International Criminal Tribunal for Rwanda

ROC curves of phenotype similarity matrices constructed with or without title portions. ROC analysis with the two benchmark datasets (A: Phenotypic Series, B: Linked OMIM Record Pairs) suggested that the similarity matrix constructed with both the text and title portions of OMIM records outperformed the matrix constructed with the text portion only. The range of false positive rates was restricted to (0, 0.1) in order to highlight the differences between each curve. (PDF 270 kb

Presentation_1_HLA-DPB1 genotype variants predict DP molecule cell surface expression and DP donor specific antibody binding capacity.pptx

The contribution of alloresponses to mismatched HLA-DP in solid organ transplantation and hematopoietic stem cell transplantation (HCT) has been well documented. Exploring the regulatory mechanisms of DPB1 alleles has become an important question to be answered. In this study, our initial investigation focused on examining the correlation between the rs9277534G/A SNP and DPB1 mRNA expression. The result showed that there was a significant increase in DPB1 mRNA expression in B lymphoblastoid cell lines (BLCLs) with the rs9277534GG genotype compared to rs9277534AA genotype. In addition, B cells with the rs9277534GG exhibited significantly higher DP protein expression than those carrying the rs9277534AA genotype in primary B cells. Furthermore, we observed a significant upregulation of DP expression in B cells following treatment with Interleukin 13 (IL-13) compared to untreated B cells carrying rs9277534GG-linked DPB1 alleles. Fluorescence in situ hybridization (FISH) analysis of DPB1 in BLCL demonstrated significant differences in both the cytoplasmic (p=0.0003) and nuclear (p=0.0001) localization of DP mRNA expression comparing DPB1*04:01 (rs9277534AA) and DPB1*05:01 (rs9277534GG) homozygous cells. The study of the correlation between differential DPB1 expression and long non-coding RNAs (lncRNAs) showed that lnc-HLA-DPB1-13:1 is strongly associated with DP expression (r=0.85), suggesting the potential involvement of lncRNA in regulating DP expression. The correlation of DP donor specific antibody (DSA) with B cell flow crossmatch (B-FCXM) results showed a better linear correlation of DP DSA against GG and AG donor cells (R2 = 0.4243, p=0.0025 and R2 = 0.6172, p=0.0003, respectively), compared to DSA against AA donor cells (R2 = 0.0649, p=0.4244). This explained why strong DP DSA with a low expression DP leads to negative B-FCXM. In conclusion, this study provides evidence supporting the involvement of lncRNA in modulating HLA-DP expression, shedding lights on the intricate regulatory mechanisms of DP, particularly under inflammatory conditions in transplantation.</p

Development and Evaluation of a Parallel Reaction Monitoring Strategy for Large-Scale Targeted Metabolomics Quantification

Recent advances in mass spectrometers which have yielded higher resolution and faster scanning speeds have expanded their application in metabolomics of diverse diseases. Using a quadrupole-Orbitrap LC–MS system, we developed an efficient large-scale quantitative method targeting 237 metabolites involved in various metabolic pathways using scheduled, parallel reaction monitoring (PRM). We assessed the dynamic range, linearity, reproducibility, and system suitability of the PRM assay by measuring concentration curves, biological samples, and clinical serum samples. The quantification performances of PRM and MS1-based assays in Q-Exactive were compared, and the MRM assay in QTRAP 6500 was also compared. The PRM assay monitoring 237 polar metabolites showed greater reproducibility and quantitative accuracy than MS1-based quantification and also showed greater flexibility in postacquisition assay refinement than the MRM assay in QTRAP 6500. We present a workflow for convenient PRM data processing using Skyline software which is free of charge. In this study we have established a reliable PRM methodology on a quadrupole-Orbitrap platform for evaluation of large-scale targeted metabolomics, which provides a new choice for basic and clinical metabolomics study

Additional file 1: Figure S1. of SoftPanel: a website for grouping diseases and related disorders for generation of customized panels

ROC curves of phenotype similarity matrices constructed with or without title portions. ROC analysis with the two benchmark datasets (A: Phenotypic Series, B: Linked OMIM Record Pairs) suggested that the similarity matrix constructed with both the text and title portions of OMIM records outperformed the matrix constructed with the text portion only. The range of false positive rates was restricted to (0, 0.1) in order to highlight the differences between each curve. (PDF 270 kb

PTEN regulates PLK1 and controls chromosomal stability during cell division

<p>PTEN functions as a guardian of the genome through multiple mechanisms. We have previously established that PTEN maintains the structural integrity of chromosomes. In this report, we demonstrate a fundamental role of PTEN in controlling chromosome inheritance to prevent gross genomic alterations. Disruption of <i>PTEN</i> or depletion of PTEN protein phosphatase activity causes abnormal chromosome content, manifested by enlarged or polyploid nuclei. We further identify polo-like kinase 1 (PLK1) as a substrate of PTEN phosphatase. PTEN can physically associate with PLK1 and reduce PLK1 phosphorylation in a phosphatase-dependent manner. We show that PTEN deficiency leads to PLK1 phosphorylation and that a phospho-mimicking PLK1 mutant causes polyploidy, imitating functional deficiency of PTEN phosphatase. Inhibition of PLK1 activity or overexpression of a non-phosphorylatable PLK1 mutant reduces the polyploid cell population. These data reveal a new mechanism by which PTEN controls genomic stability during cell division.</p

Inactivation of the epithelial innate immune response to pore-formation is mediated by phosphatase activity.

<p>(A) Confluent monolayers of A549 cells were stimulated for the indicated times with 200 ng/ml of purified Ply after pretreatment with either 100 µM sodium orthovanadate (lanes 6, 7) or vehicle control (lanes 4, 5) for 30 min. As corresponding negative controls, cells were left untreated (lane 1) or treated with sodium orthovanadate alone for 60 and 90 min (lanes 2 and 3, respectively). Sodium orthovanadate leads to enhanced Ply-induced phosphorylation of p38, JNK, and their respective upstream kinases, MKK3/6 and SEK1. (B) Pretreatment of A549s with 100 µM sodium orthovanadate (30 min) leads to significantly higher production of Ply-induced IL-8 than vehicle control (*  =  ≤0.05, ***  =  ≤0.001 by ANOVA with Tukey post-test).</p

Inhibition of serine/threonine phosphatases leads to increased Ply-induced epithelial MAPK phosphorylation.

<p>Confluent monolayers of A549 cells were stimulated for the indicated times with 100 ng/ml of purified Ply after pretreatment with either 5 nM calyculin A for 30 mins (lanes 8, 9), 250 nM okadaic acid for 60 mins (lanes 10, 11), or vehicle control (lanes 6, 7) for 30 mins. As corresponding negative controls, cells were left untreated (lane 1) or treated with calyculin A alone for 30 and 60 mins (lanes 2 and 3, respectively) or okadaic acid alone for 60 and 90 mins (lanes 4 and 5, respectively). In both cases, pretreatment with serine/threonine phosphatase inhibitors leads to prolonged and enhanced p38 and JNK phosphorylation in response to Ply.</p

PP1 and PP2A mediate inactivation of the epithelial MAPK response to pore-formation.

<p>(A)A549 cells were transfected with 2 µg PP1 or scrambled siRNA per 1×10⁶ cells and stimulated 54 hrs post-transfection with 100 ng/ml of purified Ply. Transfection with PP1 siRNA leads to reduced PP1 expression and a corresponding increase in Ply-induced p38 and JNK phosphorylation. (B) A549 cells were transfected with 6 µg PP2Aα/β or control shRNA per 1×10⁶ cells and stimulated 60 hrs post-transfection with 100 ng/ml of purified Ply. Transfection with PP2Aα/β shRNA leads to reduced PP2A expression and a moderate increase in p38 phosphorylation.</p

Regulation of the MAPK response to pore-formation is MKP1 independent.

<p>(A) A549 cells were transfected with 2.5 µg of MKP1 or scrambled siRNA per 1×10⁶ cells and stimulated 24 hrs post-transfection with 200 ng/ml of purified Ply for the indicated times. Transfection with MKP1 siRNA inhibited MKP1 expression but did not have an effect on Ply-induced MAPK phosphorylation.</p

Subcytolytic pore-formation <i>by S. pneumoniae</i> leads to temporary activation of epithelial MAP kinases.

<p>(A) Confluent monolayers of A549 cells were stimulated for the indicated times with 4×10⁴ cfu/ml sonicated <i>S. pneumoniae</i> D39 or its isogenic Ply-deficient mutant, D39<i>ply</i>. Cells were subsequently lysed, and total and phosphorylated MAPK detected by western blot. Stimulation with D39, but not D39<i>ply</i>, leads to temporary phosphorylation of p38 and JNK MAPKs. (B) Stimulation of A549 and D562 respiratory epithelial cells for the indicated times with 200 ng/ml of purified Ply toxin but not its toxoid, PdB, induces MAPK activation. (C) A549 Cell lysis was measured by LDH assay of cell supernatants with triplicate samples. (D) Dose-dependent MAPK phosphorylation in A549 cells treated with indicated concentrations of Ply for 30 min.</p