Biology subjects

Cruz, M.

Publications and source records attributed to Cruz, M..

16 recordsLinked to original sources

bioRxiv2025

Transcriptomic and network analyses of an alcohol-induced peripheral neuropathy model identify putative role for histone demethylase Jmjd1c

BackgroundAlcohol-induced peripheral neuropathy (AIPN) is a painful and prevalent condition associated with chronic alcohol use, yet its molecular underpinnings remain poorly understood. Because the analgesic effects of ethanol may reinforce alcohol consumption, elucidating the mechanisms driving AIPN is essential. This study aimed to identify ethanol-regulated gene expression patterns in the nervous system of a mouse model of AIPN. MethodsMale (n = 10) and female (n = 12) C57BL/6J mice were administered either an ethanol-containing Lieber-DeCarli liquid diet at 5% or an isocaloric control diet for four weeks. Ethanol consumption was recorded daily for the experimental group. After the drinking protocol, spinal cord and dorsal root ganglia tissues were collected for RNA sequencing. ResultsEthanol-regulated genes were identified for each sex-tissue group using DESeq2, and results were compared to known rodent neuropathic pain gene signatures. Weighted gene co-expression network analysis (WGCNA) identified modules of co-expressed genes associated with ethanol administration. Hub genes with high intramodular connectivity were identified for ethanol-correlated modules. Of the 14 identified hub genes, 10 have been previously implicated in pain or neuropathy, including Jmjd1c, Phf8, and Gas6, which emerged as particularly strong candidates for involvement in AIPN pathophysiology. ConclusionsThese findings provide novel insights into the gene networks underlying AIPN and nominate specific genes for future functional studies.

neuroscience↗

bioRxiv2025

LCR-modules: a collection of workflows for cancer genome analysis

The surge of genomic data from advanced sequencing technologies is outpacing current analytical pipelines. We introduce LCR-modules, an open-source suite of bioinformatics tools designed for flexible and automated cancer genome data analysis. LCR-modules enables reproducible analysis of diverse cancer genomics data at scale. The suite comprises 49 Snakemake-based workflows organized into three levels, facilitating tasks from low-level quality control to complex cohort-level analyses. LCR-modules supports various sequencing types and integrates pipelines such as mutation calling, expression quantification, and cohort-level aggregation, ensuring flexibility and reproducibility. LCR-modules represents a significant advancement in genomic data analysis, reducing barriers in reproducibility and scalability and has already been applied to a combination of exomes and genomes from over 10,800 samples.

bioinformatics↗

bioRxiv2025

Peripheral Neuropathy After Chronic Alcohol Exposure in Mice: Impact of sex, total intake and duration and alcohol metabolism.

Alcohol induced peripheral neuropathy (AIPN) is a neurodegenerative disease caused by chronic alcohol intake and is associated with peripheral nerve damage and somatosensory symptoms, such as allodynia. Current treatments lack efficacy and do not target underlying pathology emphasizing the need for preclinical models of AIPN to elucidate mechanisms and novel targets. Thus, we performed a detailed characterization of a mouse model of AIPN and candidate mechanistic associations including the role of neuroinflammation and acetaldehyde. Our studies showed chronic alcohol induced mechanical and cold hypersensitivity and deficits in spontaneous behaviors in EtOH concentration-, time- and sex-dependent manners. Female mice drank more alcohol and developed more rapid and severe hypersensitivity but less robust deficits of spontaneous behaviors. The grimace test demonstrated chronic alcohol promoted spontaneous pain independent of sex. Duration of intake impacted alcohol-induced deficits in peripheral nerve electrophysiology amplitude and intra-epidermal nerve fiber density. We characterized an extensive time-course of chronic alcohol-induced neuroinflammation in the DRG and spinal cord and found significant time, sex and tissue effects. Polymorphisms of ALDH2 have been associated with alcohol-induced neuroinflammation, alcohol-related pain, and alcohol-induced peripheral neuropathy. We investigated the role of acetaldehyde, via inhibition of ALDH2, in the development of AIPN and showed that ALDH2 inhibition accelerated and exacerbated development of chronic alcohol-induced hypersensitivity in male and female mice. Overall, our studies in a well-controlled model of AIPN strongly point to neuroinflammation and inflammatory modulators such as acetaldehyde as important mechanistic targets for possible intervention in AIPN.

pharmacology and toxicology↗

bioRxiv2024

Killer Toxin K28 resistance in yeast relies on COG complex mediated trafficking of the defence factor Ktd1

A/B toxins are a diverse family of protein toxins that enter host cells via endocytosis and induce cell death. In yeast, the A/B toxin K28 is internalised to endosomes of susceptible yeast, before following the retrograde trafficking pathway and ultimately triggering cell cycle arrest. The endolysosomal defence factor Ktd1 protects against K28, but its regulation remains unclear. Cog7, a subunit of the conserved oligomeric Golgi (COG) tethering complex, has been implicated in K28 defence, though the mechanism is unknown. We developed a high throughput K28 sensitivity assay and bespoke analysis package to show that all lobe B COG subunits (Cog5 - 8) are required for K28 resistance. Although the COG complex modulates glycosylation of the surface molecules required to bind extracellular K28, our experiments reveal that the hypersensitivity of cog mutants is primarily explained by defects in Ktd1 trafficking. Ktd1 mis-localisation in cog mutants is reminiscent to disruptions in Snc1, a surface cargo that recycles multiple times via the Golgi. This work suggests not only that the COG complex is responsible for the precise trafficking Ktd1 required to mediate toxin defence, but that Ktd1 may survey endolysosomal compartments for internalised K28. This work underpins the importance of Ktd1 in defence against the A/B toxin K28, and implies various membrane trafficking regulators might influence toxin effects in other eukaryotic systems.

cell biology↗

bioRxiv2024

Senescent fibroblasts support tumor growth by inducing immune cell death in humanized models

Syngeneic mouse tumor models have shown that senescence influences the tumor immune response in multiple ways, including the induction of an immunosuppressive microenvironment or the promotion of immune cell recruitment. Yet, the impact of senescence on the tumor immune response in a humanized setting remains largely unexplored. To address this, we employed tumor spheroids and mice bearing tumors immunogenic to human immune cells derived from the same donor. We found that senescent fibroblasts exert a dual effect by enhancing the recruitment of immune cells into the tumor microenvironment while simultaneously promoting the apoptosis of T and NK cells. Mechanistically, we demonstrate that this apoptosis is primarily due to increased Fas ligand (FasL) expression on the surface of senescent fibroblasts. Deletion of FasL on fibroblasts was sufficient to prevent immune cell death and increase tumor cell killing. Our results highlight the importance of evaluating the impact of therapy-induced senescence in humanized models to understand and predict the outcome of cancer treatments.

cancer biology↗

bioRxiv2024

HIV-1 infection is associated with depletion of germinal center B cells and a decrease in IgA+ plasma cells in the GI tract

Gastrointestinal (GI) B cells and plasma cells (PCs) are critical to mucosal homeostasis and the host response to HIV-1 infection. Here, high resolution mapping of human B cells and PCs sampled from the colon and ileum during both viremic and suppressed HIV-1 infection identified a reduction in germinal center (GC) B cells and follicular dendritic cells (FDCs) during HIV-1 viremia. IgA+ PCs are the major cellular output of intestinal GCs and were significantly reduced during viremic HIV-1 infection. PC-associated transcriptional perturbations, including type I interferon signaling, persisted in antiretroviral therapy (ART)-treated individuals, suggesting ongoing disruption of the intestinal immune milieu during ART. GI humoral immune perturbations were associated with changes in the intestinal microbiome composition and systemic inflammation. These findings highlight a key immune defect in the GI mucosa due to HIV-1 viremia. One Sentence SummaryIntestinal germinal center B cell reduction in HIV-1 infection linked to reduced IgA+ plasma cells and systemic inflammation.

immunology↗

bioRxiv2023

Revisiting Reddy: A DLBCL Do-over

The 2017 study by Reddy et al described the comprehensive characterization of somatic drivers of diffuse large B-cell lymphoma using whole exome sequencing.1 After additional large studies relying on exome or whole genome sequencing were published, several oddities unique to the Reddy results have emerged. Seeking to explain the discrepancies, we reanalyzed their data using established open-source pipelines. This revealed thousands of mutations that could not be independently reproduced by these pipelines and a larger set of high-quality mutations that were not reported by Reddy. This caused an artificial under-representation of the mutation prevalence in many genes including clinically relevant hot spots affecting EZH2 and CD79B. More generally, the study had an under-representation of mutations in DLBCL genes that disproportionately affected genes known to have the highest mutation rates. The missing variants and the spurious variants can be attributed to distinct problems with the analytical approaches employed in that study. Our analysis also identified strong associations between mutations and patient outcome including TP53, KMT2D and PIM1, which were not found in the Reddy study. Overall, we demonstrate that this combination of errors influenced many of the central novel findings from their study rendering their results largely non-replicable. The full results of our analyses are included as supplemental items as a resource for other researchers with an interest in the genetics of B-cell lymphomas.

genomics↗

bioRxiv2023

Membrane Lipids Augment Cell Envelope Stress Signaling and Resistance to Antibiotics and Antimicrobial Peptides in Enterococcus faecalis

Enterococci have evolved resistance mechanisms to protect their cell envelopes against bacteriocins and host cationic antimicrobial peptides (CAMPs) produced in the gastrointestinal environment. Activation of the membrane stress response has also been tied to resistance to the lipopeptide antibiotic daptomycin. However, the actual effectors mediating resistance have not been elucidated. Here, we show that the MadRS (formerly YxdJK) membrane antimicrobial peptide defense system controls a network of genes, including a previously uncharacterized three gene operon (madEFG) that protects the E. faecalis cell envelope from antimicrobial peptides. Constitutive activation of the system confers protection against CAMPs and daptomycin in the absence of a functional LiaFSR system and leads to persistence of cardiac microlesions in vivo. Moreover, changes in the lipid cell membrane environment alter CAMP susceptibility and expression of the MadRS system. Thus, we provide a framework supporting a multilayered envelope defense mechanism for resistance and survival coupled to virulence.

microbiology↗

bioRxiv2023

Cisplatin Induces BDNF Downregulation in Middle-Aged Female Rat Model while BDNF Enhancement Attenuates Cisplatin Neurotoxicity

Cancer-related cognitive impairments (CRCI) are debilitating consequences of cancer treatment with platinum agents (e.g., cisplatin) that greatly alter cancer survivors health-related quality of life. Brain-derived neurotrophic factor (BDNF) plays an essential role in neurogenesis, learning, and memory, and the reduction of BDNF is associated with the development of cognitive impairment in various neurological disorders, including CRCI. Our previous CRCI rodent studies have shown that cisplatin reduces hippocampal neurogenesis and BDNF expression and increases hippocampal apoptosis, which is associated with cognitive impairments. Few studies have reported on the effects of chemotherapy and medical stress on serum BDNF levels and cognition in middle-aged female rat models. The present study aimed to compare the effects of medical stress and cisplatin on serum BDNF levels and cognitive performance in 9-month-old female Sprague Dawley rats to age-matched controls. Serum BDNF levels were collected longitudinally during cisplatin treatment, and cognitive function was assessed by novel object recognition (NOR) 14 weeks post-cisplatin initiation. Terminal BDNF levels were collected ten weeks after cisplatin completion. We also screened three BDNF-augmenting compounds, riluzole, ampakine CX546, and CX1739, for their neuroprotective effects on hippocampal neurons, in vitro. We assessed dendritic arborization by Sholl analysis and dendritic spine density by quantifying postsynaptic density-95 (PSD95) puncta. Cisplatin and exposure to medical stress reduced serum BDNF levels and impaired object discrimination in NOR compared to age-matched controls. Pharmacological BDNF augmentation protected neurons against cisplatin-induced reductions in dendritic branching and PSD95. Ampakines (CX546 and CX1739) but not riluzole altered the antitumor efficacy of cisplatin in two human ovarian cancer cell lines, OVCAR8 and SKOV3.ip1, in vitro. In conclusion, we established the first middle-aged rat model of cisplatin-induced CRCI, assessing the contribution of medical stress and longitudinal changes in BDNF levels with cognitive function. We conducted an in vitro screening of BDNF-enhancing agents to evaluate their potential neuroprotective effects against cisplatin-induced neurotoxicity and their effect on ovarian cancer cell viability.

neuroscience↗

bioRxiv2022

Engineered retrovirus-like Arc extracellular vesicles for the in vivo targeted delivery of mRNA into the brain

Systemic delivery of mRNAs into disease neurons is first limited by the blood-brain-barrier (BBB). Leukocyte-derived extracellular vesicles (EVs) can cross the BBB at inflammatory sites, emerging as promising carriers to target the disease brain. However, efficient mRNA loading into EVs and their uptake by neurons remain challenges. Here we incorporated inside EVs the endogenous retrovirus-like Arc protein capsids, stabilized by Arc 5UTR RNA elements, to effectively load and deliver mRNAs. Produced from self-derived leukocytes, engineered retrotransposon Arc EVs (eraEVs) are immunologically inert with minimal clearance. Equipped with endothelial adhesion molecules from donor leukocytes, circulating eraEVs enter the brain enriching at neuro-inflammatory sites. During self-assembly, Arc recruits enveloping proteins onto eraEVs further promoting neuronal uptake. Possessing high effectiveness like viral vectors and biocompatibility as natural vesicles, eraEV-nanocarriers can be produced from virtually all donor cell types, potentially leading to the development of future clinical therapies for a range of diseases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/518870v2_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@260a4aorg.highwire.dtl.DTLVardef@16da754org.highwire.dtl.DTLVardef@4a79dorg.highwire.dtl.DTLVardef@1983da8_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

A more complete story of the genetic bases of resistance to the rice hoja blanca disease

Rice hoja blanca (RHB) is one of the most serious diseases in rice growing areas in tropical Americas. Its causal agent is Rice hoja blanca virus (RHBV), transmitted by the planthopper Tagosodes orizicolus Muir. Genetic resistance is the most effective and environment-friendly way of controlling the disease. So far, only one major quantitative trait locus (QTL) of Oryza sativa ssp. japonica origin, qHBV4.1, that alters incidence of the virus symptoms in two Colombian cultivars has been reported. This resistance has already started to be broken, stressing the urgent need for diversifying the resistance sources. In the present study we performed a search for new QTLs of O. sativa indica origin associated with RHB resistance. We used four F2:3 segregating populations derived from indica resistant varieties crossed with a highly susceptible japonica pivot parent. Beside the standard method for measuring disease incidence, we developed a new method based on computer-assisted image processing to determine the affected leaf area (ALA) as a measure of symptom severity. Based on the disease severity and incidence scores in the F3 families under greenhouse conditions, and SNP genotyping of the F2 individuals, we identified four new indica QTLs for RHB resistance on rice chromosomes 4, 6 and 11, namely qHBV4.2WAS208, qHBV6.1PTB25, qHBV11.1 and qHBV11.2. We also confirmed the wide-range action of qHBV4.1. Among the five QTLs, qHBV4.1 and qHBV11.1 had the largest effects on incidence and severity, respectively. These results provide a more complete understanding of the genetic bases of RHBV resistance in the cultivated rice gene pool, and can be used to develop marker-aided breeding strategies to improve RHB resistance. The power of joint-and meta-analyses allowed precise mapping and candidate gene identification, providing the basis for positional cloning of the two major QTLs qHBV4.1 and qHBV11.1.

genetics↗

bioRxiv2022

Synovial fibroblasts assume distinct functional identities and secrete R-spondin 2 to drive osteoarthritis

ObjectivesSynovium is acutely affected following joint trauma and contributes to post-traumatic osteoarthritis (PTOA) progression. Little is known about discrete cell types and molecular mechanisms in PTOA synovium. We aimed to describe synovial cell populations and their dynamics in PTOA, with a focus on fibroblasts. We also sought to define mechanisms of synovial Wnt/{beta}-catenin signaling, given its emerging importance in arthritis. MethodsWe subjected mice to non-invasive anterior cruciate ligament rupture as a model of human joint injury. We performed single-cell RNA-sequencing to assess synovial cell populations, subjected Wnt-GFP reporter mice to joint injury to study Wnt-active cells, and performed intra-articular injections of the Wnt agonist R-spondin 2 (Rspo2) to assess whether gain-of-function induced pathologies characteristic of PTOA. Lastly, we used cultured fibroblasts, macrophages, and chondrocytes to study how Rspo2 orchestrates crosstalk between joint cell types. ResultsWe uncovered seven distinct functional subsets of synovial fibroblasts in healthy and injured synovium, and defined their temporal dynamics in early and established PTOA. Wnt/{beta}-catenin signaling was overactive in PTOA synovium, and Rspo2 was strongly induced after injury and secreted exclusively by Prg4hi lining fibroblasts. Trajectory analyses predicted that Prg4hi lining fibroblasts arise from a pool of Dpp4+ mesenchymal progenitors in synovium, with SOX5 identified as a potential regulator of this emergence. We also showed that Rspo2 orchestrated pathological crosstalk between synovial fibroblasts, macrophages, and chondrocytes. ConclusionsSynovial fibroblasts assume distinct functional identities during PTOA, and Prg4hi lining fibroblasts secrete the Wnt agonist Rspo2 to drive pathological crosstalk in the joint after injury.

cell biology↗

bioRxiv2021

Anti-SARS-CoV-2 hyperimmune immunoglobulin provides potent and robust neutralization capacity and antibody-dependent cellular cytotoxicity and phagocytosis induction through N and S proteins

BackgroundAlthough progressive COVID-19 vaccinations provide a significant reduction of infection rate in the short-to mid-term, effective COVID-19 treatments will continue to be an urgent need. MethodsWe have functionally characterized the anti-SARS-CoV-2 hyperimmune immunoglobulin (hIG) prepared from human COVID-19 convalescent plasma. SARS-CoV-2 virus neutralization was evaluated by four different methodologies (plaque reduction, virus induced cytotoxicity, TCID50 reduction and immunofluorimetry-based methodology) performed at four different laboratories and using four geographically different SARS-CoV-2 isolates (one each from USA and Italy; two from Spain). Two of the isolates contained the D614G mutation. Neutralization capacity against the original Wuhan SARS-CoV-2 straom and variants (D614G mutant, B.1.1.7, P.1 and B.1.351 variants) was evaluated using a pseudovirus platform expressing the corresponding spike (S) protein. The capacity to induce antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) was also evaluated. ResultsAll the SARS-CoV-2 isolates tested were effectively neutralized by hIG solutions. This was confirmed by all four methodologies showing potent neutralization capacity. Wild-type SARS-CoV-2 and variants were effectively neutralized as demonstrated using the pseudovirus platform. The hIG solutions had the capacity to induce ADCC and ADCP against SARS-CoV-2 N and S proteins but not the E protein. Under our experimental conditions, very low concentrations (25-100 {micro}g IgG/mL) were required to induce both effects. Besides the S protein, we observed a clear and potent effect triggered by antibodies in the hIG solutions against the SARS-CoV-2 N protein. ConclusionsThese results show that, beyond neutralization, other IgG Fc-dependent pathways may play a role in the protection from and/or resolution of SARS-CoV-2 infection when using hIG COVID-19 products. This could be especially relevant for the treatment of more neutralization resistant SARS-CoV-2 variants of concern.

immunology↗

bioRxiv2020

Production of anti-SARS-CoV-2 hyperimmune globulin from convalescent plasma

BACKGROUNDIn late 2019, the SARS-CoV-2 virus emerged in China and quickly spread into a world-wide pandemic. Prior to the development of specific drug therapies or a vaccine, more immediately available treatments were sought including convalescent plasma. A potential improvement from convalescent plasma could be the preparation of anti-SARS-CoV-2 hyperimmune globulin (hIVIG). STUDY DESIGN AND METHODSConvalescent plasma was collected from an existing network of plasma donation centers. A caprylate/chromatography purification process was used to manufacture hIVIG. Initial batches of hIVIG were manufactured in a versatile, small-scale facility designed and built to rapidly address emerging infectious diseases. RESULTSProcessing convalescent plasma into hIVIG resulted in a highly purified IgG product with more concentrated neutralizing antibody activity. hIVIG will allow for the administration of greater antibody activity per unit of volume with decreased potential for several adverse events associated with plasma administration. IgG concentration and IgG antibody specific to SARS-CoV-2 were increased over 10-fold from convalescent plasma to the final product. Normalized ELISA activity (per mg/mL IgG) was maintained throughout the process. Protein content in these final product batches was 100% IgG, consisting of 98% monomer and dimer forms. Potentially hazardous proteins (IgM, IgA, and anti-A, anti-B and anti-D antibodies) were reduced to minimal levels. CONCLUSIONSMultiple batches of anti-SARS-CoV-2 hyperimmune globulin (hIVIG) that met regulatory requirements were manufactured from human convalescent plasma. The first clinical study in which the hIVIG will be evaluated will be Inpatient Treatment with Anti-Coronavirus Immunoglobulin (ITAC) [NCT04546581].

immunology↗

bioRxiv2019

A CyclinB2-Cas9 fusion promotes the homology-directed repair of double-strand breaks

The discovery of clustered regularly interspaced palindromic repeats (CRISPR), a defense system against viruses found in bacteria, launched a new era in gene targeting. The key feature of this technique is the guiding of the endonuclease Cas9 by single guide RNAs (sgRNA) to specific sequences, where a DNA lesion is introduced to trigger DNA repair. The CRISPR/Cas9 system may be extremely relevant for gene therapy, but the technique needs improvement to become a safe and fully effective tool. The Cas9-induced double-strand break (DSB) is repaired by one of two pathways, the error-prone Non-homologous end joining (NHEJ) or the high-fidelity Homology Direct Repair (HDR). Shifting the repair of the DSB to HDR is challenging, given the efficiency of NHEJ. Here we describe an engineered protein approach to increase knock-in efficiency by promoting the relative increase in Cas9 activity in G2, the phase of the cell cycle where HDR is more active. Cas9 was fused to the degradation domain of proteins known to be degraded in G1. The activity of two chimeric proteins, Geminin-Cas9 and CyclinB2-Cas9, is demonstrated, as well as their cell-cycle-dependent degradation. The chimeras shifted the repair of the DSBs to the HDR repair pathway compared to the commonly used Cas9. The application of cell cycle specific degradation tags could pave the way for more efficient and secure gene editing applications of the CRISPR/Cas9 system.

molecular biology↗

bioRxiv2018

Mitotic progression, arrest, exit or death is determined by centromere integrity and independent of de novo transcription

Recent studies have challenged the prevailing dogma that transcription is repressed during mitosis. Transcription was also proposed to sustain the spindle assembly checkpoint (SAC) for several hours in response to unattached kinetochores. Here we used live-cell imaging of human cells in culture, combined with RNA-seq and qPCR, to investigate the requirement for de novo transcription during mitosis. Under conditions of persistently unattached kinetochores, transcription inhibition with actinomycin D, or treatment with other DNA-intercalating drugs, delocalized the chromosomal passenger complex (CPC) protein Aurora B from centromeres, compromising SAC robustness and cell fate. However, we were unable to detect significant changes in transcript levels. Moreover, inhibition of transcription independently of DNA intercalation had no effect on SAC response, mitotic progression, exit or death. Mechanistically, we show that DNA intercalating agents reduce the interaction of the CPC with nucleosomes. Thus, the capacity of human cells to progress, sustain, exit or die in mitosis relies on centromere integrity, rather than de novo transcription.

cell biology↗