miR-6087 Might Regulate Cell Cycle-Related mRNAs During Cardiomyogenesis of hESCs.

MicroRNAs (miRNAs) are small noncoding RNAs that act as negative regulators of gene expression at the post-transcriptional level, promoting mRNA degradation or translation repression. Despite the well-described presence of miRNAs in various human tissues, there is still a lack of information about the relationship between miRNAs and the translation regulation in human embryonic stem cells (hESCs) during cardiomyogenesis. Here, we investigate RNA-seq data from hESCs, focusing on distinct stages of cardiomyogenesis and searching for polysome-bound miRNAs that could be involved in translational regulation. We identify miR-6087 as a differentially expressed miRNA at latest steps of cardiomyocyte differentiation. We analyzed the coexpression pattern between the differentially expressed mRNAs and miR-6087, evaluating whether they are predicted targets of the miRNA. We arranged the genes into an interaction network and identified BLM, RFC4, RFC3, and CCNA2 as key genes of the network. A post hoc analysis of the key genes suggests that miR-6087 could act as a regulator of the cell cycle in hESC during cardiomyogenesis.

Genotypic Diversity of Candida parapsilosis Complex in Invasive Candidiasis at a Pediatric Tertiary Hospital: A 5-Year Retrospective Study.

Invasive candidiasis (IC) contributes to the morbidity and mortality of hospitalized patients and represents a significant burden to the healthcare system. Previous Brazilian studies have reported the presence of endemic Candida parapsilosis sensu stricto genotypes causing candidemia and clonal transmission involving fluconazole-resistant isolates. We performed a 5-year retrospective analysis of IC cases in a Brazilian tertiary pediatric hospital and conducted a molecular investigation of C. parapsilosis sensu stricto. Non-duplicate C. parapsilosis sensu stricto genotyping was performed by microsatellite analysis. Antifungal susceptibility and biofilm formation were also evaluated. A total of 123 IC episodes were identified, with an IC incidence of 1.24 cases per 1000 hospital admissions and an overall mortality of 34%. The main species were the C. parapsilosis complex (35.8%), Candida albicans (29.2%), and Candida tropicalis (21.9%). All C. parapsilosis sensu stricto were recovered from blood cultures, and 97.5% were biofilm producers. Microsatellite typing identified high genotypic diversity among the isolates. We observed that all isolates were sensitive to amphotericin B, and although one isolate was non-sensitive to fluconazole, only a silent mutation on ERG11 gene was identified. No clear evidence of clonal outbreak or emergence of fluconazole-resistant isolates was found, suggesting that multiple sources may be involved in the epidemiology of IC in children.

Dysregulation of Transcription Factor Networks Unveils Different Pathways in Human Papillomavirus 16-Positive Squamous Cell Carcinoma and Adenocarcinoma of the Uterine Cervix.

Squamous cell carcinoma (SCC) and adenocarcinoma (ADC) are the most common histological types of cervical cancer (CC). The worse prognosis of ADC cases highlights the need for better molecular characterization regarding differences between these CC types. RNA-Seq analysis of seven SCC and three ADC human papillomavirus 16-positive samples and the comparison with public data from non-tumoral human papillomavirus-negative cervical tissue samples revealed pathways exclusive to each histological type, such as the epithelial maintenance in SCC and the maturity-onset diabetes of the young (MODY) pathway in ADC. The transcriptional regulatory network analysis of cervical SCC samples unveiled a set of six transcription factor (TF) genes with the potential to positively regulate long non-coding RNA genes DSG1-AS1, CALML3-AS1, IGFL2-AS1, and TINCR. Additional analysis revealed a set of MODY TFs regulated in the sequence predicted to be repressed by miR-96-5p or miR-28-3p in ADC. These microRNAs were previously described to target LINC02381, which was predicted to be positively regulated by two MODY TFs upregulated in cervical ADC. Therefore, we hypothesize LINC02381 might act by decreasing the levels of miR-96-5p and miR-28-3p, promoting the MODY activation in cervical ADC. The novel TF networks here described should be explored for the development of more efficient diagnostic tools.

Nucleosome landscape reflects phenotypic differences in Trypanosoma cruzi life forms.

Trypanosoma cruzi alternates between replicative and nonreplicative life forms, accompanied by a shift in global transcription levels and by changes in the nuclear architecture, the chromatin proteome and histone posttranslational modifications. To gain further insights into the epigenetic regulation that accompanies life form changes, we performed genome-wide high-resolution nucleosome mapping using two T. cruzi life forms (epimastigotes and cellular trypomastigotes). By combining a powerful pipeline that allowed us to faithfully compare nucleosome positioning and occupancy, more than 125 thousand nucleosomes were mapped, and approximately 20% of them differed between replicative and nonreplicative forms. The nonreplicative forms have less dynamic nucleosomes, possibly reflecting their lower global transcription levels and DNA replication arrest. However, dynamic nucleosomes are enriched at nonreplicative regulatory transcription initiation regions and at multigenic family members, which are associated with infective-stage and virulence factors. Strikingly, dynamic nucleosome regions are associated with GO terms related to nuclear division, translation, gene regulation and metabolism and, notably, associated with transcripts with different expression levels among life forms. Finally, the nucleosome landscape reflects the steady-state transcription expression: more abundant genes have a more deeply nucleosome-depleted region at putative 5' splice sites, likely associated with trans-splicing efficiency. Taken together, our results indicate that chromatin architecture, defined primarily by nucleosome positioning and occupancy, reflects the phenotypic differences found among T. cruzi life forms despite the lack of a canonical transcriptional control context.

The impact of blood-processing time on the proteome of human peripheral blood mononuclear cells.

Human peripheral blood mononuclear cells (PBMC) are key to several diagnostics assays and basic science research. Blood pre-analytical variations that occur before obtaining the PBMC fraction can significantly impact the assays results, including viability, composition, integrity, and gene expression changes of immune cells. With this as motivation, we performed a quantitative shotgun proteomics analysis using Isobaric Tag for Relative and Absolute Quantitation (iTRAQ 8plex) labeling to compare PBMC obtained from 24 h-stored blood at room temperature versus freshly isolated. We identified a total of 3195 proteins, of which 245 were differentially abundant (101 upregulated and 144 downregulated). Our results revealed enriched pathways of downregulated proteins related to exocytosis, localization, vesicle-mediated transport, cell activation, and secretion. In contrast, pathways related to exocytosis, neutrophil degranulation and activation, granulocyte activation, leukocyte degranulation, and myeloid leukocyte activation involved in immune response were enriched in upregulated proteins, which may indicate probable granulocyte contamination and activation due to blood storage time and temperature. Examples of upregulated proteins in the 24 h-PBMC samples are CAMP, S100A8, LTA4H, RASAL3, and S100A6, which are involved in an adaptive immune system and antimicrobial activity, proinflammatory mediation, aminopeptidase activities, and naïve T cells survival. Moreover, examples of downregulated proteins are NDUFA5, TAGLN2, H3C1, TUBA8, and CCT2 that are related to the cytoskeleton, cell junction, mitochondrial respiratory chain. In conclusion, the delay in blood-processing time directly impacts the proteomic profile of human PBMC, possibly through granulocyte contamination and activation.

Ribosome profiling reveals translation control as a key mechanism generating differential gene expression in Trypanosoma cruzi.

BACKGROUND: Due to the absence of transcription initiation regulation of protein coding genes transcribed by RNA polymerase II, posttranscriptional regulation is responsible for the majority of gene expression changes in trypanosomatids. Therefore, cataloging the abundance of mRNAs (transcriptome) and the level of their translation (translatome) is a key step to understand control of gene expression in these organisms. RESULTS: Here we assess the extent of regulation of the transcriptome and the translatome in the Chagas disease causing agent, Trypanosoma cruzi, in both the non-infective (epimastigote) and infective (metacyclic trypomastigote) insect's life stages using RNA-seq and ribosome profiling. The observed steady state transcript levels support constitutive transcription and maturation implying the existence of distinctive posttranscriptional regulatory mechanisms controlling gene expression levels at those parasite stages. Meanwhile, the downregulation of a large proportion of the translatome indicates a key role of translation control in differentiation into the infective form. The previously described proteomic data correlate better with the translatomes than with the transcriptomes and translational efficiency analysis shows a wide dynamic range, reinforcing the importance of translatability as a regulatory step. Translation efficiencies for protein families like ribosomal components are diminished while translation of the transialidase virulence factors is upregulated in the quiescent infective metacyclic trypomastigote stage. CONCLUSIONS: A large subset of genes is modulated at the translation level in two different stages of Trypanosoma cruzi life cycle. Translation upregulation of virulence factors and downregulation of ribosomal proteins indicates different degrees of control operating to prepare the parasite for an infective life form. Taking together our results show that translational regulation, in addition to regulation of steady state level of mRNA, is a major factor playing a role during the parasite differentiation.

[Allochthonous cases of canine visceral leishmaniasis in Paraná, Brazil: epidemiological implications]. / Casos alóctones de leishmaniose visceral canina no Paraná, Brasil: implicações epidemiológicas.

The south region is the only area in Brazil that does not present autochthonous cases of visceral leishmaniasis (VL), however, in the state of Paraná, dogs and humans have been found showing a VL compatible clinical profile. In view of this problem, the present work aimed at isolating and identifying the parasite and determining the cases autochthony. All animals clinically suspect of VL were clinically evaluated, and had samples of their blood collected for hemoculture (NNN culture medium), serology, PCR and RAPD-PCR, hemogram, and biochemical assays. The dogs presenting VL clinical profile had their lymph nodes and/or bone marrow punctured and their content inoculated in NNN culture medium. The protozoan isolated was identified by PCR and PCR-RAPD. Strains of Leishmania were isolated in 19 out of the 24 studied animals. Fourteen isolates were identified as L. (Leishmania) infantum, and five were L. (Viannia) braziliensis. In the epidemiological analysis, it was possible to determine that all dogs with L. (L.) infantum being allochthonous cases. Leishmaniasis is a zoonose that has the domestic dog as reservoir, the migration of such animals can disseminate the parasite to other regions, provided the agent finds an adequate ecotope and a specific vector (Lutzomyia longipalpis).

Casos alóctones de leishmaniose visceral canina no Paraná, Brasil: implicações epidemiológicas / Allochthonous cases of canine visceral leishmaniasis in Paraná, Brazil: epidemiological implications

O presente trabalho teve como objetivo pesquisar a ocorrência de leishmaniose visceral em cães com sinais clínicos compatíveis, procedentes de clínicas veterinárias das diferentes regiões do Estado do Paraná e em caso positivo verificar a autoctonia dos mesmos. Dos animais com suspeita clínica e epidemiológica de LV, foi coletada amostra de sangue para realização de hemograma, provas bioquímicas, sorologia, cultura do parasito, PCR e RAPD. Os cães portadores de quadro clínico, como febre, hiperqueratose, onicogrifose e emagrecimento exagerado, tiveram gânglio poplíteo e/ou medula óssea puncionados e o conteúdo inoculado em meio de cultivo NNN. A extração de DNA do parasito em sangue e cultura foi realizada pelo método fenol/clorofórmio. A amplificação de DNA do protozoário foi feita por PCR e RAPD. Dos 24 animais analisados, o parasito foi isolado em 19 cães. As técnicas moleculares permitiram identificar 14 isolados como L. (Leihmania) infantum e cinco como L. (Viannia) braziliensis. A análise epidemiológica dos casos permitiu determinar o local de transmissão e definir que todos os cães com LV diagnosticados eram casos alóctones, ou seja eram importados de regiões endêmicas. Sugere-se que a migração dos cães de regiões endêmicas para regiões indenes poderá permitir a instalação de novos focos, desde que o agente encontre ecótopo adequado e vetor específico (Lutzomyia longipalpis).

The south region is the only area in Brazil that does not present autochthonous cases of visceral leishmaniasis (VL), however, in the state of Parana, dogs and humans have been found showing a VL compatible clinical profile. In view of this problem, the present work aimed at isolating and identifying the parasite and determining the cases autochthony. All animals clinically suspect of VL were clinically evaluated, and had samples of their blood collected for hemoculture (NNN culture medium), serology, PCR and RAPD-PCR, hemogram, and biochemical assays. The dogs presenting VL clinical profile had their lymph nodes and/or bone marrow punctured and their content inoculated in NNN culture medium. The protozoan isolated was identified by PCR and PCR-RAPD. Strains of Leishmania were isolated in 19 out of the 24 studied animals. Fourteen isolates were identified as L. (Leishmania) infantum, and five were L. (Viannia) braziliensis. In the epidemiological analysis, it was possible to determine that all dogs with L. (L.) infantum being allochthonous cases. Leishmaniasis is a zoonose that has the domestic dog as reservoir, the migration ofsuch animals can disseminate the parasite to other regions, provided the agent finds an adequate ecotope and a specific vector (Lutzomyia longipalpis).