Biology subjects

Machado, J.

Publications and source records attributed to Machado, J..

6 recordsLinked to original sources

bioRxiv2024

Gut microbiota and type 2 diabetes associations: A meta-analysis of 16s studies and their methodological challenges

Diabetes mellitus is a prevalent chronic non-communicable disease, and recent studies have explored the link between gut microbiota and its development. Despite some evidence suggesting an association, the influence of gut microbiota on type 2 diabetes DM2 remains unclear. A systematic search of PubMed Janeiro 2016- 10 December 2023 using the keywords "16S" and "diabetes" or "DM2" and "gut microbiota" and "diabetes" or "DM2". The studies included compared gut microbiome diversity between diabetic and non-diabetic adults using 16S rRNA sequencing, excluding children, interventions, and type 1 diabetes. Alpha diversity indices and bacterial mean abundance were analyzed, with statistical assessments using a random-effects model and I2 for heterogeneity. Thirteen studies met the criteria, with the Shannon index being the most commonly used measure. Results showed significant heterogeneity I2 > 75% and no notable differences between diabetic and non-diabetic groups. Other indices, such as Chao1 and phylogenetic whole tree, similarly showed no consistent differences. Taxonomic analysis also failed to find phyla consistently correlated with DM2, with variability across studies. The relationship between gut microbiota and diabetes remains uncertain due to technical and biological factors that are often overlooked. The inconsistencies across studies highlight the low reproducibility common in microbiota research.

bioinformatics↗

bioRxiv2023

Unravelling the regulatory function of a short 3'UTR sequence in zebrafish and in human cells

The mechanisms of gene expression regulation are essential for cell identity and function, and their disruption usually leads to human disease. The 3 untranslated region (UTR) of mRNA contains important regulatory elements of gene expression, including upstream sequence elements (USEs) that are cis-regulatory sequences localized upstream of polyA signals (PAS). One of the best functionally characterised USEs is located in the 3UTR of the Drosophilas polo gene, which disruption leads to critical phenotypes in adult flies. In this work we found that the USE of the Drosophilas polo gene (DplUSE) is also found in in the 3UTR of vertebrate genes, including zebrafish, mouse and human genes, showing higher levels of conservation than the whole 3UTR sequence. Using reporter assays, we show that DplUSE is able to increase gene expression in vitro in human cell lines and in vivo in zebrafish embryos. Importantly, in humans, the DplUSE containing genes are enriched for genes associated to serious diseases such as Congenital abnormalities and Malignant neoplasms, illustrating the potential of this sequence to modulate genes with relevant biological functions and related with human health. Concomitantly, when sequestering the molecular machinery that operates at the DplUSE using a dominant negative strategy, we show that this is enough to dysregulate DplUSE containing genes in human cells and disrupt proper embryo development in zebrafish. Aiming to understand the molecular mechanism operating at the DplUSE, we identified three RNA binding proteins (RBP) that specifically bind to the DplUSE in vertebrates. Importantly, one of such RBPs is PTBP1, the vertebrate orthologue of the fruit flys RBP Heph, that was demonstrated to be required for the DplUSE function in Drosophila. To test if PTBP1is essential for the DplUSE function, as observed in Drosophila, we depleted PTBP1 from human cells and observed a downregulation of the expression of DplUSE containing genes, demonstrating that the molecular mechanisms that operate at DplUSE are ultra-conserved. Finally, we explored if variants in DplUSE consensus could be associated to human disease. We found a reported single nucleotide polymorphism (SNP; rs3087967) that is associated with malignant tumor of colon and generates an ectopic consensus of DplUSE in the 3 UTR of the tumorigenic POU2AF2/C11orf53 gene. We further show that this ectopic DplUSE motif causes a gain-of-function in vivo in zebrafish gut cells, suggesting its involvement in colon cancer development. These results show that a short motif present in the 3UTR of genes from phylogenetically distant bilaterians, from fruit flies to humans, control genes expression through an ultra-conserved mechanism involving RBPs binding and its dysregulation might impact in human disease.

molecular biology↗

bioRxiv2023

Gadusol is a maternally provided sunscreen that protects fish embryos from DNA damage

Ultraviolet radiation (UVR) and its deleterious effects on living cells selects for UVR-protective mechanisms. Organisms across the tree of life evolved a variety of natural sunscreens to prevent UVR-induced cellular damage and stress. However, in vertebrates, only melanin is known to act as a sunscreen. Here we demonstrate that gadusol, a transparent compound discovered over 40 years ago in fish eggs, is a maternally provided sunscreen required for survival of embryonic and larval zebrafish exposed to UVR. Mutating an enzyme involved in gadusol biosynthesis increases the formation of cyclobutane pyrimidine dimers, a hallmark of UVB-induced DNA damage. Compared to the contributions of melanin and the chorion, gadusol is the primary sunscreening mechanism in embryonic and larval fish. The gadusol biosynthetic pathway is retained in the vast majority of teleost genomes but is repeatedly lost in species whose young are no longer exposed to UVR. Our data demonstrate that gadusol is a maternally provided sunscreen that is critical for early-life survival in the most species-rich branch of the vertebrate phylogeny.

developmental biology↗

bioRxiv2020

Endogenous Galectin-3 is Required for Skeletal Muscle Repair

Skeletal muscle has the intrinsic ability to self-repair through a multifactorial process, but many aspects of its cellular and molecular mechanisms are not fully understood. There is increasing evidence that some members of the mammalian {beta}-galactoside-binding protein family (galectins) are involved in the muscular repair process (MRP), including galectin-3 (Gal-3). However, there are many questions about the role of this protein on muscle self-repair. Here, we demonstrate that endogenous Gal-3 is required for: i) muscle repair in vivo using a chloride-barium myolesion mouse model, and ii) mouse primary myoblasts myogenic programming. Injured muscle from Gal-3 knockout mice (GAL3KO) showed persistent inflammation associated with compromised muscle repair and the formation of fibrotic tissue on the lesion site. In GAL3KO mice, osteopontin expression remained high even after 7 and 14 days of the myolesion, while MyoD and myogenin had decreased their expression. In GAL3KO mouse primary myoblast cell culture, Pax7 detection seems to sustain even when cells are stimulated to differentiation and MyoD expression is drastically reduced. These findings suggest that the detection and temporal expression levels of these transcriptional factors appear to be altered in Gal-3-deficient myoblast cell culture compared to Wild Type (WT) cells. We observed Gal-3 expression in WT states, both in vivo and in vitro, in sarcoplasm/cytoplasm and myonuclei; as differentiation proceeds, Gal-3 expression is drastically reduced, and its location is confined to the sarcolemma/plasma cell membrane. We also observed a change in the temporal-spatial profile of Gal-3 expression and muscle transcription factors levels during the myolesion. Overall, these results demonstrate that endogenous Gal-3 is required for the skeletal muscle repair process.

cell biology↗

bioRxiv2020

Mitogenome diversity of Aedes (Stegomyia) albopictus: Detection of multiple introduction events in Portugal and potential within-country dispersal

Aedes albopictus, along with Ae. aegypti, are key arbovirus vectors that have been expanding their geographic range over the last decades. In 2017, Ae. albopictus was detected for the first time at two distinct locations in Portugal. In order to understand how the Ae. albopictus populations recently introduced in Portugal are genetically related and which is their likely route of invasion, we performed an integrative cytochrome C oxidase I gene (COI)- and mitogenome-based phylogeographic analysis of mosquitoes samples collected in Portugal in 2017 and 2018 in the context of the global Ae. albopictus diversity. COI-based analysis (31 partial sequences obtained from 83 mosquitoes) revealed five haplotypes (1 to 5), with haplotype 1 (which is widely distributed in temperate areas worldwide) being detected in both locations. Haplotypes 2 and 3 were exclusively found in Southern region (Algarve), while haplotype 4 and 5 were only detected in the North of Portugal (Penafiel, Oporto region). Subsequent high discriminatory analyses based on Ae. albopictus mitogenome (17 novel sequences) not only confirmed a high degree of genetic variability within and between populations at both geographic locations (compatible with the Ae. albopictus mosquito populations circulating in Europe), but also revealed two mitogenome mutational signatures not previously reported at worldwide level. While our results generally sustain the occurrence of multiple introduction events, fine mitogenome sequence inspection further indicates a possible Ae. albopictus migration within the country, from the Northern introduction locality to the Southern region. In summary, the observed scenario of high Ae. albopictus genetic diversity in Portugal, together with the detection of mosquitoes in successive years since 2017 in Algarve and Penafiel, points that both Ae. albopictus populations seem to be already locally establish, as its presence has been reported for three consecutive years, raising the public health awareness for future mosquito-borne diseases outbreaks. Author SummaryIn 2017, Aedes albopictus was reported for the first time in Portugal at two distinct locations, in the premises of a tyre company in Penafiel, in the North, and nearby a golf course in Algarve, a tourism destination in the southernmost country region. The geographical spread of this species is boosted by larvae and desiccation-resistant eggs transport in aquatic trade goods, as tires and aquatic plants, and adult anthropophilic behavior that favors passive land transportation. In Portugal, especially in the Southern region, temperate climate conditions are adequate for adult mosquitoes survival most of the year. In a way to understand the genetic variability of Ae. albopictus populations introduced in Portugal, we analyzed 31 cytochrome C oxidase I gene (COI) partial sequences and 17 mitogenome sequences, integrating them in the context of the global Ae. albopictus phylogeographic diversity (i.e., 183 COI and 26 mitogenome sequences previously reported at worldwide level). Although COI haplotype 1 predominated, four additional haplotypes (2 to 5) were detected in Portugal. Subsequent in-depth mitogenome analysis revealed considerable genetic diversity, including not only sequences relating to mitogenomes reported mainly from Italy, Japan and China, but also two novel mitogenome mutational signatures. Our study indicate that Ae. albopictus is locally established in Portugal and intra-country dispersal may have already happened, highlighting the challenges for vector surveillance and control programs aiming at restraining arbovirus disease burden in the future.

genomics↗

bioRxiv2019

Alternative splicing regulation by GWAS risk loci for breast cancer

Recent genome-wide association studies (GWAS) have revealed the association of hundreds of single nucleotide polymorphisms (SNPs) with breast cancer (BC) risk, which mostly locate in non-coding regions, suggesting regulatory roles to the causal variants. Functional characterisation of GWAS loci has been biased towards the effect of regulatory SNPs on transcription factor binding. Here we set out to determine the extent of the contribution of breast cancer risk-associated SNPs to alternative splicing (AS).\n\nWe screened genome-wide significant (P [≤] 5 x 10-8) BC risk SNPs for association with AS, using expression and genotype data from normal breast samples, from the GTEx project. We identified four splicing quantitative trait loci (sQTL). In locus 6p22.1, rs6456883 is a significant cis-sQTL for the expression of ZNF311 gene isoforms. Three SNPs in locus 8p23.3, rs6682326, rs3008282 and rs2906324, were also identified as significant cis-sQTLs/svQTLs for the expression of RPL23AP53 gene isoforms. In-silico functional analysis revealed that these variants can potentially alter enhancer splicing elements within the target genes.\n\nOur work shows that BC risk-associated variants at two loci are associated with AS isoforms in normal breast tissue, thus demonstrating that AS plays an important role in breast cancer susceptibility. Furthermore, it supports that all cis-regulatory mechanisms should be considered in the functional characterisation of risk loci.

cancer biology↗