Experimental determination of translational start sites resolves uncertainties in genomic open reading frame predictions – application to Mycobacterium tuberculosis

Correct identification of translational start sites is important for understanding protein function and transcriptional regulation. The annotated translational start sites contained in genome databases are often predicted using bioinformatics and are rarely verified experimentally, and so are not all accurate. Therefore, we devised a simple approach for determining translational start sites using a combination of epitope tagging and frameshift mutagenesis. This assay was used to determine the start sites of three Mycobacterium tuberculosis proteins: LexA, SigC and Rv1955. We were able to show that proteins may begin before or after the predicted site. We also found that a small, non-annotated open reading frame upstream of Rv1955 was expressed as a protein, which we have designated Rv1954A. This approach is readily applicable to any bacterial species for which plasmid transformation can be achieved

Procjena cito-/genotoksičnosti irinotekana u V79-stanicama primjenom komet-testa, mikronukleus-testa i testa kromosomskih aberacija

Irinotecan is a topoisomerase I interactive agent, widely used in the treatment of metastatic colorectal cancer. The genotoxic effects of the maximum single dose (18 μg mL-1), recommended monotherapy dose (9 μg mL-1), and recommended combined therapy dose (4.5 μg mL-1) of irinotecan were studied on V79 cells using the comet assay, chromosome aberration assay, and micronucleus test. The cells were treated with irinotecan for 2 h or 24 h. The statistical signifi cance of the results was determined using the one-way ANOVA test and a nonparametric Mann Whitney U test. The comet assay did not show dose-dependent or time-dependent effects. The chromosome aberration analysis showed large DNA rearrangements, i.e., chromosome exchanges. Although the exposed cultures showed a signifi cant increase in micronucleated cells in respect to control, no dose-dependent relation was established among the treated cultures. Timedependent effect was also not observed.Irinotekan je citotoksični lijek koji inhibira enzim DNA-topoizomerazu I. U širokoj je primjeni u terapiji metastatskog karcinoma kolona i rektuma. U uvjetima in vitro primjenom komet-testa, analize kromosomskih aberacija i mikronukleus-testa na V79-stanicama istražili smo genotoksični učinak maksimalne pojedinačne doze (18 μg mL-1), preporučene monoterapijske doze (9 μg mL-1) i preporučene doze irinotekana za kombiniranu terapiju (4,5 μg mL-1). Kulture stanica bile su tretirane irinotekanom 2 h i 24 h. Statistička značajnost određivana je jednosmjernim ANOVA-testom i neparametrijskim Mann Whitneyevim U-testom. Komet-testom nije utvrđen učinak koncentracije i/ili vremena izloženosti. Analiza kromosomskih aberacija pokazala je prisutnost izmjena kromatida, tj. porast broja triradijusa i tetraradijusa. Iako je u kulturama stanica izloženi irinotekanu opažen značajan porast broja mikronukleusa u odnosu na kontrolu, nije uočena ovisnost o dozi lijeka ni o vremenu izloženosti u opisanim eksperimentalnim uvjetima. Dobiveni rezultati upućuju na genotoksičnost irinotekana za V79-stanice. Nijednom od primijenjenih metoda nije utvrđena ovisnost učinka irinotekana o vremenu ili dozi

An integrative multi-omics analysis to identify candidate DNA methylation biomarkers related to prostate cancer risk

It remains elusive whether some of the associations identified in genome-wide association studies of prostate cancer (PrCa) may be due to regulatory effects of genetic variants on CpG sites, which may further influence expression of PrCa target genes. To search for CpG sites associated with PrCa risk, here we establish genetic models to predict methylation (N = 1,595) and conduct association analyses with PrCa risk (79,194 cases and 61,112 controls). We identify 759 CpG sites showing an association, including 15 located at novel loci. Among those 759 CpG sites, methylation of 42 is associated with expression of 28 adjacent genes. Among 22 genes, 18 show an association with PrCa risk. Overall, 25 CpG sites show consistent association directions for the methylation-gene expression-PrCa pathway. We identify DNA methylation biomarkers associated with PrCa, and our findings suggest that specific CpG sites may influence PrCa via regulating expression of candidate PrCa target genes

Identification of Novel Susceptibility Loci and Genes for Prostate Cancer Risk: A Transcriptome-Wide Association Study in over 140,000 European Descendants

Genome-wide association study–identified prostate cancer risk variants explain only a relatively small fraction of its familial relative risk, and the genes responsible for many of these identified associations remain unknown. To discover novel prostate cancer genetic loci and possible causal genes at previously identified risk loci, we performed a transcriptome-wide association study in 79,194 cases and 61,112 controls of European ancestry. Using data from the Genotype-Tissue Expression Project, we established genetic models to predict gene expression across the transcriptome for both prostate models and cross-tissue models and evaluated model performance using two independent datasets. We identified significant associations for 137 genes at P < 2.61 × 10−6, a Bonferroni-corrected threshold, including nine genes that remained significant at P < 2.61 × 10−6 after adjusting for all known prostate cancer risk variants in nearby regions. Of the 128 remaining associated genes, 94 have not yet been reported as potential target genes at known loci. We silenced 14 genes and many showed a consistent effect on viability and colony-forming efficiency in three cell lines. Our study provides substantial new information to advance our understanding of prostate cancer genetics and biology. SIGNIFICANCE: This study identifies novel prostate cancer genetic loci and possible causal genes, advancing our understanding of the molecular mechanisms that drive prostate cancer

Izloženost genotoksičnim agensima iz životnog okoliša tijekom prenatalnog razvoja i djetinjstva

Health disorders and diseases related to environmental exposure in children such as cancer and immunologic disturbances (asthma, allergies) are on the rise. However, complex transplacental and prepubertal genotoxicology is given very limited consideration, even though intrauterine development and early childhood may be critical for elucidating the cancer aetiology. The foetus is transplacentally exposed to contaminants in food and environment such as various chemicals, drugs, radiochemically contaminated water and air. Target organs of xenobiotic action may differ between the mother and the foetus due to specific stage of developmental physiology and enzyme distribution. This in turn may lead to different levels of clastogenic and aneugenic metabolites of the same xenobiotic in the mother and the foetus. Adult’s protective behaviour is not sufficient to isolate children from radioisotopes, pesticides, toxic metals and metalloids, environmental tobacco smoke, endocrine disrupting chemicals, and various food contaminants, which are just a part of the stressors present in a polluted environment. In order to improve legislation related to foetus and child exposure to genotoxic and possibly carcinogenic agents, oncologists, paediatricians, environmental health specialists, and genotoxicologists should work together much more closely to make a more effective use of accumulated scientific data, with the final aim to lower cancer incidence and mortality.Unatoč velikim naporima da se smanji okolišna izloženost u djece se dalje bilježi trend porasta pojavnosti karcinoma i imunosnih poremećaja (astma, alergije). Premda su intrauterini razvoj i rano djetinjstvo kritično razdoblje za tumačenje etiologije nastanka karcinoma, transplacentalna i prepubertetna genotoksikologija do danas su slabo istražene. Fetus je transplacentalno izložen brojnim fizikalnim i kemijskim čimbenicima: kontaminantima iz hrane i okoliša, radiokemijski kontaminiranoj vodi, zraku te lijekovima. Ciljna tkiva za djelovanje ksenobiotika mogu biti različita u majke i fetusa zbog različitosti u razvojnoj fiziologiji i distribuciji enzima. Zbog toga u organizmu majke i fetusa mogu nastati različite razine klastogenih i aneugenih metabolita istog ksenobiotika. Zaštitna uloga odraslih u namjeri da spriječe negativne utjecaje onečišćenog okoliša na djetetovo zdravlje često je ograničena jer su radioizotopi, olovo, PCB, pasivno pušenje, živa, endokrino aktivne tvari, pesticidi i kontaminanti prisutni u svim životnim područjima tijekom razvoja i rasta djeteta. Kako bi se poboljšalo zakonodavstvo vezano uz izloženost djece genotoksičnim i vjerojatno kancerogenim tvarima, tijekom razvoja potrebna je bolja suradnja onkologa, pedijatara, stručnjaka zdravstvene ekologije i genotoksikologa. Na taj način ostvarilo bi se uspješnije iskorištavanje postojećih znanstvenih podataka u cilju smanjenja incidencije karcinoma i mortaliteta

Association analyses identify 31 new risk loci for colorectal cancer susceptibility

Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide, and has a strong heritable basis. We report a genome-wide association analysis of 34,627 CRC cases and 71,379 controls of European ancestry that identifies SNPs at 31 new CRC risk loci. We also identify eight independent risk SNPs at the new and previously reported European CRC loci, and a further nine CRC SNPs at loci previously only identified in Asian populations. We use in situ promoter capture Hi-C (CHi-C), gene expression, and in silico annotation methods to identify likely target genes of CRC SNPs. Whilst these new SNP associations implicate target genes that are enriched for known CRC pathways such as Wnt and BMP, they also highlight novel pathways with no prior links to colorectal tumourigenesis. These findings provide further insight into CRC susceptibility and enhance the prospects of applying genetic risk scores to personalised screening and prevention

A Novel Soluble Immune-Type Receptor (SITR) in Teleost Fish: Carp SITR Is Involved in the Nitric Oxide-Mediated Response to a Protozoan Parasite

Background- The innate immune system relies upon a wide range of germ-line encoded receptors including a large number of immunoglobulin superfamily (IgSF) receptors. Different Ig-like immune receptor families have been reported in mammals, birds, amphibians and fish. Most innate immune receptors of the IgSF are type I transmembrane proteins containing one or more extracellular Ig-like domains and their regulation of effector functions is mediated intracellularly by distinct stimulatory or inhibitory pathways. Methodology/Principal Findings - Carp SITR was found in a substracted cDNA repertoire from carp macrophages, enriched for genes up-regulated in response to the protozoan parasite Trypanoplasma borreli. Carp SITR is a type I protein with two extracellular Ig domains in a unique organisation of a N-proximal V/C2 (or I-) type and a C-proximal V-type Ig domain, devoid of a transmembrane domain or any intracytoplasmic signalling motif. The carp SITR C-proximal V-type Ig domain, in particular, has a close sequence similarity and conserved structural characteristics to the mammalian CD300 molecules. By generating an anti-SITR antibody we could show that SITR protein expression was restricted to cells of the myeloid lineage. Carp SITR is abundantly expressed in macrophages and is secreted upon in vitro stimulation with the protozoan parasite T. borreli. Secretion of SITR protein during in vivo T. borreli infection suggests a role for this IgSF receptor in the host response to this protozoan parasite. Overexpression of carp SITR in mouse macrophages and knock-down of SITR protein expression in carp macrophages, using morpholino antisense technology, provided evidence for the involvement of carp SITR in the parasite-induced NO production. Conclusion/Significance - We report the structural and functional characterization of a novel soluble immune-type receptor (SITR) in a teleost fish and propose a role for carp SITR in the NO-mediated response to a protozoan parasite