Biology subjects

Morgado, L.

Publications and source records attributed to Morgado, L..

8 recordsLinked to original sources

bioRxiv2025

Preliminary characterization of CbcS from Geobacter sulfurreducens ' Cbc4 complex: a putative novel respiratory pathway

Electroactive bacteria mediate electron exchange with external surfaces through a process known as extracellular electron transfer (EET). A key step in EET is the transfer of electrons from the menaquinone pool to inner membrane-associated quinone-cytochrome c oxidoreductase complexes, which subsequently relay electrons to periplasmic redox partners. Gene knockout and proteomic analyses have identified several critical components involved in EET in Geobacter sulfurreducens, including six inner membrane oxidoreductase gene clusters. Of these, three - CbcL, ImcH, and CbcBA - have been linked to specific respiratory pathways depending on the redox potential of the terminal electron acceptor. Cbc4 is one of the other inner membrane oxidoreductase complexes and is composed by a membrane-anchored tetraheme c-type cytochrome (CbcS), an iron-sulfur protein containing four [4Fe-4S] clusters (CbcT), and an integral membrane subunit (CbcU). In this study, the sequence and AlphaFold model of CbcS were analyzed and its cytochrome domain was produced, and structurally and functionally characterized. Nuclear Magnetic Resonance spectroscopy data validated the hemecore arrangement predicted by AlphaFold, showing that, despite the differences in axial ligands (CbcS has four bis-histidine low-spin hemes), CbcS hemecore is homologous to CymA and NrfH from Shewanella and Desulfovibrio species, respectively. Potentiometric titrations showed that CbcS redox active window superimposes with the ones of its putative redox partners from the triheme periplasmic cytochrome family PpcA-E; however, electron transfer reactions monitored by NMR revealed that CbcS is able to transfer electrons to PpcA. Furthermore, NMR redox titrations allowed to identify heme IV as the exit gate for electrons. Together, these findings contribute to the understanding of the molecular mechanisms of EET and provide insights on a putative new respiratory pathway in G. sulfurreducens.

biochemistry↗

bioRxiv2024

Yeasts prefer daycares and molds prefer private homes

Indoor fungi can cause negative health effects due to the production of toxins or volatiles that trigger the immune system of the occupants. To what degree indoor fungi (mycobiomes) differ between buildings with different usage is poorly known. Here, we compare the indoor mycobiomes in 123 childrens daycare centers and 214 private homes throughout Norway, as revealed by metabarcoding of DNA extracted from dust samples collected by community scientists. Although the fungal richness per se was similar in dust samples from daycares and homes, the fungal community composition differed. Yeast fungi, distributed mainly across the orders Saccharomycetales, Filobasidiales and Tremellales, were proportionally more abundant in the daycares, while filamentous fungi, including spore-producing molds such as Aspergillus, Penicillum and Cladosporium, were relatively more abundant in homes. Number of occupants, which is considerably higher in daycares, correlated significantly with the fungal community shift. We hypothesize that the density of occupants and their age distribution drive the systematic difference of yeasts and filamentous fungi in the two building types. ImportanceWorldwide, people spend most of their time indoors; in their homes, workplaces, schools and daycares. The indoor environment can thus largely influence our health. In this study, we revealed that number of occupants and possibly their age distribution are important explanatory variables for the fungal communities of the indoor environments in homes and daycares. Knowledge of these communities are important for further studies on how fungal diversity affect our health.

microbiology↗

bioRxiv2024

PhotoFiTT: A Quantitative Framework for Assessing Phototoxicity in Live-Cell Microscopy Experiments

Phototoxicity in live-cell fluorescence microscopy can compromise experimental outcomes, yet quantitative methods to assess its impact remain limited. Here we present PhotoFiTT (Phototoxicity Fitness Time Trial), an integrated framework combining a standardised experimental protocol with advanced image analysis to quantify light-induced cellular stress in label-free settings. PhotoFiTT leverages machine learning and cell cycle dynamics to analyse mitotic timing, cell size changes, and overall cellular activity in response to controlled light exposure. Using adherent mammalian cells, we demonstrate PhotoFiTTs ability to detect wavelength- and dose-dependent effects, showcasing that near-UV light induces significant mitotic delays at doses as low as 0.6 J/cm2, while longer wavelengths require higher doses for comparable effects. PhotoFiTT enables researchers to establish quantitative benchmarks for acceptable levels of photodamage, facilitating the optimisation of imaging protocols that balance image quality with sample health.

cell biology↗

bioRxiv2024

An iron-sulfur cluster as a new metal centre in a flavodiiron protein

Syntrophomonas wolfei contains two distinct multiple domain flavodiiron proteins (FDPs), of Classes H and E, presumably acting as oxygen reductases to protect this anaerobic bacterium from oxidative stress due to exposure to environments containing, even if only transiently, oxygen. The Class E FDP was predicted to have, besides the two core domains characteristic of this type of enzymes, an extra C- terminal domain putatively harbouring an iron-sulfur centre. Bioinformatic analyses showed that, thus far, Class E FDPs are only present in three other bacteria of the Syntrophomonas genus: Syntrophomonas palmitatica, Syntrophomonas zenhnderi and Thermosyntropha lipolytica. In this work, we extensively characterized the enzyme from Syntrophomonas wolfei (wild type, site directed mutants and truncated domains) and showed unequivocally, using EPR and Resonance Raman spectroscopies, that indeed it contains a [3Fe- 4S]1+/0 centre, a novelty in the field of FDPs. Structure prediction using Alphafold indicated some similarities to [3Fe4S]1+/0 containing ferredoxins. The reduction potentials of each cofactor were determined: +70 mV, -5/-70 mV and -90 mV for the FeS, diiron centre and flavin, respectively.

biochemistry↗

bioRxiv2023

Role of the inner membrane cytochrome ImcH in Geobacter extracellular electron transfer and energy conservation

Electroactive bacteria combine the oxidation of carbon substrates with an extracellular electron transfer (EET) process that discharges electrons to an electron acceptor outside the cell. This process involves electron transfer through consecutive redox proteins that efficiently connect the inner membrane to the cell exterior. In this study, we isolated and characterized the quinone-interacting membrane cytochrome c ImcH from Geobacter sulfurreducens, which is involved in the EET process to high redox potential acceptors. Our work provides evidence that ImcH is electroneutral, as it transfers electrons and protons to the same side of the membrane, contributing to the maintenance of a proton motive force, and plays a central role in recycling the menaquinone pool. ImportanceGeobacter sulfurreducens is a model electroactive bacterium, widespread in the environment and of significant interest for biotechnological applications. Its ability to form thick and conductive biofilms on top of conducting surfaces makes this microbe very useful in bioelectrochemical systems for the production of energy or added value products. To explore Geobacter spp. as a biocatalyst it is essential to understand its metabolism, particularly the molecular mechanisms for extracellular electron transfer and energy conservation. Our results reveal the importance of ImcH in both processes, identifying this protein as a major player on Geobacter metabolism.

biochemistry↗

bioRxiv2021

The indoor mycobiome of daycare centers is affected by occupancy and climate

Many children spend considerable time in daycare centers and may here be influenced by indoor microorganisms, including fungi. In this study, we investigate the indoor mycobiome of 125 daycare centers distributed along strong environmental gradients throughout Norway. Dust samples were collected from doorframes outside and inside buildings using a citizen science sampling approach. Fungal communities in the dust samples were analyzed using DNA metabarcoding of the ITS2 region. We observed a marked difference between the outdoor and indoor mycobiomes. The indoor mycobiome included considerably more yeasts and molds compared to the outdoor samples, with Saccharomyces, Mucor, Malassezia and Penicillium among the most dominant fungal genera. Changes in the indoor fungal richness and composition correlated to numerous variables related to both outdoor and indoor conditions; there was a clear geographic structure in the indoor mycobiome composition that mirrored the outdoor climate, ranging from humid areas in western Norway to drier and colder areas in eastern Norway. Moreover, the number of children in the daycare centers, as well as various building features, influenced the indoor mycobiome composition. We conclude that the indoor mycobiome in Norwegian daycare centers is structured by multiple factors and is dominated by yeasts and molds. This study exemplifies how citizen science sampling enables DNA-based analyses of a high number of samples covering wide geographic areas. ImportanceWith an alarming increase in chronic diseases like childhood asthma and allergies, there is an increased focus on the exposure of young children to indoor biological and chemical air pollutants. Our study of 125 daycares throughout Norway demonstrates that the indoor mycobiome not only reflects co-occurring outdoor fungi but includes a high abundance of yeast and mold fungi with an affinity for indoor environments. A multitude of factors influence the indoor mycobiome in daycares, including building type, inhabitants, as well as the outdoor environment. Many of the detected yeasts and molds are likely associated with the human body, where some have been coupled to allergies and respiratory problems. Our results call for further studies investigating the potential impact of the identified daycare-associated mycobiomes on children health.

microbiology↗

bioRxiv2017

Learning sequence patterns of AGO-sRNA affinity from high-throughput sequencing libraries to improve in silico functional small RNA detection and classification in plants

The loading of small RNA (sRNA) into Argonaute (AGO) complexes is a crucial step in all regulatory pathways identified so far in plants that depend on such non-coding sequences. Important transcriptional and post-transcriptional silencing mechanisms can be activated depending on the specific AGO protein to which sRNA bind. It is known that sRNA-AGO associations are at least partly encoded in the sRNA primary structure, but the sequence features that drive this association have not been fully explored. Here we train support vector machines (SVM) on sRNA sequencing data obtained from AGO-immunoprecipitation experiments to identify features that determine sRNA affinity to specific AGOs. Our SVM reveal that AGO affinity is strongly determined by complex k-mers in the 5 and 3 ends of sRNA, in addition to well-known features such as sRNA length and the base composition of the first nucleotide. Moreover, we find that these k-mers tend to overlap known transcription factor (TF) binding motifs, thus highlighting a close interplay between TF and sRNA-mediated transcriptional regulation. We embedded the learned SVM in a computational pipeline that can be used for de novo functional classification of sRNA sequences. This tool, called SAILS, is provided as a web portal accessible at http://sails.eu.nu.

bioinformatics↗

bioRxiv2017

Small RNAs reflect grandparental environments in apomictic dandelion

Plants can show long-term effects of environmental stresses and in some cases a stress memory has been reported to persist across generations, potentially mediated by epigenetic mechanisms. However, few documented cases exist of transgenerational effects that persist for multiple generations and it remains unclear if or how epigenetic mechanisms are involved. Here we show that the composition of small regulatory RNAs in apomictic dandelion lineages reveals a footprint of drought stress and salicylic acid treatment experienced two generations ago. Overall proportions of 21nt and 24nt RNA pools were shifted due to grandparental treatments. While individual genes did not show strong up- or downregulation of associated sRNAs, the subset of genes that showed the strongest shifts in sRNA abundance was significantly enriched for several GO terms including stress-specific functions. This indicates that a stress-induced signal was transmitted across multiple unexposed generations leading to persistent and functional changes in epigenetic gene regulation.

genetics↗