Reconstructing Prehistoric Viral Genomes from Neanderthal Sequencing Data.

DNA viruses that produce persistent infections have been proposed as potential causes for the extinction of Neanderthals, and, therefore, the identification of viral genome remnants in Neanderthal sequence reads is an initial step to address this hypothesis. Here, as proof of concept, we searched for viral remnants in sequence reads of Neanderthal genome data by mapping to adenovirus, herpesvirus and papillomavirus, which are double-stranded DNA viruses that may establish lifelong latency and can produce persistent infections. The reconstructed ancient viral genomes of adenovirus, herpesvirus and papillomavirus revealed conserved segments, with nucleotide identity to extant viral genomes and variable regions in coding regions with substantial divergence to extant close relatives. Sequence reads mapped to extant viral genomes showed deamination patterns of ancient DNA, and these ancient viral genomes showed divergence consistent with the age of these samples (≈50,000 years) and viral evolutionary rates (10-5 to 10-8 substitutions/site/year). Analysis of random effects showed that the Neanderthal mapping to genomes of extant persistent viruses is above what is expected by random similarities of short reads. Also, negative control with a nonpersistent DNA virus does not yield statistically significant assemblies. This work demonstrates the feasibility of identifying viral genome remnants in archaeological samples with signal-to-noise assessment.

Convergent Mutations and Single Nucleotide Variants in Mitochondrial Genomes of Modern Humans and Neanderthals.

The genetic contributions of Neanderthals to the modern human genome have been evidenced by the comparison of present-day human genomes with paleogenomes. Neanderthal signatures in extant human genomes are attributed to intercrosses between Neanderthals and archaic anatomically modern humans (AMHs). Although Neanderthal signatures are well documented in the nuclear genome, it has been proposed that there is no contribution of Neanderthal mitochondrial DNA to contemporary human genomes. Here we show that modern human mitochondrial genomes contain 66 potential Neanderthal signatures, or Neanderthal single nucleotide variants (N-SNVs), of which 36 lie in coding regions and 7 result in nonsynonymous changes. Seven N-SNVs are associated with traits such as cycling vomiting syndrome, Alzheimer's disease and Parkinson's disease, and two N-SNVs are associated with intelligence quotient. Based on recombination tests, principal component analysis (PCA) and the complete absence of these N-SNVs in 41 archaic AMH mitogenomes, we conclude that convergent evolution, and not recombination, explains the presence of N-SNVs in present-day human mitogenomes.

Whole-genome analysis of monozygotic Brazilian twins discordant for type 1 narcolepsy: a case report.

BACKGROUND: Narcolepsy type 1 (NT1) is a rare and chronic neurological disease characterized by sudden sleep attacks, overwhelming daytime drowsiness, and cataplexy. When associated with a sudden loss of muscle tone (cataplexy) narcolepsy is classified as type 1, while the absence of cataplexy indicates type 2. Genetic, degenerative, and immunological hypotheses to explain the pathophysiology of NT1 are still a matter of debate. To contribute to the understanding of NT1 genetic basis, here we describe, for the first time, a whole genome analysis of a monozygotic twin pair discordant for NT1. CASE PRESENTATION: We present the case of a pair of 17-year-old male, monozygotic twins discordant for NT1. The affected twin had Epworth Sleepiness Scale (ESS) of 20 (can range from 0 to 24), cataplexy, hypnagogic hallucinations, polysomnography without abnormalities, multiple sleep latency tests (MSLT) positive for narcolepsy, a mean sleep latency of 3 min, sleep-onset REM periods SOREMPs of 5, presence of allele HLA-DQB1*06:02, and Hypocretin-1 level of zero pg/mL (normal values are > 200 pg/mL). The other twin had no narcolepsy symptoms (ESS of 4), normal polysomnography, MSLT without abnormalities, presence of allele HLA-DQB1*06:02, and Hypocretin-1 level of 396,74 pg/mL. To describe the genetic background for the NT1 discordant manifestations in this case, we present the whole-genome analysis of this monozygotic twin pair. The whole-genome comparison revealed that both twins have identical NT1 pathogenic mutations in known genes, such as HLA-DQB1*06:02:01, HLA-DRB1*11:01:02/*15:03:01. The affected twin has the expected clinical manifestation while the unaffected twin has an unexpected phenotype. The unaffected twin has significantly more frameshift mutations as compared to the affected twin (108 versus 75) and mutations that affect stop codons (61 versus 5 in stop gain, 26 versus 2 in start lost). CONCLUSIONS: The differences observed in frameshift and stop codon mutations in the unaffected twin are consistent with loss-of-function effects and protective alleles, that are almost always associated with loss-of-function rare alleles. Also, overrepresentation analysis of genes containing variants with potential clinical relevance in the unaffected twin shows that most mutations are in genes related to immune regulation function, Golgi apparatus, MHC, and olfactory receptor. These observations support the hypothesis that NT1 has an immunological basis although protective mutations in non-HLA alleles might interfere with the expression of the NT1 phenotype and consequently, with the clinical manifestation of the disease.

The epitranscriptome of Vero cells infected with SARS-CoV-2 assessed by direct RNA sequencing reveals m6A pattern changes and DRACH motif biases in viral and cellular RNAs.

The epitranscriptomics of the SARS-CoV-2 infected cell reveals its response to viral replication. Among various types of RNA nucleotide modifications, the m6A is the most common and is involved in several crucial processes of RNA intracellular location, maturation, half-life and translatability. This epitranscriptome contains a mixture of viral RNAs and cellular transcripts. In a previous study we presented the analysis of the SARS-CoV-2 RNA m6A methylation based on direct RNA sequencing and characterized DRACH motif mutations in different viral lineages. Here we present the analysis of the m6A transcript methylation of Vero cells (derived from African Green Monkeys) and Calu-3 cells (human) upon infection by SARS-CoV-2 using direct RNA sequencing data. Analysis of these data by nonparametric statistics and two computational methods (m6anet and EpiNano) show that m6A levels are higher in RNAs of infected cells. Functional enrichment analysis reveals increased m6A methylation of transcripts involved in translation, peptide and amine metabolism. This analysis allowed the identification of differentially methylated transcripts and m6A unique sites in the infected cell transcripts. Results here presented indicate that the cell response to viral infection not only changes the levels of mRNAs, as previously shown, but also its epitranscriptional pattern. Also, transcriptome-wide analysis shows strong nucleotide biases in DRACH motifs of cellular transcripts, both in Vero and Calu-3 cells, which use the signature GGACU whereas in viral RNAs the signature is GAACU. We hypothesize that the differences of DRACH motif biases, might force the convergent evolution of the viral genome resulting in better adaptation to target sequence preferences of writer, reader and eraser enzymes. To our knowledge, this is the first report on m6A epitranscriptome of the SARS-CoV-2 infected Vero cells by direct RNA sequencing, which is the sensu stricto RNA-seq.

Direct RNA Sequencing Reveals SARS-CoV-2 m6A Sites and Possible Differential DRACH Motif Methylation among Variants.

The causative agent of COVID-19 pandemic, SARS-CoV-2, has a 29,903 bases positive-sense single-stranded RNA genome. RNAs exhibit about 150 modified bases that are essential for proper function. Among internal modified bases, the N6-methyladenosine, or m6A, is the most frequent, and is implicated in SARS-CoV-2 immune response evasion. Although the SARS-CoV-2 genome is RNA, almost all genomes sequenced thus far are, in fact, reverse transcribed complementary DNAs. This process reduces the true complexity of these viral genomes because the incorporation of dNTPs hides RNA base modifications. Here, we present an initial exploration of Nanopore direct RNA sequencing to assess the m6A residues in the SARS-CoV-2 sequences of ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N, ORF10 and the 3'-untranslated region. We identified fifteen m6A methylated positions, of which, six are in ORF N. Additionally, because m6A is associated with the DRACH motif, we compared its distribution in major SARS-CoV-2 variants. Although DRACH is highly conserved among variants, we show that variants Beta and Eta have a fourth position C > U change in DRACH at 28,884b that could affect methylation. This is the first report of direct RNA sequencing of a Brazilian SARS-CoV-2 sample coupled with the identification of modified bases.

Hardy-Weinberg Equilibrium in different mitochondrial haplogroups of four genes associated with neuroprotection and neurodegeneration.

BACKGROUND: Malfunctioning or damaged mitochondria result in altered energy metabolism, redox equilibrium, and cellular dynamics and is a central point in the pathogenesis of neurological disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease and Amyotrophic Lateral Sclerosis. Therefore, it is of utmost importance to identify mitochondrial genetic susceptibility markers for neurodegenerative diseases. Potential markers include the respiratory chain enzymes Riboflavin kinase (RFK), Flavin adenine dinucleotide synthetase (FAD), Succinate dehydrogenase B subunit (SDHB), and Cytochrome C1 (CYC1). These enzymes are associated with neuroprotection and neurodegeneration. OBJECTIVE: To test if variants in genes RFK, FAD, SDHB and CYC1 deviate from Hardy-Weinberg Equilibrium (HWE) in different human mitochondrial haplogroups. METHODS: Sequence variants in genes RFK, FAD, SDHB and CYC1 of 2,504 non-affected individuals of the 1,000 genomes project were used for mitochondrial haplogroup assessment and HWE calculations in different mitochondrial haplogroups. RESULTS: We show that RFK variants deviate from HWE in haplogroups G, H, L, V and W, variants of FAD in haplogroups B, J, L, U, and C, variants of SDHB in relation to the C, W, and A and CYC1 variants in B, L, U, D, and T. HWE deviation indicates action of selective pressures and genetic drift. CONCLUSIONS: HWE deviation of particular variants in relation to global populational HWE, could be, at least in part, associated with the differential susceptibility of specific populations and ethnicities to neurodegenerative diseases. Our data might contribute to the epidemiology and diagnostic/prognostic methods for neurodegenerative diseases.

Iniquidades nas contribuições das fontes de financiamento da saúde na Pesquisa de Orçamentos Familiares no Brasil / Inequities in the contributions of health financing sources in the Family Budget Survey in Brazi / Inequidades en las contribuciones de las fuentes de financiamiento de la salud en la Encuesta de Presupuestos Familiares en Brasil

Objetivo:analisar a incidência no financiamento (FIA, em inglês), tanto estrutural como efetiva, das fontes de financiamento da saúde, encontradas na Pesquisa de Orçamentos Familiares (POF)de 2008/2009 e a distribuição dessas contribuições na população segundo seu poder de consumo. Metodologia:foram utilizados os dados da Pesquisa de Orçamentos Familiares de 2008/2009 como fonte de dados, de onde foram obtidas as fontes de financiamento (públicas e privadas) do sistema de saúde no Brasil. Essas fontes foram analisadas em referência ao poder de consumo per capitada população para avaliar a progressividade, regressividade ou proporcionalidade delas, mediante o uso de índices de concentração,Gini e Kakwani. Para isso, o estudo usou o pacote estatístico Stata 12e o programa estatístico ADePTdo Banco Mundial.Resultados:os pagamentos diretos em saúde mantiveram uma distribuição regressiva, enquanto os pagamentos com planos de saúde e impostos diretos foram progressivos e proporcionais,respetivamente. A consolidação das três fontes de financiamento foi avaliada como proporcional, por ter um índice de Kakwani de -0,0349. O índice de Gini demonstrou que os gastos feitos com saúde são ainda menos equitativos (0,609) que a distribuição geral dos recursos (0,598) dentre a população. Conclusões: o orçamento público não é suficiente para suprir todas as necessidades do SUS e as contribuições das fontes de financiamento não são progressivas,e essa é uma das possíveis causas do aumento da pobreza e das catástrofes financeiras da população brasileira.

Objective: to analyze the Incidence in Financing (FIA), both structural and effective, from the sources of health financing, found in the Family Budget Survey2008/2009 and the distribution of these contributions in the population according to their power of consumption. Methodology:The data from the Family Budget Survey2008/2009 were used as a data source, from where the sources of financing (public and private) of the health system in Brazil were obtained. These sources were analyzed in reference to the per capita consumption power of the population to assess their progressivity, regressivity or proportionality, by using indicesof concentration,Gini and Kakwani. For this, the study used the Stata 12and ADePTstatistical programs of the World Bank. Results:direct payments in health maintained a regressive distribution, while payments with health insurance and direct taxes were progressive and proportional respectively. The consolidation of the three financing sources was determined as proportional by having a Kakwani index of -0.0349. TheGini index showed that health expenditures are even less equitable than the general distribution of resources among the population (0.598). Conclusions:the public budget is not sufficient to meet all the needs of the Unified Health System and the contributions of the financing sources are not progressive and this may be one of the causes of the increase in poverty and financial catastrophes of the Brazilian population.

Objetivo: analizar la Incidencia en el Financiamiento (FIA, en inglés), tanto estructural como efectivo, de las fuentes de financiamiento de la salud, encontradas en la Encuesta de PresupuestosFamiliaresde 2008/2009 y la distribución de esas contribuciones en la población según su poder de consumo. Metodología: fueron usados los datos de la Encuesta de Presupuestos Familiaresde 2008/2009 como fuente de datos, dondese obtuvo las fuentes de financiamiento (públicas y privadas) del sistema de salud en Brasil. Esas fuentes fueron analizadas en referencia al poder de consumo per cápita de la población para evaluar la progresividad, regresividad o proporcionalidad de estas, mediante el uso de índices de concentración,Gini y Kakwani. Para esto, el estudio utilizó los programas estadísticos Stata 12y ADePTdel Banco Mundial. Resultados: los pagos directos en salud mantuvieron una distribución regresiva, mientras que los pagos con seguros de salud e impuestos directos resultaron ser progresivos y proporcionales respectivamente. La consolidación de las tres fuentes de financiamiento fue determinada como proporcional por tener un índice de Kakwani de -0.0349. Elíndice de Gini demostró que los gastos en salud son aún menos equitativos que la distribución general de los recursos entre la población (0.598).Conclusiones: el presupuesto público no es suficiente para suplir todas las necesidades del Sistema Único de Salud y las contribuciones de las fuentes de financiamiento no son progresivas y esto puede ser una de las causas del aumento de la pobreza y de catástrofes financieras de la población brasileña.

Hardy-Weinberg Equilibrium in different mitochondrial haplogroups of four genes associated with neuroprotection and neurodegeneration / Equilíbrio de Hardy-Weinberg em diferentes haplogrupos mitocondriais de quatro genes associados à neuroproteção e neurodegeneração

ABSTRACT Background: Malfunctioning or damaged mitochondria result in altered energy metabolism, redox equilibrium, and cellular dynamics and is a central point in the pathogenesis of neurological disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease and Amyotrophic Lateral Sclerosis. Therefore, it is of utmost importance to identify mitochondrial genetic susceptibility markers for neurodegenerative diseases. Potential markers include the respiratory chain enzymes Riboflavin kinase (RFK), Flavin adenine dinucleotide synthetase (FAD), Succinate dehydrogenase B subunit (SDHB), and Cytochrome C1 (CYC1). These enzymes are associated with neuroprotection and neurodegeneration. Objective: To test if variants in genes RFK, FAD, SDHB and CYC1 deviate from Hardy-Weinberg Equilibrium (HWE) in different human mitochondrial haplogroups. Methods: Sequence variants in genes RFK, FAD, SDHB and CYC1 of 2,504 non-affected individuals of the 1,000 genomes project were used for mitochondrial haplogroup assessment and HWE calculations in different mitochondrial haplogroups. Results: We show that RFK variants deviate from HWE in haplogroups G, H, L, V and W, variants of FAD in haplogroups B, J, L, U, and C, variants of SDHB in relation to the C, W, and A and CYC1 variants in B, L, U, D, and T. HWE deviation indicates action of selective pressures and genetic drift. Conclusions: HWE deviation of particular variants in relation to global populational HWE, could be, at least in part, associated with the differential susceptibility of specific populations and ethnicities to neurodegenerative diseases. Our data might contribute to the epidemiology and diagnostic/prognostic methods for neurodegenerative diseases.

RESUMO Introdução: Mitocôndrias defeituosas ou danificadas resultam em alterações do metabolismo energético, equilíbrio redox e dinâmica celular e são, portanto, identificadas como o ponto central da patogênese em muitos distúrbios neurológicos, como a doença de Alzheimer, a doença de Parkinson, a doença de Huntington e a Esclerose Lateral Amiotrófica. Portanto, é de fundamental importância identificar marcadores de susceptibilidade genética mitocondrial para doenças neurodegenerativas. Entre os potenciais marcadores relevantes estão as enzimas da cadeia respiratória riboflavina quinase (RFK), flavina adenina dinucleotídeo sintetase (FAD), succinato desidrogenase subunidade B (SDHB) e citocromo C1 (CYC1). Estas enzimas estão associadas à neuroproteção e à neurodegeneração. Objetivo: Testar se variantes nas sequências dos genes RFK, FAD, SDHB e CYC1 desviam do Equilíbrio de Hardy-Weinberg (HWE) em diferentes haplogrupos mitocondriais humanos. Métodos: Neste trabalho utilizamos os variantes nos genes RFK, FAD, SDHB e CYC1 de sequências de 2.504 indivíduos não afetados do projeto de 1.000 genomas para o cálculo dos valores de HWE em diferentes haplogrupos mitocondriais. Resultados: As variantes de RFK desviam de HWE nos haplogrupos G, H, L, V e W, variantes de FAD nos haplogrupos B, J, L, U e C, variantes de SDHB em relação às variantes C, W e A e CYC1 em B, L, U, D e T. O desvio de HWE indica a ação de pressões seletivas e desvio genético. Conclusões: O desvio do HWE de variantes particulares em relação ao HWE populacional global poderia estar, pelo menos em parte, associado à suscetibilidade diferencial de populações e etnias específicas a doenças neurodegenerativas. Nossos dados podem contribuir para a epidemiologia e métodos diagnósticos/prognósticos para doenças neurodegenerativas.

Aumento de la pobreza e inequidad en el financiamiento del sistema de salud de Ecuador / Increase in the poverty and inequity in the funding of the Ecuadorian health system

RESUMEN Objetivo El objetivo de este estudio es medir el aumento de la pobreza debido a los gastos directos en salud y analizar la equidad del financiamiento del sistema de salud ecuatoriano, con base en datos de encuestas nacionales representativas del país. Método Fue realizado con datos de la "Encuesta de condiciones de vida 2013-2014" (ECV) y utilizó líneas de pobreza, con enfoques relativo y absoluto para medir el aumento de la pobreza y, mediante Análisis de Incidencia en el financiamiento, fueron medidas las desigualdades en la distribución del financiamiento. Resultados La pobreza aumentó 2,2% debido al gasto de las familias en salud, especialmente en gastos de medicamentos y consultas médicas, que representaron 36,7% y 14,6% del gasto total en pagos directos. Además, las fuentes más importantes de financiamiento resultaron ser regresivas, hecho que afecta principalmente a la clase media. Cuando fueron consolidadas, las fuentes de financiamiento analizadas resultaron ser proporcionales. Este, aunque no es el peor escenario, no es el esperado para un sistema de salud que debe garantizar protección financiera a sus usuarios. Discusión Aunque existen metas de financiamiento de difícil alcance, al menos las leyes del país establecen la búsqueda de ese fin. Sin embargo, pérdidas de recursos financieros dificultan el logro de los objetivos trazados.(AU)

ABSTRACT Objective The objective of this study is to measure the increase in poverty due to direct health expenditures and to analyze the equity of financing of the Ecuadorian health system, based on data from representative national surveys of the country. Method It was carried out with data from the 2013-2014 Living Conditions Survey and used poverty lines, with relative and absolute approaches to measure the increase in poverty and, through Analysis of Incidence in Financing, inequalities in the financing distribution. Results Poverty increased 2.2% due to families spending on health, especially on expenses for medications and medical consultations, which represented 36.7% and 14.6% of total expenditure on direct payments. Furthermore, the most important sources of financing turned out to be regressive, mainly affecting the middle class. When they were consolidated, the sources of financing analyzed turned out to be proportional that, although it is not the worst-case scenario, it is not the expected one for a health system that must guarantee financial protection to its users. Discussion Although there are financing goals that are difficult to achieve, at least the country's laws establish the pursuit of this goal. However, losses of financial resources make it difficult to achieve the objectives set.(AU)

[Increase in the poverty and inequity in the funding of the Ecuadorian health system]. / Aumento de la pobreza e inequidad en el financiamiento del sistema de salud de Ecuador.

OBJECTIVE: The objective of this study is to measure the increase in poverty due to direct health expenditures and to analyze the equity of financing of the Ecuadorian health system, based on data from representative national surveys of the country. METHOD: It was carried out with data from the 2013-2014 Living Conditions Survey and used poverty lines, with relative and absolute approaches to measure the increase in poverty and, through Analysis of Incidence in Financing, inequalities in the financing distribution. RESULTS: Poverty increased 2.2% due to families spending on health, especially on expenses for medications and medical consultations, which represented 36.7% and 14.6% of total expenditure on direct payments. Furthermore, the most important sources of financing turned out to be regressive, mainly affecting the middle class. When they were consolidated, the sources of financing analyzed turned out to be proportional that, although it is not the worst-case scenario, it is not the expected one for a health system that must guarantee financial protection to its users. DISCUSSION: Although there are financing goals that are difficult to achieve, at least the country's laws establish the pursuit of this goal. However, losses of financial resources make it difficult to achieve the objectives set.

OBJETIVO: El objetivo de este estudio es medir el aumento de la pobreza debido a los gastos directos en salud y analizar la equidad del financiamiento del sistema de salud ecuatoriano, con base en datos de encuestas nacionales representativas del país. MÉTODO: Fue realizado con datos de la "Encuesta de condiciones de vida 2013-2014" (ECV) y utilizó líneas de pobreza, con enfoques relativo y absoluto para medir el aumento de la pobreza y, mediante Análisis de Incidencia en el financiamiento, fueron medidas las desigualdades en la distribución del financiamiento. RESULTADOS: La pobreza aumentó 2,2% debido al gasto de las familias en salud, especialmente en gastos de medicamentos y consultas médicas, que representaron 36,7% y 14,6% del gasto total en pagos directos. Además, las fuentes más importantes de financiamiento resultaron ser regresivas, hecho que afecta principalmente a la clase media. Cuando fueron consolidadas, las fuentes de financiamiento analizadas resultaron ser proporcionales. Este, aunque no es el peor escenario, no es el esperado para un sistema de salud que debe garantizar protección financiera a sus usuarios. DISCUSIÓN: Aunque existen metas de financiamiento de difícil alcance, al menos las leyes del país establecen la búsqueda de ese fin. Sin embargo, pérdidas de recursos financieros dificultan el logro de los objetivos trazados.

Prolonged growth of Candida albicans reveals co-isolated bacteria from single yeast colonies.

Bacterial species are associated with Candida albicans in at least 25% of patients with bloodstream infection (Candidemia). These polymicrobial infections are usually caused by coagulase-negative staphylococci, most commonly Staphylococcus epidermidis and are associated with significantly worse clinical outcomes as compared to monomicrobial infections. Here we show that bacteria are present in C. albicans cultures started from isolated single colonies. These bacteria can only be detected by the use of specific media, and prolonged incubation periods of at least 8 days. The detection of these bacteria is sensitive to the polymerase enzyme used for 16S rDNA gene amplification and is often missed in clinical laboratory analysis because of short incubation periods, media and temperatures, used in mycology clinical routine, that are unfavorable for bacterial growth. We identified bacteria in cultures of different C. albicans isolates in long-term, continuous growth by molecular analysis and microscopy. Also, we confirmed the presence of these bacteria by identification of S. epidermidis genome segments in sequencing reads of the C. albicans reference strain SC5314 genome sequencing project raw data deposited in GenBank. Our results show that the presence of associated bacteria correlates with antifungal resistance alterations observed in growth under hypoxia. Our findings reveal the intense interaction between C. albicans yeasts and bacteria and have direct implications in yeast clinical procedures, especially concerning patient treatment.

Genome Sequence of a Staphylococcus epidermidis Strain (GTH12) Associated with Candida albicans SC5314 Cultured under Hypoxia at 37°C in Glycerol for 12 Weeks.

Polymicrobial infections with mixed-species biofilms are important health problems because of increased antimicrobial resistance and worse patient outcomes than with monomicrobial infections. Here, we present the whole-genome sequence of Staphylococcus epidermidis strain GTH12, which was cocultured with the yeast Candida albicans SC5314 (generating C. albicans strain SC5314 GTH12), thus providing genomic information on polymicrobial infections.

Evidence for Mitochondrial Genome Methylation in the Yeast Candida albicans: A Potential Novel Epigenetic Mechanism Affecting Adaptation and Pathogenicity?

The commensal yeast Candida albicans is an opportunistic pathogen. In order to successfully colonize or infect the human body, the fungus must adapt to the host's environmental conditions, such as low oxygen tension (hypoxia), temperature (37°C), and the different carbon sources available. Previous studies demonstrated the adaptive importance of C. albicans genetic variability for its pathogenicity, although the contributions of epigenetic and the influence of environmental factors are not fully understood. Mitochondria play important roles in fungal energetic metabolism, regulation of nuclear epigenetic mechanisms and pathogenicity. However, the specific impact of inter-strain mitochondrial genome variability and mitochondrial epigenetics in pathogenicity is unclear. Here, we draw attention to this relevant organelle and its potential role in C. albicans pathogenicity and provide preliminary evidence, for the first time, for methylation of the yeast mitochondrial genome. Our results indicate that environmental conditions, such as continuous exposure for 12 weeks to hypoxia and 37°C, decrease the mitochondrial genome methylation in strains SC5314 and L757. However, the methylation decrease is quantitatively different in specific genome positions when strains SC5314 and L757 are compared. We hypothesize that this phenomenon can be promising for future research to understand how physical factors of the host affect the C. albicans mitochondrial genome and its possible impact on adaptation and pathogenicity.

A Possible Role for Platelet-Activating Factor Receptor in Amyotrophic Lateral Sclerosis Treatment.

Amyotrophic lateral sclerosis (ALS) is the third most prevalent neurodegenerative disease affecting upper and lower motor neurons. An important pathway that may lead to motor neuron degeneration is neuroinflammation. Cerebrospinal Fluids of ALS patients have increased levels of the inflammatory cytokine IL-18. Because IL-18 is produced by dendritic cells stimulated by the platelet-activating factor (PAF), a major neuroinflammatory mediator, it is expected that PAF is involved in ALS. Here we show pilot experimental data on amplification of PAF receptor (PAFR) mRNA by RT-PCR. PAFR is overexpressed, as compared to age matched controls, in the spinal cords of transgenic ALS SOD1-G93A mice, suggesting PAF mediation. Although anti-inflammatory drugs have been tested for ALS before, no clinical trial has been conducted using PAFR specific inhibitors. Therefore, we hypothesize that administration of PAFR inhibitors, such as Ginkgolide B, PCA 4248 and WEB 2086, have potential to function as a novel therapy for ALS, particularly in SOD1 familial ALS forms. Because currently there are only two approved drugs with modest effectiveness for ALS therapy, a search for novel drugs and targets is essential.

Whole-Genome Sequences and Annotation of the Opportunistic Pathogen Candida albicans Strain SC5314 Grown under Two Different Environmental Conditions.

The genetic variability of the opportunistic pathogen Candida albicans is an important adaptive mechanism. Here, we present the whole-genome sequences of the C. albicans SC5314 strain under two different growth conditions, providing useful information for comparative genomic studies and further intraspecific analysis.

A Kolmogorov-Smirnov test for the molecular clock based on Bayesian ensembles of phylogenies.

Divergence date estimates are central to understand evolutionary processes and depend, in the case of molecular phylogenies, on tests of molecular clocks. Here we propose two non-parametric tests of strict and relaxed molecular clocks built upon a framework that uses the empirical cumulative distribution (ECD) of branch lengths obtained from an ensemble of Bayesian trees and well known non-parametric (one-sample and two-sample) Kolmogorov-Smirnov (KS) goodness-of-fit test. In the strict clock case, the method consists in using the one-sample Kolmogorov-Smirnov (KS) test to directly test if the phylogeny is clock-like, in other words, if it follows a Poisson law. The ECD is computed from the discretized branch lengths and the parameter λ of the expected Poisson distribution is calculated as the average branch length over the ensemble of trees. To compensate for the auto-correlation in the ensemble of trees and pseudo-replication we take advantage of thinning and effective sample size, two features provided by Bayesian inference MCMC samplers. Finally, it is observed that tree topologies with very long or very short branches lead to Poisson mixtures and in this case we propose the use of the two-sample KS test with samples from two continuous branch length distributions, one obtained from an ensemble of clock-constrained trees and the other from an ensemble of unconstrained trees. Moreover, in this second form the test can also be applied to test for relaxed clock models. The use of a statistically equivalent ensemble of phylogenies to obtain the branch lengths ECD, instead of one consensus tree, yields considerable reduction of the effects of small sample size and provides a gain of power.

Histone Deacetylase 1 Is Essential for Rod Photoreceptor Differentiation by Regulating Acetylation at Histone H3 Lysine 9 and Histone H4 Lysine 12 in the Mouse Retina.

Histone acetylation has a regulatory role in gene expression and is necessary for proper tissue development. To investigate the specific roles of histone deacetylases (HDACs) in rod differentiation in neonatal mouse retinas, we used a pharmacological approach that showed that inhibition of class I but not class IIa HDACs caused the same phenotypic changes seen with broad spectrum HDAC inhibitors, most notably a block in the differentiation of rod photoreceptors. Inhibition of HDAC1 resulted in increase of acetylation of lysine 9 of histone 3 (H3K9) and lysine 12 of histone 4 (H4K12) but not lysine 27 of histone 3 (H3K27) and led to maintained expression of progenitor-specific genes such as Vsx2 and Hes1 with concomitant block of expression of rod-specific genes. ChiP experiments confirmed these changes in the promoters of a group of progenitor genes. Based on our results, we suggest that HDAC1-specific inhibition prevents progenitor cells of the retina from exiting the cell cycle and differentiating. HDAC1 may be an essential epigenetic regulator of the transition from progenitor cells to terminally differentiated photoreceptors.

A model of gene expression based on random dynamical systems reveals modularity properties of gene regulatory networks.

Here we propose a new approach to modeling gene expression based on the theory of random dynamical systems (RDS) that provides a general coupling prescription between the nodes of any given regulatory network given the dynamics of each node is modeled by a RDS. The main virtues of this approach are the following: (i) it provides a natural way to obtain arbitrarily large networks by coupling together simple basic pieces, thus revealing the modularity of regulatory networks; (ii) the assumptions about the stochastic processes used in the modeling are fairly general, in the sense that the only requirement is stationarity; (iii) there is a well developed mathematical theory, which is a blend of smooth dynamical systems theory, ergodic theory and stochastic analysis that allows one to extract relevant dynamical and statistical information without solving the system; (iv) one may obtain the classical rate equations form the corresponding stochastic version by averaging the dynamic random variables (small noise limit). It is important to emphasize that unlike the deterministic case, where coupling two equations is a trivial matter, coupling two RDS is non-trivial, specially in our case, where the coupling is performed between a state variable of one gene and the switching stochastic process of another gene and, hence, it is not a priori true that the resulting coupled system will satisfy the definition of a random dynamical system. We shall provide the necessary arguments that ensure that our coupling prescription does indeed furnish a coupled regulatory network of random dynamical systems. Finally, the fact that classical rate equations are the small noise limit of our stochastic model ensures that any validation or prediction made on the basis of the classical theory is also a validation or prediction of our model. We illustrate our framework with some simple examples of single-gene system and network motifs.

MIA: Mutual Information Analyzer, a graphic user interface program that calculates entropy, vertical and horizontal mutual information of molecular sequence sets.

BACKGROUND: Short and long range correlations in biological sequences are central in genomic studies of covariation. These correlations can be studied using mutual information because it measures the amount of information one random variable contains about the other. Here we present MIA (Mutual Information Analyzer) a user friendly graphic interface pipeline that calculates spectra of vertical entropy (VH), vertical mutual information (VMI) and horizontal mutual information (HMI), since currently there is no user friendly integrated platform that in a single package perform all these calculations. MIA also calculates Jensen-Shannon Divergence (JSD) between pair of different species spectra, herein called informational distances. Thus, the resulting distance matrices can be presented by distance histograms and informational dendrograms, giving support to discrimination of closely related species. RESULTS: In order to test MIA we analyzed sequences from Drosophila Adh locus, because the taxonomy and evolutionary patterns of different Drosophila species are well established and the gene Adh is extensively studied. The search retrieved 959 sequences of 291 species. From the total, 450 sequences of 17 species were selected. With this dataset MIA performed all tasks in less than three hours: gathering, storing and aligning fasta files; calculating VH, VMI and HMI spectra; and calculating JSD between pair of different species spectra. For each task MIA saved tables and graphics in the local disk, easily accessible for future analysis. CONCLUSIONS: Our tests revealed that the "informational model free" spectra may represent species signatures. Since JSD applied to Horizontal Mutual Information spectra resulted in statistically significant distances between species, we could calculate respective hierarchical clusters, herein called Informational Dendrograms (ID). When compared to phylogenetic trees all Informational Dendrograms presented similar taxonomy and species clusterization.

Genetic diversity of medically important and emerging Candida species causing invasive infection.

BACKGROUND: Genetic variation in the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has been studied among fungi. However, the numbers of ITS sequence polymorphisms in the various Candida species and their associations with sources of invasive fungal infections remain poorly investigated. Here, we characterized the intraspecific and interspecific ITS diversity of Candida spp. strains collected from patients with bloodstream or oroesophageal candidiasis. METHODS: We selected cultures of representative medically important species of Candida as well as some rare and emerging pathogens. Identification was performed by micromorphology and by biochemical testing using an ID32C system, as well as by the sequencing of rDNA ITS. The presence of intraspecific ITS polymorphisms was characterized based on haplotype networks, and interspecific diversity was characterized based on Bayesian phylogenetic analysis. RESULTS: Among 300 Candida strains, we identified 76 C. albicans, 14 C. dubliniensis, 40 C. tropicalis, 47 C. glabrata, 34 C. parapsilosis (sensu stricto), 31 C. orthopsilosis, 3 C. metapsilosis, 21 Meyerozyma guilliermondii (C. guilliermondii), 12 Pichia kudriavzevii (C. krusei), 6 Clavispora lusitaniae (C. lusitaniae), 3 C. intermedia, 6 Wickerhamomyces anomalus (C. pelliculosa), and 2 C. haemulonii strains, and 1 C. duobushaemulonii, 1 Kluyveromyces marxianus (C. kefyr), 1 Meyerozyma caribbica (C. fermentati), 1 Pichia norvegensis (C. norvegensis), and 1 Lodderomyces elongisporus strain. Out of a total of seven isolates with inconsistent ID32C profiles, ITS sequencing identified one C. lusitaniae strain, three C. intermedia strains, two C. haemulonii strains and one C. duobushaemulonii strain. Analysis of ITS variability revealed a greater number of haplotypes among C. albicans, C. tropicalis, C. glabrata and C. lusitaniae, which are predominantly related to endogenous sources of acquisition. Bayesian analysis confirmed the major phylogenetic relationships among the isolates and the molecular identification of the different Candida spp. CONCLUSIONS: Molecular studies based on ITS sequencing are necessary to identify closely related and emerging species. Polymorphism analysis of the ITS rDNA region demonstrated its utility as a genetic marker for species identification and phylogenetic relationships as well as for drawing inferences concerning the natural history of hematogenous infections caused by medically important and emerging Candida species.