A Rapid Molecular Approach for Chromosomal Phasing

Determining the chromosomal phase of pairs of sequence variants – the arrangement of specific alleles as haplotypes – is a routine challenge in molecular genetics. Here we describe Drop-Phase, a molecular method for quickly ascertaining the phase of pairs of DNA sequence variants (separated by 1-200 kb) without cloning or manual single-molecule dilution. In each Drop-Phase reaction, genomic DNA segments are isolated in tens of thousands of nanoliter-sized droplets together with allele-specific fluorescence probes, in a single reaction well. Physically linked alleles partition into the same droplets, revealing their chromosomal phase in the co-distribution of fluorophores across droplets. We demonstrated the accuracy of this method by phasing members of trios (revealing 100% concordance with inheritance information), and demonstrate a common clinical application by phasing CFTR alleles at genomic distances of 11–116 kb in the genomes of cystic fibrosis patients. Drop-Phase is rapid (requiring less than 4 hours), scalable (to hundreds of samples), and effective at long genomic distances (200 kb).     

Cytoplasmic PELP1 and ERRgamma Protect Human Mammary Epithelial Cells from Tam-Induced Cell Death

Tamoxifen (Tam) is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a woman''s risk of developing estrogen receptor positive (ER+) breast cancer, the molecular mechanisms associated with risk reduction are poorly understood. Prior studies have shown that cytoplasmic proline, glutamic acid and leucine rich protein 1 (PELP1) promotes Tam resistance in breast cancer cell lines. Herein, we tested for PELP1 localization in breast epithelial cells from women at high risk for developing breast cancer and found that PELP1 was localized to the cytoplasm in 36% of samples. In vitro, immortalized HMECs expressing a nuclear localization signal (NLS) mutant of PELP1 (PELP1-cyto) were resistant to Tam-induced death. Furthermore, PELP1-cyto signaling through estrogen-related receptor gamma (ERRγ) promoted cell survival in the presence of Tam. Overexpression of ERRγ in immortalized HMECs protected cells from Tam-induced death, while knockdown of ERRγ sensitized PELP1-cyto expressing HMECs to Tam. Moreover, Tam-induced HMEC cell death was independent of apoptosis and involved accumulation of the autophagy marker LC3-II. Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam. Additionally, PELP1-cyto expression led to the upregulation of MMP-3 and MAOB, known PELP1 and ERRγ target genes, respectively. Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam. These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.     

Prenatal influenza vaccination and allergic and autoimmune diseases in childhood: A longitudinal,population-based linked cohort study

BackgroundFew studies have evaluated the effect of maternal influenza vaccination on the development of allergic and autoimmune diseases in children beyond 6 months of age. We aimed to investigate the association between in utero exposure to seasonal inactivated influenza vaccine (IIV) and subsequent diagnosis of allergic and autoimmune diseases.Methods and findingsThis longitudinal, population-based linked cohort study included 124,760 singleton, live-born children from 106,206 mothers in Western Australia (WA) born between April 2012 and July 2016, with up to 5 years of follow-up from birth. In our study cohort, 64,169 (51.4%) were male, 6,566 (5.3%) were Aboriginal and/or Torres Strait Islander children, and the mean age at the end of follow-up was 3.0 (standard deviation, 1.3) years. The exposure was receipt of seasonal IIV during pregnancy. The outcomes were diagnosis of an allergic or autoimmune disease, including asthma and anaphylaxis, identified from hospital and/or emergency department (ED) records. Inverse probability of treatment weights (IPTWs) accounted for baseline probability of vaccination by maternal age, Aboriginal and/or Torres Strait Islander status, socioeconomic status, body mass index, parity, medical conditions, pregnancy complications, prenatal smoking, and prenatal care. The models additionally adjusted for the Aboriginal and/or Torres Strait Islander status of the child. There were 14,396 (11.5%) maternally vaccinated children; 913 (6.3%) maternally vaccinated and 7,655 (6.9%) maternally unvaccinated children had a diagnosis of allergic or autoimmune disease, respectively. Overall, maternal influenza vaccination was not associated with diagnosis of an allergic or autoimmune disease (adjusted hazard ratio [aHR], 1.02; 95% confidence interval [CI], 0.95 to 1.09). In trimester-specific analyses, we identified a negative association between third trimester influenza vaccination and the diagnosis of asthma (n = 40; aHR, 0.70; 95% CI, 0.50 to 0.97) and anaphylaxis (n = 36; aHR, 0.67; 95% CI, 0.47 to 0.95).We did not capture outcomes diagnosed in a primary care setting; therefore, our findings are only generalizable to more severe events requiring hospitalization or presentation to the ED. Due to small cell sizes (i.e., <5), estimates could not be determined for all outcomes after stratification.ConclusionsIn this study, we observed no association between in utero exposure to influenza vaccine and diagnosis of allergic or autoimmune diseases. Although we identified a negative association of asthma and anaphylaxis diagnosis when seasonal IIV was administered later in pregnancy, additional studies are needed to confirm this. Overall, our findings support the safety of seasonal inactivated influenza vaccine during pregnancy in relation to allergic and autoimmune diseases in early childhood and support the continuation of current global maternal vaccine programs and policies.

Damien Foo and colleagues evaluate the association between prenatal influenza vaccination and diagnosis of allergic and autoimmune diseases in childhood.     

Fatty acid synthase (FASN) regulates the mitochondrial priming of cancer cells

Inhibitors of the lipogenic enzyme fatty acid synthase (FASN) have attracted much attention in the last decade as potential targeted cancer therapies. However, little is known about the molecular determinants of cancer cell sensitivity to FASN inhibitors (FASNis), which is a major roadblock to their therapeutic application. Here, we find that pharmacological starvation of endogenously produced FAs is a previously unrecognized metabolic stress that heightens mitochondrial apoptotic priming and favors cell death induction by BH3 mimetic inhibitors. Evaluation of the death decision circuits controlled by the BCL-2 family of proteins revealed that FASN inhibition is accompanied by the upregulation of the pro-death BH3-only proteins BIM, PUMA, and NOXA. Cell death triggered by FASN inhibition, which causally involves a palmitate/NADPH-related redox imbalance, is markedly diminished by concurrent loss of BIM or PUMA, suggesting that FASN activity controls cancer cell survival by fine-tuning the BH3 only proteins-dependent mitochondrial threshold for apoptosis. FASN inhibition results in a heightened mitochondrial apoptosis priming, shifting cells toward a primed-for-death state “addicted” to the anti-apoptotic protein BCL-2. Accordingly, co-administration of a FASNi synergistically augments the apoptosis-inducing activity of the dual BCL-X_L/BCL-2 inhibitor ABT-263 (navitoclax) and the BCL-2 specific BH3-mimetic ABT-199 (venetoclax). FASN inhibition, however, fails to sensitize breast cancer cells to MCL-1- and BCL-X_L-selective inhibitors such as {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540"}}S63845 and A1331852. A human breast cancer xenograft model evidenced that oral administration of the only clinically available FASNi drastically sensitizes FASN-addicted breast tumors to ineffective single-agents navitoclax and venetoclax in vivo. In summary, a novel FASN-driven facet of the mitochondrial priming mechanistically links the redox-buffering mechanism of FASN activity to the intrinsic apoptotic threshold in breast cancer cells. Combining next-generation FASNis with BCL-2-specific BH3 mimetics that directly activate the apoptotic machinery might generate more potent and longer-lasting antitumor responses in a clinical setting.Subject terms: Cancer metabolism, Lipid signalling     

Splice Junction Identification using Long Short-Term Memory Neural Networks

BackgroundSplice junctions are the key to move from pre-messenger RNA to mature messenger RNA in many multi-exon genes due to alternative splicing. Since the percentage of multi-exon genes that undergo alternative splicing is very high, identifying splice junctions is an attractive research topic with important implications.ObjectiveThe aim of this paper is to develop a deep learning model capable of identifying splice junctions in RNA sequences using 13,666 unique sequences of primate RNA.MethodsA Long Short-Term Memory (LSTM) Neural Network model is developed that classifies a given sequence as EI (Exon-Intron splice), IE (Intron-Exon splice), or N (No splice). The model is trained with groups of trinucleotides and its performance is tested using validation and test data to prevent bias.ResultsModel performance was measured using accuracy and f-score in test data. The finalized model achieved an average accuracy of 91.34% with an average f-score of 91.36% over 50 runs.ConclusionComparisons show a highly competitive model to recent Convolutional Neural Network structures. The proposed LSTM model achieves the highest accuracy and f-score among published alternative LSTM structures.     

Degradation of biological macromolecules supports uncultured microbial populations in Guaymas Basin hydrothermal sediments

Hydrothermal sediments contain large numbers of uncultured heterotrophic microbial lineages. Here, we amended Guaymas Basin sediments with proteins, polysaccharides, nucleic acids or lipids under different redox conditions and cultivated heterotrophic thermophiles with the genomic potential for macromolecule degradation. We reconstructed 20 metagenome-assembled genomes (MAGs) of uncultured lineages affiliating with known archaeal and bacterial phyla, including endospore-forming Bacilli and candidate phylum Marinisomatota. One Marinisomatota MAG had 35 different glycoside hydrolases often in multiple copies, seven extracellular CAZymes, six polysaccharide lyases, and multiple sugar transporters. This population has the potential to degrade a broad spectrum of polysaccharides including chitin, cellulose, pectin, alginate, chondroitin, and carrageenan. We also describe thermophiles affiliating with the genera Thermosyntropha, Thermovirga, and Kosmotoga with the capability to make a living on nucleic acids, lipids, or multiple macromolecule classes, respectively. Several populations seemed to lack extracellular enzyme machinery and thus likely scavenged oligo- or monomers (e.g., MAGs affiliating with Archaeoglobus) or metabolic products like hydrogen (e.g., MAGs affiliating with Thermodesulfobacterium or Desulforudaceae). The growth of methanogens or the production of methane was not observed in any condition, indicating that the tested macromolecules are not degraded into substrates for methanogenesis in hydrothermal sediments. We provide new insights into the niches, and genomes of microorganisms that actively degrade abundant necromass macromolecules under oxic, sulfate-reducing, and fermentative thermophilic conditions. These findings improve our understanding of the carbon flow across trophic levels and indicate how primary produced biomass sustains complex and productive ecosystems.Subject terms: Water microbiology, Environmental sciences     

p53 prevents progression of nevi to melanoma predominantly through cell cycle regulation

p53 is the central member of a critical tumor suppressor pathway in virtually all tumor types, where it is silenced mainly by missense mutations. In melanoma, p53 predominantly remains wild type, thus its role has been neglected. To study the effect of p53 on melanocyte function and melanomagenesis, we crossed the ‘high-p53’Mdm4^+/− mouse to the well-established TP-ras^0/+ murine melanoma progression model. After treatment with the carcinogen dimethylbenzanthracene (DMBA), TP-ras^0/+ mice on the Mdm4^+/− background developed fewer tumors with a delay in the age of onset of melanomas compared to TP-ras^0/+ mice. Furthermore, we observed a dramatic decrease in tumor growth, lack of metastasis with increased survival of TP-ras^0/+: Mdm4^+/− mice. Thus, p53 effectively prevented the conversion of small benign tumors to malignant and metastatic melanoma. p53 activation in cultured primary melanocyte and melanoma cell lines using Nutlin-3, a specific Mdm2 antagonist, supported these findings. Moreover, global gene expression and network analysis of Nutlin-3-treated primary human melanocytes indicated that cell cycle regulation through the p21^WAF1/CIP1 signaling network may be the key anti-melanomagenic activity of p53.