Biology subjects

Alves, M. J.

Publications and source records attributed to Alves, M. J..

7 recordsLinked to original sources

bioRxiv2026

Intra-slide calibration technology improves immunohistochemical harmonization within and between anatomic pathology laboratories

The reproducibility of immunohistochemistry in tumor tissue analysis across reference labs remains a persistent challenge. We tested the extent to which an intra-slide calibration technology mitigated discprepencies in inter-laboratory assays of p53 immunohistochemical (IHC) reactions in brain biopsies of glioblastoma (GB), IDH-wildtype. Intra-slide calibration technologies apply a 0-100% concentration scale incorporating primary surrogate and secondary antibodies to generate a standardized curve for DAB precipitation. IHC from GB samples was performed independently by pathology departments from two different hospital laboratories and were digitalized at 40x magnification using Aperio Image Scope software. Feature extraction, including intensity and texture parameters was performed using the EBImage package in R, followed by UMAP dimensionality reduction and DBSCAN clustering analysis. Our results show significant differences in intensity and texture clustering patterns between laboratory tissue samples and intra-slide calibration technology ruler caused by the different laboratories. Intra-slide calibration technology coupled with polynomial regression analysis improved ~90% the data harmonization. Our findings demonstrate a key role for computational pathology using intra-slide calibration technology to enable intra-laboratory consistency and inter-laboratory reproducibility. These advances strengthen the reproducibility of diagnostic assessments and support more objective, data-driven decision-making in neuro-oncology.

bioinformatics↗

bioRxiv2025

Freshwater fish community assessment using eDNA metabarcoding vs. capture-based methods: differences in efficiency and resolution coupled to habitat and ecology

Environmental DNA (eDNA) metabarcoding has revolutionized ecological and environmental research by describing communities without relying on direct observations, making it a powerful, non-invasive, and cost-effective tool in biodiversity monitoring. However, implementation of eDNA as a standard protocol in long-term monitoring programs, that have traditionally relied on capture-based methods, poses challenges in terms of data comparability. Here, we compared freshwater fish communities assessed through eDNA metabarcoding and electrofishing, across 35 sampling sites in the lower Tagus River basin, Portugal. For most species or species-groups analyzed individually (13 out of 17), there was a significant correspondence between electrofishing and eDNA metabarcoding detections. The correspondence was weaker when comparing the number of specimens captured by electrofishing with the number of eDNA metabarcoding reads, with seven out of 13 taxa showing significant relationships. Species richness estimates based on the two methods were very similar at the basin level. The methods yielded significantly different species compositions, although these differences were driven by samples collected in the Tagus main channel, which is wider and has higher flow rates than tributaries. Benthic and shoreline fish communities showed similar species composition in the two methods, but this was not the case for pelagic communities, probably due to the higher water turnover of the pelagic zone and electrofishing inefficiency. Our results highlight the high potential of eDNA metabarcoding as a complementary method to electrofishing for freshwater fish monitoring, though further validation is needed to assess biases related to site-specific hydrological conditions and the ecology of the target species.

ecology↗

bioRxiv2024

Improving whole biodiversity monitoring and discovery with environmental DNA metagenomics

Environmental DNA (eDNA) metagenomics sequences all DNA molecules present in environmental samples and has the potential of identifying virtually any organism from which they are derived. However, due to unacceptable levels of false positives and negatives, this approach is underexplored as a tool for biodiversity monitoring across the tree of life, particularly for non-microscopic eukaryotes. We present SEQIDIST, a framework that combines multilocus BLAST matches against several reference databases followed by analysis of sequence identity distribution patterns to disentangle false positives while revealing new biodiversity and increasing the accuracy of metagenomic approaches. We tested SEQIDIST on an eDNA metagenomic dataset from a riverine site and compare the results to those obtained with an eDNA metabarcoding approach for benchmarking purposes. We start by characterizing the biological community ([~] 2000 taxa) across the tree of life at low taxonomic levels and show that eDNA metagenomics has a higher sensitivity than eDNA metabarcoding in discovering new diversity. We show that limited representation of whole genome sequences in reference databases can lead to false positives. For non-microscopic eukaryotes, eDNA metagenomic data often consist of a few sparse, anonymous sequences scattered across the genome, making metagenome assembly methods unfeasible. Finally, we infer eDNA source and residency time using read length distributions as a measure of decay status. The higher accuracy of SEQIDIST opens the discussion of the archival potential of eDNA metagenomics and its implementation in biodiversity monitoring actions at large planetary and temporal scales.

ecology↗

bioRxiv2023

Telomerase reverse transcriptase (TERT)-expressing cells mark a novel stem cell population in the adult mouse brain

Telomerase reverse transcriptase (TERT) is expressed by quiescent adult stem cells (ASC) in numerous adult murine and human tissues, but has never been explored in the adult brain. Here, we demonstrate that TERT+ cells in the adult mouse brain represent a novel population of multipotent ASCs that are localized to numerous classical neuro/gliogenic niches (including the ventricular-subventricular zone, hypothalamus, and olfactory bulb), as well as more recently described regions of adult brain plasticity such as the meninges and choroid plexus. Using a direct-reporter mouse line, we found that TERT+ cells expressed known neural stem cell markers such as Nestin and Sox2, but not markers of committed stem/progenitor cells, nor markers of mature neuronal or glial cells. TERT+ ASCs rarely expressed the proliferation marker Ki67, and in vitro TERT+ cells lost TERT expression when activated by growth factors, together indicating a quiescent phenotype similar to what has been observed in other tissues. When cultured, TERT+ cells behaved like neural stem cells by forming neurospheres, which could proliferate and become more metabolically active once stimulated by growth factors. TERT+ cells were observed in numerous brain niches, particularly near the ventricles and cerebrospinal fluid barriers, but notably, TERT+ cells were never observed in the hippocampus. Lineage tracing of TERT+ cells in adult transgenic mice (mTERTrtTA::oTET-Cre::RosamTmG) revealed large-scale expansion of TERT+ progeny and differentiation to diverse cell types in multiple brain regions. For example, lineage-traced cells expressed markers of mature neurons, oligodendrocytes, astrocytes, ependymal cells, and choroid epithelial cells, thus demonstrating the striking multipotency of this stem cell population in basal tissue turnover of the adult brain. Together, these data demonstrate that TERT+ cells represent a novel population of multipotent stem cells that contribute to basal plasticity and regeneration in the adult mouse brain. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/527879v1_ufig1.gif" ALT="Figure 1"> View larger version (94K): org.highwire.dtl.DTLVardef@94c0dborg.highwire.dtl.DTLVardef@185dborg.highwire.dtl.DTLVardef@1fb1e1borg.highwire.dtl.DTLVardef@17580a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Widespread hybridization between invasive bleak (Alburnus alburnus) and native Iberian chubs (Squalius spp.): a neglected threat

Hybridization between native and exotic species is a major conservation issue. In Iberian rivers, which are simultaneously among the most invaded and diverse ecosystems, harboring a high proportion of endemic fishes, this issue is becoming highly concerning. To date, hybridization has been reported to occur between the invasive bleak Alburnus alburnus and the endemic chubs Squalius alburnoides and S. pyrenaicus, in some scattered locations. However, the bleak is increasingly spreading in the region, potentially increasing the risks of hybridization with these and other Squalius species. To gather a more comprehensive picture on the current extension of hybridization, we compiled and mapped records on hybrids between bleak and chubs collected by multiple research teams in different ongoing projects in the Portuguese territory, and genetically assessed the hybrid identity of specimens in an area of sympatry between bleak and S. carolitertii, using nuclear and mitochondrial markers. We found that hybridization with bleak is widespread in several Portuguese river basins and involves at least three endemic chubs, S. alburnoides, S. pyrenaicus and S. carolitertii. This may have serious consequences, including not only waste of reproductive effort and damage of genetic integrity, but also putative shifts on the reproductive dynamics of the Squalius system, which includes endemic hybrids, reproducing sexually and nonsexually. Hybridization should be recognized as a significant impact of biological invasions, and we recommend that future studies should characterize the fitness of hybrids and their ecological and genetic interactions with parental Iberian fish, to elucidate effective conservation measures.

zoology↗

bioRxiv2021

Mutation rate of SARS-CoV-2 and emergence of mutators during experimental evolution

Background and objectivesTo understand how organisms evolve, it is fundamental to study how mutations emerge and establish. Here, we estimated the rate of mutation accumulation of SARS-CoV-2 in vitro and investigated the repeatability of its evolution when facing a new cell type but no immune or drug pressures. MethodologyWe performed experimental evolution with two strains of SARS-CoV-2, one carrying the originally described spike protein (CoV-2-D) and another carrying the D614G mutation that has spread worldwide (CoV-2-G). After 15 passages in Vero cells and whole genome sequencing, we characterized the spectrum and rate of the emerging mutations and looked for evidences of selection across the genomes of both strains. ResultsFrom the mutations accumulated, and excluding the genes with signals of selection, we estimate a spontaneous mutation rate of 1.25x10-6 nt-1 per infection cycle for both lineages of SARS-CoV-2. We further show that mutation accumulation is heterogeneous along the genome, with the spike gene accumulating mutations at rate five-fold higher than the genomic average. We also observe the emergence of mutators in the CoV-2-G background, likely linked to mutations in the RNA-dependent RNA polymerase and/or in the error-correcting exonuclease protein. Conclusions and implicationsThese results provide valuable information on how spontaneous mutations emerge in SARS-CoV-2 and on how selection can shape its genome towards adaptation to new environments. Lay summaryMutation is the ultimate source of variation. We estimated how the SARS-COV-2 virus--cause of the COVID-19 pandemic--mutates. Upon infecting cells, its genome can change at a rate of 0.04 per replication. We also find that this rate can change and that its spike protein can adapt, even within few replications.

evolutionary 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↗