Biology subjects

Gonzalez, M.

Publications and source records attributed to Gonzalez, M..

At least 19 recordsLinked to original sources

bioRxiv2026

The Salmonella effector SspH2 disrupts F-actin cytoskeletal organization to inhibit macrophage migration

The facultative intracellular pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) causes gastroenteritis in humans and persistent infections in animals, enabling widespread environmental reservoirs. Intracellular survival of S. Typhimurium depends on a suite of type III secretion system effectors encoded within Salmonella Pathogenicity Island 2 (SPI-2), many of which also impair host cell migration. Among these, SspH2 is a novel E3 ubiquitin ligase (NEL) effector, but how it inhibits host cell movement has remained unclear. By using a combination of time-lapse microscopy and wound-healing assays, we show that SspH2 is required for S. Typhimurium to fully suppress migration of macrophage-like RAW264.7 cells and that conditional expression of SspH2 alone is sufficient to restrict their movement. SspH2 expression led to a marked reduction in F-actin and fewer cells containing higher-order F-actin structures, indicating cytoskeletal remodeling. In contrast, the catalytically inactive mutant SspH2C580A had no effect, demonstrating that E3 ligase activity is essential for these phenotypes. Knockdown of the host cytoskeletal factor vinculin, a core component of podosomes and focal adhesions, mimicked SspH2's effect on cell migration. When vinculin expression was reduced, SspH2 did not further inhibit migration, suggesting that SspH2 requires vinculin-associated F-actin-rich structures to block cell migration. These findings reveal a new pathway by which S. Typhimurium limits host cell movement, potentially enhancing immune evasion and fitness within the host.

microbiology↗

bioRxiv2026

Extracellular GPX3 drives insulin receptor expression in adipose tissue independent of its peroxidase activity

Obesity-associated insulin resistance is driven in part by adipose tissue dysfunction and oxidative stress, yet the role of extracellular mechanisms in modulating insulin signaling remains poorly understood. Here, we show that extracellular Glutathione Peroxidase 3 (GPX3) enhances adipocyte insulin sensitivity and functionalities independent of its canonical peroxidase function. GPX3, a secreted selenoprotein highly expressed in white adipose tissue (WAT), is suppressed in obesity, but whether it actively regulates insulin sensitivity had not been established. We find that WAT GPX3 expression closely mirrors insulin receptor (InsR) expression across a broad range of metabolic parameters in humans and increases dynamically following weight loss. Prolonged therapeutic selenium supplementation in diet-induced obese mice selectively suppressed epididymal WAT oxidative damage, restored GPX3 and InsR expression, and enhanced insulin-stimulated AKT phosphorylation, improving oral glucose tolerance. Mechanistically, recombinant GPX3 extracellularly administered at physiological concentrations induced InsR expression across multiple adipocyte models, including pre-adipocytes, epididymal WAT explants, and adipocyte organoids, by promoting binding of the transcription factor Specificity Protein 1 (SP1) to the Insr promoter, while knockdown of GPX3 completely blocks SVF adipogenesis. These effects rely on the structural integrity, rather than selenocysteine-dependent activity. Thus, we identify a GPX3-SP1-InsR signaling axis in adipose tissue insulin sensitivity, suggesting GPX3 is a novel paracrine mediator of metabolic health and a potential therapeutic target to reinstate WAT insulin sensitivity in obesity and type 2 diabetes.

physiology↗

bioRxiv2026

Individual differences in novelty-seeking behavior are modulated by GRK2/3 inhibition in rats

Background and Purpose High novelty-seeking is an endophenotype associated with greater vulnerability to drug addiction, marked by a hyperdopaminergic state and reduced dopamine D2 receptor (D2R) levels in the dorsal striatum (DS). G protein-coupled receptor kinases (GRKs) regulate desensitization, internalization, and recycling of these receptors, including the D2R. This study investigated the relevance of GRK2 and 3 (GRK2/3) in novelty-seeking behavior and striatal dopamine dynamics. Experimental Approach Individual novelty-seeking trait was assessed in Sprague Dawley rats using an open-field test. D2R, GRK2, and GRK5 protein levels were measured in synaptosomal DS samples using Western Blot. Pharmacological inhibition of GRK2/3 was assessed using Cmpd101. We tested the effect of systemic Cmpd101 administration on contextual novelty discrimination and local Cmpd101 perfusion on baseline and stimulus-evoked extracellular dopamine (DA) levels in the DS. Key Results Locomotor activity in a novel environment inversely correlated with the synaptosomal GRK2 and D2R protein levels in the DS. Systemic Cmpd101 treatment modulated the locomotor discrimination index in a novelty phenotype-dependent manner. Moreover, local Cmpd101 perfusion in the DS elicited a sustained elevation in basal extracellular dopamine levels and significantly blunted subsequent stimulus-evoked DA release, both effects independent of novelty-seeking phenotype. Conclusion and Implications Together, our findings link GRK2/3 signaling to individual differences in novelty-seeking and striatal dopaminergic function. These results suggest a potential relevance to drug addiction vulnerability.

neuroscience↗

bioRxiv2026

Modeling human embryonic adrenogenesis in pluripotentstem cell-derived corticomedullar-like assembloids

During embryogenesis, the cortical and medullary compartments of the adrenal gland emerge from two distinct sources: intermediate mesoderm-derived steroidogenic progenitors that comprise the cortex, and migratory neural crest-derived catecholaminergic progenitors that give rise to the medulla. The distinct cellular origins and paracrine signaling environments that foster induction, differentiation, and/or migration of cortical and medullary layers present challenges to recapitulating their co-emergence and incorporation in a human pluripotent stem cell (hPSC) based system. To address this, we developed an approach for modeling the adreno-gonadal primordium (AGP) in compound organoids that are organized along a gradient of NR5A1 expression and which unexpectedly incorporate SOX10-expressing Schwann cell precursors that ultimately generate chromaffin cells. Deconstruction of paracrine signaling within AGP-like organoids (AGPLOs) identified a fibroblast growth factor 9 (FGF9)-driven mechanism that expands bridge cell-like chromaffin progenitors and correlates to paracrine signaling cues encountered by physiological correlates along their migration corridor to the cortex in vivo. Combination of NR5A1-expressing cells with chromaffin cell clusters generated corticomedullar-like assembloids (CMLAs) that generated steroids and catecholamines and were responsive to adrenocorticotropic hormone. This work establishes a platform for modeling the reciprocal signaling relationships that drive adrenal gland development and function in health and disease and provides an approach for generating hPSC-derived adrenal cells/tissues that can be applied therapeutically.

developmental biology↗

bioRxiv2026

Body condition as an indicator of long-term ecosystem change: evidence from common dolphins

As climate change continues to disrupt marine ecosystems, reliable body condition indices (BCIs) for sentinel megafauna species such as dolphins will become increasingly important tools for long-term ecosystem monitoring. We developed novel BCIs from routinely collected morphometric measurements and applied them to a 22-year dataset (1998-2019) of 597 stranded common dolphins (Delphinus delphis) from Northwest Spain. BCIs were calculated as residuals from Generalized Additive Models relating body length to girth and blubber thickness measurements, thereby controlling for body size effects. The indices were first validated against visually assessed nutritional status and then used to investigate long-term trends and potential environmental and trophic drivers of body condition. The girth-based BCI outperformed the blubber thickness-based index in predicting nutritional status. The indices differed in their sensitivity to confounding variables. Both indices revealed an increase in male body condition until 2009-2010 followed by a sustained decline through 2019, whereas no long-term trend was detected in females. By integrating data on climate variability (North Atlantic Oscillation), oceanographic conditions (upwelling intensity, sea surface temperature, chlorophyll-a), prey availability, and prey quality, we found evidence that changes in body condition was associated with climate- and oceanography-driven changes in prey communities and reductions in the quality of prey available to dolphins. Our findings suggest that body condition in common dolphins integrates signals across multiple components of marine ecosystems and can serve as a sensitive indicator of ecosystem change. Given growing reports of declining body condition in marine predators worldwide, the approach presented here provides a practical and transferable framework for long-term ecosystem monitoring using existing stranding network datasets.

ecology↗

bioRxiv2026

Region-specific patterns of sexual shape variation in the human bony labyrinth: 3D geometric morphometric analysis of a sample with known genomic sex

The human bony labyrinth has attracted increasing interest because of its taxonomic, evolutionary, and functional significance. Although sexual dimorphism has been reported in several aspects of the temporal bone, the extent to which sex, age, size, and allometry contribute to labyrinth shape variation remains poorly understood. Here, we investigated patterns of sexual shape variation in the human bony labyrinth using three-dimensional geometric morphometrics in a sample of 98 archaeological individuals from South America with known genomic sex. Centroid size and allometric effects were assessed in a subset of 90 individuals with comparable metric scaling. In addition to analysing the complete labyrinth, the cochlea and semicircular canals were examined separately to evaluate region-specific patterns of sexual shape variation. Principal Component Analysis showed extensive overlap between females and males, and overall labyrinth shape did not differ significantly between sexes. Males exhibited significantly larger labyrinths than females, and centroid size explained a small but significant proportion of overall shape variation. Regional analyses showed no evidence of significant sexual shape differences in the cochlea or in any individual semicircular canal when analysed separately. In contrast, the combined semicircular canal system exhibited subtle but significant sexual shape variation independent of centroid size, whereas morphological disparity did not differ between sexes. The geometric comparison of the female and male consensus configurations further showed that sexual shape variation was regionally heterogeneous. Whereas the cochlea exhibited a pattern of localized changes with low directional coherence, the semicircular canals displayed more coordinated regional shape changes. The male consensus also exhibited slightly higher canal circularity across all three semicircular canals, particularly the posterior canal, while differences in canal-plane orientation remained minimal. These findings demonstrate that sexual shape variation in the human bony labyrinth is subtle and anatomically partitioned among its components. Although significant sex differences in centroid size were detected across most anatomical regions, overall labyrinth shape and cochlear morphology were primarily influenced by allometry, whereas significant sex-related shape differences were detected only when the semicircular canals were considered as an integrated anatomical system. These findings demonstrate that sexual dimorphism in the human bony labyrinth is subtle but regionally heterogeneous, with the cochlea and semicircular canals exhibiting distinct patterns of shape variation, suggesting that these structures are influenced by different developmental, functional, and evolutionary processes.

evolutionary biology↗

bioRxiv2026

Senescence targeting re-enables injury-responsive repair in a human RPE aging model

Aging is associated with progressive tissue dysfunction and impaired repair after injury. In the retinal pigment epithelium (RPE), these changes contribute to age-related macular degeneration (AMD), yet the mechanisms limiting repair remain incompletely understood. Here, we establish a longitudinal human embryonic stem cell (hESC)-derived RPE aging model that recapitulates key features of aged human donor RPE and combine it with mosaic cell ablation to assess injury-responsive repair. Although aged RPE cells initiate DNA synthesis after injury, they exhibit impaired mitotic progression, uncoupling S-phase entry from epithelial repopulation. Transcriptomic profiling links this defect to a senescence-associated program marked by inflammatory signaling and suppressed mitotic networks. Pharmacologic reduction of senescent cells with Navitoclax shifts aged RPE toward a younger transcriptional profile but does not induce repopulation by itself. Instead, senolytic treatment primes aged RPE for repair, improving epithelial density and homeostatic function only in response to injury, a strategy we term "senolytic priming." These findings establish a human stem-cell-derived platform for investigating age-associated epithelial repair failure and show that aged human RPE retains latent repair capacity that can be re-enabled by targeting cellular senescence.

Cell Biology↗

bioRxiv2026

Limited Volumetric Separation Across CDR Groups in OASIS-1

Clinical Dementia Rating (CDR) scores are used to classify the cognitive state of patients and are provided within neuroimaging datasets. This is achieved through a standardized clinical assessment that evaluates participants cognitive and functional abilities in everyday life, after which they are given a score ranging from 0 to 3. Where 0 represents no signs of dementia and three represents severe dementia1. These scores are then used to track the progression of dementia over time2. This study explored if these CDR labels within the OASIS-1 dataset produced consistent volumetric separation across the hippocampus, amygdala, and cortex.

neuroscience↗

bioRxiv2026

Behavioral, hormonal, and chemical responses to seasonality in poison frogs with divergent reproductive strategies

Seasonal rainfall shapes biological responses in tropical ecosystems, yet how tropical organisms integrate behavioral and physiological responses to cope with seasonality remains poorly understood. We assessed how four poison frog species with contrasting reproductive strategies respond to dry and wet season environmental conditions. We quantified spatial behavior, microhabitat use, hormone release rates, and chemical defenses in two seasonal breeders (Allobates femoralis and Ameerega trivittata) and two year-round breeders (Ameerega macero and Ameerega shihuemoy). Seasonal breeders exhibited pronounced sex-specific shifts in space use, where males expanded their space use during the wet season, likely to track reproductive opportunities, while A. femoralis females increased their spatial use during the dry season, likely responding to foraging demands when prey resources are sparse. Year-round breeders maintained similar space use across seasons, likely reflecting their ability to access key resources within the same space to reproduce year-round. Microhabitat use was flexible, as seasonal breeders shifted toward humid refugia during the dry season and reproduction-associated microhabitats during the wet season, whereas year-round breeders selected microhabitats that facilitate continuous reproduction across seasons. Despite these behavioral responses, water-borne corticosterone and testosterone, as well as chemical defenses, showed no consistent seasonal variation, suggesting that behavioral responses may be partially decoupled from shifts in endocrine and chemical defenses. These results support a role for behavioral buffering in mediating responses to seasonal environmental variation. However, given the increasing unpredictability in rainfall timing and intensity as a result of climate change, how these coping strategies will function in the long term is uncertain.

animal behavior and cognition↗

bioRxiv2026

Individualized and stereotypical seizure semiology in a porcine model of post-traumatic epilepsy.

Large-animal models of traumatic brain injury may yield translatable data on epileptogenesis, given their similarities in anatomy, brain size, and immune systems to humans. Adult male and female swine received bilateral cortical impact (N=16) or sham surgery (N=6) and were screened for convulsions via video-EEG for up to one year. Post-traumatic epilepsy (PTE) was defined as 2 seizures after 1-week post-injury. Nine out of sixteen pigs (56%) receiving bilateral cortical impact developed PTE, with an average 4.6 months ({+/-}3.4,SD) latent period. Seizures (N = 199) began focally, sometimes with motor onset including automatisms, before becoming generalized, with tonic-clonic or tonic convulsions. We defined a library differentiating peri-ictal behaviors (N = 31) from rhythmic/odd behaviors typical in healthy pigs (N = 12). Seizures had an average of 7.3 behaviors per seizure (max 26) lasting an average of 1.8 minutes (max 7.9). For seizures comprised of multiple convulsive episodes, the first convulsion had a greater number of peri-ictal behaviors than subsequent convulsions (P < 0.001). The array of peri-ictal behaviors displayed was pig-specific, with many behaviors consistently observed across seizures. The seizure frequency detected was 0.38/day. This large-brain model of PTE exhibits a variable period of epileptogenesis, a substantial rate of PTE, and an expansive repertoire of ictal behaviors. This first description of semiology in this species will serve as a guide for other porcine epilepsy models. Biofidelic models of PTE are expected to increase our understanding of the pathophysiology, enabling the identification and testing of therapeutics that translate into human patients. HighlightsO_LIThe average time from bilateral cortical impact to post-traumatic epilepsy in swine is 6 months, and is highly variable, ranging from 2 to 47 weeks post-TBI. C_LIO_LISwine with post-traumatic epilepsy display an array of specific behaviors around convulsions, distinct from pigs without post-traumatic epilepsy. C_LIO_LIThough the duration of convulsion was typically a few seconds, the entire seizure, with the associated peri-ictal behaviors, lasts up to 7.9 minutes. C_LIO_LIThe complexity of behaviors around convulsions tended to increase from early traumatic seizures to post-traumatic seizures. C_LIO_LIPeri-ictal behaviors observed around convulsions in an individual were often displayed prior to the first convulsion. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/708000v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@9523aborg.highwire.dtl.DTLVardef@158cfa6org.highwire.dtl.DTLVardef@1a8b499org.highwire.dtl.DTLVardef@e8c86f_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2026

Bacterial wilt resistance is correlated with rhizosphere bacterial communities in wild potato Solanum malmeanum

Wild potatoes are long-evolving relatives of the cultivated potatoes we have today. These wild Solanum species harbor traits that can be exploited to develop more nutritive and resilient potato varieties, providing the genetic basis for resistance to abiotic and biotic stresses such as drought, low temperatures, diseases and pests. Wild potato species are widely used as valuable genetic resources in breeding programs, including efforts aimed at improving resistance to bacterial wilt caused by Ralstonia solanacearum. Among the wild species native to Uruguay, Solanum malmeanum has emerged as a particularly valuable source of resistance. The aim of this work was to investigate weather differences in bacterial wilt resistance among S. malmeanum accessions are associated with structural and compositional changes of rhizosphere bacterial communities. Two S. malmeanum accessions were compared, one susceptible (RN9P2) and the other resistant (A11P1) to bacterial wilt. The impact of plant resistance and pathogen colonization on the structure of rhizosphere bacterial communities were evaluated using high throughput 16S rRNA gene amplicon-sequencing. Significant differences were observed between accessions and pronounced shifts in rhizosphere bacterial communities were detected in response to pathogen inoculation. Cryseobacterium, Sphingobacterium, Komagataeibacter, Gluconobacter, Lactobacillus and Dyella were differential genera and enriched in the rhizosphere of the resistant accession. Several of these genera have been previously associated with disease suppression. Overall, these results suggest that the rhizosphere bacterial community associated with resistant S. malmeanum accessions may contribute to protection against R. solanacearum infection.

microbiology↗

bioRxiv2026

Cancer drives atherosclerotic plaque vulnerability by inducing pathological angiogenesis

Cardiovascular disease (CVD) is the leading cause of death for many cancer survivors, a phenomenon traditionally attributed to shared risk factors and cardiotoxic chemotherapies. Here, we hypothesized that cancer may also directly promote atherosclerosis. Propensity-matched analyses confirmed significantly elevated cardiovascular event rates amongst cancer patients, independent of comorbidities such as smoking. Atheroprone mice implanted with colorectal tumors demonstrated accelerated features of plaque vulnerability, driven by pathological angiogenesis and intraplaque hemorrhage. Mechanistically, tumor-secreted TNF- induced the pro-angiogenic factor LRG1 across multiple murine models and human plaques. Therapeutic interventions targeting these pathways, including with FDA-approved cytokine inhibitors or tumor resection, prevented plaque destabilization in mice and reduced coronary revascularization rates in patients. Together, these findings suggest that cancer may causally promote CVD and unveil novel translational strategies for cancer survivors.

pathology↗

bioRxiv2025

Dysregulated lymphocyte localization in idiopathic multicentric Castleman disease

Idiopathic multicentric Castleman disease (iMCD) is a rare and life-threatening hematologic disease involving episodic flares of uncontrollable systemic inflammation by unknown causes. Hallmark features of iMCD include multiple enlarged lymph nodes with characteristic histopathological phenotypes and a potentially fatal, cytokine release syndrome. The key pathophysiologic drivers of disease are poorly understood and few effective treatment options exist. Here, we discovered an association between elevated chemokines, namely CXCL13, and lymph node size in iMCD, providing one possible explanation for the lymphadenopathy observed clinically. Instead of a concurrent increase in circulating CXCL13 and CXCR5-expressing cells that has been described in other contexts, during active disease, chemokine-responsive lymphocytes downregulated CXCR5 levels in iMCD. Despite heightened chemokine production by lymph node stromal cells, T and B cells failed to appropriately respond to their cues locally within the tissue and were particularly scarce within CXCL13-expressing germinal centers (GC). Inflammatory signals in iMCD lymph nodes appeared to restrict the production and movement of T follicular helper cells, which play an important role in facilitating appropriate GC responses. Together, these data provide a link between dysregulated chemokine production and germinal center lymphocyte trafficking, highlighting a potential mechanism and therapeutic target in iMCD lymphadenopathy. One Sentence SummaryLymphocyte chemotaxis to discrete areas of lymphoid tissue is disrupted in idiopathic multicentric Castleman disease.

immunology↗

bioRxiv2025

Sex-dependent hypothalamic microinflammation and microglial polarization: The role of JNK in high-fat diet-induced insulin resistance

Obesity and its associated metabolic disorders, including insulin resistance and type 2 diabetes, are major global health challenges. Early hypothalamic microinflammation is emerging as a key contributor to the onset of metabolic dysfunction, but its temporal dynamics, underlying mechanisms, and sex-specific differences remain unclear. Here, we examined the effects of short-term high-fat diet (HFD) exposure on hypothalamic microinflammation, glial activation, and insulin signaling in male and female mice. Males rapidly developed hypothalamic microinflammation, characterized by increased pro-inflammatory cytokines, reactive gliosis, and impaired insulin signaling, whereas females did not show these alterations. Interestingly, we also observed a sex-specific pattern in microglial M2 polarization: females maintained a sustained M2 response throughout the experimental period, while males exhibited only a transient peak that declined in the following days. These sex-specific differences in microglial dynamics and polarization may be linked to the c-Jun N-terminal kinase pathway, a classical mediator of inflammatory signaling. To further explore this, we analyzed mice lacking JNK3, the CNS-enriched isoform of JNK. Loss of JNK3 disrupted early microglial polarization, abolished the transient M2 response, and predisposed males to enhanced gliosis and partial hypothalamic insulin resistance, while females retained partial resilience. These findings indicate that JNK3 contributes to the regulation of microglial dynamics and polarization in response to metabolic stress. Overall, our study highlights a sex-dependent role of microglia and JNK in shaping early hypothalamic microinflammation and central insulin sensitivity, providing potential targets for intervention in obesity-associated metabolic disease.

neuroscience↗

bioRxiv2025

The rhizosphere of Pappostipa frigida as a hotspot of active bacterial communities in the Andean steppe of the Atacama Desert

BackgroundThe rhizosphere is a resource-rich microenvironment where plants, soil nutrients, and microorganisms interact. In arid and semi-arid regions, this tripartite relationship must withstand long periods of drought followed by brief periods of rainfall. In the present study, we employed a combination of RNA/DNA metabarcoding and shotgun metagenomic sequencing to provide insights into the functional capabilities and activity levels of the bacterial communities present in the bulk soil and rhizosphere samples of Pappostipa frigida, a grass species endemic to the Andean steppe of the Atacama Desert. ResultsThe active bacterial community in the rhizosphere of P. frigida exhibited greater diversity and a higher Shannon index than the total bacterial community. In terms of beta diversity, the structures of the total and active communities differed markedly between the BS and the RZ. Furthermore, active bacteria in the RZ showed a stronger correlation with total bacterial populations than those in the BS. This finding is consistent with the low proportion of ASVs derived from RNA extractions detected in the BS. Notably, 64% of these putative inactive bacterial populations were identified as active RZ members and 73% grew in culture media, suggesting they were likely dormant. The bacterial communities of the BS exhibited higher abundances of sporulation genes. In contrast, active bacterial communities in the RZ consistently contained higher abundances of genes associated with halotolerance, siderophore synthesis, and resuscitation-promoting factors. ConclusionsThe results emphasize the importance of the conditions created by plants in recruiting bacterial populations from the soil and provide insights into how the rhizosphere of arid native plants influences the activity and functional traits of soil microorganisms in their natural habitat. Additionally, this study advances our understanding of the mechanisms employed by soil microorganisms to cope with desiccation in natural environments, establishing P. frigida as a model in plant science for studying grass traits and responses to extreme environments.

microbiology↗

bioRxiv2025

Lack of tetrodotoxin analogues and individual metabolomic profiling of the cryptic frog Colostethus imbricolus

Poison frogs (Dendrobatoidea) are characterized by the great diversity of alkaloids discovered in their skin. However, most of these alkaloids have been found in brightly colored species and there is a wide lack of knowledge of alkaloid profiles in the less colorful species. Previous finding of paralytic tetrodotoxins (TTXs) in only two cryptically colored species from the genus Colostethus, establishes the unique occurrence of hydrophilic alkaloids in the superfamily Dendrobatoidea. Unpublished results using extracts from Colostethus imbricolus, demonstrated that this species contains paralysis-producing substances, after intraperitoneal injection of mice. To analyze their skin metabolites and to determine if they correspond to TTX, or TTX analogues, we have employed a TTX-targeted separation in normal phase gradient, and an untargeted profiling in reversed-phase gradient. After performing both analyses, neither TTX nor TTX-analogues were detected in C. imbricolus. In contrast, other metabolites were separated, allowing the extraction of 76 adducts common to both analyses, being 33 of them tentatively annotated as amphibian alkaloids, eight as amphibian metabolites different from alkaloids and 25 that matched with natural products from the DNP. A total of 10 common molecular formulas remained non-annotated. The absence of MS/MS spectra for these adducts requires their structures to be confirmed in future analyses, following the completion of targeted MS/MS acquisition. After analyzing the inter-individual variation of six specimens, it was demonstrated that the skin metabolome differs between males and females of C. imbricolus. Our results lead us to conclude that TTX is not the only paralyzing compound in dendrobatid frogs and that more work should be undergone to identify this phenomenon. A notable additional outcome of this study is the first successful separation of TTX on an SB-CN column using a normal-phase gradient, enabling a novel method for TTX-targeted separation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/655531v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@dc4cf1org.highwire.dtl.DTLVardef@13f816eorg.highwire.dtl.DTLVardef@4a6ed6org.highwire.dtl.DTLVardef@b2b323_HPS_FORMAT_FIGEXP M_FIG C_FIG

zoology↗

bioRxiv2025

Mobile-CRISPRi as a tool for genetic manipulation in the intracellular pathogen Piscirickettsia salmonis

Piscirickettsia salmonis is the causative agent of Salmonid Rickettsial Septicemia (SRS), the main bacterial disease affecting the salmon industry in Chile. In this work, we implemented a Mobile-CRISPRi system to generate gene silencing using a catalytically inactive dCas9 protein and an IPTG-inducible single-guide RNA (sgRNA). We demonstrate the efficacy of the CRISPRi system in P. salmonis by silencing an exogenous gene encoding green fluorescent protein (sfGFP), and the endogenous homolog of the fur gene, whose gene product regulates intracellular iron homeostasis in bacteria. The inducible expression of dcas9 and the sfGFP-directed sgRNA caused a 98.7% decrease in fluorescence in the knockdown strain. This silencing system was effective in seven P. salmonis strains from both genogroups. Furthermore, the same system was used to construct fur knockdown strains. A 50-fold decrease in fur expression level was determined in these strains, when the expression of the fur gRNA was induced with IPTG. By RNA-seq we detected a significant increase in the expression of genes encoding the Fe+2 and Fe+3 acquisition systems and iron mobilization in the fur1 knockdown, after IPTG induction. All the genes with over two-fold increased expression in the RNA-seq presented the Fur box consensus sequence in their regulatory region. The successful implementation of the Mobile-CRISPRi system in P. salmonis paves the way for systematic analysis of gene function in this pathogen We anticipate that these analyses will be very valuable in identifying genes involved in the mechanisms of pathogenesis of P. salmonis. ImportanceSalmon Rickettsial Septicemia (SRS) is an infectious disease caused by the marine bacterium Piscirickettsia salmonis. This Gamma-proteobacteria is a fastidious and facultative intracellular pathogen that has a nearly worldwide distribution, particularly impacting Chilean salmonid aquaculture. Its fastidious nature has made it hard to grow in labs, hindering research into its virulence and treatment, especially because of the lack of molecular techniques to study gene function. We show here the successful implementation of the Mobile-CRISPRi system for gene silencing. Significantly, we have adapted this technique for use with the marine pathogen P. salmonis, inserting exogenous genes into the bacteriums chromosome to ensure their constitutive and inducible expression, and silencing both exogenous and endogenous gene expression. The Mobile-CRISPRi system was also used to study the iron regulator Fur, confirming Furs relevance to the iron metabolism in the pathogen.

microbiology↗

bioRxiv2025

Development of a Plant Growth Promoting Bacterial EcoBiome Derived from Desert Soil Isolates

The application of plant growth-promoting (PGP) bacteria is increasingly studied for its potential to improve plant tolerance to biotic and abiotic stress. Developing synthetic microbial consortia represents a promising strategy, as it can enhance colonization success and functional synergy within the rhizosphere. In this study, we designed a stable EcoBiome derived from a synthetic community (SynCom) of 17 bacterial isolates obtained from three desert environments. We evaluated their PGP traits, including siderophore production, indoleacetic acid (IAA) synthesis, phosphate solubilization, and nitrogen fixation. Using Oxford Nanopore Technologies (ONT) sequencing of 16S rRNA genes, we tracked changes in relative abundance across successive subcultures under four temperature conditions. From this analysis, Erwinia rhapontici 1SR, Pseudomonas yamanorum RZ5, and Plantibacter sp. RU18 were identified as the dominant isolates and subsequently selected to construct the EcoBiome. Functional characterization showed that these isolates exhibited complementary PGP traits, biofilm formation capacity, and tolerance to water stress, both individually and in combinations. These findings highlight the potential of desert- derived bacterial consortia as microbial resources for developing biostimulants to enhance plant resilience under environmental stress conditions. ImportanceSeveral studies have focused on obtaining bacterial isolates with plant growth promoting traits, however, their success as microbial inoculants with plant stimulant activity is diminished (or null) because they do not have the ability to compete efficiently with the natural soil microbiome. Thus, in this study we designed a synthetic community of 17 bacteria from desert environments and individually characterized their plant growth-promoting attributes, and in parallel we subjected this synthetic community to co-culture, subsequently evaluating its temporal prevalence (24 and 48 hours of culture) and at four different temperatures. With this, we developed and presented a stable EcoBiome of three isolates (one from each desert), stable over time, with attributes that promote plant growth and proliferation in hostile conditions, such as drought, with the purpose of being used as microbial inoculants as a whole, capable of competing, proliferating and forming part of the rhizosphere microbiome.

microbiology↗