Mining the bitter melon (momordica charantia l.) seed transcriptome by 454 analysis of non-normalized and normalized cDNA populations for conjugated fatty acid metabolism-related genes

Abstract Background Seeds of Momordica charantia (bitter melon) produce high levels of eleostearic acid, an unusual conjugated fatty acid with industrial value. Deep sequencing of non-normalized and normalized cDNAs from developing bitter melon seeds was conducted to uncover key genes required for biotechnological transfer of conjugated fatty acid production to existing oilseed crops. It is expected that these studies will also provide basic information regarding the metabolism of other high-value novel fatty acids. Results Deep sequencing using 454 technology with non-normalized and normalized cDNA libraries prepared from bitter melon seeds at 18 DAP resulted in the identification of transcripts for the vast majority of known genes involved in fatty acid and triacylglycerol biosynthesis. The non-normalized library provided a transcriptome profile of the early stage in seed development that highlighted the abundance of transcripts for genes encoding seed storage proteins as well as for a number of genes for lipid metabolism-associated polypeptides, including Δ12 oleic acid desaturases and fatty acid conjugases, class 3 lipases, acyl-carrier protein, and acyl-CoA binding protein. Normalization of cDNA by use of a duplex-specific nuclease method not only increased the overall discovery of genes from developing bitter melon seeds, but also resulted in the identification of 345 contigs with homology to 189 known lipid genes in Arabidopsis. These included candidate genes for eleostearic acid metabolism such as diacylglycerol acyltransferase 1 and 2, and a phospholipid:diacylglycerol acyltransferase 1-related enzyme. Transcripts were also identified for a novel FAD2 gene encoding a functional Δ12 oleic acid desaturase with potential implications for eleostearic acid biosynthesis. Conclusions 454 deep sequencing, particularly with normalized cDNA populations, was an effective method for mining of genes associated with eleostearic acid metabolism in developing bitter melon seeds. The transcriptomic data presented provide a resource for the study of novel fatty acid metabolism and for the biotechnological production of conjugated fatty acids and possibly other novel fatty acids in established oilseed crops.</p

Hepatic P450 Enzyme Activity, Tissue Morphology and Histology of Mink (Mustela vison) Exposed to Polychlorinated Dibenzofurans

Dose- and time-dependent effects of environmentally relevant concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQ) of 2,3,7,8-tetrachlorodibenzofuran (TCDF), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or a mixture of these two congeners on hepatic P450 enzyme activity and tissue morphology, including jaw histology, of adult ranch mink were determined under controlled conditions. Adult female ranch mink were fed either TCDF (0.98, 3.8, or 20 ng TEQTCDF/kg bw/day) or PeCDF (0.62, 2.2, or 9.5 ng TEQPeCDF/kg bw/day), or a mixture of TCDF and PeCDF (4.1 ng TEQTCDF/kg bw/day and 2.8 ng TEQPeCDF/kg bw/day, respectively) for 180 days. Doses used in this study were approximately eight times greater than those reported in a parallel field study. Activities of the cytochrome P450 1A enzymes, ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-deethylase (MROD) were significantly greater in livers of mink exposed to TCDF, PeCDF, and a mixture of the two congeners; however, there were no significant histological or morphological effects observed. It was determined that EROD and MROD activity can be used as sensitive biomarkers of exposure to PeCDF and TCDF in adult female mink; however, under the conditions of this study, the response of EROD/MROD induction occurred at doses that were less than those required to cause histological or morphological changes

The Public Repository of Xenografts enables discovery and randomized phase II-like trials in mice

More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publicly available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment, called the Public Repository of Xenografts (PRoXe). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional, and proteomic biomarkers in both treatment-naive and relapsed/refractory disease

Myocardial infarction and atrial fibrillation: Importance of atrial ischemia

Background-Myocardial infarction (MI) is associated with the development of atrial fibrillation (AF). We aimed to characterize the atrial abnormalities because of MI and determine the role of ischemia to the AF substrate. Methods and Results-Forty-four s

Genomic analyses of PMBL reveal new drivers and mechanisms of sensitivity to PD-1 blockade

Primary mediastinal large B-cell lymphomas (PMBLs) are aggressive tumors that typically present as large mediastinal masses in young women. PMBLs share clinical, transcriptional, and molecular features with classical Hodgkin lymphoma (cHL), including constitutive activation of nuclear factor κB (NF-κB), JAK/STAT signaling, and programmed cell death protein 1 (PD-1)-mediated immune evasion. The demonstrated efficacy of PD-1 blockade in relapsed/refractory PMBLs led to recent approval by the US Food and Drug Administration and underscored the importance of characterizing targetable genetic vulnerabilities in this disease. Here, we report a comprehensive analysis of recurrent genetic alterations -somatic mutations, somatic copy number alterations, and structural variants-in a cohort of 37 newly diagnosed PMBLs. We identified a median of 9 genetic drivers per PMBL, including known and newly identified components of the JAK/STAT and NF-κB signaling pathways and frequent B2M alterations that limit major histocompatibility complex class I expression, as in cHL. PMBL also exhibited frequent, newly identified driver mutations in ZNF217 and an additional epigenetic modifier, EZH2. The majority of these alterations were clonal, which supports their role as early drivers. In PMBL, we identified several previously uncharacterized molecular features that may increase sensitivity to PD-1 blockade, including high tumor mutational burden, microsatellite instability, and an apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) mutational signature. The shared genetic features between PMBL and cHL provide a framework for analyzing the mechanism of action of PD-1 blockade in these related lymphoid malignancies

The national ambulance surveillance system:A novel method for monitoring acute alcohol, illicit and pharmaceutical drug related-harms using coded Australian ambulance clinical records

Although harmful consumption of alcohol and other drugs (both illicit and pharmaceutical) significantly contribute to global burden of disease, not all harms are captured within existing morbidity data sources. Indeed, harms occurring in the community may be missed or under-reported. This paper describes the National Ambulance Surveillance System, a unique Australian system for monitoring and mapping acute harms related to alcohol and other drug consumption. Data are sourced from paramedic electronic patient care records provided by ambulance services from across Australia. Coding occurs in a purpose-built system, by a team of specialised research assistants. Alcohol, and specific illicit and pharmaceutical drugs, rather than broad drug classes, are manually coded and the dataset is reviewed and cleaned prior to analysis. The National Ambulance Surveillance System is an ongoing, dynamic surveillance system of alcohol and other drug-related harms across Australia. The data includes more than 140 output variables per attendance, including individual substances, demographics, temporal, geospatial, and clinical data (e.g., Glasgow Coma Scale score, naloxone provision and response, outcome of attendance). The National Ambulance Surveillance System is an internationally unique population-level surveillance system of acute harms arising from alcohol and other drug consumption. Dissemination of National Ambulance Surveillance System data has been used to inform and evaluate policy approaches and potential points of intervention, as well as guide workforce development needs and clinical practice at the local and national level. This methodology could be replicated in other countries

Genomic analyses of flow-sorted Hodgkin Reed-Sternberg cells reveal complementary mechanisms of immune evasion

Classical Hodgkin lymphoma (cHL) is composed of rare malignant Hodgkin Reed-Sternberg (HRS) cells within an extensive, but ineffective, inflammatory/immune cell infiltrate. HRS cells exhibit near-universal somatic copy gains of chromosome 9p/9p24.1, which increase expression of the programmed cell death protein 1 (PD-1) ligands. To define genetic mechanisms of response and resistance to PD-1 blockade and identify complementary treatment targets, we performed whole-exome sequencing of flow cytometry-sorted HRS cells from 23 excisional biopsies of newly diagnosed cHLs, including 8 Epstein-Barr virus-positive (EBV+) tumors. We identified significantly mutated cancer candidate genes (CCGs) as well as somatic copy number alterations and structural variations and characterized their contribution to disease-defining immune evasion mechanisms and nuclear factor κB (NF-κB), JAK/STAT, and PI3K signaling pathways. EBV- cHLs had a higher prevalence of genetic alterations in the NF-κB and major histocompatibility complex class I antigen presentation pathways. In this young cHL cohort (median age, 26 years), we identified a predominant mutational signature of spontaneous deamination of cytosine- phosphate-guanines ("Aging"), in addition to apolipoprotein B mRNA editing catalytic polypeptide-like, activation-induced cytidine deaminase, and microsatellite instability (MSI)-associated hypermutation. In particular, the mutational burden in EBV- cHLs was among the highest reported, similar to that of carcinogen-induced tumors. Together, the overall high mutational burden, MSI-associated hypermutation, and newly identified genetic alterations represent additional potential bases for the efficacy of PD-1 blockade in cHL. Of note, recurrent cHL alterations, including B2M, TNFAIP3, STAT6, GNA13, and XPO1 mutations and 2p/2p15, 6p21.32, 6q23.3, and 9p/9p24.1 copy number alterations, were also identified in >20% of primary mediastinal B-cell lymphomas, highlighting shared pathogenetic mechanisms in these diseases

Targetable genetic features of primary testicular and primary central nervous system lymphomas

Primary central nervous system lymphomas (PCNSLs) and primary testicular lymphomas (PTLs) are extranodal large B-cell lymphomas (LBCLs) with inferior responses to current empiric treatment regimens. To identify targetable genetic features of PCNSL and PTL, we characterized their recurrent somatic mutations, chromosomal rearrangements, copy number alterations (CNAs), and associated driver genes, and compared these comprehensive genetic signatures to those of diffuse LBCL and primary mediastinal large B-cell lymphoma (PMBL). These studies identify unique combinations of genetic alterations in discrete LBCL subtypes and subtype-selective bases for targeted therapy. PCNSLs and PTLs frequently exhibit genomic instability, and near-uniform, often biallelic, CDKN2A loss with rare TP53 mutations. PCNSLs and PTLs also use multiple genetic mechanisms to target key genes and pathways and exhibit near-uniform oncogenic Toll-like receptor signaling as a result of MYD88 mutation and/or NFKBIZ amplification, frequent concurrent B-cell receptor pathway activation, and deregulation of BCL6. Of great interest, PCNSLs and PTLs also have frequent 9p24.1/PD-L1/PD-L2 CNAs and additional translocations of these loci, structural bases of immune evasion that are shared with PMB