Biology subjects

Salazar, M. G.

Publications and source records attributed to Salazar, M. G..

6 recordsLinked to original sources

bioRxiv2025

Immunoglobulin sub-class levels define inter-donor plasma variability: a longitudinal dual-lab study

Advancements in mass spectrometry have transformed plasma proteomics, allowing for high-throughput analysis of large cohorts. This study utilized the TIMES cohort, consisting of 51 healthy participants monitored monthly over 12 months, to evaluate intra- and inter-individual variability in the plasma proteome. Approximately 600 samples were analyzed in two independent laboratories, revealing strong correlations despite methodological differences. The study focused on the stability of the plasma proteome within donors over a year, with larger differences observed between donors. A support vector machine model achieved a 98% median classification accuracy, confirming stable, donor-specific proteomic profiles. Immunoglobulins, often overlooked in plasma proteomics, were found to contribute significantly to donor-specific signatures, showing pronounced inter-individual variability but remarkable intra-donor stability. In contrast, C-reactive protein and other inflammation markers exhibited significant temporal fluctuations and donor-specific baseline levels. This inter-laboratory study highlights the importance of longitudinal sampling for biomarker discovery, the robustness of MS-based proteomic workflows, and provides new insights into immunoglobulin dynamics and variability in human plasma. Synopsis O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/686385v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1e7b501org.highwire.dtl.DTLVardef@11c85d7org.highwire.dtl.DTLVardef@1971a1dorg.highwire.dtl.DTLVardef@1338061_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LILongitudinal analysis of individual plasma proteomes over a one-year period revealed low intra-individual variability albeit high inter-individual differences. C_LIO_LICross-site laboratory comparisons confirmed strong concordance in analytical performance and measurement consistency. C_LIO_LIConversion of relative protein intensities to absolute concentrations revealed immunoglobulin levels on par with established clinical reference ranges. C_LIO_LIDonors were distinguishable based on their unique plasma proteome. C_LIO_LIThe immunoglobulin sub-classes are key discriminators defining inter- and intra-donor variability C_LI

molecular biology↗

bioRxiv2025

Requirement of ClpX for CtsR dissociation from its operator elements upon heat stress in Bacillus subtilis

A sudden increase in temperature triggers Bacillus subtilis to activate expression of stress-specific heat shock proteins of the CtsR (class three stress gene repressor) regulon to withstand the adverse conditions. Key members of this regulon, such as ATPases, proteolytic subunits and their adaptors, which can assemble to the functional Clp protease system, perform crucial roles in maintaining cellular proteostasis, while their transcription is repressed by CtsR during vegetative growth. Upon heat shock, a conformational change in a thermosensing glycine-rich loop causes CtsR to detach from its DNA operators, enabling the transcriptional activation of the regulon. Novel data from a clpX-deficient strain demonstrated that in addition, the presence of the ATPase ClpX is essential for the CtsR dissociation from its DNA binding site. To further elucidate this role of ClpX, we constructed a conditional clpX strain, in which clpX induction is decoupled from its native transcriptional control. This conditional expression system mimicked a clpX-deficient phenotype under non-inducing conditions and restored the wild-type phenotype upon induction. Our results indicate that the full induction of the CtsR regulon, particularly clpE, requires both heat and the presence of ClpX, thereby extending the current model for the transcriptional activation of genes repressed by CtsR.

microbiology↗

bioRxiv2025

Temperature-dependent regulation of bacterial cell division hydrolases by the coordinated action of a regulatory RNA and the ClpXP protease

A defining feature of bacteria is the peptidoglycan cell wall which provides structural integrity and prevents osmotic lysis. While peptidoglycan hydrolases are required for daughter cell separation, dysregulated cell wall degradation may result in cell lysis. The mechanisms allowing bacteria to control these deadly enzymes in response to environmental changes remain incompletely understood. Here, we find that in Staphylococcus aureus, temperature-dependent regulation of such hydrolases occurs by the coordinated action of a CHAP domain-specific regulatory RNA and the ClpXP protease. Using a proteomics approach, we identify a hitherto uncharacterized ClpXP controlled autolysin, CxcA, with a catalytic CHAP domain and show that it contributes to separation of daughter cells. CxcA is positively controlled by a non-coding RNA, named Rbc1 (for RNA binding to CHAP domain) transcribed from the antisense strand of cxcA. Notably, Rbc1 is capable of base pairing with RNAs encoding the CHAP domains of numerous cell wall hydrolases and we show that Rbc1 works in trans to upregulate the cell division hydrolase Sle1. Specifically, Rbc1 functions as a thermosensor allowing for upregulation of CxcA and Sle1 at low temperature where daughter cell separation is impeded. Interestingly, the Rbc1-mediated up-regulation of CxcA and Sle1 does not involve mRNA stabilization or increased translation; instead, Rbc1 depletion increases ClpXP-mediated degradation. In conclusion, we identify a novel cell division hydrolase that is highly conserved in Staphylococci and show that it is co-regulated with enzymes containing the catalytic CHAP domain via transcriptional regulation, an RNA-RNA temperature sensory mechanism and the ClpXP protease.

microbiology↗

bioRxiv2023

Multi-omics investigations uncover unique pathogenic markers in clinical Klebsiella pneumoniae that could be leveraged as novel antimicrobial targets

BackgroundKlebsiella pneumoniae (KP), often multidrug-resistant (MDR), is a significant public health concern and frequently associated with various diseases including urinary-tract infection. In addition, in recent years, an increasing number of studies reports on the emergence of convergent KP that combine MDR with hypervirulence leading to severely limited treatment options and thus calling for alternative approaches. MethodsIn this study, we compared high-risk clonal KP lineages with less pathogenic Klebsiella variicola (KV) and Klebsiella quasipneumoniae (KQ) strains on multiple-omics levels and performed integrative data analysis to identify unique markers that could be subsequently leveraged as novel targets in alternative treatment strategies. ResultsOur initial genomic analysis revealed 107 genes as part of the patho-core genome in eight clinical KP that were associated with different metabolic pathways. Subsequent transcriptome and proteome analyses in infection-mimicking media demonstrated similar regulatory patterns among KP vs. other Klebsiella strains, again with metabolic responses playing a pivotal role. In total, we identified 193 KP-specific, differentially expressed genes on transcriptomic and/or proteomic levels. When then comparing these regulated genes to over 6,000 publicly available Klebsiella genomes, we identified unique markers either in KP genomes or adaptively regulated on transcriptomics and/or proteomics levels. An example for the latter was a gene cluster for the cellobiose phosphotransferase system that has been previously described in the context of bacterial virulence and biofilm formation. ConclusionIn conclusion, our study not only highlights that KP strains demonstrate metabolic flexibility in response to particular environmental conditions, which is potentially important for their success as opportunistic pathogens, but identified unique KP-markers. Subsequent studies are needed to explore whether these markers might be prospectively used as novel anti-virulence targets, providing alternatives to traditional antibiotics.

microbiology↗

bioRxiv2023

Automated Maternal Behavior during Early life in Rodents (AMBER) pipeline

Mother-infant interactions during the early postnatal period are critical for infant survival and the scaffolding of infant development. Rodent models are used extensively to understand how these early social experiences influence neurobiology across the lifespan. However, methods for measuring postnatal dam-pup interactions typically involve time-consuming manual scoring, vary widely between research groups, and produce low density data that limits downstream analytical applications. To address these methodological issues, we developed the Automated Maternal Behavior during Early life in Rodents (AMBER) pipeline for quantifying home-cage maternal and mother-pup interactions using open-source machine learning tools. DeepLabCut was used to track key points on rat dams (32 points) and individual pups (9 points per pup) in postnatal day 1-10 video recordings. Pose estimation models reached key point test errors of approximately 4.1-10 mm (14.39 pixels) and 3.44-7.87 mm (11.81 pixels) depending on depth of animal in the frame averaged across all key points for dam and pups respectively. Pose estimation data and human-annotated behavior labels from 38 videos were used with Simple Behavioral Analysis (SimBA) to generate behavior classifiers for dam active nursing, passive nursing, nest attendance, licking and grooming, self-directed grooming, eating, and drinking using random forest algorithms. All classifiers had excellent performance on test frames, with F1 scores above .886. Performance on hold-out videos remained high for nest attendance (F1=.990), active nursing (F1 =.828), and licking and grooming (F1=.766) but was lower for eating, drinking, and self-directed grooming (F1=.534-.554). A set of 242 videos was used with AMBER and produced behavior measures in the expected range from postnatal 1-10 home-cage videos. This pipeline is a major advancement in assessing home-cage dam-pup interactions in a way that reduces experimenter burden while increasing reproducibility, reliability, and detail of data for use in developmental studies without the need for special housing systems or proprietary software.

animal behavior and cognition↗

bioRxiv2021

ACTN2 missense variant causes proteopathy in human iPSC-derived cardiomyocytes

Genetic variants in -actinin-2 (ACTN2) are associated with several forms of (cardio)myopathy. We previously reported a heterozygous missense (c.740C>T) ACTN2 gene variant, associated with hypertrophic cardiomyopathy, and characterized by an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we created with CRISPR/Cas9 genetic tools two heterozygous functional knock-out hiPSC lines with a second wild-type (ACTN2wt) and missense ACTN2 (ACTN2mut) allele, respectively. We evaluated their impact on cardiomyocyte structure and function, using a combination of different technologies, including immunofluorescence and live cell imaging, RNA-seq, and mass spectrometry. This study showed that ACTN2mut present a higher percentage of multinucleation, protein aggregation, hypertrophy, myofibrillar disarray and activation of both the ubiquitin-proteasome system and the autophagy-lysosomal pathway as compared to ACTN2wt in 2D-cultured hiPSC-CMs. Furthermore, the expression of ACTN2mut was associated with a marked reduction of sarcomere-associated protein levels in 2D-cultured hiPSC-CMs and force impairment in engineered heart tissues. In conclusion, our study highlights the activation of proteolytic systems in ACTN2mut hiPSC-CMs likely to cope with ACTN2 aggregation and therefore directs towards proteopathy as an additional cellular pathology caused by this ACTN2 variant, which may contribute to human ACTN2-associated cardiomyopathies.

cell biology↗