Biology subjects

Pereira, F. C.

Publications and source records attributed to Pereira, F. C..

10 recordsLinked to original sources

bioRxiv2026

Reversible fixation of microbial cells for targeted genomics

Phylogeny- or activity-based fluorescent labelling of microorganisms coupled with fluorescence activated cell sorting (FACS) is a powerful approach to rapidly isolate cells of interest from complex communities for targeted genomics. However, the method has not been widely adopted because standard paraformaldehyde or ethanol fixation procedures hamper nucleic acid recovery and/or long-term sample storage. Here, we present dithiobis(succinimidyl propionate) (DSP) as a reversible fixative for microbial cells that enables fluorescence imaging and DNA extraction and amplification for sequencing. DSP fixation is compatible with widely used fluorescence in situ hybridization (FISH) approaches and bioorthogonal non-canonical amino acid tagging, and can be applied to diverse sample types, where it consistently performed as well as paraformaldehyde. Furthermore, high-quality genomes were obtained from individual and multiple bacterial cells selectively extracted from complex samples using FISH-FACS. Thus, DSP is a dual-use fixative for microbiome imaging and sequencing, improving targeted microbial genomics.

microbiology↗

bioRxiv2025

Rapid genetic diversification of Bacteroides thetaiotaomicron in mono-associated mice revealed through deep population-level sequencing

Bacteria often feature short generation times and large populations, thereby allowing them to quickly evolve and adapt to new environments. Although it is known that gut bacteria can evolve on relatively short time scales, the extent of genetic diversification of bacteria in the gut environment remains underexplored. Here, we characterize the genetic diversification of the gut commensal Bacteroides thetaiotaomicron during 28 days of colonization of germ-free mice using deep shotgun sequencing as well as genome analysis of evolved isolates. We detect thousands of genetic polymorphisms as early as three days post inoculation and observe highly dynamic genetic diversity in the distal gut. We identify multiple haplotypes of a phase-variable polysaccharide utilization locus (BT2260 - BT2268) and propose that phase variation may be an important mechanism for diversification and adaptation in the gut. In addition, we find evidence that hybrid two-component system (HTCS) regulators are mutational hotspots. We identify multiple persistent and parallelly evolved genetic polymorphisms in genes, including the TonB-dependent transporter BT0867 - a homolog of BF3581 from the commensal colonization factor (ccf) in B. fragilis. Lastly, we find that the small intestine accumulated approximately 20 times more polymorphisms compared to the large intestine, highlighting overall the importance of studying spatiotemporal distribution of genetic variants. These results underscore the prevalence of rapid genetic diversification of gut bacteria, which may have important implications for adaptation as well as interactions in the microbiome and with the host. ImportanceStudying the within-host evolution of gut commensals is an essential step for understanding the role of microbiome in health and disease. It can provide insights into the mechanisms underlying the development of various gastrointestinal disorders, metabolic conditions, autoimmune diseases, and other health disorders. Additionally, this kind of research can further drive the development of personalized therapies, such as strain-level or gene specific interventions for improving health outcomes. Here, we report extensive genetic variation within days upon colonization of mice with B. thetaiotaomicron and identify genes that accumulate persistent and highly prevalent genetic polymorphisms across a mouse population. We also detect several haplotypes in phase-variable loci. Altogether, our findings underscore the rapid pace of genetic diversification and phase variation upon colonization of the gut environment.

microbiology↗

bioRxiv2025

Nutrient landscape shapes the genetic diversification of the human gut commensal Bacteroides thetaiotaomicron

Bacteroides thetaiotaomicron is a prominent member of the human gut microbiome that has evolved a suite of polysaccharide utilization loci (PUL) to break down a range of diet- and host-derived glycans. To gain insight into the evolution of this bacterium on ecologically meaningful time scales, we carried out an in vitro evolution study in which B. thetaiotaomicron was cultivated on media of different carbohydrate complexity for three months. Shotgun sequencing of the evolved populations revealed an increased number of single nucleotide polymorphisms with increased medium complexity, suggesting that genetic diversification is driven in part by the nutrient landscape. We also observed high-frequency reversible DNA inversions mediated by site-specific DNA integrases, which may be important to production and maintenance of phenotypic heterogeneity in the population. Competition experiments against the ancestor revealed adaption to the experimental conditions, with a fitness gain of the evolved populations and lineages thereof. This fitness advantage was accompanied by an increase in cell size, faster glucose depletion rates, and increased amylopectin degradation in the presence of glucose. In conclusion, rapid adaptive genetic diversification in B. thetaiotaomicron is induced and maintained in part by a complex nutritional environment.

microbiology↗

bioRxiv2025

Robust functional ultrasound imaging in the awake and behaving brain: a systematic framework for motion artifact removal

Functional ultrasound imaging (fUSI) is a promising tool for studying brain activity in awake and behaving animals, offering insights into neural dynamics that are more naturalistic than those obtained under anesthesia. However, motion artifacts pose a significant challenge, introducing biases that can compromise the integrity of the data. This study provides a comprehensive evaluation and benchmarking of strategies for detecting and removing motion artifacts in transcranial fUSI acquisitions of awake mice. We evaluated 792 denoising strategies across four datasets, focusing on clutter filtering, scrubbing, frequency filtering, and confound regression methods. Our findings highlight the superior performance of adaptive clutter filtering and aCompCor confound regression in mitigating motion artifacts while preserving functional connectivity patterns. We also demonstrate that high-pass filtering is generally more effective than band-pass filtering in the presence of motion artifacts. Additionally, we show that with effective clutter filtering, scrubbing may become optional, which is particularly beneficial for experimental designs where motion correlates with conditions of interest. Based on these insights, we propose four optimized denoising paradigms tailored to different experimental constraints, providing practical recommendations for enhancing the reliability and reproducibility of fUSI data. Our findings challenge current practices in the field and have immediate practical implications for existing fUSI analysis workflows, paving the way for more sophisticated applications of fUSI in studying complex brain functions and dysfunctions in awake experimental paradigms.

neuroscience↗

bioRxiv2025

Edited Filamin A in myeloid cells reduces intestinal inflammation and protects from colitis

Patho-mechanistic origins and disease dynamics of ulcerative colitis are still poorly understood. The actin-crosslinker Filamin A (FLNA) impacts cellular responses through interaction with cytosolic proteins. FLNA exists in two forms that differ only in one amino acid: genome-encoded FLNAQ and FLNAR - generated by post-transcriptional A-to-I editing. FLNA is edited in fibroblasts, smooth muscle- and endothelial cells in the colon. We identified the FLNA editing status as a key determinant of colitis severity. FLNA editing was highest in healthy colons and reduced during acute murine and human colitis. Mice that exclusively express edited FLNAR and do not downregulate editing upon challenge were highly resistant to DSS-induced colitis, whereas fully unedited FLNAQ animals developed severe inflammation. While the genetic induction of FLNA editing influenced transcriptional states of structural cells and the microbiome composition, we found that FLNAR exerts protection specifically via its influence on myeloid cells, which are not edited under physiological conditions. Introducing fixed, fully edited FLNAR did not hamper normal cell migration but reduced macrophage inflammation and rendered neutrophils less prone to NETosis. In conclusion, loss of FLNA editing correlates with colitis severity, and targeted FLNA editing of myeloid cells might serve as a novel therapeutic approach in intestinal inflammation. SummaryIn this study, Gawish et al. show that RNA editing of the actin cross-linker FLNA is similarly regulated in mice and humans and that the targeted induction of edited FLNAR in myeloid cells governs resistance to DSS-induced colitis, revealing its potential in IBD therapy.

molecular biology↗

bioRxiv2024

PhysiofUS : a tissue-motion based method for heart and breathing rate assessment in neurofunctional ultrasound imaging

Recent studies have shown growing evidence that brain function is closely synchronised with global physiological parameters. Heart rate is linked to various cognitive processes and previous research has also demonstrated a strong correlation between neuronal activity and breathing. These findings highlight the significance of monitoring these key physiological parameters during neuroimaging as they provide valuable insights into the overall brain function. Today, in neuroimaging, assessing these parameters required additional cumbersome devices or implanted electrodes. In this work, we performed ultrafast ultrasound imaging both in rodents and human neonates, and we extracted heart and breathing rates from local tissue motion assessed by raw ultrasound data processing. Such PhysiofUS automatically select two specific and optimal brain regions with pulsatile tissue signals to monitor such parameters. We validated the correspondence of these periodic signals with heart and breathing rates assessed using gold-standard electrodes in various conditions in rodents. We also validated Physio-fUS imaging in a clinical environment using conventional ECG. We show the potential of fUS imaging as an integrative tool for simultaneously monitoring physiological parameters during neurofunctional imaging. Beyond the technological improvement, this innovation could enhance our understanding of the link between breathing, heart rate and neurovascular activity both anesthetised in preclinincal research and clinical functional ultrasound imaging.

bioengineering↗

bioRxiv2024

Human-derived microRNA 21 regulates indole and L-tryptophan biosynthesis transcripts in a prominent gut symbiont

In the gut, microRNAs (miRNAs) produced by intestinal epithelial cells are secreted into the lumen and can shape the composition and function of the gut microbiome. Crosstalk between gut microbes and the host plays a key role in irritable bowel syndrome (IBS) and inflammatory bowel diseases, yet little is known about how the miRNA-gut microbiome axis contributes to the pathogenesis of these conditions. In this study, we aimed to explore the ability of miR-21, a miRNA that we found decreased in stool samples from IBS patients, to associate with and regulate gut microbiome function. Incubation of human faecal microbiota with miR-21 revealed a rapid association with microbial cells, reproducible across multiple donor samples. Fluorescence-activated cell sorting and sequencing of microbial cells incubated with fluorescently-labelled miR-21 identified organisms belonging to the genera Bacteroides, Limosilactobacillus, Ruminococcus, or Coprococcus which predominantly interacted with miR-21. Surprisingly, these and other genera also interacted with a miRNA scramble control, suggesting that physical interaction and/or uptake of these miRNAs by gut microbiota is not sequence-dependent. Nevertheless, transcriptomic analysis of the gut commensal Bacteroides thetaiotaomicron revealed a miRNA sequence-specific effect on bacterial transcript levels. Supplementation of miR-21, but not of small RNA controls resulted in significantly altered levels of many cellular transcripts and increased transcription of a biosynthetic operon for indole and L-tryptophan, metabolites known to regulate host inflammation and colonic motility. Our study identifies a novel putative miR-21-dependent pathway of regulation of intestinal function through the gut microbiome with implications for gastrointestinal conditions.

microbiology↗

bioRxiv2023

The Parkinson's drug entacapone disrupts gut microbiome homeostasis via iron sequestration

Increasing evidence shows that many human-targeted drugs alter the gut microbiome, leading to implications for host health. However, much less is known about the mechanisms by which drugs target the microbiome and how drugs affect microbial function. Here we combined quantitative microbiome profiling, long-read metagenomics, stable isotope probing and single cell chemical imaging to investigate the impact of two widely prescribed nervous system targeted drugs on the gut microbiome. Ex vivo supplementation of physiologically relevant concentrations of entacapone or loxapine succinate to faecal samples significantly impacted the abundance of up to one third of the microbial species present. Importantly, we demonstrate that the impact of these drugs on microbial metabolism is much more pronounced than their impact on abundances, with low concentrations of drugs reducing the activity, but not the abundance of key microbiome members like Bacteroides, Ruminococcus or Clostridium species. We further demonstrate that entacapone impacts the microbiome due to its ability to complex and deplete available iron, and that microbial growth can be rescued by replenishing levels of microbiota-accessible iron. Remarkably, entacapone-induced iron starvation selected for iron-scavenging organisms carrying antimicrobial resistance and virulence genes. Collectively, our study unveils the impact of two under-investigated drugs on whole microbiomes and identifies metal sequestration as a mechanism of drug-induced microbiome disturbance.

microbiology↗

bioRxiv2021

SRS-FISH: High-Throughput Platform Linking Microbiome Function to Identity at the Single Cell Level

One of the biggest challenges in microbiome research in environmental and medical samples is to better understand functional properties of microbial community members at a single cell level. Single cell isotope probing has become a key tool for this purpose, but the currently applied detection methods for measuring isotope incorporation into single cells do not allow high-throughput analyses. Here, we report on the development of an imaging-based approach termed stimulated Raman scattering - two-photon fluorescence in situ hybridization (SRS-FISH) for high-throughput structure-function analyses of microbial communities with single cell resolution. SRS-FISH has an imaging speed of 10 to 100 milliseconds per cell, which is two to three orders of magnitude faster than spontaneous Raman-FISH. Using this technique, we delineated metabolic responses of thirty thousand individual cells to various mucosal sugars in the human gut microbiome via incorporation of deuterium from heavy water as an activity marker. Application of SRS-FISH to investigate the utilization of host-derived nutrients by two major human gut microbiome taxa revealed that response to mucosal sugars tends to be dominated by Bacteroidales, with an unexpected finding that Clostridia can outperform Bacteroidales at foraging fucose.

microbiology↗

bioRxiv2018

Structure and assembly of a Clostridioides difficile spore polar appendage

Clostridioides difficile, a strict anaerobic spore-former, is the main cause of nosocomial disease associated to antibiotic therapy in adults and a growing concern in the community. Spores are the main infectious, persistence and transmission vehicle. Spore germination occurs in the intestine and the resulting vegetative cells will produce the toxins responsible for the disease symptoms, and spores. During sporulation, a wild type population bifurcates into two main spore morphotypes, with or without a thick exosporium. We show that this bifurcation extends to the formation of spores with a robust polar appendage or spores with a short appendage or that lack this structure. The cysteine-rich CdeM protein localizes to the appendage and around the entire surface of the spore, and is a major structural component of the exosporium, which we show is continuous with the appendage. In a CdeM mutant, when present, the polar appendage is short and disorganized. We show that wild type and cdeM spores with a short or no appendage germinate poorly in response to taurocholate, compared to those with an appendage. cdeM spores of the two types, however, germinate faster than their wild type counterparts. Thus, while the absence of CdeM may increase the permeability of spores to taurocholate, proper assembly of the appendage is also important for germination. Consistent with an overall enhancement of germination, a cdeM mutant shows increased virulence in a hamster model of disease. For a wild type population, spores with a short or no appendage germinate slower than the appendage-bearing spores. Differences in transmission, persistence and disease severity may result, in part, from their proportion in a spore population.

microbiology↗