Novel Approaches for Fungal Transcriptomics from Host Samples.

Candida albicans adaptation to the host requires a profound reprogramming of the fungal transcriptome as compared to in vitro laboratory conditions. A detailed knowledge of the C. albicans transcriptome during the infection process is necessary in order to understand which of the fungal genes are important for host adaptation. Such genes could be thought of as potential targets for antifungal therapy. The acquisition of the C. albicans transcriptome is, however, technically challenging due to the low proportion of fungal RNA in host tissues. Two emerging technologies were used recently to circumvent this problem. One consists of the detection of low abundance fungal RNA using capture and reporter gene probes which is followed by emission and quantification of resulting fluorescent signals (nanoString). The other is based first on the capture of fungal RNA by short biotinylated oligonucleotide baits covering the C. albicans ORFome permitting fungal RNA purification. Next, the enriched fungal RNA is amplified and subjected to RNA sequencing (RNA-seq). Here we detail these two transcriptome approaches and discuss their advantages and limitations and future perspectives in microbial transcriptomics from host material

RNA Enrichment Method for Quantitative Transcriptional Analysis of Pathogens In Vivo Applied to the Fungus Candida albicans.

UNLABELLED: In vivo transcriptional analyses of microbial pathogens are often hampered by low proportions of pathogen biomass in host organs, hindering the coverage of full pathogen transcriptome. We aimed to address the transcriptome profiles of Candida albicans, the most prevalent fungal pathogen in systemically infected immunocompromised patients, during systemic infection in different hosts. We developed a strategy for high-resolution quantitative analysis of the C. albicans transcriptome directly from early and late stages of systemic infection in two different host models, mouse and the insect Galleria mellonella. Our results show that transcriptome sequencing (RNA-seq) libraries were enriched for fungal transcripts up to 1,600-fold using biotinylated bait probes to capture C. albicans sequences. This enrichment biased the read counts of only ~3% of the genes, which can be identified and removed based on a priori criteria. This allowed an unprecedented resolution of C. albicans transcriptome in vivo, with detection of over 86% of its genes. The transcriptional response of the fungus was surprisingly similar during infection of the two hosts and at the two time points, although some host- and time point-specific genes could be identified. Genes that were highly induced during infection were involved, for instance, in stress response, adhesion, iron acquisition, and biofilm formation. Of the in vivo-regulated genes, 10% are still of unknown function, and their future study will be of great interest. The fungal RNA enrichment procedure used here will help a better characterization of the C. albicans response in infected hosts and may be applied to other microbial pathogens. IMPORTANCE: Understanding the mechanisms utilized by pathogens to infect and cause disease in their hosts is crucial for rational drug development. Transcriptomic studies may help investigations of these mechanisms by determining which genes are expressed specifically during infection. This task has been difficult so far, since the proportion of microbial biomass in infected tissues is often extremely low, thus limiting the depth of sequencing and comprehensive transcriptome analysis. Here, we adapted a technology to capture and enrich C. albicans RNA, which was next used for deep RNA sequencing directly from infected tissues from two different host organisms. The high-resolution transcriptome revealed a large number of genes that were so far unknown to participate in infection, which will likely constitute a focus of study in the future. More importantly, this method may be adapted to perform transcript profiling of any other microbes during host infection or colonization

Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models.

The aim of the present study was to identify Candida albicans transcription factors (TFs) involved in virulence. Although mice are considered the gold-standard model to study fungal virulence, mini-host infection models have been increasingly used. Here, barcoded TF mutants were first screened in mice by pools of strains and fungal burdens (FBs) quantified in kidneys. Mutants of unannotated genes which generated a kidney FB significantly different from that of wild-type were selected and individually examined in Galleria mellonella. In addition, mutants that could not be detected in mice were also tested in G. mellonella. Only 25% of these mutants displayed matching phenotypes in both hosts, highlighting a significant discrepancy between the two models. To address the basis of this difference (pool or host effects), a set of 19 mutants tested in G. mellonella were also injected individually into mice. Matching FB phenotypes were observed in 50% of the cases, highlighting the bias due to host effects. In contrast, 33.4% concordance was observed between pool and single strain infections in mice, thereby highlighting the bias introduced by the "pool effect." After filtering the results obtained from the two infection models, mutants for MBF1 and ZCF6 were selected. Independent marker-free mutants were subsequently tested in both hosts to validate previous results. The MBF1 mutant showed impaired infection in both models, while the ZCF6 mutant was only significant in mice infections. The two mutants showed no obvious in vitro phenotypes compared with the wild-type, indicating that these genes might be specifically involved in in vivo adapt

Condition-specific series of metabolic sub-networks and its application for gene set enrichment analysis.

Genome-scale metabolic networks and transcriptomic data represent complementary sources of knowledge about an organism's metabolism, yet their integration to achieve biological insight remains challenging. We investigate here condition-specific series of metabolic sub-networks constructed by successively removing genes from a comprehensive network. The optimal order of gene removal is deduced from transcriptomic data. The sub-networks are evaluated via a fitness function, which estimates their degree of alteration. We then consider how a gene set, i.e. a group of genes contributing to a common biological function, is depleted in different series of sub-networks to detect the difference between experimental conditions. The method, named metaboGSE, is validated on public data for Yarrowia lipolytica and mouse. It is shown to produce GO terms of higher specificity compared to popular gene set enrichment methods like GSEA or topGO. The metaboGSE R package is available at https://CRAN.R-project.org/package=metaboGSE. Supplementary data are available at Bioinformatics online

Effects of the functional HOTAIR rs920778 and rs12826786 genetic variants in glioma susceptibility and patient prognosis

Abnormal expression of the long non-coding RNA HOX transcript antisense intergenic RNA (HOTAIR) is oncogenic in several human cancers, including gliomas. The HOTAIR single nucleotide polymorphisms (SNPs) rs920778 (C > T) and rs12826786 (C > T) present in the intronic enhancer and promoter regions of HOTAIR, respectively, are associated with expression, cancer susceptibility, and patient prognosis in some tumor types. However, the relevance of these HOTAIR SNPs has not been studied in glioma. Here, we report a case-control study comprising 177 Portuguese glioma patients and 199 cancer-free controls. All subjects were genotyped by PCR and restriction fragment length polymorphism (RFLP). No statistically significant differences were found in the genotype or allele distributions of either rs920778 or rs12826786 between glioma patients and controls, suggesting these SNPs are not associated with glioma risk. No significant associations were found between rs920778 variants and HOTAIR expression levels, while rs12826786 CT genotype was associated with increased intratumoral HOTAIR RNA levels when compared to TT genotype (p-value = 0.04). Univariate (Log-rank) and multivariate (Cox proportional) analyses showed both rs920778 CT and rs12826786 CT genotypes were significantly associated with longer overall survival of WHO grade III anaplastic oligodendroglioma patients. Our results suggest that HOTAIR SNPs rs920778 and rs12826786 do not play a significant role in glioma susceptibility, but may be important prognostic factors in anaplastic oligodendroglioma patients. Future studies are warranted to validate and expand these findings, and to further dissect the importance of these SNPs in glioma.info:eu-repo/semantics/publishedVersio

Genetic diversity of carotenoid-rich bananas evaluated by Diversity Arrays Technology (DArT)

The aim of this work was to evaluate the carotenoid content and genetic variability of banana accessions from the Musa germplasm collection held at Embrapa Cassava and Tropical Fruits, Brazil. Forty-two samples were analyzed, including 21 diploids, 19 triploids and two tetraploids. The carotenoid content was analyzed spectrophotometrically and genetic variability was estimated using 653 DArT markers. The average carotenoid content was 4.73 μg.g -1 , and ranged from 1.06 μg.g -1 for the triploid Nanica (Cavendish group) to 19.24 μg.g -1 for the triploid Saney. The diploids Modok Gier and NBA-14 and the triploid Saney had a carotenoid content that was, respectively, 7-fold, 6-fold and 9-fold greater than that of cultivars from the Cavendish group (2.19 μg.g -1). The mean similarity among the 42 accessions was 0.63 (range: 0.24 to 1.00). DArT analysis revealed extensive genetic variability in accessions from the Embrapa Musa germplasm bank

Birth defects in newborns and stillborns: an example of the Brazilian reality

<p>Abstract</p> <p>Background</p> <p>This study constitutes a clinical and genetic study of all newborn and stillborn infants with birth defects seen in a period of one year in a medical school hospital located in Brazil. The aims of this study were to estimate the incidence, causes and consequences of the defects.</p> <p>Methods</p> <p>For all infants we carried out physical assessment, photographic records, analysis of medical records and collection of additional information with the family, besides the karyotypic analysis or molecular tests in indicated cases.</p> <p>Result</p> <p>The incidence of birth defects was 2.8%. Among them, the etiology was identified in 73.6% (ci95%: 64.4-81.6%). Etiology involving the participation of genetic factors single or associated with environmental factors) was more frequent 94.5%, ci95%: 88.5-98.0%) than those caused exclusively by environmental factors (alcohol in and gestational diabetes mellitus). The conclusive or presumed diagnosis was possible in 85% of the cases. Among them, the isolated congenital heart disease (9.5%) and Down syndrome (9.5%) were the most common, followed by gastroschisis (8.4%), neural tube defects (7.4%) and clubfoot (5.3%). Maternal age, parental consanguinity, exposure to teratogenic agents and family susceptibility were some of the identified risk factors. The most common observed consequences were prolonged hospital stays and death.</p> <p>Conclusions</p> <p>The current incidence of birth defects among newborns and stillbirths of in our population is similar to those obtained by other studies performed in Brazil and in other underdeveloped countries. Birth defects are one of the major causes leading to lost years of potential life. The study of birth defects in underdeveloped countries should continue. The identification of incidence, risk factors and consequences are essential for planning preventive measures and effective treatments.</p

Omega-3 supplementation from pregnancy to postpartum to prevent depressive symptoms: a randomized placebo-controlled trial

Background: Low n-3 polyunsaturated fatty acids (PUFAs) has been linked to depression, but the preventive effect of n-3PUFAs supplementation on maternal depression needs further investigation. We aimed to evaluate the efficacy of a daily dose of n-3 PUFAs supplementation (fish oil) on the prevention of postpartum depression (PPD). Methods: A randomized, placebo-controlled, double blind trial was designed and nested into a cohort study conducted in Rio de Janeiro, Brazil. Sixty pregnant women identified as being at risk for PPD were invited and randomly assigned to receive fish oil capsules [1.8 g (1.08 g of Eicosapentaenoic (EPA) and 0.72 g of Docosapentaenoic (DHA) acids)] or placebo (control). The Edinburgh Postnatal Depression Scale (EPDS) was scored at 5–13 (T0, baseline), 22–24 (T1), 30–32 weeks of gestation (T2) and 4–6 weeks’ postpartum (T3). Supplementation started at week 22–24 of gestation (T1) and lasted for 16 weeks. Serum fatty acids were assayed to evaluate compliance. Prevalence of EPDS ≥11 was the primary outcome, and mean and changes in EPDS score, length of gestation, and birth weight the secondary outcomes. Linear mixed-effect (LME) and random-intercept logistic regression models were performed to test the effect of fish oil supplementation on prevalence of EPDS ≥11 and EPDS scores variation. Results: In intention-to-treat (ITT) analysis, at 30–32 weeks’ gestation women in the fish oil presented higher serum concentration of EPA, DHA and lower n-6/n-3 ratio comparing to the control group. There were no differences between intervention and control groups in the prevalence of EPDS ≥11, EPDS scores over time, or in changes in EPDS scores from pregnancy to postpartum in either the ITT or per-protocol analyses. Women in the fish oil group with previous history of depression presented a higher reduction on the EPDS score from the second to the third trimester in the fish oil comparing to the control group in the ITT analyses [−1.0 (−3.0–0.0) vs. -0.0 (−1.0–3.0), P = 0.038). These results were confirmed on the LME model (β = −3.441; 95%CI: -6.532– -0.350, P = 0.029). Conclusion: Daily supplementation of 1.8 g of n-3 PUFAs during 16 weeks did not prevent maternal depressive symptoms in a sample of Brazilian women