Biology subjects

Cruz, M. A.

Publications and source records attributed to Cruz, M. A..

14 recordsLinked to original sources

bioRxiv2026

Caloric Restriction Promotes Ischemia/Reperfusion Cardioprotection Through Increased Mitochondrial Na+/Ca2+ Exchange

Caloric restriction (CR) protects against cardiac ischemia/reperfusion (I/R) injury, but the underlying mechanisms remain incompletely understood. Since mitochondrial Ca2+ overload and redox imbalance is a major driver of cardiac damage during reperfusion, we investigated if enhanced mitochondrial Ca2+ efflux contributes toward CR-induced cardioprotection. Rats were subjected to 16 weeks of ad libitum (AL) feeding or 40% caloric restriction. CR significantly increased mitochondrial Ca2+ retention capacity when Na+ ions were present, reduced H2O2 release, and increased the expression of proteins related to mitochondrial Ca2+ extrusion. In cardiomyocytes exposed to serum from CR rats, Ca2+ retention capacity also markedly increased, as well as Ca2+ efflux and the expression of extrusion proteins NCLX and TMEM65. Following I/R, CR hearts exhibited improved functional recovery accompanied by enhanced retention and increased mitochondrial Ca2+ efflux activity, as well as reduced H2O2 release compared to AL controls. Inhibition of mitochondrial Na+/Ca2+ exchange abolished the mitochondrial adaptive effects of CR, eliminating its protection against damage in cardiomyocytes and perfused hearts. These findings demonstrate that CR protects the heart from I/R injury by enhancing CGP-sensitive Na+-dependent mitochondrial Ca2+ efflux, preserving mitochondrial function, limiting redox imbalance, and improving post-ischemic cardiac recovery.

biochemistry↗

bioRxiv2026

Unexpected changes in reproductive barriers between incipient species after experimental evolution in sympatry

Hybridization is generally considered a temporary phenomenon, but it is actually widespread and may last for large time periods between species that stably coexist. Here, to test whether evolving with a closely-related species modifies or maintains partial reproductive isolation, we performed experimental evolution in artificial sympatry vs. allopatry with two closely-related colour forms of spider mites (Tetranychus urticae) that exhibit an asymmetrical pattern of pre-mating isolation despite almost complete post-zygotic isolation. We assessed whether evolutionary changes occurred in traits associated to (i) pre-mating isolation, (ii) post-mating pre-zygotic and early post-zygotic isolation, and (iii) late post-zygotic isolation. Our results revealed that reinforcement did not occur even under forced long-term sympatric evolution. Instead, the strength of some reproductive barriers decreased (e.g., premating isolation and fertilization failure), and some trait changes indicated convergence rather than divergence between species (e.g., mating propensity, latency to copulation). In fact, both types of males showed the same decreased preference for red-form females across generations in sympatry. In line with this, traits underlying fertilization success evolved in the same direction and with similar amplitude in heterotypic crosses and in their homotypic control, as the offspring sex ratio of green-form females decreased in sympatry irrespective of the male they mated with. Finally, other changes in reproductive barriers resulted from trait correlations (e.g., decreased zygote mortality but increased juvenile mortality). Hence, despite very high costs of hybridization, responses occurring following evolution in sympatry were unrelated to selection directly associated to hybridization, but rather the by-product of other evolutionary forces, with cascading consequences for reproductive barriers. In particular, these results support the underappreciated hypothesis that within-species sexual interactions can constrain population diver-gence, or even drive trait convergence between species, thereby playing a role in the maintenance of partial reproductive isolation.

evolutionary biology↗

bioRxiv2025

Engineering the citrus phyllosphere microbiome for enhanced disease resistance to bacterial canker

Engineering phyllosphere microbiomes toward plant protection against pathogens on crops has shown promising effects. Here we tested the effect of application of beneficial bacteria individually and as a synthetic community (SynCom) on the phyllosphere microbiome and management of bacterial canker caused by Xanthomonas citri subsp. citri (Xcc). Foliar spray of a SynCom consisting of three citrus endophytic bacterial strains (Paenibacillus sp. ATY16, Bacillus megaterium PT6, and Bacillus subtilis PT26A, SynCom1) with mechanistically complemented plant-beneficial properties exerted a more profound effect on the leaf microbiota and suppression of citrus canker than single strains. Cultivation-dependent and bacterial 16S rRNA gene-sequence profiling analyses revealed that leaf endophytic Bradyrhizobium, Brevibacillus, Cellvibrio, Flavobacterium, and Pseudomonas were significantly enriched in the SynCom treated plants, in addition to Bacillus and Paenibacillus. The enriched Brevibacillus and Pseudomonas spp. were isolated, whole-genome sequenced, and confirmed to possess multiple beneficial traits including antimicrobial activity against Xcc, Moreover, the expression of citrus defense genes was induced by the SynCom1 inoculation. Foliar application of SynCom1 and a newly assembled SynCom (SynCom2; SynCom1 with representative Brevibacillus and Pseudomonas isolates) prior to Xcc inoculation significantly reduced the citrus canker disease severity in greenhouse assays, and SynCom2 had a better efficacy, comparable to a copper bactericide. Three-year field tests showed that both SynCom1 and SynCom2 effectively controlled citrus canker, with a 50% reduction of foliar and fruit canker incidence. Collectively, these findings provide novel insights and valuable clues for microbiome engineering to serve as a sustainable strategy for the control of phyllosphere pathogens on crops.

plant biology↗

bioRxiv2025

Genetic variance in spider mites reveals trade-offs: lines with higher growth rate suffer stronger competition but exert stronger reproductive interference

O_LIWithin-species individual variation has been gaining increased attention in ecological studies of species interactions. Yet, these studies rarely consider genetic variation and potential genetic correlations among traits. Moreover, traits beyond those associated with trophic interactions are mostly overlooked. Filling these gaps is important to fully understand the relevance of trait variation in both the ecology and evolution of species interactions. C_LIO_LIWe addressed this using inbred lines of Tetranychus cinnabarinus, a spider mite species that often engages in antagonistic interactions with its congeneric T. urticae. Specifically, we measured (a) the intrinsic growth rate and the sex ratio in the absence of food competition and reproductive interference; (b) food competition against a density gradient of con- or heterospecific competitors in absence of reproductive interference; (c) reproductive interference in absence of food competition; and (d) the body size of adult females and males. C_LIO_LIWe found significant genetic variance (i.e., broad-sense heritability) among lines for all variables except for the effect of T. cinnabarinus on its heterospecific competitor via food competition. Moreover, we showed that lines with higher intrinsic growth were more sensitive to intra- and inter-specific competition for food but exerted stronger reproductive interference on heterospecifics. In addition, the higher the proportion of sons produced by a line, the more resilient it was to reproductive interference but also the lower its growth rate. No genetic correlations were found between any variable and body size. C_LIO_LIOur results indicate that considering the genetic correlations among traits involved in both trophic and sexual interactions is key to understanding the role of individual variation in species interactions and their evolution. C_LI

evolutionary biology↗

bioRxiv2024

The probability of cryptic pocket opening controls functional tradeoffs in filovirus immune evasion.

Cryptic pockets are of growing interest as potential drug targets, particularly to control protein-nucleic acid interactions that often occur via flat surfaces. However, it remains unclear whether cryptic pockets contribute to protein function or if they are merely happenstantial features that can easily be evolved away to achieve drug resistance. Here, we explore whether a cryptic pocket in the Interferon Inhibitory Domain (IID) of viral protein 35 (VP35) of Zaire ebolavirus aids its ability to bind double-stranded RNA (dsRNA). We use simulations and experiments to study the relationship between cryptic pocket opening and dsRNA binding of the IIDs of two other filoviruses, Reston and Marburg. These homologs have nearly identical structures but block different interferon pathways due to different affinities for blunt ends and backbone of the dsRNA. Simulations and thiol-labeling experiments demonstrate that the homologs have varying probabilities of pocket opening. Subsequent dsRNA-binding assays suggest that closed conformations preferentially bind dsRNA blunt ends while open conformations prefer binding the backbone. Point mutations that modulate pocket opening proteins further confirm this preference. These results demonstrate the open cryptic pocket has a function, suggesting cryptic pockets are under selective pressure and may be difficult to evolve away to achieve drug resistance.

biophysics↗

bioRxiv2024

Wolbachia strengthens the match between pre-mating and early post-mating isolation in spider mites

Endosymbiotic reproductive manipulators are widely studied as sources of post-zygotic isolation in arthropods, but their effect on pre-zygotic isolation between genetically differentiated populations has garnered less attention. We tested this using two partially isolated populations of the red and green colour forms of Tetranychus urticae, either uninfected or infected with different Wolbachia strains, one inducing cytoplasmic incompatibility and the other not. We first investigated male and female preferences, and found that, in absence of infection, females were not choosy but all males preferred red-form females. Wolbachia effects were more subtle, with only the CI-inducing strain slightly strengthening colour-form based preferences. We then performed a double-mating experiment to test how incompatible matings affect subsequent mating behaviour and offspring production, as compared to compatible matings. Females mated with an incompatible male (infected and/or heterotypic) were more attractive and/or receptive to subsequent (compatible) matings, although analyses of offspring production revealed no clear benefit for this re-mating behaviour (i.e., apparently unaltered first male sperm precedence). Finally, by computing the relative contributions of each reproductive barrier to total isolation, we showed that pre-mating isolation matches both host-associated and Wolbachia-induced post-mating isolation, suggesting that Wolbachia could contribute to reproductive isolation in this system.

evolutionary biology↗

bioRxiv2023

Sex in the kitchen: non-additive effects of competition for food and reproductive interference on coexistence outcomes between closely related species.

Competition for food and reproductive interference (negative interspecific sexual interactions) have been identified as major drivers of species exclusion. Still, how these biotic interactions jointly determine competitive dominance remains largely unknown. We tackle this by coupling population models and laboratory experiments with two sibling species of spider mites. Using experiments specifically designed to measure the single and combined effects of food competition and reproductive interference, we first show that the strength and symmetry of reproductive interference between species changes in presence of food competition. Next, we show that population models incorporating each type of interaction alone lead to markedly different predictions, from exclusion of the inferior competitor for food, to priority effects instead favouring the latter under the sole effect of reproductive interference. Moreover, accounting for the observed reduction in the strength of reproductive interference in the presence of food competition changes the threshold frequency determining the dominant competitor when both interactions are at play, from equal chances for the two species to exclude the other depending on their initial frequency, to favouring the superior competitor for food except when it is extremely rare. Finally, we showed that the model generates accurate predictions for population dynamics in an independent population cage experiment, indicating that our approach captures the most relevant processes governing the outcome of interactions between competing spider mite species. Altogether, our results suggest that trophic interactions can modulate sexual interactions, significantly impacting population dynamics and competitive outcomes. Hence, the joint consideration of food competition and reproductive interference is critical to accurately predict and understand species coexistence.

ecology↗

bioRxiv2023

Evolution in abiotic environments shapes coexistence between two spider mite species

Adaptation to abiotic stresses generally relies on traits that are not independent from those affecting species interactions. Still, the impact of such evolutionary processes on coexistence remains elusive. Here, we studied two spider mite species evolving separately on tomato plants that hyper-accumulated cadmium, a stressful environment for herbivores, or on plants without cadmium. Through experimental evolution and structural stability theory, we found that both species coexist in the cadmium environment, but evolution of a single species in cadmium leads to exclusion. However, when both species evolve in cadmium they can coexist. This shift occured due to a simultaneous increase in intra and a decrease in interspecific competition in that environment. These predictions were further confirmed with complementary experiments of population dynamics. Therefore, population shifts to novel environments, even in absence of interspecific competitors, may have unforeseen evolutionary consequences for community composition and the maintenance of species diversity.

evolutionary biology↗

bioRxiv2023

Disease Modifying Osteoarthritis Drug Discovery Using A Temporal Phenotypic Reporter In Primary Human Chondrocytes

Osteoarthritis is a significant and growing problem with no disease modifying drugs in the clinic. Current screening platforms typically use 2D culture, immortalized or non-human cells in a hyperoxic environment. To challenge this paradigm and identify new drugs, we engineered primary human chondrocytes with a secreted luciferase reporter under the control of the articular cartilage marker, type II collagen. We then successfully screened a natural product library using a high throughput model with COL2A1-Gaussia luciferase primary human chondrocyte reporter cells in 3D aggregates under physioxia. We identified several candidate compounds that increased type II collagen over controls, with aromoline being the best candidate. Aromoline is a bisbenzylisoquinoline alkaloid that has been studied for its anti-proliferative, anti-inflammatory, and anti-microbial properties, and we are the first to explore its effects on chondrocytes and chondrogenesis. In silico analysis of predicted targets narrowed by RNA-Seq data on expression provided an unexpected initial candidate target protein: the dopamine receptor D4 (DRD4). The researchers confirmed upregulation in the expression of DRD4 and type II collagen after treatment with aromoline. This novel approach combining in silico and in vitro methods provides a platform for drug discovery in a challenging and under-researched area. In conclusion, a novel drug (aromoline) and target receptor (dopamine receptor D4) were identified as stimulating type II collagen, with the goal to treat or prevent osteoarthritis.

pharmacology and toxicology↗

bioRxiv2022

Micronutrient Optimization Using Design of Experiments Approach in Tissue Engineered Articular Cartilage for Production of Type II Collagen

Tissue Engineering of cartilage has been hampered by the inability of engineered tissue to express native levels of type II collagen in vitro. Inadequate levels of type II collagen are, in part, due to a failure to recapitulate the physiological environment in culture. In this study, we engineered primary rabbit chondrocytes to express a secreted reporter, Gaussia Luciferase, driven by the type II collagen promoter, and applied a Design of Experiments approach to assess chondrogenic differentiation in micronutrient-supplemented medium. Using a Response Surface Model, 240 combinations of micronutrients absent in standard chondrogenic differentiation medium, were screened and assessed for type II collagen expression. Five conditions predicted to produce the greatest Luciferase expression were selected for further study. Validation of these conditions in 3D aggregates identified an optimal condition for type II collagen expression. Engineered cartilage grown in this condition, showed a 170% increase in type II collagen expression (Day 22 Luminescence) and in Youngs tensile modulus compared to engineered cartilage in basal media alone. Collagen cross-linking analysis confirmed formation of type II-type : II collagen and type II-type : IX collagen cross-linked heteropolymeric fibrils, characteristic of mature native cartilage. Combining a Design of Experiments approach and secreted reporter cells in 3D aggregate culture enabled a high-throughput platform that can be used to identify more optimal physiological culture parameters for chondrogenesis.

cell biology↗

bioRxiv2022

Optimizing bioink composition for human chondrocyte expression of lubricin

The surface zone of articular cartilage is the first area impacted by cartilage defects, commonly resulting in osteoarthritis. Chondrocytes in the surface zone of articular cartilage synthesize and secrete lubricin, a proteoglycan that functions as a lubricant protecting the deeper layers from shear stress. 3D bioprinting is a tissue engineering technique that uses cells encapsulated in biomaterials to fabricate 3D constructs. Gelatin methacrylate (GelMA) is a frequently used biomaterial for 3D bioprinting cartilage. Oxidized methacrylated alginate (OMA) is a chemically modified alginate designed for its tunable degradation rate and mechanical properties. To determine an optimal combination of GelMA and OMA for lubricin expression, we used our novel high-throughput human articular chondrocyte reporter system. Primary human chondrocytes were transduced with PRG4 (lubricin) promoter-driven Gaussia luciferase, allowing for temporal assessment of lubricin expression. A lubricin expression driven Design of Experiment screen and subsequent validation identified 14% GelMA/2% OMA for further study. Therefore, 14% GelMA/2% OMA, 14% GelMA and 16% GelMA were 3D bioprinted. The combination of lubricin protein expression and shape retention over the 22 days in culture, determined the 14% GelMA/2%OMA to be the optimal formulation for lubricin secretion.

bioengineering↗

bioRxiv2020

Wolbachia and host intrinsic reproductive barriers contribute additively to post-mating isolation in spider mites

AO_SCPLOWBSTRACTC_SCPLOWWolbachia are widespread maternally-inherited bacteria suggested to play a role in arthropod host speciation through induction of cytoplasmic incompatibility, but this hypothesis remains controversial. Most studies addressing Wolbachia-induced incompatibilities concern closely-related populations, which are intrinsically compatible. Here, we used three populations of two genetically differentiated colour forms of the haplodiploid spider mite Tetranychus urticae to dissect the interaction between Wolbachia-induced and host-associated incompatibilities, and to assess their relative contribution to post-mating isolation. We found that these two sources of incompatibility act through different mechanisms in an additive fashion. Host-associated incompatibility contributes 1.5 times more than Wolbachia-induced incompatibility in reducing hybrid production, the former through an overproduction of haploid sons at the expense of diploid daugters (ca. 75% decrease) and the latter by increasing the embryonic mortality of daughters (by ca. 49%). Furthermore, regardless of cross direction, we observed near-complete F1 hybrid sterility and complete F2 hybrid breakdown between populations of the two forms, but that Wolbachia did not contribute to this outcome. This study identifies the mechanistic independence and additive nature of host-intrinsic and Wolbachia-induced sources of isolation. It suggests that Wolbachia could drive reproductive isolation in this system, thereby potentially affecting host differentiation and distribution in the field.

evolutionary biology↗

bioRxiv2020

Creating outbred and inbred populations of haplodiploid mites to measure adaptive responses in the lab

Laboratory studies are often criticized for not being representative of processes occurring in natural populations. This can be partially mitigated by using lab populations that capture large amounts of variation. Additionally, many studies addressing adaptation of organisms to their environment are done with laboratory populations, using quantitative genetics or experimental evolution methodologies. Such studies rely on populations that are either highly outbred or inbred. However, the methodology underlying the generation of such biological resources are usually not explicitly documented. Given their small size, short generation time, amenability to laboratory experimentation and knowledge of their ecological interactions, haplodiploid spider mites are becoming a widely used model organism. Here, we describe the creation of outbred populations of two species of spider mites, Tetranychus urticae and T. evansi, obtained by performing controlled crosses between individuals from field-collected populations. Subsequently, from the outbred population of T. evansi, we derived inbred lines, by performing several generations of sib-mating. These can be used to measure broad-sense heritability as well as correlations among traits. Finally, we outline an experimental evolution protocol that can be widely used in other systems. Sharing these biological resources with other laboratories and combining them with the available powerful genetic tools for T. urticae (and other species) will allow consistent and comparable studies that greatly contribute to our understanding of ecological and evolutionary processes.

evolutionary biology↗

bioRxiv2020

Discovery of a cryptic allosteric site in Ebola's 'undruggable' VP35 protein using simulations and experiments

Protein-protein and protein-nucleic acid interactions are often considered difficult drug targets because the surfaces involved lack obvious druggable pockets. Cryptic pockets could present opportunities for targeting these interactions, but identifying and exploiting these pockets remains challenging. Here, we apply a general pipeline for identifying cryptic pockets to the interferon inhibitory domain (IID) of Ebola viral protein 35 (VP35). VP35 plays multiple essential roles in Ebolas replication cycle but lacks pockets that present obvious utility for drug design. Using adaptive sampling simulations and machine learning algorithms, we predict VP35 harbors a cryptic pocket that is allosterically coupled to a key dsRNA-binding interface. Thiol labeling experiments corroborate the predicted pocket and mutating the predicted allosteric network supports our model of allostery. Finally, covalent modifications that mimic drug binding allosterically disrupt dsRNA binding that is essential for immune evasion. Based on these successes, we expect our pipeline is equally applicable to other proteins.

biochemistry↗