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1.
Immunity ; 42(6): 1143-58, 2015 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-26070485

RESUMO

Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8(+) T cell immunity and promoted CD4(+)Foxp3(+) Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages in vivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN(+) suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic.


Assuntos
Moléculas de Adesão Celular/metabolismo , Rejeição de Enxerto/prevenção & controle , Transplante de Coração , Lectinas Tipo C/metabolismo , Macrófagos/imunologia , Receptores de Superfície Celular/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Moléculas de Adesão Celular/genética , Células Cultivadas , Fatores de Transcrição Forkhead/metabolismo , Rejeição de Enxerto/etiologia , Tolerância Imunológica , Interleucina-10/metabolismo , Lectinas Tipo C/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terapia de Alvo Molecular , Receptores de Superfície Celular/genética , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Tolerância ao Transplante , Regulação para Cima
2.
PLoS Pathog ; 17(8): e1009541, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34437654

RESUMO

Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2, respectively) are prevalent human pathogens of clinical relevance that establish long-life latency in the nervous system. They have been considered, along with the Herpesviridae family, to exhibit a low level of genetic diversity during viral replication. However, the high ability shown by these viruses to rapidly evolve under different selective pressures does not correlates with that presumed genetic stability. High-throughput sequencing has revealed that heterogeneous or plaque-purified populations of both serotypes contain a broad range of genetic diversity, in terms of number and frequency of minor genetic variants, both in vivo and in vitro. This is reminiscent of the quasispecies phenomenon traditionally associated with RNA viruses. Here, by plaque-purification of two selected viral clones of each viral subtype, we reduced the high level of genetic variability found in the original viral stocks, to more genetically homogeneous populations. After having deeply characterized the genetic diversity present in the purified viral clones as a high confidence baseline, we examined the generation of de novo genetic diversity under culture conditions. We found that both serotypes gradually increased the number of de novo minor variants, as well as their frequency, in two different cell types after just five and ten passages. Remarkably, HSV-2 populations displayed a much higher raise of nonconservative de novo minor variants than the HSV-1 counterparts. Most of these minor variants exhibited a very low frequency in the population, increasing their frequency over sequential passages. These new appeared minor variants largely impacted the coding diversity of HSV-2, and we found some genes more prone to harbor higher variability. These data show that herpesviruses generate de novo genetic diversity differentially under equal in vitro culture conditions. This might have contributed to the evolutionary divergence of HSV-1 and HSV-2 adapting to different anatomical niche, boosted by selective pressures found at each epithelial and neuronal tissue.


Assuntos
Evolução Biológica , Variação Genética , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/genética , Queratinócitos/virologia , Replicação Viral , Genoma Viral , Herpes Simples/genética , Herpes Simples/metabolismo , Humanos , Queratinócitos/metabolismo , Ativação Viral , Latência Viral
3.
J Gen Virol ; 103(10)2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36260063

RESUMO

The application of CRISPR/Cas9 to improve genome engineering efficiency for large dsDNA viruses has been extensively described, but a robust and versatile method for high-throughput generation of marker-free recombinants for a desired locus has not yet been reported. Cytoplasmic-replicating viruses use their own repair enzymes for homologous recombination, while nuclear-replicating viruses use the host repair machinery. This is translated into a wide range of Cas9-induced homologous recombination efficiencies, depending on the virus replication compartment and viral/host repair machinery characteristics and accessibility. However, the use of Cas9 as a selection agent to target parental virus genomes robustly improves the selection of desired recombinants across large dsDNA viruses. We used ectromelia virus (ECTV) and herpes simplex virus (HSV) type 1 and 2 to optimize a CRISPR/Cas9 method that can be used versatilely for efficient genome editing and selection of both cytoplasmic- and nuclear-replicating viruses. We performed a genome-wide genetic variant analysis of mutations located at predicted off-target sequences for 20 different recombinants, showing off-target-free accuracy by deep sequencing. Our results support this optimized method as an efficient, accurate and versatile approach to enhance the two critical factors of high-throughput viral genome engineering: generation and colour-based selection of recombinants. This application of CRISPR/Cas9 reduces the time and labour for screening of desired recombinants, allowing for high-throughput generation of large collections of mutant dsDNA viruses for a desired locus, optimally in less than 2 weeks.


Assuntos
Herpesvirus Humano 1 , Vírus , Sistemas CRISPR-Cas , Edição de Genes/métodos , Genoma Viral , Herpesvirus Humano 1/genética , Vírus/genética
4.
J Virol ; 94(20)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32669337

RESUMO

During primary infection, herpes simplex virus 2 (HSV-2) replicates in epithelial cells and enters neurites to infect neurons of the peripheral nervous system. Growth factors and attractive and repulsive directional cues influence neurite outgrowth and neuronal survival. We hypothesized that HSV-2 modulates the activity of such cues to increase neurite outgrowth. To test this hypothesis, we exposed sensory neurons to nerve growth factor (NGF) and mock- or HSV-2-infected HEK-293T cells, since they express repellents of neurite outgrowth. We show that HEK-293T cells secrete factors that inhibit neurite outgrowth, while infection with HSV-2 strains MS and 333 reduces this repelling phenotype, increasing neurite numbers. The HSV-2-mediated restoration of neurite outgrowth required the activity of NGF. In the absence of infection, however, NGF did not overcome the repulsion mediated by HEK-293T cells. We previously showed that recombinant, soluble glycoprotein G of HSV-2 (rSgG2) binds and enhances NGF activity, increasing neurite outgrowth. However, the effect of gG2 during infection has not been investigated. Therefore, we addressed whether gG2 contributes to overcoming neurite outgrowth repulsion. To do so, we generated viruses lacking gG2 expression and complemented them by exogenous expression of gG2. Overall, our results suggest that HSV-2 infection of nonneuronal cells reduces their repelling effect on neurite outgrowth in an NGF-dependent manner. gG2 contributed to this phenotype, but it was not the only factor. The enhanced neurite outgrowth may facilitate HSV-2 spread from epithelial cells into neurons expressing NGF receptors and increase HSV-2-mediated pathogenesis.IMPORTANCE Herpes simplex virus 2 (HSV-2) is a prevalent human pathogen that establishes lifelong latency in neurons of the peripheral nervous system. Colonization of neurons is required for HSV-2 persistence and pathogenesis. The viral and cellular factors required for efficient infection of neurons are not fully understood. We show here that nonneuronal cells repel neurite outgrowth of sensory neurons, while HSV-2 infection overcomes this inhibition and, rather, stimulates neurite outgrowth. HSV-2 glycoprotein G and nerve growth factor contribute to this phenotype, which may attract neurites to sites of infection and facilitate virus spread to neurons. Understanding the mechanisms that modulate neurite outgrowth and facilitate HSV-2 infection of neurons might foster the development of therapeutics to reduce HSV-2 colonization of the nervous system and provide insights on neurite outgrowth and regeneration.


Assuntos
Herpes Genital/metabolismo , Herpesvirus Humano 2/metabolismo , Fator de Crescimento Neural/metabolismo , Neuritos , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Herpesvirus Humano 2/patogenicidade , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neuritos/metabolismo , Neuritos/virologia , Células Vero
5.
J Virol ; 92(11)2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29563289

RESUMO

In this study, we have characterized quasispecies dynamics and the evolution of viral tropism in naive HIV-1-infected patients treated with a short course of maraviroc monotherapy (ClinicalTrials.gov registration no. NCT01060618) independently of the tropism of the infecting virus. We randomly selected 20 patients infected with viruses displaying different basal tropisms-10 carrying R5 and 10 carrying dual/mixed X4 (DM/X4) viruses-at recruitment as determined by phenotypic assay (Trofile). Evolution of viral quasiespecies at the end of treatment was determined by ultradeep sequencing of the V3 region using a 454 Life Sciences Platform and geno2pheno (g2p) algorithm for viral tropism prediction. The false-positive rate (FPR) that defines the probability of classifying an R5 virus falsely as X4 was set at 10%. X4-specific HIV-1 viral load (VL) was calculated from sequences with an FPR of <3.75%. Virological response as defined as >1-log10 copies/ml reduction in VL was detected in 70% of patients independently of the basal tropism of the infecting virus. Viral tropism remained stable, and nonsignificant differences in FPR values before and after treatment were found for the majority of patients in both tropism groups. Only three patients (one with R5 and two with DM/X4 viruses) showed an increased (>1 log) X4 VL, and one patient harboring a DM/X4-tropic virus displayed a significant reduction in FPR values at the end of treatment. Fast changes in the composition of viral populations were observed in all patients after 10 days of maraviroc (MVC) monotherapy treatment, and a complete replacement of viral quasiespecies was found in 3/10 patients carrying R5-using viruses and 4/10 patients carrying DM/X4-using viruses.IMPORTANCE Initiation of treatment with maraviroc requires previous determination of viral tropism by genotypic or phenotypic methods because of the risk of treatment failure and selection of DM/X4-tropic variants. In this study, we confirm previous work showing that the virologic response to maraviroc is independent of basal tropism. By deep-sequencing analysis, we determined that fast changes in viral populations were due to the emergence of minority variants in some patients whereas in others generation of new strains was detected. The risk of DM/X4 selection was very low as FPR values remained stable, and only one patient showed a detrimental switch to DM/X4 variants. Our data show that some DM/X4 viruses are sensitive to maraviroc treatment probably because only a low proportion of DM/X4 viruses use preferentially the X4 receptor and contain authentically maraviroc-resistant viruses that are not accurately detected by current assays.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Cicloexanos/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/genética , Triazóis/uso terapêutico , Tropismo Viral/genética , Adulto , Antagonistas dos Receptores CCR5/farmacologia , Feminino , Infecções por HIV/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Maraviroc , Pessoa de Meia-Idade , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Adulto Jovem
6.
Genome Res ; 25(5): 633-44, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25800673

RESUMO

Noise in gene expression is a main determinant of phenotypic variability. Increasing experimental evidence suggests that genome-wide cellular constraints largely contribute to the heterogeneity observed in gene products. It is still unclear, however, which global factors affect gene expression noise and to what extent. Since eukaryotic gene expression is an energy demanding process, differences in the energy budget of each cell could determine gene expression differences. Here, we quantify the contribution of mitochondrial variability (a natural source of ATP variation) to global variability in gene expression. We find that changes in mitochondrial content can account for ∼50% of the variability observed in protein levels. This is the combined result of the effect of mitochondria dosage on transcription and translation apparatus content and activities. Moreover, we find that mitochondrial levels have a large impact on alternative splicing, thus modulating both the abundance and type of mRNAs. A simple mathematical model in which mitochondrial content simultaneously affects transcription rate and splicing site choice can explain the alternative splicing data. The results of this study show that mitochondrial content (and/or probably function) influences mRNA abundance, translation, and alternative splicing, which ultimately affects cellular phenotype.


Assuntos
Processamento Alternativo , DNA Mitocondrial/genética , Genoma , Metabolismo Energético , Variação Genética , Células HeLa , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Mem Inst Oswaldo Cruz ; 114: e180438, 2018 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-30540030

RESUMO

Leishmania braziliensis is the etiological agent of American mucosal leishmaniasis, one of the most severe clinical forms of leishmaniasis. Here, we report the assembly of the L. braziliensis (M2904) genome into 35 continuous chromosomes. Also, the annotation of 8395 genes is provided. The public availability of this information will contribute to a better knowledge of this pathogen and help in the search for vaccines and novel drug targets aimed to control the disease caused by this Leishmania species.


Assuntos
DNA de Protozoário/genética , Leishmania braziliensis/genética , Análise de Sequência de DNA
8.
BMC Genomics ; 18(1): 7, 2017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-28049418

RESUMO

BACKGROUND: Chimeric transcripts are commonly defined as transcripts linking two or more different genes in the genome, and can be explained by various biological mechanisms such as genomic rearrangement, read-through or trans-splicing, but also by technical or biological artefacts. Several studies have shown their importance in cancer, cell pluripotency and motility. Many programs have recently been developed to identify chimeras from Illumina RNA-seq data (mostly fusion genes in cancer). However outputs of different programs on the same dataset can be widely inconsistent, and tend to include many false positives. Other issues relate to simulated datasets restricted to fusion genes, real datasets with limited numbers of validated cases, result inconsistencies between simulated and real datasets, and gene rather than junction level assessment. RESULTS: Here we present ChimPipe, a modular and easy-to-use method to reliably identify fusion genes and transcription-induced chimeras from paired-end Illumina RNA-seq data. We have also produced realistic simulated datasets for three different read lengths, and enhanced two gold-standard cancer datasets by associating exact junction points to validated gene fusions. Benchmarking ChimPipe together with four other state-of-the-art tools on this data showed ChimPipe to be the top program at identifying exact junction coordinates for both kinds of datasets, and the one showing the best trade-off between sensitivity and precision. Applied to 106 ENCODE human RNA-seq datasets, ChimPipe identified 137 high confidence chimeras connecting the protein coding sequence of their parent genes. In subsequent experiments, three out of four predicted chimeras, two of which recurrently expressed in a large majority of the samples, could be validated. Cloning and sequencing of the three cases revealed several new chimeric transcript structures, 3 of which with the potential to encode a chimeric protein for which we hypothesized a new role. Applying ChimPipe to human and mouse ENCODE RNA-seq data led to the identification of 131 recurrent chimeras common to both species, and therefore potentially conserved. CONCLUSIONS: ChimPipe combines discordant paired-end reads and split-reads to detect any kind of chimeras, including those originating from polymerase read-through, and shows an excellent trade-off between sensitivity and precision. The chimeras found by ChimPipe can be validated in-vitro with high accuracy.


Assuntos
Proteínas de Fusão Oncogênica , Recombinação Genética , Software , Transcrição Gênica , Animais , Biologia Computacional/métodos , Simulação por Computador , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Reprodutibilidade dos Testes , Análise de Sequência de RNA
9.
Appl Environ Microbiol ; 83(13)2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28455334

RESUMO

Pollen, fungi, and bacteria are the main microscopic biological entities present in outdoor air, causing allergy symptoms and disease transmission and having a significant role in atmosphere dynamics. Despite their relevance, a method for monitoring simultaneously these biological particles in metropolitan environments has not yet been developed. Here, we assessed the use of the Hirst-type spore trap to characterize the global airborne biota by high-throughput DNA sequencing, selecting regions of the 16S rRNA gene and internal transcribed spacer for the taxonomic assignment. We showed that aerobiological communities are well represented by this approach. The operational taxonomic units (OTUs) of two traps working synchronically compiled >87% of the total relative abundance for bacterial diversity collected in each sampler, >89% for fungi, and >97% for pollen. We found a good correspondence between traditional characterization by microscopy and genetic identification, obtaining more-accurate taxonomic assignments and detecting a greater diversity using the latter. We also demonstrated that DNA sequencing accurately detects differences in biodiversity between samples. We concluded that high-throughput DNA sequencing applied to aerobiological samples obtained with Hirst spore traps provides reliable results and can be easily implemented for monitoring prokaryotic and eukaryotic entities present in the air of urban areas.IMPORTANCE Detection, monitoring, and characterization of the wide diversity of biological entities present in the air are difficult tasks that require time and expertise in different disciplines. We have evaluated the use of the Hirst spore trap (an instrument broadly employed in aerobiological studies) to detect and identify these organisms by DNA-based analyses. Our results showed a consistent collection of DNA and a good concordance with traditional methods for identification, suggesting that these devices can be used as a tool for continuous monitoring of the airborne biodiversity, improving taxonomic resolution and characterization together. They are also suitable for acquiring novel DNA amplicon-based information in order to gain a better understanding of the biological particles present in a scarcely known environment such as the air.


Assuntos
Ar/análise , Bactérias/isolamento & purificação , Eucariotos/isolamento & purificação , Fungos/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Pólen/genética , Microbiologia do Ar , Bactérias/classificação , Bactérias/genética , Biodiversidade , Cidades , Eucariotos/classificação , Eucariotos/genética , Fungos/classificação , Fungos/genética , Filogenia , Estações do Ano , Esporos Fúngicos/classificação , Esporos Fúngicos/genética , Esporos Fúngicos/isolamento & purificação
10.
Microbiol Resour Announc ; 13(6): e0130023, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38651926

RESUMO

The isolation and characterization of additional phages is crucial for adding reliable viral sequences with relevant biological information to viral databases. In this study, we present the complete genomes of two Arthrobacter phages obtained from different soil samples.

11.
Microbiol Resour Announc ; 13(4): e0122023, 2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38517186

RESUMO

In the present work, we present the draft genome sequence of a new putative Arthrobacter species associated with the tomato rhizosphere.

12.
Sci Total Environ ; 952: 175866, 2024 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-39222816

RESUMO

Monitoring zoonoses in urban environments is of great relevance, where the incidence of certain pathogens may be higher and where population density makes the spread of any contagious disease more likely. In this study we applied a metabarcoding approach to study potentially zoonotic pathogens in faecal samples of 9 urban vertebrate species. We applied this methodology with two objectives. Firstly, to obtain information on potential pathogens present in the urban fauna of a large European city (Madrid, Spain) and to determine which are their main reservoirs. In addition, we tested for differences in the prevalence of these potential pathogens between urban and rural European rabbits, used as ubiquitous species. Additionally, based on the results obtained, we evaluated the effectiveness of metabarcoding as a tool for monitoring potential pathogen. Our results revealed the presence of potentially zoonotic bacterial genera in all studied host species, 10 of these genera with zoonotic species of mandatory monitoring in the European Union. Based on these results, urban birds (especially house sparrows and pigeons) and bats are the species posing the greatest potential risk, with Campylobacter and Listeria genera in birds and of Chlamydia and Vibrio cholerae in bats as most relevant pathogens. This information highlights the risk associated with fresh faeces from urban wildlife. In addition, we detected Campylobacter in >50 % of the urban rabbit samples, while we only detected it in 11 % of the rural rabbit samples. We found that urban rabbits have a higher prevalence of some pathogens relative to rural rabbits, which could indicate increased risk of pathogen transmission to humans. Finally, our results showed that metabarcoding can be an useful tool to quickly obtain a first screening of potentially zoonotic organisms, necessary information to target the monitoring efforts on the most relevant pathogens and host species.


Assuntos
Cidades , Fezes , Zoonoses , Animais , Fezes/microbiologia , Espanha , Zoonoses/microbiologia , Zoonoses/transmissão , Zoonoses/epidemiologia , Animais Selvagens/microbiologia , Código de Barras de DNA Taxonômico , Monitoramento Ambiental/métodos , Coelhos
13.
BMC Genomics ; 14: 199, 2013 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-23521802

RESUMO

BACKGROUND: Alternative splicing (AS) is a major mechanism for modulating gene expression of an organism, allowing the synthesis of several structurally and functionally distinct mRNAs and protein isoforms from a unique gene. Related to AS is the Transcription Induced Chimerism (TIC) or Tandem Chimerism, by which chimeric RNAs between adjacent genes can be found, increasing combinatorial complexity of the proteome. The Ly6g5b gene presents particular behaviours in its expression, involving an intron retention event and being capable to form RNA chimera transcripts with the upstream gene Csnk2b. We wanted to characterise these events more deeply in four tissues in six different mammals and analyse their protein products. RESULTS: While canonical Csnk2b isoform was widely expressed, Ly6g5b canonical isoform was less ubiquitous, although the Ly6g5b first intron retained transcript was present in all the tissues and species analysed. Csnk2b-Ly6g5b chimeras were present in all the samples analysed, but with restricted expression patterns. Some of these chimeric transcripts maintained correct structural domains from Csnk2b and Ly6g5b. Moreover, we found Csnk2b, Ly6g5b, and Csnk2b-Ly6g5b transcripts that present exon skipping, alternative 5' and 3' splice site and intron retention events. These would generate truncated or aberrant proteins whose role remains unknown. Some chimeric transcripts would encode CSNK2B proteins with an altered C-terminus, which could affect its biological function broadening its substrate specificity. Over-expression of human CSNK2B, LY6G5B, and CSNK2B-LY6G5B proteins, show different patterns of post-translational modifications and cell distribution. CONCLUSIONS: Ly6g5b intron retention and Csnk2b-Ly6g5b transcript chimerism are broadly distributed in tissues of different mammals.


Assuntos
Antígenos Ly/genética , Caseína Quinase II/genética , Mamíferos/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Antígenos Ly/metabolismo , Caseína Quinase II/metabolismo , Bovinos , Humanos , Íntrons , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Alinhamento de Sequência , Suínos/genética
14.
BMC Genomics ; 14: 223, 2013 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-23557257

RESUMO

BACKGROUND: Although the genome sequence of the protozoan parasite Leishmania major was determined several years ago, the knowledge of its transcriptome was incomplete, both regarding the real number of genes and their primary structure. RESULTS: Here, we describe the first comprehensive transcriptome analysis of a parasite from the genus Leishmania. Using high-throughput RNA sequencing (RNA-seq), a total of 10285 transcripts were identified, of which 1884 were considered novel, as they did not match previously annotated genes. In addition, our data indicate that current annotations should be modified for many of the genes. The detailed analysis of the transcript processing sites revealed extensive heterogeneity in the spliced leader (SL) and polyadenylation addition sites. As a result, around 50% of the genes presented multiple transcripts differing in the length of the UTRs, sometimes in the order of hundreds of nucleotides. This transcript heterogeneity could provide an additional source for regulation as the different sizes of UTRs could modify RNA stability and/or influence the efficiency of RNA translation. In addition, for the first time for the Leishmania major promastigote stage, we are providing relative expression transcript levels. CONCLUSIONS: This study provides a concise view of the global transcriptome of the L. major promastigote stage, providing the basis for future comparative analysis with other development stages or other Leishmania species.


Assuntos
Perfilação da Expressão Gênica , Leishmania major/crescimento & desenvolvimento , Leishmania major/genética , Estágios do Ciclo de Vida/genética , Anotação de Sequência Molecular , Análise de Sequência de RNA , Sequência de Aminoácidos , Animais , Sequência de Bases , Dados de Sequência Molecular , Poliadenilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
15.
Sci Total Environ ; 871: 162137, 2023 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-36775167

RESUMO

The dispersion of microorganisms through the atmosphere is a continual and essential process that underpins biogeography and ecosystem development and function. Despite the ubiquity of atmospheric microorganisms globally, specific knowledge of the determinants of atmospheric microbial diversity at any given location remains unresolved. Here we describe bacterial diversity in the atmospheric boundary layer and underlying soil at twelve globally distributed locations encompassing all major biomes, and characterise the contribution of local and distant soils to the observed atmospheric community. Across biomes the diversity of bacteria in the atmosphere was negatively correlated with mean annual precipitation but positively correlated to mean annual temperature. We identified distinct non-randomly assembled atmosphere and soil communities from each location, and some broad trends persisted across biomes including the enrichment of desiccation and UV tolerant taxa in the atmospheric community. Source tracking revealed that local soils were more influential than distant soil sources in determining observed diversity in the atmosphere, with more emissive semi-arid and arid biomes contributing most to signatures from distant soil. Our findings highlight complexities in the atmospheric microbiota that are relevant to understanding regional and global ecosystem connectivity.


Assuntos
Ecossistema , Microbiota , Solo , Bactérias , Atmosfera , Temperatura , Microbiologia do Solo
16.
Mol Ther Nucleic Acids ; 29: 769-786, 2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36159592

RESUMO

Satellite cells (SCs), muscle stem cells, display functional heterogeneity, and dramatic changes linked to their regenerative capabilities are associated with muscle-wasting diseases. SC behavior is related to endogenous expression of the myogenic transcription factor MYF5 and the propensity to enter into the cell cycle. Here, we report a role for miR-106b reinforcing MYF5 inhibition and blocking cell proliferation in a subset of highly quiescent SC population. miR-106b down-regulation occurs during SC activation and is required for proper muscle repair. In addition, miR-106b is increased in dystrophic mice, and intramuscular injection of antimiR in injured mdx mice enhances muscle regeneration promoting transcriptional changes involved in skeletal muscle differentiation. miR-106b inhibition promotes the engraftment of human muscle stem cells. Furthermore, miR-106b is also high in human dystrophic muscle stem cells and its inhibition improves intrinsic proliferative defects and increases their myogenic potential. This study demonstrates that miR-106b is an important modulator of SC quiescence, and that miR-106b may be a new target to develop therapeutic strategies to promote muscle regeneration improving the regenerative capabilities of injured dystrophic muscle.

17.
Microb Genom ; 7(6)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34170814

RESUMO

Herpes simplex virus serotype 2 (HSV-2) is a ubiquitous human pathogen that causes recurrent genital infections and ulcerations. Many HSV-2 strains with different biological properties have been identified, but only the genomes of HSV-2 strains HG52, SD90e and 333 have been reported as complete and fully characterized sequences. We de novo assembled, annotated and manually curated the complete genome sequence of HSV-2 strain MS, a highly neurovirulent strain, originally isolated from a multiple sclerosis patient. We resolved both DNA ends, as well as the complex inverted repeats regions present in HSV genomes, usually undisclosed in previous published partial herpesvirus genomes, using long reads from Pacific Biosciences (PacBio) technology. Additionally, we identified isomeric genomes by determining the alternative relative orientation of unique fragments in the genome of the sequenced viral population. Illumina short-read sequencing was crucial to examine genetic variability, such as nucleotide polymorphisms, insertion/deletions and sequence determinants of strain-specific virulence factors. We used Illumina data to fix two disrupted open reading frames found in coding homopolymers after PacBio assembly. These results support the combination of long- and short-read sequencing technologies as a precise and effective approach for the accurate de novo assembly and curation of complex microbial genomes.


Assuntos
Genoma Viral , Herpesvirus Humano 2/genética , Animais , Chlorocebus aethiops , Herpesvirus Humano 2/classificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Fases de Leitura Aberta , Filogenia , Análise de Sequência de DNA/métodos , Células Vero , Montagem de Vírus , Sequenciamento Completo do Genoma
18.
Acta Trop ; 222: 106053, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34273311

RESUMO

All trypanosomatid genomes are colonized by non-LTR retrotransposons which exhibit a highly conserved 77-nt sequence at their 5' ends, known as the Pr77-hallmark (Pr77). The wide distribution of Pr77 is expected to be related to the gene regulation processes in these organisms as it has promoter and HDV-like ribozyme activities at the DNA and RNA levels, respectively. The identification of Pr77 hallmark-bearing retrotransposons and the study of the associations of mobile elements with relevant genes have been analyzed in the genomes of six strains of Trypanosoma cruzi belonging to different discrete typing units (DTUs) and with different geographical origins and host/vectors. The genomes have been sequenced, assembled and annotated. BUSCO analyses indicated a good quality for the assemblies that were used in comparative analyses. The results show differences among the six genomes in the copy number of genes related to virulence processes, the abundance of retrotransposons bearing the Pr77 sequence and the presence of the Pr77 hallmarks not associated with retroelements. The analyses also show frequent associations of Pr77-bearing retrotransposons and single Pr77 hallmarks with genes coding for trans-sialidases, RHS, MASP or hypothetical proteins, showing variable proportion depending on the type of retroelement, gene class and parasite strain. These differences in the genomic distribution of active retroelements and other Pr77-containing elements have shaped the genome architecture of these six strains and might be contributing to the phenotypic variability existing among them.


Assuntos
Retroelementos , Trypanosoma cruzi , Regulação da Expressão Gênica , Genoma de Protozoário , Regiões Promotoras Genéticas , Retroelementos/genética , Trypanosoma cruzi/genética
19.
Genes (Basel) ; 12(9)2021 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-34573340

RESUMO

Leishmania major is the main causative agent of cutaneous leishmaniasis in humans. The Friedlin strain of this species (LmjF) was chosen when a multi-laboratory consortium undertook the objective of deciphering the first genome sequence for a parasite of the genus Leishmania. The objective was successfully attained in 2005, and this represented a milestone for Leishmania molecular biology studies around the world. Although the LmjF genome sequence was done following a shotgun strategy and using classical Sanger sequencing, the results were excellent, and this genome assembly served as the reference for subsequent genome assemblies in other Leishmania species. Here, we present a new assembly for the genome of this strain (named LMJFC for clarity), generated by the combination of two high throughput sequencing platforms, Illumina short-read sequencing and PacBio Single Molecular Real-Time (SMRT) sequencing, which provides long-read sequences. Apart from resolving uncertain nucleotide positions, several genomic regions were reorganized and a more precise composition of tandemly repeated gene loci was attained. Additionally, the genome annotation was improved by adding 542 genes and more accurate coding-sequences defined for around two hundred genes, based on the transcriptome delimitation also carried out in this work. As a result, we are providing gene models (including untranslated regions and introns) for 11,238 genes. Genomic information ultimately determines the biology of every organism; therefore, our understanding of molecular mechanisms will depend on the availability of precise genome sequences and accurate gene annotations. In this regard, this work is providing an improved genome sequence and updated transcriptome annotations for the reference L. major Friedlin strain.


Assuntos
Genoma de Protozoário/genética , Leishmania major/genética , Cromossomos/genética , Genes de Protozoários , Íntrons , Anotação de Sequência Molecular , Análise de Sequência de DNA/métodos , Sintenia , Transcriptoma
20.
PLoS Negl Trop Dis ; 14(12): e0009004, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33370288

RESUMO

A detailed understanding of the mechanisms underlying the capacity of a virus to break the species barrier is crucial for pathogen surveillance and control. New World (NW) mammarenaviruses constitute a diverse group of rodent-borne pathogens that includes several causative agents of severe viral hemorrhagic fever in humans. The ability of the NW mammarenaviral attachment glycoprotein (GP) to utilize human transferrin receptor 1 (hTfR1) as a primary entry receptor plays a key role in dictating zoonotic potential. The recent isolation of Tacaribe and lymphocytic choriominingitis mammarenaviruses from host-seeking ticks provided evidence for the presence of mammarenaviruses in arthropods, which are established vectors for numerous other viral pathogens. Here, using next generation sequencing to search for other mammarenaviruses in ticks, we identified a novel replication-competent strain of the NW mammarenavirus Tamiami (TAMV-FL), which we found capable of utilizing hTfR1 to enter mammalian cells. During isolation through serial passaging in mammalian immunocompetent cells, the quasispecies of TAMV-FL acquired and enriched mutations leading to the amino acid changes N151K and D156N, within GP. Cell entry studies revealed that both substitutions, N151K and D156N, increased dependence of the virus on hTfR1 and binding to heparan sulfate proteoglycans. Moreover, we show that the substituted residues likely map to the sterically constrained trimeric axis of GP, and facilitate viral fusion at a lower pH, resulting in viral egress from later endosomal compartments. In summary, we identify and characterize a naturally occurring TAMV strain (TAMV-FL) within ticks that is able to utilize hTfR1. The TAMV-FL significantly diverged from previous TAMV isolates, demonstrating that TAMV quasispecies exhibit striking genetic plasticity that may facilitate zoonotic spillover and rapid adaptation to new hosts.


Assuntos
Antígenos CD/metabolismo , Infecções por Arenaviridae/transmissão , Arenaviridae/genética , Receptores da Transferrina/metabolismo , Receptores Virais/metabolismo , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos/genética , Animais , Arenaviridae/isolamento & purificação , Arenavirus do Novo Mundo , Linhagem Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Insetos Vetores/virologia , Alinhamento de Sequência , Carrapatos/virologia , Células Vero , Envelope Viral/metabolismo , Zoonoses/transmissão , Zoonoses/virologia
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