In silico identification and analysis of Leishmania ncRNAs using RNA-seq data

Análises de dados gerados em larga escala revelaram que os transcriptomas são mais extensos e complexos do que inferido previamente. Hoje é evidente que a maioria dos genomas de eucariotos são quase inteiramente transcritos e sob regulação atrelada a estágios de desenvolvimento. O controle da expressão gênica envolve RNAs não codificadores (ncRNAs) regulatórios, inclusive em processos pós-transcricionais. A regulação de expressão gênica em nível pós-transcricional é crucial em diferentes organismos, mas é particularmente central nos tripanossomatídeos. Os parasitos do gênero Leishmania (Ordem Kinetoplastidae, família Trypanosomatidae) provocam doenças infecto-parasitárias conhecidas como leishmanioses e em seu ciclo de vida apresenta-se sob três formas principais de desenvolvimento: promastigotas procíclicos, promastigotas metacíclicos e os amastigotas. Este trabalho teve como objetivo identificar e caracterizar computacionalmente ncRNAs putativos de Leishmania em diferentes estágios de desenvolvimento. A associação de abordagens em larga escala e ferramentas de bioinformática possibilitaram a identificação de 11.376 ncRNAs putativos em L. braziliensis e, em um estudo preliminar, de 37 ncRNAs putativos em L. donovani. Adicionalmente, o transcriptoma de L. braziliensis foi analisado comparativamente entre os três estágios de desenvolvimento do parasito. Em L. donovani, dos 37 ncRNAs putativos identificados, 34 estavam em UTRs (Untranslated Regions) e 3 em regiões intergênicas. Preditores de características específicas de ncRNAs foram utilizados e 32 ncRNAs putativos tiveram pelo menos uma predição positiva. Todos os candidatos estão conservados inteira ou parcialmente em pelo menos 3 espécies de Leishmania. Cinco ncRNAs de L. donovani foram confirmados por experimentos de Northern blotting. A análise do transcriptoma de L. braziliensis revelou uma diferença de expressão gênica entre os estágios de desenvolvimento que variou entre 52% e 71%, dependendo dos estágios comparados. Também foram definidos os limites das 5UTRs e 3UTRs de 81% e 38% das CDSs anotadas, respectivamente. Propusemos uma metodologia para identificação de ncRNAs putativos utilizando dados de sequenciamento de RNA-total. Essa metodologia identificou 11.376 ncRNAs putativos em L. braziliensis, sendo que todos os candidatos foram analisados por programas preditores de características específicas de ncRNAs e apresentaram pelo menos uma predição positiva, além de não possuírem semelhança com domínios proteicos conhecidos. A análise de conservação demonstrou que de 27% a 41% dos ncRNAs putativos identificados são conservados em outras espécies de Leishmania. Foram encontrados de 27% a 38% de ncRNAs putativos com regulação atrelada ao estágio de desenvolvimento, dependendo dos estágios comparados. Assim, além da identificação e descrição de ncRNAs em Leishmania foram encontrados candidatos com regulação atrelada ao desenvolvimento e padrões foram descortinados com o processo de análise proposto e executado. Portanto, esse trabalho contribui significativamente para ampliar a compreensão dos processos de regulação de expressão gênica em Leishmania e oferecerá à comunidade um conjunto grande e importante de informações sobre a organização genética do parasito, diferenças genéticas e regulatórias ao longo do desenvolvimento, além de informações do transcriptoma de forma global.High-throughput data analyses indicated that transcriptomes are more extensive and complex than previously supposed. Currently, it is evident that most eukaryotic genomes are almost entirely transcribed and under regulation tied to developmental stages. Control of gene expression involves regulatory non-coding RNAs (ncRNAs), including post-transcriptional processes. Post-transcriptional regulation is particularly relevant for the regulation of gene expression in trypanosomatids, as compared to other organisms. Parasites of the genus Leishmania (Order Kinetoplastidae, family Trypanosomatidae) causes infectious-parasitic diseases known as leishmaniasis, and their life cycle comprises three development stages: procyclic promastigotes, metacyclic promastigotes and amastigotes. This study aimed to computationally identify and characterize Leishmania putative ncRNAs at different stages of development. Large-scale approaches combined with bioinformatics tools allowed the identification of 11,376 putative ncRNAs in L. braziliensis and, in a preliminary study, of 37 putative ncRNAs in L. donovani. In addition, the L. braziliensis the complete transcriptome was analyzed comparatively between the parasite development stages. In L. donovani, of the 37 putative ncRNAs identified, 34 were in UTRs (Untranslated Regions) and 3 in intergenic regions. Predictors of ncRNAs specific characteristics were used and 32 putative ncRNAs had at least one positive prediction. All candidates are conserved, partially or entirely, in at least three Leishmania species. Five L. donovani ncRNAs were confirmed by Northern blotting experiments. Analysis of the L. braziliensis transcriptome revealed differences in gene expression levels between developmental stages, ranging from 52% to 71%, depending on the compared stages. The boundaries of the 5\'UTRs and 3\'UTRs were also defined for 81% and 38% of the annotated CDSs, respectively. We developed a methodology for the identification of putative ncRNAs using total RNA sequencing data. This methodology allowed the identification of 11,376 putative ncRNAs in L. braziliensis, and all candidates were analyzed by predictive programs of ncRNAs specific characteristics and presented at least one positive prediction, in addition to bearing no similarity to known protein domains. The analysis showed that from 27% to 41% of the putative ncRNAs identified are conserved in other Leishmania species. We found 27% to 38% of putative ncRNAs with regulation associated to the developmental stage, depending on the compared stages. Consequently, besides identification and characterization of ncRNAs in Leishmania, candidates with developmental-related regulation were found and patterns were uncovered with the proposed and implemented analysis. Thus, this work contributes significantly to improve understanding of gene expression regulation processes in Leishmania and will offer to the community important information about the parasite genetics and regulatory differences along the development, besides of L. braziliensis transcriptome information

Computational analysis based on developing a pipeline of techniques for ab initio prediction of protein structural disorder in the genomes of trypanosomatids

Submitted by Repositório Arca ([email protected]) on 2019-05-07T13:26:26Z No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) patricia_cassia.pdf: 1282766 bytes, checksum: 4df9ecafbd6e40b34efc75f44fb14392 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2019-07-31T16:06:13Z (GMT) No. of bitstreams: 2 patricia_cassia.pdf: 1282766 bytes, checksum: 4df9ecafbd6e40b34efc75f44fb14392 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-07-31T16:06:13Z (GMT). No. of bitstreams: 2 patricia_cassia.pdf: 1282766 bytes, checksum: 4df9ecafbd6e40b34efc75f44fb14392 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011Fundação Oswaldo Cruz. Centro de Pesquisa Rene Rachou. Belo Horizonte, MG, Brasil.Proteínas são compostas por uma ou mais cadeias de aminoácidos e exibem vários níveis de organização estrutural. Recentemente, uma classe de proteínas conhecidas como IUPs (Intrinsically Unstructured Proteins) foi descoberta e sua principal característica é a ausência de estrutura parcial ou total em seu estado nativo. Devido à sua adaptabilidade intrínseca, tais proteínas participam em muitos processos biológicos regulatórios incluindo o escape do sistema imune. Utilizando a informação contida no proteoma predito de Leishmania braziliensis, Leishmania major, Leishmania infantum, Trypanosoma cruzi e Trypanosoma brucei, desenvolvemos um pipeline de análise computacional que tem como objetivo a identificação, caracterização e análise de IUPs. Nosso principal objetivo é investigar as correlações biológicas entre desordem estrutural e as interações parasitohospedeiro. O pipeline emprega 6 metodologias de predição de desordem, integra informações obtidas através da anotação estrutural e funcional, predição subcelular e o cálculo de propriedades físico-químicas. Como cerne do pipeline de IUPs existe um banco de dados relacional modelado de forma a viabilizar a extração das relações entre as inúmeras variáveis analisadas e gerar os relatórios. Nossos resultados demonstram que as espécies de Leishmania e Trypanosoma possuem aproximadamente 70% e 55% de IUPs, respectivamente. Nossos resultados indicam que nos tripanosomatídeos os aminoácidos promotores de ordem são: W, Y, F, V, I, L e C e os promotores de desordem são P, Q, E, R e S. A anotação funcional revelou o enriquecimento dos termos GO: transcription, ribonucleoproteins, RNA metabolic process, protein binding e ribonucleotide binding. Outra característica é a associação entre o aumento da porcentagem de resíduos desordenados, a localização subcelular e o número de regiões transmembrana. Como resultado da validação experimental feita através de técnicas de eletroforese bidimensional (2D) específicas para identificação de IUPs, 100% dos spots identificados foram preditos in silico. Até o presente momento este trabalho representa a primeira tentativa de estabelecimento das correlações entre desordem estrutural protéica e função nos tripanosomatídeos. A execução do pipeline pode ser solicitada por instituições acadêmicas através de solicitação no sitio http://iup.cpqrr.fiocruz.br/iup-pipeline.Proteins are composed of one or more chains of amino acids, and exhibit several levels of structure. Recently, a class of proteins called IUPs (Intrinsically Unstructured Proteins) has been discovered that do not fold into any particular configuration existing as dynamic ensembles in their native state. Due to their intrinsic adaptability, they participate in many regulatory biological processes including parasite immune escape. Using the information from Leishmania braziliensis, Leishmania major, Leishmania infantum, Trypanosoma cruzi and Trypanosoma brucei proteomes we developed a pipeline aiming the identification, characterization and analysis of IUPs, our main goal is to establish the biological correlations between protein structural disorder and host-parasite interactions. The pipeline employs 6 methodologies of disorder prediction, integrates information obtained through the structural and functional annotation, subcellular prediction and physicochemical properties. As the core of the IUP pipeline there is a relational database modeled to enable the extraction of relations between the numerous variables and generate reports. The results demonstrate that Leishmania and Trypanosoma species has approximately 70% and 55% of IUPs respectively. Our results indicate that in tripanosomatides IUPs have disorder promoter amino acids (P, Q, E, R and S) and order promoter amino acids (W, Y, F, V, I, L and C). The functional annotation pointed the enrichment of the following GO terms: transcription, ribonucleoproteins, RNA metabolic process, protein binding and ribonucleotide binding, among others. Another characteristic is the association between the increase of disordered residues percent with the nuclear subcelullar localization and the lack of transmembrane regions. We made an experimental validation through 2D electrophoresis that identifies IUPs and our results revealed that 100% of the identified spots were predicted in silico. Since there is no pipeline or databases addressing this issue this IUP pipeline represents the first attempt to establish the correlations between protein function and structural disorder. The execution of pipeline can be requested by academic institutions at http://iup.cpqrr.fiocruz.br/iup-pipeline

Assessing the efficiency of multiple sequence alignment programs.

Submitted by Nuzia Santos ([email protected]) on 2015-02-04T12:24:24Z No. of bitstreams: 1 2014_035.pdf: 516742 bytes, checksum: 2fa775121df1fd4b23c2255d7c7684fc (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2015-02-04T12:24:34Z (GMT) No. of bitstreams: 1 2014_035.pdf: 516742 bytes, checksum: 2fa775121df1fd4b23c2255d7c7684fc (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2015-02-04T12:30:02Z (GMT) No. of bitstreams: 1 2014_035.pdf: 516742 bytes, checksum: 2fa775121df1fd4b23c2255d7c7684fc (MD5)Made available in DSpace on 2015-02-04T12:30:02Z (GMT). No. of bitstreams: 1 2014_035.pdf: 516742 bytes, checksum: 2fa775121df1fd4b23c2255d7c7684fc (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Centro de Excelência em Bioinformática. Belo Horizonte, MG, Brazil/Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou Grupo de Genômica Biologia Computacional. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Centro de Excelência em Bioinformática. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Centro de Excelência em Bioinformática. Belo Horizonte, MG, Brazil/Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou Grupo de Genômica Biologia Computacional. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Centro de Excelência em Bioinformática. Belo Horizonte, MG, Brazil/Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou Grupo de Genômica Biologia Computacional. Belo Horizonte, MG, BrazilBACKGROUND: Multiple sequence alignment (MSA) is an extremely useful tool for molecular and evolutionary biology and there are several programs and algorithms available for this purpose. Although previous studies have compared the alignment accuracy of different MSA programs, their computational time and memory usage have not been systematically evaluated. Given the unprecedented amount of data produced by next generation deep sequencing platforms, and increasing demand for large-scale data analysis, it is imperative to optimize the application of software. Therefore, a balance between alignment accuracy and computational cost has become a critical indicator of the most suitable MSA program. We compared both accuracy and cost of nine popular MSA programs, namely CLUSTALW, CLUSTAL OMEGA, DIALIGN-TX, MAFFT, MUSCLE, POA, Probalign, Probcons and T-Coffee, against the benchmark alignment dataset BAliBASE and discuss the relevance of some implementations embedded in each program's algorithm. Accuracy of alignment was calculated with the two standard scoring functions provided by BAliBASE, the sum-of-pairs and total-column scores, and computational costs were determined by collecting peak memory usage and time of execution. RESULTS: Our results indicate that mostly the consistency-based programs Probcons, T-Coffee, Probalign and MAFFT outperformed the other programs in accuracy. Whenever sequences with large N/C terminal extensions were present in the BAliBASE suite, Probalign, MAFFT and also CLUSTAL OMEGA outperformed Probcons and T-Coffee. The drawback of these programs is that they are more memory-greedy and slower than POA, CLUSTALW, DIALIGN-TX, and MUSCLE. CLUSTALW and MUSCLE were the fastest programs, being CLUSTALW the least RAM memory demanding program. CONCLUSIONS: Based on the results presented herein, all four programs Probcons, T-Coffee, Probalign and MAFFT are well recommended for better accuracy of multiple sequence alignments. T-Coffee and recent versions of MAFFT can deliver faster and reliable alignments, which are specially suited for larger datasets than those encountered in the BAliBASE suite, if multi-core computers are available. In fact, parallelization of alignme

Adverse birth outcomes associated with Zika virus exposure during pregnancy in São José do Rio Preto, Brazil

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2017-11-17T12:41:59Z No. of bitstreams: 1 Nogueira ML (PREPRINT) Adverse birth outcomes associated with Zika virus exposure during pregnancy in São....pdf: 2093267 bytes, checksum: 6543c2d2fad9c94caa4b20a635a1eab9 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2017-11-17T12:43:36Z (GMT) No. of bitstreams: 1 Nogueira ML (PREPRINT) Adverse birth outcomes associated with Zika virus exposure during pregnancy in São....pdf: 2093267 bytes, checksum: 6543c2d2fad9c94caa4b20a635a1eab9 (MD5)Made available in DSpace on 2017-11-17T12:43:36Z (GMT). No. of bitstreams: 1 Nogueira ML (PREPRINT) Adverse birth outcomes associated with Zika virus exposure during pregnancy in São....pdf: 2093267 bytes, checksum: 6543c2d2fad9c94caa4b20a635a1eab9 (MD5) Previous issue date: 2017The São Paulo Research Foundation (FAPESP) via Grant 262 No. 2013/21719-3 and 2016/15021-1for M.L.N, Grant No. 2015/12295-0 for A.C.B.T., and Grant 263 No. 2016/05115-9 for L.C.M. P.F.C.V. was supported by the Zika Virus Fast Track program pro264 vided by the Association for the Improvement of Higher Education Personnel (CAPES) and the 265 Brazilian National Council for Scientific and Technological Development (CNPq) via Grant Nos. 266 303999/2016-0, 440405/2016-5, and 457664/2013-4. MLM is a CNPq Research Fellow.São José do Rio Preto School of Medicine. São José do Rio Preto, SP, Brazil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil.São José do Rio Preto School of Medicine. São José do Rio Preto, SP, Brazil.São José do Rio Preto School of Medicine. São José do Rio Preto, SP, Brazil.São José do Rio Preto School of Medicine. São José do Rio Preto, SP, Brazil.São José do Rio Preto School of Medicine. São José do Rio Preto, SP, Brazil.São José do Rio Preto School of Medicine. São José do Rio Preto, SP, Brazil.São José do Rio Preto School of Medicine. São José do Rio Preto, SP, Brazil.São José do Rio Preto School of Medicine. São José do Rio Preto, SP, Brazil.São Paulo State University. São José do Rio Preto, SP, Brazil.São Paulo State University. São José do Rio Preto, SP, Brazil.São Paulo State University. São José do Rio Preto, SP, Brazil.Evandro Chagas Institute. Ananindeua, PA, Brazil.Evandro Chagas Institute. Ananindeua, PA, Brazil.Health Secretariat. São José do Rio Preto, SP, Brazil.Health Secretariat. São José do Rio Preto, SP, Brazil.Health Secretariat. São José do Rio Preto, SP, Brazil.Health Secretariat. São José do Rio Preto, SP, Brazil.Health Secretariat. São José do Rio Preto, SP, Brazil.Health Secretariat. São José do Rio Preto, SP, Brazil.Health Secretariat. São José do Rio Preto, SP, Brazil.Health Secretariat. São José do Rio Preto, SP, Brazil.Health Secretariat. São José do Rio Preto, SP, Brazil.Health Secretariat. São José do Rio Preto, SP, Brazil.Health Secretariat. São José do Rio Preto, SP, Brazil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Federal University of Bahia. Salvador, BA, Brazil / Yale School of Public Health. New Haven, Connecticut, USA.Fundacao Oswaldo Cruz. Rio de Janeiro, RJ, Brazil.São José do Rio Preto School of Medicine. São José do Rio Preto, SP, Brazil.São José do Rio Preto School of Medicine. São José do Rio Preto, SP, Brazil.São José do Rio Preto School of Medicine. São José do Rio Preto, SP, Brazil.University of Texas Medical Branch. Galveston, Texas, USA.São José do Rio Preto School of Medicine. São José do Rio Preto, SP, Brazil.São José do Rio Preto School of Medicine. São José do Rio Preto, SP, Brazil.Yale School of Public Health. New Haven, Connecticut, USA.Objective: We aimed to report the first 54 cases of pregnant women infected by Zika vírus (ZIKV) and their virological and clinical outcomes, as well as the newborns’ outcomes in 2016, after the emergence of ZIKV in dengue endemic areas of São Paulo, Brazil. Methods: This is a descriptive study performed from February to October 2016 on 54 qPCR ZIKV50 positive pregnant women identified by the Public Health Authority of São Jose do Rio Preto, São Paulo, Brazil. The women were followed and had clinical and epidemiological data collected before and after birth. Adverse outcomes in newborns were analyzed and reported. Urine or blood samples from newborns were collected to identify ZIKV infection by RT-PCR. Results: 216 acute Zika-suspected pregnant women were identified, and 54 had the diagnosis con55 firmed by RT-PCR. None of the 54 women miscarried. Among the 54 newborns, 15 exhibited ad56 verse outcomes at birth. The highest number of ZIKV infections occurred during the second and third trimesters. No cases of microcephaly were reported, though the broad clinical spectrum of outcomes, as lenticulostriate vasculopathy, subependymal cysts, auditive and ophtalmological dis59 orders, were identified. ZIKV RNA was detected in 18 of 51 newborns tested and in eight of 15 newborns with adverse outcomes. Conclusions: Although other studies have associated many newborn outcomes to ZIKV infection during pregnancy, these same adverse outcomes were rare or non-existent in this study. The clinical presentation in our newborns was mild compared to other reports, suggesting that there is significant heterogeneity of Congenital Zika Infection