Biopolymeric materials used as nonviral vectors: a review

Bacterial transformation and gene transfection can be understood as being the results of introducing specific genetic material into cells, resulting in gene expression, and adding a new genetic trait to the host cell. Many studies have been carried out to investigate different types of lipids and cationic polymers as promising nonviral vectors for DNA transfer. The present study aimed to carry out a systematic review on the use of biopolymeric materials as nonviral vectors. The methodology was carried out based on searches of scientific articles and applications for patents published or deposited from 2006 to 2020 in different databases for patents (EPO, USPTO, and INPI) and articles (Scopus, Web of Science, and Scielo). The results showed that there are some deposits of patents regarding the use of chitosan as a gene carrier. The 16 analyzed articles allowed us to infer that the use of biopolymers as nonviral vectors is limited due to the low diversity of biopolymers used for these purposes. It was also observed that the use of different materials as nonviral vectors is based on chemical structure modifications of the material, mainly by the addition of cationic groups. Thus, the use of biopolymers as nonviral vectors is still limited to only a few polysaccharide types, emphasizing the need for further studies involving the use of different biopolymers in processes of gene transfer.info:eu-repo/semantics/publishedVersio

Molecular characterization of Cyclophilin (TcCyP19) in Trypanosoma cruzi populations susceptible and resistant to benznidazole

Cyclophilin (TcCyP19), a peptidyl-prolyl cis/trans isomerase, is a key molecule with diverse biological functions that include roles in molecular chaperoning, stress response, immune modulation, and signal transduction. In this respect, TcCyP19 could serve as a potential drug target in diseasecausing parasites. Previous studies employing proteomics techniques have shown that the TcCyP19 isoform was more abundant in a benznidazole (BZ)-resistant Trypanosoma cruzi population than in its susceptible counterpart. In this study, TcCyP19 has been characterized in BZ-susceptible and BZresistant T. cruzi populations. Phylogenetic analysis revealed a clear dichotomy between Cyphophilin A (CyPA) sequences from trypanosomatids and mammals. Sequencing analysis revealed that the amino acid sequences of TcCyP19 were identical among the T. cruzi samples analyzed. Southern blot analysis showed that TcCyP19 is a single-copy gene, located in chromosomal bands varying in size from 0.68 to 2.2 Mb, depending on the strain of T. cruzi. Northern blot and qPCR indicated that the levels of TcCyP19 mRNA were two-fold higher in drug-resistant T. cruzi populations than in their drugsusceptible counterparts. Similarly, as determined by two-dimensional gel electrophoresis immunoblot, the expression of TcCyP19 protein was increased to the same degree in BZ-resistant T. cruzi populations. No differences in TcCyP19 mRNA and protein expression levels were observed between the susceptible and the naturally resistant T. cruzi strains analyzed. Taken together, these data indicate that cyclophilin TcCyP19 expression is up-regulated at both transcriptional and translational levels in T. cruzi populations that were in vitro-induced and in vivo-selected for resistance to BZ.Fil: Rêgo, Juciane Vaz. Universidade Federal Do Piaui.; BrasilFil: Duarte, Ana Paula. Fundación Oswaldo Cruz; BrasilFil: Liarte, Daniel Barbosa. Universidade Federal Do Piaui.; BrasilFil: de Carvalho Sousa, Francirlene. Universidade Federal Do Piaui.; BrasilFil: Barreto, Humberto Medeiros. Universidade Federal Do Piaui.; BrasilFil: Bua, Jacqueline Elena. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romanha, Alvaro José. Fundación Oswaldo Cruz; BrasilFil: Rádis Baptista, Gandhi. Universidade Federal Do Piaui.; BrasilFil: Murta, Silvane Maria Fonseca. Universidade Federal Do Piaui.; Brasi

Genomic and transcriptomic analysis of populations of Leishmania brazilliensis sensitive and resistant to antimonials

Submitted by Repositório Arca ([email protected]) on 2019-05-07T14:21:13Z No. of bitstreams: 1 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2019-08-08T19:12:58Z (GMT) No. of bitstreams: 2 T_2010_DanielBarbosaLiarte.pdf: 7929247 bytes, checksum: 2294b00f8e9fe0291c5f58ca05342ac3 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-08-08T19:12:58Z (GMT). No. of bitstreams: 2 T_2010_DanielBarbosaLiarte.pdf: 7929247 bytes, checksum: 2294b00f8e9fe0291c5f58ca05342ac3 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2010CNPq, FAPEMIG e FIOCRUZFundação Oswaldo Cruz. Centro de Pesquisa Rene Rachou. Belo Horizonte, MG, Brasil.Na primeira parte desta tese selecionamos in vitro populações de Leishmania Viannia guyanensis, L. (V.) braziliensis, L. Leishmania amazonensis e L. (L.) infantum chagasi resistentes ao tartarato potássico de antimônio (SbIII) (Liarte & Murta, 2010). A concentração inibitória de 50% (IC50) destas populações foi de 4 a 20 vezes maior do que seus pares sensíveis. Nenhuma mudança na resistência foi observada em L. (V.) guyanensis e L. (L.) infantum chagasi após 37-47 passagens em meio na ausência de SbIII. Entretanto, uma diminuição de duas vezes foi observada no índice de resistência das populações L. (V.) braziliensis e L. (L.) amazonensis. Nenhuma das populações resistentes ao SbIII apresentou resistência cruzada a anfotericina B e miltefosina. Entretanto, as populações resistentes de L. (V.) braziliensis,L. (L.) amazonensis e L. (L.) infantum chagasi foram também resistentes a paromomicina. Uma drástica redução na infectividade de camundongos foi observada nas populações resistentes de L. (V.) guyanensis, L. (L.) amazonensis e L. (V.) braziliensis. Na segunda parte, analisamos a amplificação e deleção de genes e identificamos transcritos diferencialmente expressos nas populações de Leishmania spp. sensíveis e resistentes ao SbIII utilizando a metodologia do microarranjo de DNA. Ensaios de CGH (comparative genomic hybridization) permitiram a identificação de 124 CDS amplificadas e 128 CDS deletadas na população de L. (V.) braziliensis resistente ao SbIII. Em L. (V.) braziliensis foi observada a amplificação de genes associados à região H e amplificações de regiões dos cromossomos 17, 20 e 31. Foram identificados 560 transcritos mais expressos e 397 transcritos menos expressos na população de L. (V.) braziliensis resistente ao SbIII. Dados de anotação funcional sugerem um aumento na expressão de transcritos associados à replicação e à transcrição do DNA e uma diminuição na expressão de transcritos associados ao metabolismo de lipídios, de carboidratos e ao transporte de proteínas. Análises em bancos de dados de drogas mostraram que 247 transcritos diferencialmente expressos em L. (V.) braziliensis apresentam pelo menos um alvo para drogas. Destes 247, 15 possuem pouca similaridade com proteínas humanas sendo, portanto bons alvos para quimioterapia. Um banco de dados de resistência a drogas em Leishmania foi construído possibilitando a integração de todos os dados obtidos nesta tese. A análise integrada dos dados fenotípicos, genômicos e transcriptômicos estão permitindo estabelecer novas estratégias de pesquisa básica e aplicada, dessa forma contribuindo para o desenvolvimento de novas drogas para a quimioterapia das leishmanioses.In this thesis initially we selected in vitro populations from Leishmania iannia guyanensis, L. (V.) braziliensis, L. Leishmania amazonensis and L. (L.) nfantum chagasi that were resistant to potassium antimony tartrate (SbIII). The resistance index of these populations varied from 4- to 20-fold higher than that of their wild-type counterparts. No change in the resistance indexes of L. (V.) guyanensis and L. (L.) infantum chagasi was observed after 37-47 passages in a culture medium without SbIII. In contrast, a decrease in the resistance index was observed for L. (V.) braziliensis and L. (L.) amazonensis. None of the antimony-resistant populations exhibited cross-resistance to amphotericin B and miltefosina. However, the resistant populations of L. (V.) braziliensis, L. (L.) amazonensis and L. (L.) infantum chagasi were also resistant to paromomycin. A drastic reduction was observed in the infectivity in mice for the resistant L. (V.) guyanensis, L. (L.) amazonensis and L. (V.) braziliensis populations. In the second part of this thesis we analyzed gene amplification and deletion and identified transcripts differentially expressed in Leishmania spp. populations susceptible and resitant to SbIII using DNA microarray methodology. CGH assays (comparative genomic hybridization) allowed the identification of 124 CDS amplified and 128 CDS deleted in the SbIII-resistant L. (V.) braziliensis population. The amplification of the H region and regions from chromosomes 17, 20 and 31 was observed in the SbIII-resistant population from L. (V.) braziliensis. We identified 560 up-regulated transcripts and 397 down-regulated transcripts in SbIII-resistant L. (V.) braziliensis population. Functional annotation data suggest an increased expression of transcripts related to DNA replication and transcription and a decreased expression of transcripts associated with metabolism of lipids and carbohydrates and transport of proteins. Drug bank analysis showed that 247 transcripts differentially expressed in SbIII-resistant L. (V.) braziliensis population present at least one target for drugs. 15 out of 247 transcripts present low similarity with human proteins being then, a good target for chemotherapy. A database of drug resistance in Leishmania was constructed, allowing the integration of all data obtained in this thesis. The integrated analysis of phenotypic, genomic and transcriptomic data, is allowing to define new strategies for basic and applied research, thereby contributing to the development of new drugs for the Leishmaniasis chemotherapy

Development of a PCR multiplex capable to detect and to classify cepas of Trypanosoma cruzi in clinical samples and field

Made available in DSpace on 2012-05-07T15:26:25Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 000012.pdf: 9561271 bytes, checksum: 2b5a0bca8b621acdc87733afadb8e4b6 (MD5) Previous issue date: 2006Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil.Nesse trabalho, descrevemos a busca por marcadores moleculares que permitam simultaneamente o diagnóstico da doença de Chagas e a classificação das cepas segundo o fenótipo de susceptibilidade a drogas, zimodema e/ou grupos I e II do T. cruzi. Inicialmente, selecionamos seqüências repetitivas do DNA do T. cruzi e iniciadores descritos para o diagnóstico da doença de Chagas. Analisamos as seqüências do elemento repetitivo E13, kDNA e o DNA satélite (195 pb). Os resultados com o elemento repetitivo E13 e seqüências do kDNA, mostraram uma grande variabilidade dessas seqüências, inviabilizando a busca de marcadores. Analisamos 160 seqüências do DNA satélite de 195 pb de 12 cepas do T. cruzi previamente caracterizadas segundo o fenótipo de susceptibilidade a drogas e zimodema. Estudos de filogenia mostraram a existência de dois grupos distintos, associados com os grupos I e II do T. cruzi. Observamos a presença de 8 polimorfismos exclusivos das cepas do grupo I do T. cruzi. Desenhamos iniciadores específicos para estes polimorfismos. Um sistema de PCR multiplex constituído pelos iniciadores TcSat 4 e Diaz 7 e 8 permitiram a classificação das cepas de T. cruzi nos grupos I e II. A sensibilidade foi de 10 fg, o que corresponde a 1/30 do DNA de um parasito. Amostras de DNA de outros tripanosomatídeos não produziram produto amplificado. Comparamos o perfil de amplificação do DNA satélite de 30 cepas de T. cruzi e 24 amostras de sangue de camundongos experimentalmente infectados com as cepas Colombiana (grupo I) e Y (grupo II) em papel de filtro. Em todas as amostras positivas foi possível a identificação dos grupos I e II. Para validarmos a técnica com amostras de campo, utilizamos 7 amostras de DNA do creme leucocitário de pacientes na fase aguda da doença de Chagas e 15 amostras de fezes de Triatoma infestans em papel de filtro. As amostras de pacientes foram grupo II e as amostras de T. infestans grupo I. Esses resultados estão de acordo com os dados descritos na literatura que mostram uma associação entre cepas do grupo I do T. cruzi e o ciclo silvestre do parasito e entre cepas do grupo II e o ciclo doméstico. O PCR multiplex que desenvolvemos permite a classificação das cepas nos dois grupos principais de T. cruzi sem, entretanto correlacioná-las à resistência a drogas. Apresentamos uma metodologia sensível, específica e rápida que poderá ser utilizada em amostras clínicas e de campo como ferramenta de diagnóstico molecular e classificação das cepas de T. cruzi.In the present work, we describe our search for molecular markers that allow the diagnosis of Chagas’ disease and strain classification simultaneously according to the drug susceptibility, zymodeme and/or groups I and II of T. cruzi. We selected DNA repetitive sequences from T. cruzi and primers described previously for diagnosis of Chagas’ disease. We analyzed the sequences of the repetitive element E13, kDNA and satellite DNA (195 bp). The sequences of the repetitive element E13 and the kDNA were highly variable, making unfeasible the search for molecular markers. Polymorphism of the 160 satellite DNAs (195 bp) from 12 T. cruzi strains from different zymodemes and BZ susceptibility were determined. Phylogenetic studies showed the existence of two groups of sequences, associated with the groups I and II of T. cruzi. The existence of 8 polymorphisms exclusive to T. cruzi I strains, lead us to design specific primers for these polymorphisms. A system of PCR multiplex using primers TcSat 4, Diaz 7 and 8, allowed the strain classification of T. cruzi in the groups I and II. The sensibility was of 10 fg, that corresponds at 1/30 of the DNA of one parasite. This PCR multiplex was T. cruzi specific, did not amplifying DNA from other tripanosomatids. We compared the amplification of satellite DNA of 30 T. cruzi strains and 24 blood samples from mice experimentally infected with the Colombian (group I) and Y (group II) strains in filter paper. The samples were classified in groups I and II. In order to validate the technique with field samples, we used 7 samples of Buffy coat DNA from patients in the acute phase of Chagas’ disease, and 15 samples from naturally infected Triatoma infestans feces collected in filter paper. The patients' samples were group II and the samples of T. infestans group I. Our results are consistent with the data described in the literature that show an association between strains of T. cruzi I and the Sylvatic cycle of the parasite and between strains of T. cruzi II and the domestic cycle. The PCR multiplex that we developed allows the T. cruzi DNA detection and the strain classification without correlating them to drug resistance. We presented a sensitive, specific and a fast methodology that can be used in clinical and field samples, as a tool for molecular diagnosis and strain classification of T. cruzi simultaneously

Arylfurans as potential Trypanosoma cruzi trypanothione reductase inhibitors

Submitted by Nuzia Santos ([email protected]) on 2013-06-06T18:32:43Z No. of bitstreams: 1 87.pdf: 480919 bytes, checksum: 0ec2d7dcbb146ddb70e377368034b213 (MD5)Made available in DSpace on 2013-06-06T18:32:43Z (GMT). No. of bitstreams: 1 87.pdf: 480919 bytes, checksum: 0ec2d7dcbb146ddb70e377368034b213 (MD5) Previous issue date: 2006Fiocruz, CNPq, FapemigFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Química de Produtos Naturais. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Química de Produtos Naturais. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Parasitologia Celular e Molecular. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Parasitologia Celular e Molecular. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Exatas. Departamento de Química. NEQUIM. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Parasitologia Celular e Molecular. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Química de Produtos Naturais. Belo Horizonte, MG, Brasil.The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzitrypanothione reductase (TR) inhibi-tors as well against the parasite’s intracellular (amastigote) and bloodstream (trypomastigote) forms. Compound 12 was the most effective against TR with an IC50 of 48.5 µM while 7and 14 were active against amastigotes, inhibiting the parasite development by 60% at 20 µg/ml (59 and 90 µM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 µg/ml (0.6-1.5 mM)

Transcriptomic analysis of benznidazole-resistant and susceptible Trypanosoma cruzi populations

Abstract Background Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is a serious public health concern in Latin America. Nifurtimox and benznidazole (BZ), the only two drugs currently approved for the treatment of CD, have very low efficacies in the chronic phase of the disease and several toxic side effects. Trypanosoma cruzi strains that are naturally resistant to both drugs have been reported. We performed a comparative transcriptomic analysis of wild-type and BZ-resistant T. cruzi populations using high-throughput RNA sequencing to elucidate the metabolic pathways related to clinical drug resistance and identify promising molecular targets for the development of new drugs for treating CD. Methods All complementary DNA (cDNA) libraries were constructed from the epimastigote forms of each line, sequenced and analysed using the Prinseq and Trimmomatic tools for the quality analysis, STAR as the aligner for mapping the reads against the reference genome (T. cruzi Dm28c—2018), the Bioconductor package EdgeR for statistical analysis of differential expression and the Python-based library GOATools for the functional enrichment analysis. Results The analytical pipeline with an adjusted P-value of 1.5 identified 1819 transcripts that were differentially expressed (DE) between wild-type and BZ-resistant T. cruzi populations. Of these, 1522 (83.7%) presented functional annotations and 297 (16.2%) were assigned as hypothetical proteins. In total, 1067 transcripts were upregulated and 752 were downregulated in the BZ-resistant T. cruzi population. Functional enrichment analysis of the DE transcripts identified 10 and 111 functional categories enriched for the up- and downregulated transcripts, respectively. Through functional analysis we identified several biological processes potentially associated with the BZ-resistant phenotype: cellular amino acid metabolic processes, translation, proteolysis, protein phosphorylation, RNA modification, DNA repair, generation of precursor metabolites and energy, oxidation–reduction processes, protein folding, purine nucleotide metabolic processes and lipid biosynthetic processes. Conclusions The transcriptomic profile of T. cruzi revealed a robust set of genes from different metabolic pathways associated with the BZ-resistant phenotype, proving that T. cruzi resistance mechanisms are multifactorial and complex. Biological processes associated with parasite drug resistance include antioxidant defenses and RNA processing. The identified transcripts, such as ascorbate peroxidase (APX) and iron superoxide dismutase (Fe-SOD), provide important information on the resistant phenotype. These DE transcripts can be further evaluated as molecular targets for new drugs against CD. Graphical Abstrac

Comparative transcriptomic analysis of antimony resistant and susceptible Leishmania infantum lines

International audienceBackground: One of the major challenges to leishmaniasis treatment is the emergence of parasites resistant to antimony. To study differentially expressed genes associated with drug resistance, we performed a comparative transcriptomic analysis between wild-type and potassium antimonyl tartrate (SbIII)-resistant Leishmania infantum lines using high-throughput RNA sequencing.Methods: All the cDNA libraries were constructed from promastigote forms of each line, sequenced and analyzed using STAR for mapping the reads against the reference genome (L. infantum JPCM5) and DESeq2 for differential expression statistical analyses. All the genes were functionally annotated using sequence similarity search.Results: The analytical pipeline considering an adjusted p-value 2.0 identified 933 transcripts differentially expressed (DE) between wild-type and SbIII-resistant L. infantum lines. Out of 933 DE transcripts, 504 presented functional annotation and 429 were assigned as hypothetical proteins. A total of 837 transcripts were upregulated and 96 were downregulated in the SbIII-resistant L. infantum line. Using this DE dataset, the proteins were further grouped in functional classes according to the gene ontology database. The functional enrichment analysis for biological processes showed that the upregulated transcripts in the SbIII-resistant line are associated with protein phosphorylation, microtubule-based movement, ubiquitination, host-parasite interaction, cellular process and other categories. The downregulated transcripts in the SbIII-resistant line are assigned in the GO categories: ribonucleoprotein complex, ribosome biogenesis, rRNA processing, nucleosome assembly and translation.Conclusions: The transcriptomic profile of L. infantum showed a robust set of genes from different metabolic pathways associated with the antimony resistance phenotype in this parasite. Our results address the complex and multifactorial antimony resistance mechanisms in Leishmania, identifying several candidate genes that may be further evaluated as molecular targets for chemotherapy of leishmaniasis