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Domingues, C.

Publications and source records attributed to Domingues, C..

4 recordsLinked to original sources

The Rare Plasmid Biosphere: A Hidden Reservoir of Genetic Diversity

Bacterial communities typically display highly uneven abundance patterns, with a few dominant taxa and many low-abundance ones contributing to extensive genetic diversity 1-4. Notably, this rare biosphere 5 includes species performing critical ecological functions, such as biogeochemical cycling and resisting invasions 6-9. While bacterial abundance patterns have been extensively studied, the distribution of plasmids-extrachromosomal, self-replicating genetic elements ubiquitous in prokaryotes-- remains poorly understood. Using a dataset of 52,909 plasmids from isolates of bacteria and archaea 10, we found that their 16,547 Plasmid Taxonomic Units (PTUs) 11,12 exhibit a distribution with a fat tail, whether in rank abundance or relative abundance distributions: a few highly prevalent PTUs and many rare. The relative abundance distributions are well described by a Poisson log-normal distribution, consistent with recent findings for species abundance distributions across several taxonomic groups 13. The host distribution also presents a heavy tail; however, we show that rare PTUs are not necessarily associated with rare bacterial species, nor are common PTUs exclusively found in common hosts. This indicates that PTUs distribution is not a direct consequence of hosts distribution. Per plasmid, the host range of rare PTUs is higher than that of common PTUs, at all taxa levels, from species to phyla. Yet, plasmids from common PTUs are more mobile, likely explaining their success. The large group of rare PTUs constitutes a much more diverse reservoir of genetic material than the group of common PTUs. Under appropriate selective pressures, some of these rare plasmids could spread not only by hitchhiking with their hosts but also through horizontal transfer. Therefore, this work opens new paths into plasmid research.

microbiology↗

Readaptation of mesenchymal stem cells to high stiffness and oxygen environments modulate the extracellular matrix

The therapeutic potential of mesenchymal stem cells (MSCs) has been explored over the past decades due to their ability to modulate the microenvironment through paracrine signaling. Consequently, the secretome of MCSs has emerged as a cell-free therapy rather than a cell therapy, offering the advantages of being readily commercialized as an off-the-shelf product without immunogenicity compatibility issues. As a result, strategies to manipulate and enhance the secretory profile of MSCs secretome are emerging. MSCs from the Whartons jelly niche are accommodated to the stiffness and oxygen level found at the umbilical cord (UC), which are 2 to 5kPa (Youngs modulus) and 2.4% to 3.8% O2, respectively. However in vitro culture conditions (2-3 GPa and 18.5% O2) are largely different from the one observed in vivo. Here, we present a proteomic characterization of the secretome of MSCs primed (48h) or readapted (7-10 days) to soft (3kPa) (mechanomodulated) or low oxygen levels (5% O2) (physioxia). Maintaining MSCs on soft platforms for long periods increased the secretion of proteins associated with cell redox homeostasis, such as protein disulfide isomerases and mitochondrial proteins, while physioxia enhanced the secretion of immunomodulatory proteins. The high secretion of these proteins might confer a therapeutical advantage by favoring a regenerative environment at the injury site. Interestingly, lowering the stiffness or oxygen converged on the downregulation of several extracellular matrix proteins (ECM), particularly collagen fibrils, on primed and readapted cells. These results suggest that a massive reorganization of the extracellular space occurs upon culturing MSCs on conventional culture conditions, which may affect not only matrix stiffness but also several signaling pathways initiated at the cell membrane, such as PDGF signaling pathways (e.g., PI3K-AKT), consequently biasing stem cell fate. In conclusion, mimicking physiological culture conditions in vitro modulates secretome composition, which may empower its therapeutical properties by enriching proteins that promote cell survival. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/609692v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@16350corg.highwire.dtl.DTLVardef@1f4c18borg.highwire.dtl.DTLVardef@1ce9c0aorg.highwire.dtl.DTLVardef@1666d0b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

Mimicking physiological stiffness or oxygen levels in vitro reorganizes mesenchymal stem cells machinery toward a more naive phenotype

Mesenchymal stem cells (MSCs) offer a promising therapeutic potential for a wide variety of pathologies. However, obtaining minimal effective doses requires an extensive in vitro expansion, which compromises their stemness and therapeutic properties. The stiffness of the umbilical cord ranges between 2 and 5kPa, and the oxygen levels fluctuate from 2.4% to 3.8%, differing from the standard in vitro culture conditions where MSCs are exposed to the stiffness of the Petri dish (2-3 GPa) and near atmospheric oxygen levels (18.5% O2). Since MSCs can sense and respond to biomechanical and chemical characteristics of the microenvironment, it was hypothesized that expanding MSCs on 3kPa platforms - mechanomodulation - or at 5% O2 levels - physioxia - could potentially impact the cellular proteome of MSCs, for long (7-10 days) or short (48h) periods. Data analysis has unveiled that culturing MSCs on soft substrates for long periods promotes the expression of various proteins related to cell redox homeostasis, such as thioredoxins and peroxiredoxins. Conversely, culturing these cells during the same period but under low oxygen levels leads to an increase in chaperone machinery proteins, such as HSP90 or TRiC. These proteins can favor the clearance of misfolded proteins and telomerase maintenance processes, possibly preventing MSCs from being driven to a senescent phenotype. Although mechanomodulation and physioxia are two distinct stimuli, both converge in downregulating the expression of histones and several ribosomal subunits, possibly decreasing translational complexity, which could hypothetically favor a more naive phenotype for MSCs. Interestingly, priming UC-MSCs (48h) leads to a differential expression of proteins of the extracellular matrix and histone subtypes. Understanding the role of these proteins in transducing environmental cues might provide insights into how conventional culture conditions significantlyalter fundamental cellular processes and support the development of a more efficient protocol to expand and empower the therapeutic potential of MSCs. In the future, employing a combination of reduced stiffness and lower oxygen levels may present a promising strategic approach. HighlightsO_LICulturing MSCs on a soft substrate (3kPa) enhances the expression of antioxidant proteins, such as thioredoxins and peroxiredoxins C_LIO_LIProtein homeostasis is remodeled in MSCs cultured under physiological levels of oxygen (5% O2) through the differential expression of the chaperone machinery C_LIO_LILowering stiffness or oxygen levels during in vitro MSCs expansion decreases histones and ribosomal subunits expression, possibly favoring a more naive phenotype C_LI

cell biology↗

Identification and quantification of α- and β-amanitin in wild mushrooms by HPLC-UV-EC and HPLC-DAD-MS detection

Amatoxins are a group of highly toxic peptides, which include - and {beta}-amanitin, found in several species of mushrooms (e.g. Amanita phalloides). Due to their high hepatotoxicity, they account for most deaths occurring after mushrooms ingestion. The determination of - and {beta}- amanitin content in wild mushrooms is invaluable for treating cases involving poisoning. In the present study, we have developed and validated an analytical method based on high-performance liquid chromatography, with in-line ultraviolet and electrochemical detection (HPLC-UV-EC), for the rapid quantification of - and {beta}-amanitin in wild mushroom samples collected from the Inner Center of Portugal. A reproducible and simple solid-phase extraction (SPE) using OASIS(R) PRIME HLB cartridges was used for sample pre-treatment, followed by chromatographic separation based on the RP-C18 column. The UV and EC chromatograms of - and {beta}-amanitin were recorded at 305 nm and +0.600 V vs. Ag/AgCl, respectively. The linear quantification for both amanitins was in the range of 0.5-20.0 g{middle dot}mL-1 (R2 > 0.999). The LOD, calculated based on the calibration curve, was similar for UV and EC detection (0.12-0.33 g ml.-1). Intra-day and inter-day precision were less than 13%, and the recovery ratios ranged from 89% to 117%. Nine Amanita species and five edible mushrooms were analysed by HPLC-UV-EC, and HPLC-DAD-MS confirmed the identification of amatoxins. We find high - and {beta}-amanitin content in A. phalloides and not in the other species analysed. In sum, the developed and validated method provides a simple and fast analysis of - and {beta}-amanitins contents in wild mushrooms and is suitable for screening and routine assessment of mushroom intoxication. HighlightsO_LINew validated method using HPLC-UV-EC to determine - and {beta}-amanitin in wild mushrooms. C_LIO_LIReproducible and fast SPE procedure for small samples. C_LIO_LIEffective sample pre-treatment with the OASIS(R) PRIME HLB SPE cartridge. C_LIO_LIIdentification and quantification of - and {beta}-amanitin in wild mushroom samples from Portugal. C_LIO_LIHPLC-DAD-MS confirmation of amatoxins present in mushroom samples. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/483521v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@734f7aorg.highwire.dtl.DTLVardef@66d9fforg.highwire.dtl.DTLVardef@720f39org.highwire.dtl.DTLVardef@458e64_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗