Intraspecific comparative genomics of Candida albicans mitochondria reveals non-coding regions under neutral evolution

The opportunistic fungal pathogen Candida albicans causes serious hematogenic hospital acquired candidiasis with worldwide impact on public health. Because of its importance as a nosocomial etiologic agent, C albicans genome has been largely studied to identify intraspecific variation and several typing methods have been developed to distinguish closely related strains. Mitochondrial DNA can be useful for this purpose because, as compared to nuclear DNA, its higher mutational load and evolutionary rate readily reveals microvariants. Accordingly, we sequenced and assembled, with 8-fold coverage, the mitochondria( genomes of two C albicans clinical isolates (L296 and L757) and compared these sequences with the genome sequence of reference strain SC5314. the genome alignment of 33,928 positions revealed 372 polymorphic sites being 230 in coding and 142 in non-coding regions. Three intergenic regions located between genes tRNAGly/COX1, NAD3/COB and ssurRNA/NAD4L, named IG1, IG2 and IG3, respectively, which showed high number of neutral substitutions, were amplified and sequenced from 18 clinical isolates from different locations in Latin America and 2 ATCC standard C albicans strains. High variability of sequence and size were observed, ranging up to 56 bp size difference and phylogenies based on IG1, IG2 and IG3 revealed three groups. Insertions of up to 49 bp were observed exclusively in Argentinean strains relative to the other sequences which could suggest clustering by geographical polymorphism. Because of neutral evolution, high variability, easy isolation by PCR and full length sequencing these mitochondrial intergenic regions can contribute with a novel perspective in molecular studies of C albicans isolates, complementing well established multilocus sequence typing methods. (C) 2012 Elsevier B.V. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)International Program of the Howard Hughes Medical InstituteUniversidade Federal de São Paulo, Lab Genom Evolut & Biocomplexidade, Dept Microbiol Imunol & Parasitol, Disciplina Microbiol, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Infectol, Lab Especial Micol, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Lab Genom Evolut & Biocomplexidade, Dept Microbiol Imunol & Parasitol, Disciplina Microbiol, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Infectol, Lab Especial Micol, BR-04039032 São Paulo, BrazilWeb of Scienc

Detecting ovarian cancer using extracellular vesicles: Progress and possibilities

Ovarian cancer (OC) is the deadliest gynecological malignancy. Most patients are diagnosed when they are already in the later stages of the disease. Earlier detection of OC dramatically improves the overall survival, but this is rarely achieved as there is a lack of clinically implemented biomarkers of early disease. Extracellular vesicles (EVs) are small cell-derived vesicles that have been extensively studied in recent years. They contribute to various aspects of cancer pathology, including tumour growth, angiogenesis and metastasis. EVs are released from all cell types and the macromolecular cargo they carry reflects the content of the cells from which they were derived. Cancer cells release EVs with altered cargo into biofluids, and so they represent an excellent potential source of novel biomarkers for the disease. In this review we describe the latest developments in EVs as potential biomarkers for earlier detection of OC. The field is still relatively young, but a number of studies have shown that EVs and the cargo they carry, including miRNAs and proteins, can be used to detect OC. They could also give insight into the stage of the disease and predict the likely therapeutic outcome. There remain a number of challenges to the use of EVs as biomarkers, but through ongoing research and innovation in this exciting field there is great potential for the development of diagnostic assays in the clinic that could improve patient outcome

Identificacao de dominios de evolucao neutra no DNA mitocondrial de Candida albicans e suas possiveis aplicacoes em tipagem

fungo comensal humano Candida albicans e um patogeno oportunista que diante de imunossupressao do hospedeiro pode causar desde infeccoes muco-cutaneas simples ate graves infeccoes hematogenicas hospitalares. Por ser um agente etiologico importante de infeccoes nosocomiais, sao necessarios metodos eficientes de tipagem que permitam a distincao entre cepas proximamente relacionadas e consequentemente a identificacao da fonte de infeccao hospitalar. O DNA mitocondrial e indicado para estudos filogeneticos de organismos muito proximos (microevolucao) por apresentar taxa de mutacao mais elevada e evolucao mais rapida que o DNA nuclear. Para identificar regioes hipervariaveis intraespecificas no DNA mitocondrial, neste trabalho foram sequenciados, com COBertura de oito vezes, os genomas mitocondriais de dois isolados clinicos de C. albicans e suas sequencias foram comparadas com a do genoma da linhagem de referencia SC5314. Como revelado pelo alinhamento dos genomas, dos 33.928 sitios analisados, 372 foram polimorficos (variacao global de 1,1 %), sendo 230 localizados em suas regioes codificadoras e 142 nas nao codificadoras. Tres regioes intergenicas, localizadas entre os genes tRNA-Gly / COX1, NAD3 / COB e ssuRNAr / NAD4L, apresentaram maior numero de substituicoes neutras e foram investigadas quanto ao seu potencial para utilizacao em tipagem molecular. Para isto, estas tres regioes intergenicas mitocondriais foram amplificadas e sequenciadas em 18 isolados clinicos de C. albicans de diferentes locais da America Latina, alem de duas linhagens padrao ATCC. Nestas regioes, foi observada grande variacao tanto na sequencia nucleotidica quanto no tamanho, com variacao de ate 56pb no tamanho da sequencia entre as cepas e frequencia de alteracao nucleotidica de 4,24%. Arvores filogeneticas das linhagens revelaram a existencia de tres grupos, sendo um deles formado quase exclusivamente por cepas originarias da Argentina. As sequencias dessas cepas apresentaram insercoes de ate 49pb, indicando que estes segmentos podem ser utilizados como marcadores geograficos. Estes resultados indicam que o DNA mitocondrial, em especial as regioes intergenicas, sao ferramentas moleculares uteis para diferenciacao de diferentes isolados clinicos proximamente relacionados e estudos populacionais de C. albicans, devido a sua grande variabilidade e facilidade de obtencao por PCR e sequenciamentoBV UNIFESP: Teses e dissertaçõe

Dinâmica mutacional e epigenética do genoma de candida albicans durante evolução in vitro sob hipóxia e choque térmico

The commensal fungus Candida albicans colonizes several niches in the human body and is part of the microbiota of healthy individuals. It is also an opportunistic pathogen and causative agent of superficial and systemic infections in immunocompromised hosts, with a high mortality rate. To effectively colonize or infect a host, these fungi must adapt to physical constraints in the human body, such as its low oxygen tension (hypoxia, 5%CO2), and temperature (37°C heat shock). Previous studies have demonstrated that the genetic variability of C. albicans isolates is an important adaptive mechanism, although little is known about the effect of environmental conditions on their mitochondrial genomes. To simulate the physical environmental factors affecting C. albicans, two strains, SC5314 and L757, were subjected to an in vitro evolution scheme under hypoxia and 37°C for up to 48 weeks in order to evaluate the synergistic effect of oxygen tension and temperature on C. albicans mitochondrial genome (mtDNA). Sequencing of mitochondrial fragments over different periods (6, 12, 24 and 48 weeks) of strain SC5314 or sequencing the complete mitochondrial genome of both strains after 12 weeks grown in non-fermentative medium (GTH12) showed no sequence variation and/or lesions in mtDNA sequences, which kept their integrity even after 48 weeks. Under these different conditions, the mtDNA copy number mean values were invariant (nonsignificant differences in mean and variance). On the other hand, sequencing of nuclear gene fragments of strain SC5314 showed loss of heterozygosity after 48 weeks, on samples cultured in normoxia (normal oxygen tension) and 28°C (GVN48) and at 37 °C and hypoxia (DTH48). In addition, sequencing of the complete nuclear genome of strain SC5314 GTH12, revealed microvariations as compared to the same strain before in vitro evolution, with at least 155 variable alleles involved with regulatory processes and pathogenicity (adhesion and hyphal growth), including 19 involved in mitochondrial functions. Although we have not identified sequence changes in the mtDNA during in vitro evolution, we have described, for the first time, the whole mitochondrial genome methylation map of C. albicans. Our results indicate that environmental conditions, such as continuous exposure to hypoxia and 37°C affect the methylation patterns in a strain-specific manner, therefore providing a clue on how C. albicans employs epigenetic mechanisms for its survival in mammalian hosts.O fungo comensal Candida albicans coloniza diversos nichos no corpo humano e é componente da microbiota de diversos indivíduos saudáveis, no entanto, é também um patógeno oportunista que causa infecções de mucosas ou infecções sistêmicas em hospedeiros imunocomprometidos, com alta taxa de mortalidade. Para colonizar ou infectar com eficiência um hospedeiro, esses fungos precisam se adaptar às condições encontradas no corpo humano, onde a tensão de oxigênio é menor que a atmosférica (hipóxia, 5% CO2), e a temperatura é de aproximadamente 37°C, que desencadeia no fungo a resposta celular ao choque térmico. Um dos mecanismos de adaptação dessa espécie é o desenvolvimento de alta variabilidade genética no seu genoma nuclear, que lhe confere vantagens adaptativas principalmente quanto ao seu metabolismo e virulência. No entanto, pouco se sabe sobre os efeitos das condições ambientais em seu genoma mitocondrial. Com o intuito de simularmos os fatores ambientais que afetam C. albicans, cultivamos duas linhagens de C. albicans, SC5314 e L757, sob hipóxia e 37°C por até 48 semanas, com o intuito de avaliarmos o efeito concomitante da tensão de oxigênio e temperatura sobre o DNA mitocondrial (mtDNA) desse fungo. O sequenciamento de fragmentos mitocondriais ao longo de diferentes tempos (6, 12, 24 e 48 semanas) de SC5314 ou do genoma mitocondrial completo das duas linhagens após 12 semanas em meio não fermentativo (GTH12) não mostrou polimorfismos e/ou lesões nas sequências de mtDNA, que manteve sua integridade mesmo após 48 semanas de evolução in vitro. O número de cópias de mtDNA teve variações sutis e se manteve estável entre as linhagens nas diferentes condições e tempos. Por outro lado, o sequenciamento de fragmentos de genes nucleares de SC5314 mostrou perda de heterozigose após 48 semanas, tanto em amostras cultivadas em condições normais de oxigênio (normóxia) e 28°C (GVN48) quanto a 37°C e hipóxia (DTH48). Ainda, o sequenciamento do genoma nuclear completo da linhagem SC5314 GTH12, apresentou microvariações em relação à linhagem no tempo zero, com pelo menos 155 alelos variáveis, incluindo 19 envolvidos com funções mitocondriais e os demais envolvidos principalmente com processos regulatórios celulares e mecanismos de patogenicidade, como adesão e formação de hifas. Embora não tenhamos identificado alterações nas sequências do mtDNA durante a evolução in vitro, neste trabalho caracterizamos pela primeira vez o metiloma mitocondrial de C. albicans. Nossos resultados indicam que condições ambientais, como exposição contínua à hipóxia e 37°C, podem influenciar seu padrão de metilação de uma maneira linhagem-específica, indicando que C. albicans usa mecanismos epigenéticos para sobrevivência e adaptação ao seu hospedeiro.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Mutational Signatures Driven by Epigenetic Determinants Enable the Stratification of Patients with Gastric Cancer for Therapeutic Intervention

DNA mismatch repair deficiency (dMMR) is associated with the microsatellite instability (MSI) phenotype and leads to increased mutation load, which in turn may impact anti-tumor immune responses and treatment effectiveness. Various mutational signatures directly linked to dMMR have been described for primary cancers. To investigate which mutational signatures are associated with prognosis in gastric cancer, we performed a de novo extraction of mutational signatures in a cohort of 787 patients. We detected three dMMR-related signatures, one of which clearly discriminates tumors with MLH1 gene silencing caused by promoter hypermethylation (area under the curve = 98%). We then demonstrated that samples with the highest exposure of this signature share features related to better prognosis, encompassing clinical and molecular aspects and altered immune infiltrate composition. Overall, the assessment of the prognostic value and of the impact of modifications in MMR-related genes on shaping specific dMMR mutational signatures provides evidence that classification based on mutational signature exposure enables prognosis stratification

A global metagenomic map of urban microbiomes and antimicrobial resistance

We present a global atlas of 4,728 metagenomic samples from mass-transit systems in 60 cities over 3 years, representing the first systematic, worldwide catalog of the urban microbial ecosystem. This atlas provides an annotated, geospatial profile of microbial strains, functional characteristics, antimicrobial resistance (AMR) markers, and genetic elements, including 10,928 viruses, 1,302 bacteria, 2 archaea, and 838,532 CRISPR arrays not found in reference databases. We identified 4,246 known species of urban microorganisms and a consistent set of 31 species found in 97% of samples that were distinct from human commensal organisms. Profiles of AMR genes varied widely in type and density across cities. Cities showed distinct microbial taxonomic signatures that were driven by climate and geographic differences. These results constitute a high-resolution global metagenomic atlas that enables discovery of organisms and genes, highlights potential public health and forensic applications, and provides a culture-independent view of AMR burden in cities.Funding: the Tri-I Program in Computational Biology and Medicine (CBM) funded by NIH grant 1T32GM083937; GitHub; Philip Blood and the Extreme Science and Engineering Discovery Environment (XSEDE), supported by NSF grant number ACI-1548562 and NSF award number ACI-1445606; NASA (NNX14AH50G, NNX17AB26G), the NIH (R01AI151059, R25EB020393, R21AI129851, R35GM138152, U01DA053941); STARR Foundation (I13- 0052); LLS (MCL7001-18, LLS 9238-16, LLS-MCL7001-18); the NSF (1840275); the Bill and Melinda Gates Foundation (OPP1151054); the Alfred P. Sloan Foundation (G-2015-13964); Swiss National Science Foundation grant number 407540_167331; NIH award number UL1TR000457; the US Department of Energy Joint Genome Institute under contract number DE-AC02-05CH11231; the National Energy Research Scientific Computing Center, supported by the Office of Science of the US Department of Energy; Stockholm Health Authority grant SLL 20160933; the Institut Pasteur Korea; an NRF Korea grant (NRF-2014K1A4A7A01074645, 2017M3A9G6068246); the CONICYT Fondecyt Iniciación grants 11140666 and 11160905; Keio University Funds for Individual Research; funds from the Yamagata prefectural government and the city of Tsuruoka; JSPS KAKENHI grant number 20K10436; the bilateral AT-UA collaboration fund (WTZ:UA 02/2019; Ministry of Education and Science of Ukraine, UA:M/84-2019, M/126-2020); Kyiv Academic Univeristy; Ministry of Education and Science of Ukraine project numbers 0118U100290 and 0120U101734; Centro de Excelencia Severo Ochoa 2013–2017; the CERCA Programme / Generalitat de Catalunya; the CRG-Novartis-Africa mobility program 2016; research funds from National Cheng Kung University and the Ministry of Science and Technology; Taiwan (MOST grant number 106-2321-B-006-016); we thank all the volunteers who made sampling NYC possible, Minciencias (project no. 639677758300), CNPq (EDN - 309973/2015-5), the Open Research Fund of Key Laboratory of Advanced Theory and Application in Statistics and Data Science – MOE, ECNU, the Research Grants Council of Hong Kong through project 11215017, National Key RD Project of China (2018YFE0201603), and Shanghai Municipal Science and Technology Major Project (2017SHZDZX01) (L.S.