Biology subjects

Santos, J.

Publications and source records attributed to Santos, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Reconstructing the hyphosphere using a hyphal release-capture soil microcosm

Fungal hyphae form spatially confined interfaces in soil that mediate close associations with bacteria, collectively referred to as the hyphosphere. Despite its recognized ecological importance, experimental access to hyphosphere-associated microbial communities under realistic soil and plant-associated conditions has remained limited. Here, we present a soil-mimetic microcosm that enables controlled reconstruction and recovery of hyphosphere bacterial communities embedded within plant-associated soil. The system integrates field-derived soil, a native soil microbial inoculum, living cotton seedlings, and a spatially constrained fungal inoculum housed within sterile cell-strainer assemblies, permitting hyphal extension into soil while preserving a recoverable fungal-soil boundary. Using the soil-borne plant pathogen Fusarium oxysporum f. sp. vasinfectum as a model filamentous fungus, we show that the microcosm enables reproducible recovery of hypha-associated soil microaggregates containing physically attached bacterial cells. Full-length 16S rRNA profiling revealed pronounced reductions in bacterial richness and evenness in hyphosphere samples relative to bulk and rhizosphere soils, consistent with recruitment of a restricted subset of the surrounding microbiota. Ordination analyses demonstrated clear compositional separation between soil and hyphosphere compartments, with convergence of hypha-associated communities across bulk and rhizosphere contexts. Phylogenetic turnover analyses indicated phylogenetic structuring, whereas taxonomic analyses identified a conserved set of bacterial genera consistently associated with hyphae alongside compartment-specific taxa influenced by soil and plant context. Together, these findings establish the novel hyphal release-and-capture microcosm as a reproducible, ecologically grounded platform for studying hyphosphere-associated bacterial communities in plant-associated soils.

microbiology↗

bioRxiv2026

High-Resolution 3D Histology of the Murine Kidney Using Synchrotron X-Ray Micro-CT

Conventional two-dimensional (2D) histology relies upon destructive sample preparation and stereological estimation, frequently leading to sampling bias and loss of critical spatial context required for understanding renal structure relationships. Here, we detail a novel pipeline for high-resolution 3D histology of ex vivo murine kidneys using X-ray micro-computed tomography (micro-CT) at the high flux of a synchrotron light source, the architecture of the nephron and associated microvasculature necessitates three-dimensional (3D) analysis to accurately characterize its complexity. Soft-tissue contrast was optimized through an established phosphotungstic acid (PTA) staining protocol, enabling robust mapping of macro and microstructures via absorption contrast. Multi-scale imaging was performed, providing whole-organ context at resolutions around 3 m and achieving sub-micron detail (down to 400 nm) in targeted regions of interest (ROI) of the renal cortex. Utilizing machine learning segmentation pipelines optimized for large volumetric datasets, we extracted crucial 3D quantitative morphometric data. The results presented herein demonstrate accuracy and morphological insight achievable through synchrotron-based 3D imaging, establishing a robust method for quantitative preclinical research.

systems biology↗

bioRxiv2025

Machine Learning-Guided Synthetic Microbial Communities Enable Functional and Sustainable Degradation of Persistent Environmental Pollutants

Persistent environmental pollutants demand the use of diverse microbial metabolic capabilities for effective degradation. While naturally occurring consortia or single strains often fall short in efficiency, synthetic microbial communities (SynComs) hold greater promise for enhanced degradation. To address this challenge, we developed GENIA (Genomically and Environmentally Networked Intelligent Assemblies), a genome-informed and machine learning-guided framework for the rational design of SynComs capable of multi-pollutant degradation under simulated environmental conditions. Using a microfluidic high-throughput cultivation platform, 2,155 bacterial strains were isolated from xenobiotic-enriched environments and screened for pollutant-specific growth. Whole-genome sequencing and functional annotation of 45 prioritized strains revealed metabolic traits associated with the potential degradation of challenging persistent environmental pollutants as proof of concept, i.e., lignin oxidation, atrazine dechlorination, and PFAS defluorination. These genomic profiles were encoded into spline-based graph representations and integrated within the GENIA pipeline, which combines graph neural networks, pathway complementarity modeling, and functional redundancy minimization to predict optimal community assemblies. The resulting nine-member community--comprising Pantoea dispersa, Atlantibacter hermannii, Pseudomonas fulva, Paenibacillus polymyxa, Bacillus cabrialesii, Micrococcus luteus, Bacillus pseudomycoides, Bacillus licheniformis, and Pseudomonas pergaminensis--was predicted to exhibit broad catabolic capacity and minimal intra-community competition. Kinetic experiments in minimal medium demonstrated simultaneous multi-pollutant degradation: lignin (91.6% removal by day 5), atrazine (91.4% removal by day 3), and PFOS (93.1% removal within seven days), representing a 2-4-fold improvement over existing approaches. GENIA establishes a scalable and generalizable framework that integrates systems-level genomics, phenotypic screening, and predictive modeling to engineer ecologically coherent microbial consortia with application to complex environmental bioremediation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/677392v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@1beee39org.highwire.dtl.DTLVardef@a0a1corg.highwire.dtl.DTLVardef@11ddc4aorg.highwire.dtl.DTLVardef@169ba57_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Antiretroviral therapy in the peripartum period impairs post pregnancy cardiac reverse remodeling with early signs of heart failure with preserved ejection fraction

Cardiovascular (CV) disease in pregnancy is the leading cause of maternal mortality worldwide, often associated with the cardiometabolic remodeling needed for fetal growth. In healthy pregnancies these changes reverse postpartum with no consequence, but improper reverse remodeling increases long-term CV risk. Pregnant patients with HIV are at further risk, but it remains unclear whether this is due to chronic low viremia or administration of antiretrovirals (ART). ART used in pregnancy includes nucleoside reverse transcriptase inhibitors (NRTIs), which have known mitochondrial toxicity, and may impact cardiac function. Here, we tested the hypothesis that NRTI-containing ART negatively impacts the maternal heart. Using rats, we show that pregnancy-associated reverse remodeling was impaired by ART. Molecularly, we found mitochondrial changes in the heart and in the liver, leading to dyslipidemia that collectively affected the heart. Our data suggest caution in the choice of ART prescribed for women with HIV during pregnancy and highlight the importance of studies focusing on maternal health.

physiology↗

bioRxiv2025

NAC promotes co-translational folding at the ribosomal tunnel exit

The nascent polypeptide-associated complex (NAC) coordinates enzymatic modifications and membrane targeting of nascent chains during translation. While NACs function as a dynamic hub for other factors is well-established, its direct role in co-translational folding is unclear. By proteome-wide profiling NAC co-translational interactions in human cells, we found that NAC recognizes emerging segments enriched in hydrophobicity and -helical propensity, within folded domains of cytonuclear proteins. Single-molecule and structural analyses reveal that NAC, via its {beta}-barrel domain, dynamically interacts with nascent chains at the ribosomal tunnel exit and is capable of promoting on-pathway folding. Compartment-specific nascent chain interactions of NAC further elucidate its role in targeting to the endoplasmic reticulum and mitochondrial membrane protein biogenesis. Together, these findings show that NAC acts as a bona fide co-translational chaperone that facilitates early protein folding at the ribosomal tunnel exit, expanding its functional repertoire in protein biogenesis.

biochemistry↗

bioRxiv2025

Production of high-affinity glycosylated anti-mouse conjugated nanobodies in Pichia pastoris

Nanobodies (NBs) are small antibody fragments derived from camelid heavy-chain antibodies which represent the minimal functional domain capable of antigen recognition and binding. NB are ten times smaller than conventional antibodies, exhibit a compact structure and high stability, making them ideal for recombinant production. The eukaryotic unicellular system Pichia pastoris provides multiple advantages for protein expression, including the ability to perform several eukaryotic posttranslational modifications. In this work, we engineered a modular plasmid sequence that, through specific restriction enzyme cuts and ligations, codes the expression of a secreted anti-mouse kappa chain NB fused with various accessory peptides in P. pastoris. This system enables the incorporation of a plastic binding sequence, a histidine tag (Hisx6) for purification, the horseradish peroxidase (HRP) enzyme for chemiluminescence detection, or the biotinylatable AviTag sequence, in multiple combinations. We successfully expressed and purified anti-kappa NBs fused to a Hisx6-tag ({kappa}NB) and to HRP -Hisx6-tag ({kappa}NB-HRP), with subsequent structural and functional characterization revealing high affinity for mouse immunoglobulins. The {kappa}NB-kappa light chain domain complex was modeled showing a fitted surface interaction of CDR3 domain. The position of a glycan in the complex was modeled predicting that glycan addition would not affect the interaction surface. Accordingly, no functional differences were observed in {kappa}NB after deglycosylation, indicating that high mannose glycan addition has not interfered with its binding capability. Moreover, glycosylated {kappa}NB fused to HRP was expressed with retained HRP activity, and proved to be functional as a secondary antibody, demonstrating the systems versatility in producing NBs and conjugated NBs with posttraslational modification that may be required for diverse biotechnological applications.

molecular biology↗

bioRxiv2025

A simple, fast and cost-efficient protocol for ultra-sensitive ribosome profiling

Ribosome profiling has become an essential tool for studying mRNA translation in cells with codon-level resolution. However, its widespread application remains hindered by the labour-intensive workflow, low efficiency and high costs associated with sequencing sample preparation. Here, we present a new cost-effective and ultra-sensitive library preparation method that significantly advances the applicability of ribosome profiling. By implementing bead-coupled enzymatic reactions and product purifications, our approach increases both yield and throughput while maintaining high reproducibility. Demonstrating the sensitivity of the protocol we prepared libraries from as little as 12 fmol of RNA, which expands the feasibility of ribosome profiling from minimal input samples, such as derived from small populations, stressed cells, or patient-derived specimens. Additionally, we validate the versatility of the protocol across multiple species and demonstrate its applicability for RNA-seq library preparation. Altogether, this protocol provides a highly accessible and efficient alternative to existing ribosome profiling workflows, facilitating research in previously challenging experimental contexts.

systems biology↗

bioRxiv2024

Design of Novel Dehalogenases using Protein Large Language Models

Per- and polyfluoroalkyl substances (PFAS) are toxic compounds linked to cancers, infertility, and vaccine resistance at concentrations above 1 part per trillion. Owing to strong carbon-fluorine bonds, they are persistent in the environment, taking centuries to millennia to degrade. As part of the 2024 iGEM competition, several high school students local to the Pasadena area leveraged recently-developed bioinformatic tools and large language models to discover and design novel reductive dehalogenases predicted to degrade perfluorooctanoic acid (PFOA), a long chain PFAS whose manufacture is prohibited, yet still persists in the environment and drinking water. The team identified 68 enzymes with structural similarity to Acidimicrobium sp. Strain A6 RdhA, the only specific known PFAS degrading enzyme in nature, expressed and refolded 5 of these enzymes in addition to 1 rational, and 3 large language model designs. These designs all have diverse sequences yet all are predicted to retain key substrate and cofactor binding pockets. These enzymes will be assayed on the ability to defluorinate PFOA.

bioengineering↗

bioRxiv2024

Circulating microRNAs as biomarkers of chronic kidney disease and its association with renal and cardiovascular outcomes in non-dialysis patients

Chronic kidney disease (CKD) affects over 10% of the population worldwide and entails a significant risk for cardiovascular disease (CVD), leading to a 500-fold increase in cardiovascular mortality in advanced stages. Still, the pathophysiological mechanisms underlying kidney-heart intercommunication remain largely unknown. In recent years, microRNAs (miRNAs) emerged as important key regulators of gene expression that may serve as biomarkers for several diseases, however, their role in kidney-heart crosstalk in CKD remains underexplored. In this study, we evaluated the expression of a miRNA panel in plasma samples from non-dialysis CKD patients and explored their association with main comorbidities and significant outcomes in a 5-year follow-up. Results show that miR-30c-5p and miR-132-3p were downregulated in CKD patients presenting a significant power to discriminate the disease state. Importantly, only miR-30c-5p was downregulated in early glomerular filtration rate (GFR) categories, being able to discriminate these CKD patients from non-diseased individuals. Concerning cardiovascular outcomes, miR-199a-5p was found to be associated with an increased frequency of CVD. When analyzing the major disease outcomes in a 5-year time, miR-199a-5p upregulation at baseline was associated with increased mortality, while miR-324-3p was downregulated in patients who progressed to more advanced stages of the disease. These findings highlight the involvement of novel circulating miRNAs in CKD onset and progression, and identify, for the first time, the enrollment of miR-199a-5p in kidney-heart pathophysiological crosstalk, paving the way for the establishment of new biomarkers and therapeutic targets for CKD and its outcomes.

molecular biology↗

bioRxiv2024

Mapping multiple RNA modifications simultaneously by proximity barcode sequencing

RNA is subject to a multitude of different chemical modifications that collectively represent the epitranscriptome. Individual RNA modifications including N6-methyladenosine (m6A) on mRNA play essential roles in the posttranscriptional control of gene expression. Recent technological advances have enabled the transcriptome-wide mapping of certain RNA modifications, to reveal their broad relevance and characteristic distribution patterns. However, convenient methods that enable the simultaneous mapping of multiple different RNA marks within the same sample are generally lacking. Here we present EpiPlex RNA modification profiling, a bead-based proximity barcoding assay with sequencing readout that expands the scope of molecular recognition-based RNA modification detection to multiple targets, while providing relative quantification and enabling low RNA input. Measuring signal intensity against spike-in controls provides relative quantification, indicative of the RNA mod abundance at each locus. We report on changes in the modification status of HEK293T cells upon treatment with pharmacological inhibitors separately targeting METTL3, the dominant m6A writer enzyme, and the EIF4A3 component of the exon junction complex (EJC). The treatments resulted in decreased or increased m6A levels, respectively, without effect on inosine levels. Inhibiting the helicase activity of EIF4A3 and EIF4A3 knockdown both cause a significant increase of m6A sites near exon junctions, consistent with the previously reported role of EIF4A3 in shaping the m6A landscape. Thus, EpiPlex offers a reliable and convenient method for simultaneous mapping of multiple RNA modifications to facilitate epitranscriptome studies.

genomics↗

bioRxiv2024

Mitochondrial membrane potential regulates nuclear DNA methylation and gene expression through phospholipid remodeling

Maintenance of the mitochondrial inner membrane potential ({Delta}{Psi}M) is critical for many aspects of mitochondrial function, including mitochondrial protein import and ion homeostasis. While {Delta}{Psi}M loss and its consequences are well studied, little is known about the effects of increased {Delta}{Psi}M. In this study, we used cells deleted of ATPIF1, a natural inhibitor of the hydrolytic activity of the ATP synthase, as a genetic model of mitochondrial hyperpolarization. Our data show that chronic {Delta}{Psi}M increase leads to nuclear DNA hypermethylation, regulating transcription of mitochondria, carbohydrate and lipid metabolism genes. Surprisingly, remodeling of phospholipids, but not metabolites or redox changes, mechanistically links the {Delta}{Psi}M to the epigenome. These changes were also observed upon chemical exposures and reversed by decreasing the {Delta}{Psi}M, highlighting them as hallmark adaptations to chronic mitochondrial hyperpolarization. Our results reveal the {Delta}{Psi}M as the upstream signal conveying the mitochondrial status to the epigenome to regulate cellular biology, providing a new framework for how mitochondria can influence health outcomes in the absence of canonical dysfunction. HighlightsO_LIMitochondria hyperpolarization leads to nuclear DNA hypermethylation C_LIO_LIDNA methylation regulates expression of mitochondrial and lipid metabolism genes C_LIO_LIPhospholipid remodeling mediates the epigenetic effects of mitochondrial hyperpolarization C_LI

genomics↗

bioRxiv2023

Toxicogenomic analysis of the carcinogenic potential of plastic additives

Plastics are the most prevalent human-made substance in the world and are ubiquitous throughout all ecosystems. Microscopic plastic particles are routinely ingested and inhaled by humans and other organisms. Despite the frequency of plastic exposures, little is known about their health consequences. Of particular concern are plastic additives -chemical compounds that are mixed into plastics to improve functionality or are unintentionally acquired during plastic production and use. Additives are loosely bound to the plastic polymer and may be released during plastic exposures. These compounds may pose health concerns, such as cancer, but little is known about the potential impact of these chemicals on health. To better understand the health effects of plastic additives, we performed an integrated toxicogenomic analysis on 2,712 additives, focusing on cancer as a well-studied toxicological endpoint. Screening these substances across three chemical databases revealed two key observations: 1) over 150 plastic additives have known carcinogenicity and 2) the majority ([~]90%) of plastic additives lack data on carcinogenic endpoints. Analyses of additive usage patterns pinpointed specific polymers, functions, and products in which carcinogenic additives reside. Based on published chemical-gene interactions, both carcinogenic additives and additives with unknown carcinogenicity impacted similar biological pathways. The predominant pathways involved DNA damage, apoptosis, immune response, viral diseases, and cancer. This study underscores the urgent need for systematic and comprehensive carcinogenicity assessment of plastic additives and regulatory responses to mitigate the potential health risks of plastic exposure.

pharmacology and toxicology↗

bioRxiv2023

CRISPR/Cas9-based edition of frataxin gene in Dictyostelium discoideum for Friedreich's Ataxia disease modeling

In this paper we describe the development of a new model system for Friedreichs Atax- ia (FA) using Dictyostelium discoideum. We investigated the conservation of function between humans and D. discoideum and showed that DdFXN can substitute the human version in the interaction and activation of the Fe-S assembly supercomplex. We edited the fxn locus and isolated a defective mutant, clone 8, which presents landmarks of frataxin deficiency, such as a decrease in Fe-S cluster-dependent enzymatic functions, growth rate reduction, and increased sensitivity to oxidative stress. In addition multicellular development is affected as well as grow on bacterial lawn. We also assessed the rescuing capacity of DdFXN-G122V, a version that mimics a human variant present in some FA patients. While the expression of DdFXN-G122V rescues growth and enzymatic activity defects, as DdFXN does, multicellular development defects were only partially rescued The results of the study suggest that this new model system offers a wide range of pos- sibilities to easily explore diverse phenotypes in FA and develop drug or treatment screenings for designing and evaluating therapeutic strategies.

cell biology↗

bioRxiv2023

The structural architecture of an α-synuclein toxic oligomer

Oligomeric species populated during -synuclein aggregation are considered key drivers of neurodegeneration in Parkinsons disease. However, the development of oligomer-targeting therapeutics is constrained by our limited knowledge of their structure and the molecular determinants driving their conversion to fibrils. PSM3 is a nanomolar peptide binder of -synuclein oligomers that inhibits aggregation by blocking oligomer to fibril conversion. Here, we investigate the binding of PSM3 to -synuclein oligomers to discover the mechanistic basis of this protective activity. We find that PSM3 selectively targets an -synuclein N-terminal motif (residues 36-61) that populates a distinct conformation in the monomeric and oligomeric states. This -synuclein region plays a pivotal role in oligomer to fibril conversion, as its absence renders the central NAC domain insufficient to prompt this structural transition. The hereditary mutation G51D, associated with early-onset Parkinsons disease, causes a conformational fluctuation in this region, leading to delayed oligomer to fibril conversion and an accumulation of oligomers that are resistant to remodeling by molecular chaperones. Overall, our findings unveil a new targetable region in -synuclein oligomers, advance our comprehension of oligomer to amyloid fibril conversion and reveal a new facet of -synuclein pathogenic mutations.

biophysics↗

bioRxiv2022

Paradox Found: Global Accounting of Lymphocyte Protein Synthesis

Rapid lymphocyte cell division places enormous demands on the protein synthesis machinery. Flow cytometric measurement of puromycylated ribosome-associated nascent chains after treating cells or mice with translation initiation inhibitors reveals that ribosomes in resting lymphocytes in vitro and in vivo elongate at typical rates for mammalian cells. Intriguingly, elongation rates can be increased up to 30% by activation in vivo or fever temperature in vitro. Resting and activated lymphocytes possess abundant monosome populations, most of which actively translate in vivo, while in vitro, nearly all can be stalled prior to activation. Quantitating lymphocyte protein mass and ribosome count reveals a paradoxically high ratio of cellular protein to ribosomes insufficient to support their rapid in vivo division, suggesting that the activated lymphocyte proteome in vivo may be generated in an unusual manner. Our findings demonstrate the importance of a global understanding of protein synthesis in lymphocytes and other rapidly dividing immune cells.

immunology↗

bioRxiv2022

Pan-genome inversion index reveals evolutionary insights into the subpopulation structure of Asian rice (Oryza sativa)

Understanding and exploiting genetic diversity is a key factor for the productive and stable production of rice. Utilizing 16 high-quality genomes that represent the subpopulation structure of Asian rice (O. sativa), plus the genomes of two close relatives (O. rufipogon and O. punctata), we built a pan-genome inversion index of 1,054 non-redundant inversions that span an average of [~] 14% of the O. sativa cv. Nipponbare reference genome sequence. Using this index we estimated an inversion rate of 1,100 inversions per million years in Asian rice, which is 37 to 73 times higher than previously estimated for plants. Detailed analyses of these inversions showed evidence of their effects on gene regulation, recombination rate, linkage disequilibrium and agronomic trait performance. Our study uncovers the prevalence and scale of large inversions ([≥] 100 kb) across the pan-genome of Asian rice, and hints at their largely unexplored role in functional biology and crop performance.

genomics↗

bioRxiv2022

Microbes are potential key players in the evolution of life histories and aging in Caenorhabditis elegans

Microbes can have profound effects on host fitness and health and the appearance of late-onset diseases. Host-microbe interactions thus represent a major environmental context for healthy aging of the host and might also mediate trade-offs between life-history traits in the evolution of host senescence. Here, we have used the nematode Caenorhabditis elegans to study how host-microbe interactions may modulate the evolution of life histories and aging. We first characterized the effects of two non-pathogenic and one pathogenic Escherichia coli strains, together with the pathogenic Serratia marcescens DB11 strain, on population growth rates and survival of C. elegans from five different genetic backgrounds. We then focused on an outbred C. elegans population, to understand if microbe-specific effects on the reproductive schedule and in traits such as developmental rate and survival were also expressed in the presence of males and standing genetic variation, which could be relevant for the evolution of C. elegans and other nematode species in nature. Our results show that host-microbe interactions have a substantial host-genotype-dependent impact on the reproductive aging and survival of the nematode host. Although both pathogenic bacteria reduced host survival in comparison with benign strains, they differed in how they affected other host traits. Host fertility and population growth rate were affected by S. marcescens DB11 only during early adulthood, whereas this occurred at later ages with the pathogenic E. coli IAI1. In both cases, these effects were largely dependent on the host genotypes. Given such microbe-specific genotypic differences in host life history, we predict that the evolution of reproductive schedules and senescence might be critically contingent on host-microbe interactions in nature.

evolutionary biology↗

bioRxiv2022

Selection of Synthetic Proteins to Modulate the Human Frataxin Function

Frataxin is a kinetic activator of the mitochondrial supercomplex for iron-sulfur cluster assembly. Low frataxin expression or a decrease in its functionality results in Friedreichs Ataxia (FRDA). With the aim of creating new molecular tools to study this metabolic pathway, and ultimately, to explore new therapeutic strategies, we have investigated the possibility of obtaining small proteins exhibiting a high affinity for frataxin. In this study, we applied the ribosome display approach, using human frataxin as the target. We focused on Affi_224, one of the proteins that we were able to select after five selection rounds. We have studied the interaction between both proteins and discussed some applications of this specific molecular tutor, concerning the modulation of supercomplex activity. Affi_224 and frataxin showed a KD value in the nanomolar range, as judged by surface plasmon resonance analysis. Most likely, it binds to the frataxin acidic ridge, as suggested by the analysis of chemical shift perturbations (NMR) and computational simulations. Affi_224 was able to increase Cys NFS1 desulfurase activation exerted by the FRDA frataxin variant G130V. Our results suggest quaternary addition may be a new tool to modulate frataxin function in vivo. Nevertheless, more functional experiments under physiological conditions should be carried out to evaluate Affi_224 effectiveness in FRDA cell models.

biochemistry↗