Biology subjects

Carvalho, G.

Publications and source records attributed to Carvalho, G..

13 recordsLinked to original sources

bioRxiv2024

The specific AMPK activator A-769662 ameliorates pathological phenotypes following mitochondrial DNA depletion

AMP-activated protein kinase (AMPK) is a master regulator of cellular energy homeostasis that also plays a role in preserving mitochondrial function and integrity. Upon a disturbance in the cellular energy state that increases AMP levels, AMPK activity promotes a switch from anabolic to catabolic metabolism to restore energy homeostasis. However, it is currently unclear how severe of a mitochondrial dysfunction is required to trigger AMPK activation, and whether stimulation of AMPK using specific agonists can improve the cellular phenotype following mitochondrial dysfunction. Using a cell model of mitochondrial disease characterized by progressive mitochondrial DNA (mtDNA) depletion and deteriorating mitochondrial metabolism, we show that mitochondria-associated AMPK becomes activated early in the course of the advancing mitochondrial dysfunction, before any quantifiable decrease in the ATP/(AMP+ADP) ratio or respiratory chain activity. Moreover, stimulation of AMPK activity using the specific small-molecule agonist A-769662 alleviated the mitochondrial phenotypes caused by the mtDNA depletion and restored normal mitochondrial membrane potential. Notably, the agonist treatment was able to partially restore mtDNA levels in cells with severe mtDNA depletion, while it had no impact on mtDNA levels of control cells. The beneficial impact of the agonist was also observed in cells from patients suffering from mtDNA depletion. However, the positive effects of A-769662 in the two experimental cell models appeared to involve at least partially different mechanisms. These findings improve our understanding of the effects of specific small-molecule activators of AMPK on mitochondrial and cellular function, and suggest a potential utility for these compounds in disease states involving mtDNA depletion.

molecular biology↗

bioRxiv2023

Manipulation of natural transformation by AbaR-type islands promotes fixation of antibiotic resistance in Acinetobacter baumannii populations

The opportunistic pathogen Acinetobacter baumannii, a major global public health concern, has developed multiple variants of AbaR-type genomic islands that confer multidrug resistance. The mechanisms facilitating the persistence of these potentially costly islands within A. baumannii populations have remained enigmatic. In this study, we employed a combination of investigative methods to shed light on the factors contributing to their selective advantage and long-term persistence. The dissemination of AbaR islands is intricately linked to their horizontal transfer via natural transformation, a process through which bacteria can import and recombine exogenous DNA, facilitating allelic recombination, genetic acquisition, and deletion. In experimental populations, we first quantified the rate at which natural transformation events occur between individuals. Our findings indicate that the rate of AbaR deletion events is marginally higher than the rate of their acquisition. When this result is integrated into a model of population dynamics not exposed to antibiotic selection pressure, it leads to the swift removal of AbaRs from the population, a pattern that stands in contrast to AbaR prevalence in genomes. Yet, genomic analyses show that nearly all AbaRs-carrying A. baumannii have at least one AbaR disrupting comM, a gene encoding a helicase critical for natural transformation. We discovered that such disruption differentially inhibits the rate of genetic acquisition and deletion. Specifically, they significantly impede the removal of AbaRs while still enabling the host cell to acquire and recombine short sequences, such as allelic variants. Through mathematical evolutionary modeling, we demonstrate that AbaRs inserted into comM gain a selective advantage over AbaRs inserted in sites that do not inhibit or completely inhibit transformation, in line with the genomic observations. The persistence of AbaRs within populations can be ascribed to their targeted integration into a gene, substantially diminishing the likelihood of their removal from the bacterial genome. In contrast, this integration enables the host cell to preserve the ability to acquire and eliminate various short heterologous sequences, enabling the host bacterium - and thus its AbaR - to adapt to the unpredictability of the environment and persist over the long term. This work underscores how AbaRs, and potentially other Mobile Genetic Elements (MGEs), can manipulate natural transformation to ensure their persistence in populations, ultimately leading to the high prevalence of multidrug resistance.

microbiology↗

bioRxiv2023

Comparative population genomics of manta rays has global implications for management

Understanding population connectivity and genetic diversity is of fundamental importance to conservation. However, in globally threatened marine megafauna, challenges remain due to their elusive nature and wide-ranging distributions. As overexploitation continues to threaten biodiversity across the globe, such knowledge gaps compromise both the suitability and effectiveness of management actions. Here, we use a comparative framework to investigate genetic differentiation and diversity of manta rays, one of the most iconic yet vulnerable groups of elasmobranchs on the planet. Despite their recent divergence, we show how oceanic manta rays (Mobula birostris) display significantly higher heterozygosity than reef manta rays (Mobula alfredi) and that M. birostris populations display higher connectivity worldwide. Through inferring modes of colonisation, we reveal how both contemporary and historical forces have likely influenced these patterns, with important implications for population management. Our findings highlight the potential for fisheries to disrupt population dynamics at both local and global scales and therefore have direct relevance for international conservation of marine species.

evolutionary biology↗

bioRxiv2022

CRAM compression: practical across-technologies considerations for large-scale sequencing projects

CRAM is an efficient format to store high-throughput sequencing data and it has been widely adopted. We thus plan to use CRAM for the Emirati Genome Program, which aims to sequence the genomes of ~1 million nationals in the United Arab Emirates using short- and long-read sequencing technologies (Illumina, MGI and Oxford Nanopore Sequencing). We conducted a pilot study on the three technologies before start using CRAM at scale. We found CRAM achieved 40-70% compression depending on the sequencing platform. As expected, CRAM compression was data lossless and did not alter variant calls. In our cloud, we observed compression speeds 0.7-1.4 GB per minute, varying on the sequencing platform too. This translates into ~1-2 hours using a single CPU to compress a ~30X human whole-genome sequencing sample. Despite its wide use, we found little publicly available information about CRAM compression rate, speed, losslessness and parallelization, especially across many sequencing platforms. This work will have direct application for Emirati Genome Program and provide practical considerations for other large-scale sequencing efforts.

bioinformatics↗

bioRxiv2022

Mitochondrial membrane potential acts as a retrograde signal to regulate cell cycle progression

Mitochondria are central to numerous anabolic and catabolic pathways whereby mitochondrial dysfunction has a profound impact on metabolism and can manifest in disease. The consequences of mitochondrial dysfunction can be ameliorated by adaptive responses that rely on mito-cellular crosstalk to communicate mitochondrial distress to the rest of the cell. Such mito-cellular signaling slows cell cycle progression in mitochondrial-DNA deficient ({rho}0) Saccharomyces cerevisiae cells, but the initial trigger and the pathway mediating the response has remained unknown. Here, we show that decreased mitochondrial membrane potential ({Delta}{Psi}m) acts as the initial signal of mitochondrial stress that delays G1-to-S phase transition in both {rho}0 and control cells. Accordingly, experimentally increasing {Delta}{Psi}m was sufficient to restore timely cell cycle progression in {rho}0 cells. Neither the RTG retrograde pathway nor central DNA damage checkpoint kinases were involved in mediating this form of mito-cellular communication. The identification of {Delta}{Psi}m as a novel regulator of cell cycle progression may have implications for disease states involving mitochondrial dysfunction.

cell biology↗

bioRxiv2021

Managing human mediated range shifts: understanding spatial, temporal and genetic variation in marine non- native species

The use of molecular methods to manage natural resources is increasingly common. However, DNA-based methods are seldom used to understand the spatial and temporal dynamics of species range shifts. This is important when managing range-shifting species such as non-native species (NNS), which can have negative impacts on biotic communities. Here we investigated the range-shifting NNS Ciona robusta, Clavelina lepadiformis, Microcosmus squamiger and Styela plicata using a combined methodological approach. We first conducted non-molecular biodiversity surveys for these NSS along the South African coastline, and compared the results with historical surveys. We detected no consistent change in range size across species, with some displaying range stability and others showing range shifts. We then sequenced a section of cytochrome c oxidase subunit I (COI) from tissue samples and found genetic differences along the coastline but no change over recent times. Finally, we found that environmental DNA metabarcoding data showed broad congruence with both the non-molecular biodiversity and the COI datasets, but failed to capture complete incidence of all NSS. Overall, we demonstrated how a combined methodological approach can effectively detect spatial and temporal variation in genetic composition and range size, which is key for managing biodiversity changes of both threatened and NSS.

ecology↗

bioRxiv2020

Consistent marine biogeographic boundaries across the tree of life despite centuries of human impacts

Over millennia, ecological and evolutionary mechanisms have shaped macroecological distributions across the tree of life. Research describing patterns of regional and global biogeography has traditionally focussed on the study of conspicuous species. Consequently, there is limited understanding of cross-phyla biogeographic structuring, and an escalating need to understand the macroecology of both microscopic and macroscopic organisms. Here we used environmental DNA (eDNA) metabarcoding to explore the biodiversity of marine metazoans, micro-eukaryotes and prokaryotes along an extensive and highly heterogeneous coastline. Our results showed remarkably consistent biogeographic structure across the kingdoms of life, which were underpinned by environmental and anthropogenic influence. Additionally, metazoan communities displayed biographic patterns that suggest regional biotic homogenisation of conspicuous species. Against the backdrop of global pervasive anthropogenic environmental change, our work highlights the importance of considering multiple domains of life to understand the maintenance and drivers of marine biodiversity across broad taxonomic, ecological and geographical scales.

ecology↗

bioRxiv2020

Mutational, transcriptional and viral shedding dynamics of the marine turtle fibropapillomatosis tumor epizootic

Sea turtle populations are directly and indirectly under threat from a range of anthropogenic processes. Perhaps the most visibly apparent of these is the disfiguring tumor disease epizootic (animal epidemic) known as fibropapillomatosis. Fibropapillomatosis continues to spread geographically, with prevalence of the disease also growing at a number of affected sites globally. Environmental exposures seem key to inducing tumor development, possibly through weakening host immune systems to the point of enabling pathogen-induced tumor formation. However, we do not yet understand the precise molecular and mutational events driving fibropapillomatosis tumor formation and progression. Similarly, many open questions remain about the role of the herpesvirus (chelonid herpesvirus 5, ChHV5) associated with the disease as a potential co-trigger, and whether its occurrence within tumors is causative or opportunistic. Without improved understanding of the basic biology of this disease epizootic, treatment, containment and mitigation options are severely hampered. To address fundamental questions relating to the oncogenic signaling, mutational spectrum, viral load, viral transcriptional status (lytic or latent) and spread, we employed transcriptomic profiling, whole genome sequencing, immunohistochemistry and environmental (e)DNA-based monitoring of viral shedding. In particular we focused on the mutational landscape of tumors and assessing the transcriptional similarity of external (skin) and internal (visceral organs) tumors, and the oncogenic signaling events driving early stage tumor growth and post-surgical tumor regrowth. These analyses revealed that internal fibropapillomatosis tumors are molecularly distinct from the more common external tumors. However, our molecular analyses also revealed that there are a small number of conserved potentially therapeutically targetable molecular vulnerabilities in common between internal and external tumors, such as the MAPK, Wnt, TGF{beta} and TNF oncogenic signaling pathways. We also determined that the tumor genomes can harbor copy number gains, indicating potentially viral-independent oncogenic processes. Genes within such mutated genomic regions have known roles in human skin cancer, including MAPK-associated genes. Turtles attempt to mount an immune response, but in some animals this appears to be insufficient to prevent tumor development and growth. ChHV5 was transcriptionally latent in all tumor stages sequenced, including early stage and recurrent tumors. We also revealed that the tumors themselves are the primary source of viral shedding into the marine environment and, if they are surgically removed, the level of ChHV5 in the water column drops. Together, these results offer an improved understanding of fibropapillomatosis tumorigenesis and provide insights into the origins, therapeutic treatment, and appropriate quarantine responses for this wildlife epizootic. Furthermore, they provide insights into human pathogen-induced cancers, particularly mechanisms which are difficult to study in the human and terrestrial context, such as time-course quantification-based monitoring of viral shedding.

cancer biology↗

bioRxiv2019

A role for the P2Y1 receptor in nonsynaptic cross-depolarization in the rat dorsal root ganglia

Non-synaptic transmission is pervasive throughout the nervous system. It appears especially prevalent in peripheral ganglia, where non-synaptic interactions between neighboring cell bodies have been described in both physiological and pathological conditions, a phenomenon referred to as cross-depolarization (CD) and thought to play a role in sensory processing and chronic pain. CD has been proposed to be mediated by a chemical agent, but its identity has remained elusive. Here, we report that in the rat dorsal root ganglion (DRG), the P2Y1 purinergic receptor (P2RY1) plays an important role in regulating CD. The effect of P2RY1 is cell-type specific: pharmacological blockade of P2RY1 inhibited CD in A-type neurons while enhancing it in unmyelinated C-type cells. In the nodose ganglion of the vagus, CD requires extracellular calcium in a large percentage of cells. In contrast, we show that in the DRG extracellular calcium appears to play no major role, pointing to a mechanistic difference between the two peripheral ganglia. Furthermore, we show that DRG glial cells also play a cell-type specific role in CD regulation. Fluorocitrate-induced glial inactivation had no effect on A-cells but enhanced CD in C-cells. These findings shed light on the mechanism of CD in the DRG and pave the way for further analysis of non-synaptic neuronal communication in sensory ganglia.\n\nHighlightsO_LIThe purinergic receptor P2RY1 plays a regulatory role in non-synaptic crossdepolarization (CD) in the mammalian DRG\nC_LIO_LIThe effect of P2RY1 is cell type-specific: it enhances CD in myelinated A-type neurons, but inhibits it in unmyelinated C-cells\nC_LIO_LICD in the DRG does not require extracellular calcium. This is in contrast with the nodose ganglion, where extracellular calcium plays an important role in nonsynaptic interactions\nC_LIO_LICD is also modulated by DRG glial cells. Glia selectively inhibit CD in C-type neurons\nC_LI

neuroscience↗

bioRxiv2019

Bacterial transformation buffers environmental fluctuations through the reversible integration of mobile genetic elements

Horizontal gene transfer (HGT) is known to promote the spread of genes in bacterial communities, which is of primary importance to human health when these genes provide resistance to antibiotics. Among the main HGT mechanisms, natural transformation stands out as being widespread and encoded by the bacterial core genome. From an evolutionary perspective, transformation is often viewed as a mean to generate genetic diversity and mixing within bacterial populations. However, another recent paradigm proposes that its main evolutionary function would be to cure bacterial genomes from their parasitic mobile genetic elements (MGEs). Here, we propose to combine these two seemingly opposing points of view because MGEs, although costly for bacterial cells, can carry functions that are point-in-time beneficial to bacteria under stressful conditions (e.g. antibiotic resistance genes under antibiotic exposure). Using computational modeling, we show that, in stochastic environments (unpredictable stress exposure), an intermediate transformation rate maximizes bacterial fitness by allowing the reversible integration of MGEs carrying resistance genes but costly for the replication of host cells. By ensuring such reversible genetic diversification (acquisition then removal of MGEs), transformation would be a key mechanism for stabilizing the bacterial genome in the long term, which would explain its striking conservation.

microbiology↗

bioRxiv2018

Phylogenomics and species delimitation of mobulid rays reveals cryptic diversity and a new species of manta ray

Practical biodiversity conservation relies on delineation of biologically meaningful units, particularly with respect to global conventions and regulatory frameworks. Traditional approaches have typically relied on morphological observation, resulting in artificially broad delineations and non-optimal species units for conservation. More recently, species delimitation methods have been revolutionised with High-Throughput Sequencing approaches, allowing study of diversity within species radiations using genome-wide data. The highly mobile elasmobranchs, manta and devil rays (Mobula spp.), are threatened globally by targeted and bycatch fishing pressures resulting in recent protection under several global conventions. However, a lack of global data, morphological similarities, a succession of recent taxonomic changes and ineffectual traceability measures combine to impede development and implementation of a coherent and enforceable conservation strategy. Here, we generate genome-wide Single Nucleotide Polymorphism (SNP) data from among the most globally and taxonomically representative set of mobulid tissues. The resulting phylogeny and delimitation of species units represents the most comprehensive assessment of mobulid diversity with molecular data to date. We find a mismatch between current species classifications, and optimal species units for effective conservation. Specifically, we find robust evidence for an undescribed species of manta ray in the Gulf of Mexico and show that species recently synonymised are reproductively isolated. Further resolution is achieved at the population level, where cryptic diversity is detected in geographically distinct populations, and indicates potential for future traceability work determining regional location of catch. We estimate the optimal species tree and uncover substantial incomplete lineage sorting, where standing variation in extinct ancestral populations is identified as a driver of phylogenetic uncertainty, with further conservation implications. Our study provides a framework for molecular genetic species delimitation that is relevant to wide-ranging taxa of conservation concern, and highlights the potential for genomic data to support effective management, conservation and law enforcement strategies.

genomics↗

bioRxiv2018

The detection of novel and resident marine non-indigenous species using environmental DNA metabarcoding of seawater and sediment

Environmental DNA (eDNA) surveys are increasingly being used for biodiversity monitoring, principally because they are sensitive and can provide high resolution community composition data. Despite considerable progress in recent years, eDNA studies examining how different environmental sample types can affect species detectability remain rare. Comparisons of environmental samples are especially important for providing best practice guidance on early detection and subsequent mitigation of non-indigenous species. Here we used eDNA metabarcoding of COI (cytochrome c oxidase subunit I) and 18S (nuclear small subunit ribosomal DNA) genes to compare community composition between sediment and water samples in artificial coastal sites across the United Kingdom. We first detected markedly different communities and a consistently greater number of distinct operational taxonomic units in sediment compared to water. We then compared our eDNA datasets with previously published rapid assessment biodiversity surveys and found excellent concordance among the different survey techniques. Finally, our eDNA surveys detected many non-indigenous species, including several newly introduced species, highlighting the utility of eDNA metabarcoding for both early detection and temporal / spatial monitoring of non-indigenous species. We conclude that careful consideration on environmental sample type is needed when conducting eDNA surveys, especially for studies assessing community change.

molecular biology↗

bioRxiv2017

The wheat blast pathogen Pyricularia graminis-tritici has complex origins and a disease cycle spanning multiple grass hosts

The wheat blast disease has been a serious constraint for wheat production in Latin America since the late 1980s. We used a population genomics analysis including 95 genome sequences of the wheat blast pathogen Pyricularia graminis-tritici (Pygt) and other Pyricularia species to show that Pygt is a distinct, highly diverse pathogen species with a broad host range. We assayed 11 neutral SSR loci in 526 Pygt isolates sampled from wheat and other grasses distributed across the wheat-growing region of Brazil to estimate gene flow, assess the importance of sexual reproduction, and compare the genetic structures of Pygt populations infecting wheat and nearby grasses. Our results suggest a mixed reproductive system that includes sexual recombination as well as high levels of gene flow among regions, including evidence for higher gene flow from grass-infecting populations and into wheat-infecting populations than vice versa. The most common virulence groups were shared between the grass- and wheat-infecting Pygt populations, providing additional evidence for movement of Pygt between wheat fields and nearby grasses. Analyses of fruiting body formation found that proto-perithecia and perithecia developed on senescing stems of wheat and other grass hosts, suggesting that sexual reproduction occurs mainly during the saprotrophic phase of the disease cycle on dead residues. Phalaris canariensis (canarygrass) supported the fullest development of perithecia, suggesting it is a promising candidate for identifying the teleomorph in the field. Based on these findings, we formulated a more detailed disease cycle for wheat blast that includes an important role for grasses growing near wheat fields. Our findings strongly suggest that widely grown pasture grasses function as a major reservoir of wheat blast inoculum and provide a temporal and spatial bridge that connects wheat fields across Brazil.\n\nAuthor summaryAfter the first wheat blast epidemic occurred in 1985 in Parana, Brazil, the disease spread to Bolivia, Argentina, and Paraguay, and was introduced into Bangladesh in 2016 followed by India in 2017. Wheat blast is caused by Pyricularia graminis-tritici (Pygt), a highly diverse pathogen species related to the rice blast fungus P. oryzae, but with an independent origin and a broader host range. We conducted a large scale contemporary sampling of Pygt from symptomatic wheat and other grass species across Brazil and analyzed the genetic structure of Pygt populations. Pygt populations on both wheat and other grasses had high genotypic and virulence diversity, a genetic structure consistent with a mixed reproductive system that includes regular cycles of recombination. The pathogen formed sexual fruiting structures (perithecia) on senescing stems of wheat and other grasses. Historical migration analyses indicated that the majority of gene flow has been from Pygt populations on other grasses and into the Pygt population infecting wheat, consistent with the hypothesis that Pygt originated on other grasses before becoming a wheat pathogen. We found that the Pygt populations infecting wheat were indistinguishable from the Pygt populations infecting other grass species, including signal grass (Urochloa brizantha). Because U. brizantha is a widely grown grass pasture often found next to wheat fields, we propose that it functions as reservoir of Pygt inoculum that provides a temporal and spatial bridge that connects wheat fields in Brazil.

plant biology↗