fucosylated anti-CD20. In the effector side interaction, non-fucosylated anti-CD20 showed sufficiently high FcγRIIIa binding activity to overcome competition from plasma IgG for binding to FcγRIIIa on natural killer (NK) cells, whereas the binding of fucosylated anti-CD20 to FcγRIIIa was almost abolished in the presence of human plasma and failed to recruit NK cells effectively. The core fucosylation levels of individual serum IgG1 from healthy donors was found to be so slightly different that it did not affect the inhibitory effect on the ADCC of fucosylated anti-CD20.</p> <p>Conclusion</p> <p>Our results demonstrate that removal of fucosylated antibody ingredients from antibody therapeutics elicits high ADCC in human blood by two mechanisms: namely, by evading the inhibitory effects both of plasma IgG on FcγRIIIa binding (effector side interaction) and of fucosylated antibodies on antigen binding (target side interaction).</p

Effect of thrombin peptide 508 (TP508) on bone healing during distraction osteogenesis in rabbit tibia

Thrombin-related peptide 508 (TP508) accelerates bone regeneration during distraction osteogenesis (DO). We have examined the effect of TP508 on bone regeneration during DO by immunolocalization of Runx2 protein, a marker of osteoblast differentiation, and of osteopontin (OPN) and bone sialoprotein (BSP), two late markers of the osteoblast lineage. Distraction was performed in tibiae of rabbits over a period of 6 days. TP508 (30 or 300 μg) or vehicle was injected into the distraction gap at the beginning and end of the distraction period. Two weeks after active distraction, tissue samples were harvested and processed for immunohistochemical analysis. We also tested the in vitro effect of TP508 on Runx2 mRNA expression in osteoblast-like (MC3T3-E1) cells by polymerase chain reaction analysis. Runx2 and OPN protein were observed in preosteoblasts, osteoblasts, osteocytes of newly formed bone, blood vessel cells and many fibroblast-like cells of the soft connective tissue. Immunostaining for BSP was more restricted to osteoblasts and osteocytes. Significantly more Runx2- and OPN-expressing cells were seen in the group treated with 300 μg TP508 than in the control group injected with saline or with 30 μg TP508. However, TP508 failed to increase Runx2 mRNA levels significantly in MC3T3-E1 cells after 2–3 days of exposure. Our data suggest that TP508 enhances bone regeneration during DO by increasing the proportion of cells of the osteoblastic lineage. Clinically, TP508 may shorten the healing time during DO; this might be of benefit when bone regeneration is slow

A novel copper complex induces ROS generation in doxorubicin resistant Ehrlich ascitis carcinoma cells and increases activity of antioxidant enzymes in vital organs in vivo

BACKGROUND: In search of a suitable GSH-depleting agent, a novel copper complex viz., copper N-(2-hydroxyacetophenone) glycinate (CuNG) has been synthesized, which was initially found to be a potential resistance modifying agent and later found to be an immunomodulator in mice model in different doses. The objective of the present work was to decipher the effect of CuNG on reactive oxygen species (ROS) generation and antioxidant enzymes in normal and doxorubicin-resistant Ehrlich ascites carcinoma (EAC/Dox)-bearing Swiss albino mice. METHODS: The effect of CuNG has been studied on ROS generation, multidrug resistance-associated protein1 (MRP1) expression and on activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). RESULTS: CuNG increased ROS generation and reduced MRP1 expression in EAC/Dox cells while only temporarily depleted glutathione (GSH) within 2 h in heart, kidney, liver and lung of EAC/Dox bearing mice, which were restored within 24 h. The level of liver Cu was observed to be inversely proportional to the level of GSH. Moreover, CuNG modulated SOD, CAT and GPx in different organs and thereby reduced oxidative stress. Thus nontoxic dose of CuNG may be utilized to reduce MRP1 expression and thus sensitize EAC/Dox cells to standard chemotherapy. Moreover, CuNG modulated SOD, CAT and and GPx activities to reduce oxidative stress in some vital organs of EAC/Dox bearing mice. CuNG treatment also helped to recover liver and renal function in EAC/Dox bearing mice. CONCLUSION: Based on our studies, we conclude that CuNG may be a promising candidate to sensitize drug resistant cancers in the clinic

A Histone Map of Human Chromosome 20q13.12

We present a systematic search for regulatory elements in a 3.5 Mb region on human chromosome 20q13.12, a region associated with a number of medical conditions such as type II diabetes and obesity.We profiled six histone modifications alongside RNA polymerase II (PolII) and CTCF in two cell lines, HeLa S3 and NTERA-2 clone D1 (NT2/D1), by chromatin immunoprecipitation using an in-house spotted DNA array, constructed with 1.8 kb overlapping plasmid clones. In both cells, more than 90% of transcription start sites (TSSs) of expressed genes showed enrichments with PolII, di-methylated lysine 4 of histone H3 (H3K4me2), tri-methylated lysine 4 of histone H3 (H3K4me3) or acetylated H3 (H3Ac), whereas mono-methylated lysine 4 of histone H3 (H3K4me1) signals did not correlate with expression. No TSSs were enriched with tri-methylated lysine 27 of histone H3 (H3K27me3) in HeLa S3, while eight TSSs (4 expressed) showed enrichments in NT2/D1. We have also located several CTCF binding sites that are potential insulator elements.In summary, we annotated a number of putative regulatory elements in 20q13.12 and went on to verify experimentally a subset of them using dual luciferase reporter assays. Correlating this data to sequence variation can aid identification of disease causing variants

Psychiatric disorders and clinical correlates of suicidal patients admitted to a psychiatric hospital in Tokyo

<p>Abstract</p> <p>Background</p> <p>Patients admitted to a psychiatric hospital with suicidal behavior (SB) are considered to be especially at high risk of suicide. However, the number of studies that have addressed this patient population remains insufficient compared to that of studies on suicidal patients in emergency or medical settings. The purpose of this study is to seek features of a sample of newly admitted suicidal psychiatric patients in a metropolitan area of Japan.</p> <p>Method</p> <p>155 suicidal patients consecutively admitted to a large psychiatric center during a 20-month period, admission styles of whom were mostly involuntary, were assessed using Structured Clinical Interviews for DSM-IV Axis I and II Disorders (SCID-I CV and SCID-II) and SB-related psychiatric measures. Associations of the psychiatric diagnoses and SB-related characteristics with gender and age were examined.</p> <p>Results</p> <p>The common DSM-IV axis I diagnoses were affective disorders 62%, anxiety disorders 56% and substance-related disorders 38%. 56% of the subjects were diagnosed as having borderline PD, and 87% of them, at least one type of personality disorder (PD). SB methods used prior to admission were self-cutting 41%, overdosing 32%, self-strangulation 15%, jumping from a height 12% and attempting traffic death 10%, the first two of which were frequent among young females. The median (range) of the total number of SBs in the lifetime history was 7 (1-141). Severity of depressive symptomatology, suicidal intent and other symptoms, proportions of the subjects who reported SB-preceding life events and life problems, and childhood and adolescent abuse were comparable to those of the previous studies conducted in medical or emergency service settings. Gender and age-relevant life-problems and life events were identified.</p> <p>Conclusions</p> <p>Features of the studied sample were the high prevalence of affective disorders, anxiety disorders and borderline PD, a variety of SB methods used prior to admission and frequent SB repetition in the lifetime history. Gender and age appeared to have an influence on SB method selection and SB-preceding processes. The findings have important implications for assessment and treatment of psychiatric suicidal patients.</p