Biology subjects

Rivera, A.

Publications and source records attributed to Rivera, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Development of an HK-fbp1-based vaccine platform against Cryptococcus neoformans

Cryptococcal meningitis remains a leading cause of death among HIV/AIDS patients, and no antifungal vaccine is available in clinical use. We previously showed that mice immunized with a heat-killed preparation of Cryptococcus neoformans (Cn) fbp1{Delta} mutant (HK-fbp1) are protected against cryptococcosis and other invasive mycoses, suggesting that HK-fbp1 is an intriguing vaccine candidate for further study. Recent studies have reported that heat-killed wild type H99 (HK-H99) can also offer some protection in other vaccination models. Here we set out to decipher the differential protective capacity of HK-fbp1 versus HK-H99 across multiple conditions. We observed that HK-fbp1 confers superior protection in both prophylactic and therapeutic intranasal immunization settings than HK-H99, which was associated with greater activation of pulmonary CD4+ T cells and stronger airway IFN-{gamma} production. In addition, HK-fbp1 preparations were highly stable, retaining full protective efficacy even after 5 years of frozen storage. Mice intranasally immunized with two-dose HK-fbp1 were protected against challenge by up to 106 H99 cells, a 100-fold increase over the commonly used lethal inoculum. Addition of a CpG adjuvant ODN to HK-fbp1 (HK-bp1/ODN) led to a 5-fold reduction of the intranasal vaccine inoculum required for protection, and its single-dose intramuscular vaccination induced protection persisted for at least 3 months post vaccination. Together, these findings highlight the robust protective capacity and formulation versatility of HK-fbp1, supporting its further development as a stable, dose-sparing antifungal vaccine candidate.

microbiology↗

bioRxiv2026

Analysis of the spatiotemporal dynamics of behavior in domestic dogs with free locomotion under appetitive Pavlovian contingencies

Spatiotemporal dynamics of behavior is key to understanding the organism-environment relationship. While often implicitly addressed, its relation to Pavlovian contingencies remains understudied in domestic dogs. This study quantitatively examined the spatial dimension of behavior of three freely moving dogs under pairing and extinction of a tone-food Pavlovian contingency. In pairing, a tone (CS) was paired with food delivery (US) on a fixed-time 60-second (FT 60 s) schedule; in extinction, only the tone was presented. Locomotion was recorded using two-dimensional tracking based on center of mass. During pairing, dogs moved closer to the dispenser, covered greater distances, displayed extended trajectories, and showed a conditional approach pattern to the dispenser during CS presentation. In extinction, they stayed closer to the owner or room periphery, traveled shorter distances, and exhibited more restricted trajectories. These findings show that spatial segments (dispenser area) are integrated into the CS-US relationship, demonstrate the usefulness of continuous recording of spatial behavior in the analysis of Pavlovian contingencies, and suggest potential application in contexts relevant to animal welfare. HighlightsO_LISpatial behavior remains largely unexplored in studies of Pavlovian contingencies C_LIO_LIDog locomotion was continuously tracked during pairing and extinction phases C_LIO_LIDistinct spatial patterns emerged across pairing and extinction phases C_LIO_LIIncorporating space into CS-US relations clarifies behavioral organization C_LIO_LITracking tools expand the experimental and applied scope of Pavlovian research C_LI

animal behavior and cognition↗

bioRxiv2026

Multidimensional analysis of drought response in an inter-specific tomato population (ToMAGIC)

Drought stress poses a significant threat to agricultural productivity, particularly in regions with limited water availability. This study delves into the drought response in a multiparental interspecific tomato MAGIC population (ToMAGIC), developed by intercrossing Solanum pimpinellifolium (SP) and S. lycopersicum var. cerasiforme (SLC). A core collection of 139 recombinant lines, selected for their genetic diversity, was evaluated under both control and water stress conditions over two consecutive years. Phenotypic data were collected for 25 traits, including vegetative growth, flowering, fruit production, and physiological traits, providing a comprehensive assessment of drought response. Genome-wide association studies (GWAS) identified 15 significant genomic regions associated with drought response across eight chromosomes, highlighting key loci related to growth, earliness, fruit set, and physiological traits such as stomatal conductance and proline accumulation. Transgressive lines, such as S5_T_600 and S5_T_601, which exhibit enhanced drought resilience compared to the parental lines, were identified through genomic assisted selection, highlighting their potential as valuable breeding materials. The study emphasizes the importance of the ToMAGIC population in uncovering the polygenic nature of drought response. These findings offer valuable insights for developing drought-resilient tomato cultivars supporting agricultural sustainability in water-limited environments.

genomics↗

bioRxiv2025

Causal modelling reveals lower coral genetic diversity in more heterogenous reef environments

Conserving genetic diversity is crucial for maintaining species evolutionary potential and resilience to environmental change. Yet, directly measuring genetic diversity across large spatial scales remains resource-intensive and impractical for conservation planning. Identifying reliable, accessible, and cost-effective spatial predictors of genetic diversity would significantly enhance our ability to incorporate genetic diversity into conservation efforts. Caribbean coral reefs represent a compelling system for investigating such predictors, as these ecosystems face unprecedented threats from disease outbreaks and climate change. Here, we investigate the utility of several environmental proxies as surrogates for genetic diversity, which may be useful for conservation planning in resource- or data-poor regions. To achieve this goal, we used reduced-representation genomic sequencing to estimate intraspecific genetic diversity, and in doing so, identified two genetically distinct and sympatric cryptic coral species in the Agaricia tenuifolia species complex. We employed a causal inference approach to assess the relationships between genetic alpha diversity and habitat area and connectivity, habitat heterogeneity, and temperature. Statistical investigations revealed that increased habitat heterogeneity, derived from remotely sensed habitat maps, was associated with lower genetic diversity within coral populations, suggesting that habitat characteristics promoting species diversity may not maintain high genetic diversity within a single species. Habitat area and connectivity, and temperature have predominantly negative effects on genetic diversity with coral habitat area and connectivity showing markedly contrasting responses in two cryptic taxa. Thus, our findings reveal that context matters greatly, as results differ between cryptic species, across variables, and among spatial scales.

evolutionary biology↗

bioRxiv2025

Combining the alpha-2 adrenergic agonist clonidine with naloxone rescues fentanyl-induced physiologic dysfunction and increases survival

Fentanyl leads to tens of thousands of overdose deaths every year despite widespread availability of naloxone. Like other opioids, fentanyl causes respiratory depression. Unlike morphine, high dose fentanyl rapidly produces airway obstruction, muscle rigidity, and cardiovascular failure. Using a rat model of opioid overdose, we compared the physiological effects of fentanyl and morphine and studied the efficacy of a novel rescue strategy. In contrast to morphine, we report that fentanyl more frequently causes respiratory failure secondary to vocal cord closure and leads to more severe cardiovascular disruption, including the blockade of baroreflex-like rebound in blood pressure. We also show that administration of intramuscular naloxone immediately after intravenous infusion of fentanyl did not improve survival. However, combining intramuscular naloxone with the alpha-2 adrenergic agonist clonidine rescued vocal cord function and stabilized cardiovascular and respiratory physiology from fentanyl-induced effects. Our findings demonstrate that fentanyl is associated with a unique and more severe toxidrome compared to morphine. Also, supplementing naloxone with drugs targeting the adrenergic system improves survival primarily by reopening the upper airway, implicating airway obstruction as a significant component of fentanyl-induced respiratory depression. Therefore, reversal of vocal cord closure appears to be the necessary precursor to the restoration of not only respiration, but also vascular autoregulation, a significant determinant of survival from fentanyl overdose.

pharmacology and toxicology↗

bioRxiv2025

Toll-like Receptor 4 Contributes to PCOS-like Metabolic and Reproductive Pathogenesis

Polycystic ovary syndrome (PCOS) is a reproductive disorder with heterogeneous symptoms and severity. Despite extensive research documenting chronic immune dysfunction as a hallmark of PCOS, the specific molecular mechanisms driving immune activation and its connection to the syndromes diverse symptoms remain poorly understood. Emerging evidence suggests that gut-derived bacterial endotoxins, particularly lipopolysaccharide (LPS), may breach the intestinal barriers in PCOS patients and trigger systemic inflammation through Toll-like receptor 4 (TLR4), a pattern recognition receptor of the innate immune system. This study investigated whether TLR4 serves as a critical mechanistic driver of PCOS pathogenesis by examining the effect of genetic TLR4 knockout (TLR4-/-) in a letrozole (LET)-induced mouse model of PCOS. Our results demonstrate that TLR4 deficiency reduces many PCOS-like symptoms, including elevated luteinizing hormone, anovulation, and metabolic dysfunction. TLR4 knockout also preserved estrous cycling and fertility, improved glucose tolerance, maintained gut barrier integrity, and reduced inflammatory markers in LET-treated females. These findings establish TLR4 as a key mediator orchestrating PCOSs multi-system pathology, positioning TLR4 as a critical convergence point rather than affecting individual symptoms in isolation. This novel work reveals that TLR4-mediated inflammation drives multiple PCOS pathologies, opening avenues for targeted anti-inflammatory treatments in women with this disorder. Significance StatementPolycystic ovary syndrome (PCOS) affects up to 15% of reproductive-age women worldwide. This study reveals that TLR4, an innate immune receptor, is key to the pathophysiology of PCOS-like symptoms in female mice. When TLR4 was genetically deleted, mice treated with letrozole to induce PCOS-like symptoms maintained normal weight, glucose regulation, estrous cycling, and fertility. The improvements coincided with preserved gut barrier breakdown and reduced inflammation. These findings identify TLR4 as a key mediator between gut health, immune activation, and PCOS pathophysiology, suggesting that targeting TLR4 could offer new therapeutic approaches for this common but poorly understood syndrome affecting millions of women.

immunology↗

bioRxiv2025

Mannoprotein Cig1 Contributes to the Immunogenicity of a Heat-Killed F-box Protein Fbp1 Cryptococcus neoformans Vaccine Model

Currently, no fungal vaccine exists for clinical use while fungal infections are responsible for over 1.5 million deaths every year. Our previous studies identified a Cryptococcus neoformans mutant strain fbp1{Delta} as a potential vaccine candidate. This mutant strain contains a deletion of the F-box protein Fbp1, a key subunit of the SCF E3 ligase complex necessary for ubiquitin-mediated proteolysis. Vaccination with heat-killed fbp1{Delta} (HK-fbp1) can elicit protection against C. neoformans parental strain and its sibling species C. gattii in an interferon gamma (IFN-{gamma}) dependent Type 1 immune response. However, we have yet to decipher the immunogenic factor(s) expressed by the fbp1{Delta} mutant that are responsible for the induction of the protective immune response. In this study, we have identified that capsule plays an important role in HK-fbp1 vaccine mediated protection, as acapsular HK-fbp1 cells showed diminished protection against wild type challenge. Additionally, our studies have shown that Cytokine Inducing Glycoprotein 1 (Cig1), a GPI anchored mannoprotein, is regulated by Fbp1 and contributes to the immunogenicity of HK-fbp1. Deletion of Cig1 in the fbp1{Delta} background resulted in decreased recruitment of anti-fungal effector T cells and diminished production of protective inflammatory cytokines by the host. Furthermore, loss of Cig1 in the fbp1{Delta} mutant resulted in reduced protection in vaccination survival studies at lower vaccine inoculum doses compared to HK-fbp1. In aggregate, these findings demonstrate Cig1 is an antigen contributing to the immunogenicity of HK-fbp1 that may be utilized to further optimize the HK-fbp1 fungal vaccine as a tool in the arsenal against invasive fungal infections.

microbiology↗

bioRxiv2025

Basal activation of ERK1/2 blunts the antimicrobial activity of neutrophils from aged hosts against antibody opsonized Streptococcus pneumoniae

The decline in neutrophil antimicrobial function renders vaccinated aged hosts less protected against Streptococcus pneumoniae infection. In vaccinated hosts, activation of neutrophils via phagocytic receptors, like complement and Fc{gamma} receptors, mediates bacterial uptake and killing. However, the mechanisms behind changes in signaling downstream of these receptors with age is not known. Mitogen-activated protein kinases (MAPK) are signaling cascades activated in response to cell surface receptors. Using bone marrow neutrophils isolated from young and aged mice, and MAPK phosphorylation array, we found differences in MAPK activation in an opsonin dependent manner (complement vs antibody). Neutrophils from aged mice failed to increase MAPK phosphorylation upon infection with pneumococci opsonized with heat-inactivated (HI) sera from hosts immunized with the pneumococcal conjugate vaccine (PCV), indicating an age-related defect in Fc{gamma}R signaling. Neutrophils from aged mice had higher basal levels of MAPK phosphorylation when compared to young controls, including a 15-fold increase in ERK1/2 phosphorylation. Inhibition of ERK1/2 signaling blunted pneumococcal killing by PMNs from young mice, but improved killing by neutrophils from aged mice, only when the bacteria were opsonized with HI immune but not naive sera. In young human participants, in vitro inhibition of ERK1/2 in neutrophils resulted in decreased pneumococcal killing, but only following PCV vaccination, suggesting that the effects of ERK1/2 inhibition are clinically relevant in vaccinated hosts. This study identifies age-related changes in ERK1/2 activation and demonstrates that balanced activation of this pathway is crucial for neutrophil antimicrobial activity against antibody opsonized bacteria following vaccination in both mice and humans. Summary sentenceImbalanced ERK1/2 activation impairs neutrophil activity in aged hosts. LEGENDS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/656427v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@179c31forg.highwire.dtl.DTLVardef@f95d4borg.highwire.dtl.DTLVardef@15c1dcborg.highwire.dtl.DTLVardef@a8076e_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical AbstractGraphic Abstract Created in BioRender. Bou Ghanem, E. (2025) https://BioRender.com/jjoodsu.

immunology↗

bioRxiv2025

The PBAF chromatin remodeling complex contributes to metal homeostasis through Mtf1 regulation

SWI/SNF chromatin remodeling complexes regulate gene expression by modulating nucleosome positioning, yet their roles in metal homeostasis during skeletal muscle development remain unclear. Here, we uncover distinct functions of the BAF, PBAF, and ncBAF complexes in myoblast proliferation under metal stress. While knockdown (KD) of Baf250a (BAF-specific) or Brd9 (ncBAF-specific) reduces myoblast proliferation, Baf180 (PBAF-specific) KD does not impair cell proliferation under basal conditions. Interestingly, supplementation with copper (Cu) or zinc (Zn) rescues proliferation in Baf250a- and Brd9-deficient myoblasts but paradoxically inhibits growth in Baf180 KD cells. Mechanistically, Baf180 KD disrupts Cu and Zn homeostasis, leading to intracellular Cu accumulation without labile Cu pools and impaired expression of Atp7a, a key Cu exporter. Transcriptomic analyses reveal widespread gene dysregulation in metal-treated Baf180-deficient cells, while metal supplementation promotes pro-proliferative gene expression in Baf250a- and Brd9-KD myoblasts. CUT&RUN assays demonstrate that metal-responsive transcription factor Mtf1 exhibits increased chromatin binding upon Cu treatment, targeting genes involved in stress response and myogenesis. Notably, Mtf1 colocalizes with Baf180 in the nucleus and co-immunoprecipitates with both conserved SWI/SNF subunits and Baf180, suggesting a functional interplay between PBAF and Mtf1 in regulating metal-dependent gene expression. Our findings establish the PBAF complex as a crucial regulator of Cu/Zn homeostasis in myoblast proliferation via Mtf1, while metal supplementation compensates for BAF and ncBAF dysfunction but exacerbates defects in PBAF-deficient cells. This study reveals a novel link between chromatin remodeling, metal signaling, and muscle development, with implications for stress adaptation and metabolic regulation in myogenesis.

molecular biology↗

bioRxiv2025

Helminth infection favors reprogramming and proliferation of lung neutrophils

Neutrophils are a granulocytic population of myeloid cells that have critical effector functions during infectious disease but are generally thought to be short-lived and nonproliferative with markedly limited activation states. In these studies, we directly compared lung neutrophil activation following infection with different groups of pathogens including bacteria, fungi, and helminths. Our results demonstrate considerable heterogeneity depending on the type of infectious agent. In contrast to bacterial and fungal infection, after helminth infection neutrophils expressed markers associated with characteristic type 2 responses and unexpectedly upregulated genes associated with cell cycling and protein synthesis. Further studies showed reduced neutrophil cell death following helminth infection and increased proliferation, which was dependent on IL-4R signaling. This distinct subset of proliferating neutrophils expanded following helminth infection and was released from the endothelial niche to colocalize with invading parasites in the airways. These studies demonstrate a novel long-lived cycling phenotype for neutrophils following helminth infection.

immunology↗

bioRxiv2025

The diversity of cellular systems involved in carbonate precipitation by Escherichia coli

Climate change is increasing the need to limit levels of anthropogenic CO2 released into the atmosphere. One approach being investigated is to generate products based on microbially induced carbonate precipitation (MICP), which can trap CO2 as CaCO3. We recently identified a novel MICP pathway in bacteria that is initiated by Ca2+ toxicity in cells, causing extracellular CO2 to be trapped as CO 2- by Escherichia coli, although the yield of precipitated CaCO remained low (in the milligram range). In this work, we used the E. coli Keio gene knock-out library to identify 54 genes involved in MICP in E. coli, which could be broadly characterized into four groups: central metabolism, iron metabolism, cell architecture, and transport. The role of central metabolism appears to be crucial in maintaining alkaline conditions surrounding the cell that promote CaCO3 precipitation. The role of iron metabolism was less clear, although the results suggest that growth rate influences the initiation of MICP. While the impact of repeating polymeric structures on cell surfaces promoting MICP is well established, our results suggest that other structural features may play a role, including fimbriae and flagella. Finally, the results confirmed that Ca2+ transport is central to MICP under calcium stress. The results further suggest that the ZntB efflux pump may play a previously unidentified role in Ca2+ transport in E. coli. By overexpressing some of these genes, our work suggests that there are several previously unidentified cellular mechanisms that could serve as a target for enhanced MICP in E. coli. By incorporating these processes into MICP pathways in E. coli, it may be possible to increase the volume of CO2 fixed using this pathway and yield potentially new products that can replace CO2 intensive products, such as precipitated calcium carbonates (PCCs) for industry.

microbiology↗

bioRxiv2024

Structure-guided design, synthesis, and characterization of C22- and C32-modified FK520 analogs with enhanced activity against human pathogenic fungi

Invasive fungal infections are a leading cause of death worldwide. Translating molecular insights into clinical benefits is challenging because fungal pathogens and their hosts share similar eukaryotic physiology. Consequently, current antifungal treatments have limited efficacy, may be poorly fungicidal in the host, can exhibit toxicity, and are increasingly compromised by emerging resistance. We have established that the phosphatase calcineurin (CaN) is required for invasive fungal disease and an attractive target for antifungal drug development. CaN is a druggable target, and there is vast clinical experience with the CaN inhibitors FK506 and cyclosporin A (CsA). However, while FK506 and its natural analog FK520 exhibit antifungal activity, they are also immunosuppressive in the host and thus not fungal-selective. We leverage our pathogenic fungal CaN-FK506-FKBP12 complex X-ray structures and biophysical data to support structure-based ligand design as well as structure-activity relationship analyses of broad-spectrum FK506/FK520 derivatives with potent antifungal activity and reduced immunosuppressive activity. Here we apply molecular docking studies to develop antifungal C22- or C32-modified FK520 derivatives with improved therapeutic index scores. Among them, the C32-modified FK520 derivative JH-FK-44 (7) demonstrates a significantly improved therapeutic index compared to JH-FK-08, our lead compound to date. NMR binding studies with C32-derivatives are consistent with our hypothesis that C32 modifications disrupt the hydrogen bonding network in the human complex while introducing favorable electrostatic and cation-{pi} interactions with the fungal FKBP12 R86 residue. These findings further reinforce calcineurin inhibition as a promising strategy for antifungal therapy. SignificanceInvasive fungal infections cause significant mortality worldwide, and current antifungal treatments are often ineffective, toxic, or face growing resistance. This research identifies calcineurin (CaN), a critical protein for fungal survival, as a potential target for developing new antifungal drugs. Although existing CaN inhibitors such as FK506 (tacrolimus) and FK520 (ascomycin) possess antifungal properties, their immunosuppressive effects limit their clinical utility. By studying the structure of human and fungal FKBP12-FK506 or FK520 complexes with CaN, we have designed and synthesized modified FK520 derivatives with strong antifungal activity and reduced immunosuppressive effects. These new derivatives are expected to have significantly improved therapeutic profiles, offering hope for more effective and safer antifungal treatments.

microbiology↗

bioRxiv2024

Traumatic Brain Injury Exacerbates Alcohol Consumption and Neuroinflammation with Decline in Cognition and Cholinergic Activity

Traumatic brain injury (TBI) is a global health challenge, responsible for 30% of injury-related deaths and significantly contributing to disability. Annually, over 50 million TBIs occur worldwide, with most adult patients at emergency departments showing alcohol in their system. TBI is also a known risk factor for alcohol abuse, yet its interaction with alcohol consumption remains poorly understood. In this study, we demonstrate that the fluid percussion injury (FPI) model of TBI in mice significantly increases alcohol consumption and impairs cognitive function. At cellular levels, FPI markedly reduced the number and activity of striatal cholinergic interneurons (CINs) while increasing microglial cells. Notably, depleting microglial cells provided neuroprotection, mitigating cholinergic loss and enhancing cholinergic activity. These findings suggest that TBI may promote alcohol consumption and impair cognitive abilities through microglia activation and consequently reduced cholinergic function. Our research provides critical insights into the mechanisms linking TBI with increased alcohol use and cognitive deficits, potentially guiding future therapeutic strategies.

neuroscience↗

bioRxiv2024

Traffic Jam activates the Flamenco piRNA cluster locus and the Piwi pathway to ensure transposon silencing and Drosophila fertility.

Flamenco (Flam) is the most prominent piRNA cluster locus expressed in Drosophila ovarian follicle cells, and it is required for female fertility to silence gypsy/mdg4 transposons. To determine how Flam is regulated, we used promoter-bashing reporter assays in OSS cells to uncover novel enhancer sequences within the first exons of Flam. We confirmed the enhancer sequence relevance in vivo with new Drosophila Flam deletion mutants of these regions that compromised Flam piRNA expression and lowered female fertility from activated transposons. Our proteomic analysis of proteins associated with these enhancer sequences discovered the transcription factor Traffic Jam (TJ). Tj knockdowns in OSS cells caused a decrease in Flam transcripts, Flam piRNAs, and multiple Piwi pathway genes. A TJ ChIP-seq analysis from whole flies and OSS cells confirmed TJ binding exactly at the enhancer that was deleted in the new Flam mutant as well as at multiple Piwi pathway gene enhancers. Interestingly, TJ also bound the Long Terminal Repeats of transposons that had decreased expression after Tj knockdowns in OSS cells. Our study reveals the integral role TJ plays in the on-going arms race between selfish transposons and their suppression by the host Piwi pathway and the Flam piRNA cluster locus.

genetics↗

bioRxiv2024

Cysteine Rich Intestinal Protein 2 is a copper-Responsive regulator of skeletal muscle differentiation.

Copper (Cu) is an essential trace element required for respiration, neurotransmitter synthesis, oxidative stress response, and transcriptional regulation. Imbalance in Cu homeostasis can lead to several pathological conditions, affecting neuronal, cognitive, and muscular development. Mechanistically, Cu and Cu-binding proteins (Cu-BPs) have an important but underappreciated role in transcription regulation in mammalian cells. In this context, our lab investigates the contributions of novel Cu-BPs in skeletal muscle differentiation using murine primary myoblasts. Through an unbiased synchrotron X-ray fluorescence-mass spectrometry (XRF/MS) metalloproteomic approach, we identified the murine cysteine rich intestinal protein 2 (mCrip2) in a sample that showed enriched Cu signal, which was isolated from differentiating primary myoblasts derived from mouse satellite cells. Immunolocalization analyses showed that mCrip2 is abundant in both nuclear and cytosolic fractions. Thus, we hypothesized that mCrip2 might have differential roles depending on its cellular localization in the skeletal muscle lineage. mCrip2 is a LIM-family protein with 4 conserved Zn2+-binding sites. Homology and phylogenetic analyses showed that mammalian Crip2 possesses histidine residues near two of the Zn2+-binding sites (CX2C-HX2C) which are potentially implicated in Cu+-binding and competition with Zn2+. Biochemical characterization of recombinant human hsCRIP2 revealed a high Cu+-binding affinity for two and four Cu+ ions and limited redox potential. Functional characterization using CRISPR/Cas9-mediated deletion of mCrip2 in primary myoblasts did not impact proliferation, but impaired myogenesis by decreasing the expression of differentiation markers, possibly attributed to Cu accumulation. Transcriptome analyses of proliferating and differentiating mCrip2 KO myoblasts showed alterations in mRNA processing, protein translation, ribosome synthesis, and chromatin organization. CUT&RUN analyses showed that mCrip2 associates with a select set of gene promoters, including MyoD1 and metallothioneins, acting as a novel Cu-responsive or Cu-regulating protein. Our work demonstrates novel regulatory functions of mCrip2 that mediate skeletal muscle differentiation, presenting new features of the Cu-network in myoblasts.

molecular biology↗

bioRxiv2024

Matrimeres are systemic nanoscale mediators of tissue integrity and function

Tissue barriers must be rapidly restored after injury to promote regeneration. However, the mechanism behind this process is unclear, particularly in cases where the underlying extracellular matrix is still compromised. Here, we report the discovery of matrimeres as constitutive nanoscale mediators of tissue integrity and function. We define matrimeres as non-vesicular nanoparticles secreted by cells, distinguished by a primary composition comprising at least one matrix protein and DNA molecules serving as scaffolds. Mesenchymal stromal cells assemble matrimeres from fibronectin and DNA within acidic intracellular compartments. Drawing inspiration from this biological process, we have achieved the successful reconstitution of matrimeres without cells. This was accomplished by using purified matrix proteins, including fibronectin and vitronectin, and DNA molecules under optimal acidic pH conditions, guided by the heparin-binding domain and phosphate backbone, respectively. Plasma fibronectin matrimeres circulate in the blood at homeostasis but exhibit a 10-fold decrease during systemic inflammatory injury in vivo. Exogenous matrimeres rapidly restore vascular integrity by actively reannealing endothelial cells post-injury and remain persistent in the host tissue matrix. The scalable production of matrimeres holds promise as a biologically inspired platform for regenerative nanomedicine.

bioengineering↗

bioRxiv2024

GM-CSF-mediated epithelial-immune cell crosstalk orchestrates pulmonary immunity to Aspergillus fumigatus

Aspergillus fumigatus causes life-threatening mold pneumonia in immune compromised patients, particularly in those with quantitative or qualitative defects in neutrophils. While innate immune cell crosstalk licenses neutrophil antifungal activity in the lung, the role of epithelial cells in this process is unknown. Here, we find that that surfactant protein C (SPC)-expressing lung epithelial cells integrate infection-induced IL-1 and type III interferon signaling to produce granulocyte-macrophage colony-stimulating factor (GM-CSF) preferentially at local sites of fungal infection and neutrophil influx. Using in vivo models that distinguish the role of GM-CSF during acute infection from its homeostatic function in alveolar macrophage survival and surfactant catabolism, we demonstrate that epithelial-derived GM-CSF increases the accumulation and fungicidal activity of GM-CSF-responsive neutrophils, with the latter being essential for host survival. Our findings establish SPC+ epithelial cells as a central player in regulating the quality and strength of neutrophil-dependent immunity against inhaled mold pathogens. HIGHLIGHTSO_LIGM-CSF is essential for host defense against A. fumigatus in the lung C_LIO_LIIL-1 and IFN-{lambda} promote GM-CSF production by lung epithelial cells in parallel C_LIO_LIEpithelial cell-derived GM-CSF increases neutrophil accumulation and fungal killing capacity C_LIO_LIEpithelial cells preferentially upregulate GM-CSF in local sites of inflammation C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/574062v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@708497org.highwire.dtl.DTLVardef@11106f7org.highwire.dtl.DTLVardef@e08140org.highwire.dtl.DTLVardef@1459e89_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Enhanced fungal specificity and in vivo therapeutic efficacy of a C-22 modified FK520 analog against C. neoformans

Fungal infections are of mounting global concern, and the current limited treatment arsenal poses challenges when treating such infections. In particular, infections by Cryptococcus neoformans are associated with high mortality, emphasizing the need for novel therapeutic options. Calcineurin is a protein phosphatase that mediates fungal stress responses, and calcineurin inhibition by the natural product FK506 blocks C. neoformans growth at 37{degrees}C. Calcineurin is also required for pathogenesis. However, because calcineurin is conserved in humans, and inhibition with FK506 results in immunosuppression, the use of FK506 as an anti-infective agent is precluded. We previously elucidated the structures of multiple fungal calcineurin-FK506-FKBP12 complexes and implicated the C-22 position on FK506 as a key point for differential modification of ligand inhibition of the mammalian versus fungal target proteins. Through in vitro antifungal and immunosuppressive testing of FK520 (a natural analog of FK506) derivatives, we identified JH-FK-08 as a lead candidate for further antifungal development. JH-FK-08 exhibited significantly reduced immunosuppressive activity and both reduced fungal burden and prolonged survival of infected animals. JH-FK-08 exhibited additive activity in combination with fluconazole in vivo. These findings further advance calcineurin inhibition as an antifungal therapeutic approach. ImportanceFungal infections cause significant morbidity and mortality globally. The therapeutic armamentarium against these infections is limited and development of antifungal drugs has been hindered by the evolutionary conservation between fungi and the human host. With rising resistance to the current antifungal arsenal and an increasing at-risk population, there is an urgent need for the development of new antifungal compounds. The FK520 analogs described in this study display potent antifungal activity as a novel class of antifungals centered on modifying an existing orally-active FDA approved therapy. This research advances the development of much needed newer antifungal treatment options with novel mechanisms of action.

microbiology↗