Metagenomic of clinically diseased and healthy broiler affected with respiratory disease complex.

In recent past, the respiratory infection has emerged as a great challenge to the poultry farmers. Various pathogens including Avian pneumovirus (APV), Avian influenza virus (AIV), Infectious bronchitis virus (IBV) and Newcastle disease virus (NDV), Avibacterium paragallinarum, Ornithobacterium rhinotracheale (ORT), Mycoplasma synoviae (MS), Mycoplasma gallisepticum (MG) and Avian pathogenic Escherichia coli (APEC) are involved in the respiratory disease complex in birds [1], [2] (Bradbury, 1984; Roussan et al., 2008). Hence, respiratory disease complex is the most serious disease affecting to poultry and causes heavy economic losses in the poultry industry worldwide [3] (Murthy et al., 2008). In recent years, metagenomics is powerful analyzing tool for detection of pathogens directly from clinical samples without any prior knowledge of the organism in a given sample [4], [5] (Schuster, 2008; Pereira et al., 2010). High throughput Next-Generation-Sequencing technology was used for sequencing the isolated genomic DNA. These data provides an insight about taxonomic and functional status of microorganisms responsible for causing respiratory infection in broiler. The data of these metagenome are available in the BioSample Submission Portal as Bioproject PRJNA339659 and SRA accession number SRR5997823, SRR5992854, SRR6037376, SRR6024702, SRR6012248 and SRR6008913.

Genomic determinants of chronic myelomonocytic leukemia.

The biology, clinical phenotype and progression rate of chronic myelomonocytic leukemia (CMML) are highly variable due to diverse initiating and secondary clonal genetic events. To determine the effects of molecular features including clonal hierarchy in CMML, we studied whole-exome and targeted next-generation sequencing data from 150 patients with robust clinical and molecular annotation assessed cross-sectionally and at serial time points of disease evolution. To identify molecular lesions unique to CMML, we compared it to the related myeloid neoplasms (N=586), including juvenile myelomonocytic leukemia, myelodysplastic syndromes (MDS) and primary monocytic acute myeloid leukemia and discerned distinct molecular profiles despite similar pathomorphological features. Within CMML, mutations in certain pathways correlated with clinical classification, for example, proliferative vs dysplastic features. While most CMML patients (59%) had ancestral (dominant/co-dominant) mutations involving TET2, SRSF2 or ASXL1 genes, secondary subclonal hierarchy correlated with clinical phenotypes or outcomes. For example, progression was associated with acquisition of new expanding clones carrying biallelic TET2 mutations or RAS family, or spliceosomal gene mutations. In contrast, dysplastic features correlated with mutations usually encountered in MDS (for example, SF3B1 and U2AF1). Classification of CMML based on hierarchies of ancestral and subclonal mutational events may correlate strongly with clinical features and prognosis.

The complexity of interpreting genomic data in patients with acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous neoplasm characterized by the accumulation of complex genetic alterations responsible for the initiation and progression of the disease. Translating genomic information into clinical practice remained challenging with conflicting results regarding the impact of certain mutations on disease phenotype and overall survival (OS) especially when clinical variables are controlled for when interpreting the result. We sequenced the coding region for 62 genes in 468 patients with secondary AML (sAML) and primary AML (pAML). Overall, mutations in FLT3, DNMT3A, NPM1 and IDH2 were more specific for pAML whereas UTAF1, STAG2, BCORL1, BCOR, EZH2, JAK2, CBL, PRPF8, SF3B1, ASXL1 and DHX29 were more specific for sAML. However, in multivariate analysis that included clinical variables, only FLT3 and DNMT3A remained specific for pAML and EZH2, BCOR, SF3B1 and ASXL1 for sAML. When the impact of mutations on OS was evaluated in the entire cohort, mutations in DNMT3A, PRPF8, ASXL1, CBL EZH2 and TP53 had a negative impact on OS; no mutation impacted OS favorably; however, in a cox multivariate analysis that included clinical data, mutations in DNMT3A, ASXL1, CBL, EZH2 and TP53 became significant. Thus, controlling for clinical variables is important when interpreting genomic data in AML.

Incorporation of molecular data into the Revised International Prognostic Scoring System in treated patients with myelodysplastic syndromes.

The Revised International Prognostic Scoring System (IPSS-R) was developed for untreated myelodysplastic syndrome (MDS) patients based on clinical data. We created and validated a new model that incorporates mutational data to improve the predictive capacity of the IPSS-R in treated MDS patients. Clinical and mutational data from treated MDS patients diagnosed between January 2000 and January 2012 were used to develop the new prognostic system. A total of 508 patients were divided into training (n=333) and validation (n=175) cohorts. Independent significant prognostic factors for survival included age, IPSS-R, EZH2, SF3B1 and TP53. Weighted coefficients for each factor were used to build the new linear predictive model, which produced four prognostic groups: low, intermediate-1, intermediate-2 and high with a median overall survival of 37.4, 23.2, 19.9 and 12.2 months, respectively, P<0.001. Significant improvement in the C-index of the new model (0.73) was observed compared with the IPSS-R (0.69). The new model predicted outcome both in a separate validation cohort and in another cohort of patients with paired samples at different time points during their disease course. The addition of mutational data to the IPSS-R makes it dynamic and enhances its predictive ability in treated MDS patients regardless of their initial or subsequent therapies.

The G209A mutation in the alpha-synuclein gene is not detected in familial cases of Parkinson disease in non-Greek and/or Italian populations.

OBJECTIVE: To determine whether the G-to-A substitution at nucleotide 209 (G209A) mutation in the alpha-synuclein gene is responsible for familial Parkinson disease (PD) in the US population. DESIGN: Polymerase chain reaction-based DNA analysis of consecutive patients with PD and family history of PD. SETTING: A university-affiliated movement disorder clinic and a Veterans Affairs clinical research laboratory. PATIENTS: Forty-four patients with PD and family history of PD and 29 patients with sporadic PD, all with no known Greek and/or Italian background. RESULTS: None of the DNA samples showed the G209A mutation. CONCLUSION: The G209A mutation is rare in US patients with familial PD.

Ro 19-3704 directly inhibits immunoglobulin E-dependent mediator release by a mechanism independent of its platelet-activating factor antagonist properties.

Rat basophilic leukemia (RBL 2H3) cells were passively sensitized by exposure to monoclonal anti-trinitrophenol mouse immunoglobulin E (anti-trinitrophenol IgE) (0.5 microgram/ml) and triggered by exposure to a sub-optimal concentration of trinitrophenol ovalbumin conjugate (5 ng/ml). At this concentration, trinitrophenol-ovalbumin increased histamine release from a basal rate of 4.8 +/- 0.5 to 28.5 +/- 4.6% and peptidoleukotrienes from less than 0.1 to 4.2 +/- 1.3 ng/10(6) cells in the activated cells. Ro 19-3704 and Ro 19-1400, platelet activating factor (PAF) antagonists which are structural analogs of PAF, potently inhibited both the IgE-dependent release of histamine (IC50 values of 3.0 and 3.6 microM, respectively) and LT release (IC50 values of 5.0 microM for both compounds) from the cells. These effects appeared to be independent to the ability of the compounds to act as PAF antagonists since PAF on its own had no effect on mediator release, and WEB 2086 and BN 52021, structurally distinct PAF antagonists, were relatively ineffective as inhibitors of mediator release. Ro 19-3704 and Ro 19-1400 were observed to be potent inhibitors of the soluble phospholipase A2 activity in synovial fluid from rheumatoid arthritic patients (IC50 values of 6.5 and 8.4 microM, respectively). In contrast, WEB 2086 and BN 52021 had no effect on this phospholipase A2. Ro 19-3704 significantly inhibited the IgE-dependent formation of inositol phosphates in RBL 2H3 cells (IC50 value of 7.0 microM). These data suggest that the mediator release inhibitory action of these compounds may be related to the ability of these compounds to inhibit phospholipase A2 and/or phospholipase C.

Retinoids inhibit phospholipase A2 in human synovial fluid and arachidonic acid release from rat peritoneal macrophages.

Retinoids have demonstrated antiinflammatory activity in certain animal models and human disease states. The mechanism by which retinoids elicit this activity is unknown. Some retinoids are known to inhibit arachidonic acid (AA) release and metabolism in intact cells in vitro. Retinoids may exert their antiinflammatory effects by inhibiting phospholipase A2 (PLA2) and the resultant production of inflammatory AA metabolites. Retinoids were evaluated in vitro as inhibitors of the PLA2 activity in human synovial fluid (HSF-PLA2). Of the naturally occurring, nonaromatic retinoids tested, all-trans-retinal, all-trans-retinoic acid (all-trans-RA) and 13-cis-RA were the most potent inhibitors (IC50 S 6-15 microM), whereas all-trans-retinol was much less potent. Of the synthetic aromatic retinoids and arotinoids examined, the free carboxylic, sulfonic, and sulfinic acid forms were more than 15-fold more potent inhibitors of HSF-PLA2 than their corresponding ethyl esters. These retinoids also were evaluated as inhibitors of calcium ionophore A23187-induced AA release from rat peritoneal macrophages. All-trans-RA and 13-cis-RA were potent inhibitors of AA release from these cells (IC50 S 4 microM), while the other natural retinoids were inactive. Of the aromatic retinoids and arotinoids tested, the free acid forms (IC50 S 2-6 microM) were 5- to 21-fold more potent inhibitors of AA release from the macrophages than their corresponding ethyl esters. The potencies of the arotinoids as inhibitors of HSF-PLA2 appeared to correlate with their potencies as inhibitors of AA release from A23187-stimulated rat peritoneal macrophages. These data support the hypothesis that one possible mechanism for the known antiinflammatory activity of some retinoids may be by inhibition of phospholipase A2.

Case report: small bowel histiocytosis-X.

A case of histiocytosis-X with gastrointestinal involvement is described in a 4-month-old child presenting with bloody diarrhoea and obstructive symptoms. Diagnosis was made following laparotomy. The child subsequently developed classical multisystem manifestations of the disease.

Antiflammin-2 (HDMNKVLDL) does not inhibit phospholipase A2 activities.

A basic nonapeptide P2 (antiflammin-2, HDMNKVLDL) which is identical to a portion of the amino acid sequence (residues 246-254) of lipocortin I, has been described to have antiinflammatory activity in a rat paw edema model (Nature 335: 726-730 [1988]). P2 (0.05 microM) was also reported to inhibit porcine pancreatic phospholipase A2 (PLA2). The effect of synthetic P2 (98% pure) on PLA2 was evaluated in two assay systems. Using porcine pancreatic PLA2 and phosphatidylcholine/deoxycholate mixed micellar substrate, P2 (0.005-50 microM) had no effect on PLA2 activity, even in the presence of 2-mercaptoethanol to prevent peptide oxidation. In another assay, using human synovial fluid PLA2 as the enzyme and [14C]-oleate-labelled E. coli substrate, P2 (0.005-50 microM) had no significant effect on PLA2 activity. A reported PLA2 inhibitor, manoalide, was a potent inhibitor of PLA2 in both assay systems. On the basis of these results, we conclude that P2 is devoid of PLA2 inhibitory activity.

Comparison of Snellen and interferometer visual acuity in an aging noncataractous population.

Using standard clinical procedures we have compared visual acuity (VA) estimates made with a hand-held white light interferometer to those obtained with a Snellen acuity chart. Fifty noncataractous patients with a mean age of 45 years (SD = 18) were tested. Snellen and interferometric acuity measures were obtained with and without refractive correction. On average, aided Snellen VA's were better (decimal acuity = 0.98) than the unaided interferometric VA's (decimal acuity = 0.67). Although we found a statistically significant p less than 0.01) correlation between unaided interferometric and aided Snellen VA's, the correlation was poor (r = 0.36). This poor correlation may account for the often observed failure to estimate postoperative aided Snellen VA with preoperative interferometric VA in cataract patients.

Instability of the expanded (CTG)n repeats in the myotonin protein kinase gene in cultured lymphoblastoid cell lines from patients with myotonic dystrophy.

The mutation associated with myotonic dystrophy (DM) is the expansion of an unstable trinucleotide repeat, (CTG)n, in the 3'-untranslated region of the myotonin protein kinase gene. Although expanded repeats show both germline and somatic instability, the mechanisms of the instability are poorly understood. To establish a model system in which somatic instability of the DM repeat could be studied in more detail, we established lymphoblastoid cell lines (LBCL) from DM patients. Analysis of the DNA from DM LBCL using Southern blotting showed that the (CTG)n repeats were apparently stable up to 29 passages in culture. To study infrequent repeat size mutations that are undetectable due to the size heterogeneity, we established LBCL of single-cell origins by cloning using multiple steps of limiting dilution. After expansion to approximately 10(6) cells (equivalent to approximately 20 cell cycles), the DNAs of these cell lines were analyzed by the small pool PCR technique using primers flanking the (CTG)n repeat region. Two types of mutations of the expanded (CTG)n repeat alleles were detected: (1) frequent mutations that show small changes of the (CTG)n repeat size, resulting in alleles in a normal distribution around the progenitor allele, and (2) relatively rare mutations with large changes of the (CTG)n repeat size, with a bias toward contraction. The former may represent the mechanism responsible for the somatic heterogeneity of the (CTG)n repeat size observed in blood cells of DM patients. This in vitro experimental system will be useful for further studies on mechanisms involved in the regulation of the somatic stability of the (CTG)n repeats in DM.

CL1-GFP: an androgen independent metastatic tumor model for prostate cancer.

PURPOSE: The mechanisms responsible for tumor progression to androgen independence in prostate cancer (CaP) remain unknown. To characterize these changes and provide a basis for rational therapeutic strategies for advanced CaP, an in vivo model from a highly aggressive androgen independent CaP cell line with distinct cellular and molecular properties was developed. MATERIALS AND METHODS: An aggressive androgen-independent cell line designated CL1 was derived from a slow-growing, and androgen-dependent, parental LNCaP cell line through in-vitro androgen-deprivation and selection. CL1 was stably transfected with a green fluorescence protein gene (CL1-GFP) and orthotopically injected into SCID mice. The pathologic behavior, histology, and molecular determinants of CL1 tumor and metastases were determined and characterized by standard light and fluorescent microscopy, and quantitative RT-PCR analysis. RESULTS: CL1 is an anaplastic prostate cancer cell line which demonstrates extensive local invasion and metastases to various organs that can be visualized via GFP expression. When compared with parental LNCaP cells, RT-PCR analysis of the tumor revealed an over-expression of EGFR, b-FGF, VEGF, TGF-beta, IL-8, IL-6, and bcl-2 and a down regulated expression of the p53, E-cadherin and PTEN. In contrast to LNCaP cells, CL1 tumors express lower levels of androgen receptor and barely detectable PSA mRNA. CONCLUSIONS: CL1-GFP represents an aggressive androgen-independent CaP tumor model derived through androgen deprivation whose pathologic development and molecular properties in animals resembles the clinical characteristics of hormone refractory prostate cancer (HRPC). Metastatic sites of CL1-GFP can be visualized with fluorescence microscopy offering a unique therapeutic model for the evaluation of drug sensitivity and other therapeutic modalities.