Motivation: Proportional Venn diagrams provide a compact representation of the relationships between sets. Each relationship is represented with a region whose area reflects the number of elements shared by a given combination of sets. This means that the number of regions grows exponentially with the number of sets, which is why proportional Venn diagrams with more than five sets are cumbersome to interpret and seldom used. However, Venn diagrams with a large number of sets may still be legible if enough regions are empty and do not need to be represented. Results: Here, we present nVenn2, the second version of the nVenn algorithm, to create quasi-proportional Venn diagrams. This new version uses a different, more flexible approach which includes steps to minimize the complexity of the diagram. Thus, computation time for nVenn2 mainly grows with the number of non-empty diagram regions, rather than with the number of sets. This property allows users to create interpretable quasi-proportional Venn diagrams with large numbers of sets. Availability and implementation: The nVenn2 algorithm is freely available as an executable program, as a web page, as an R package (nVennR2) and as a Python package (nVennPy). All interfaces allow users to edit the appearance of the resulting diagram.
Cocoa (Theobroma cacao L.) is an economically important tropical crop, but its production is increasingly constrained by strict regulations related to heavy metal accumulation in soils. Arbuscular mycorrhizal fungi (AMF) are a key biological component of cocoa agroecosystems due to their role in soil processes and plant nutrition. However, information on AMF diversity and their response to heavy metals remains limited. In this study, AMF communities were evaluated in cocoa plantations of different ages established under contrasting heavy metal conditions. A total of 53 AMF taxa were identified, indicating high fungal richness across all plantations. No significant differences were found in α-diversity indices or community composition among plantation ages. In contrast, multivariate analyses showed that variation in AMF community structure was primarily associated with soil heavy metal gradients, particularly cadmium (Cd) and zinc (Zn). Species-level models revealed contrasting responses among taxa, with both positive and negative associations along Cd and Zn gradients, suggesting differential tolerance strategies. Overall, these results indicate that edaphic factors, especially heavy metal concentrations, play a more important role than plantation age in shaping AMF community composition. These findings provide a basis for identifying AMF taxa adapted to metal-enriched soils and for developing management strategies that support sustainable cocoa production.
Extensive research has examined extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in memory and synaptic plasticity, yet the mechanisms linking ERK activity to memory stabilization remain unclear. ERK dimerization is required for the activation of cytoplasmic targets involved in plasticity. Here, we investigated the role of ERK2 dimerization in long-term memory reconsolidation and synaptic plasticity. Reactivation of weak inhibitory avoidance (wIA) memory significantly reduced hippocampal ERK2 dimerization. Post-reactivation, intrahippocampal infusion of the ERK dimerization inhibitor DEL-22379 (DEL) produced bidirectional effects, impairing reconsolidation of strong inhibitory avoidance (sIA) memory, while enhancing reconsolidation of wIA memory. DEL administration efficiently blocked hippocampal ERK2 dimerization and impaired maintenance of high-frequency stimulation-induced long-term potentiation (LTP) in the hippocampal slices. These findings demonstrate that ERK2 dimerization occurs in the intact mouse brain and plays a critical role in IA memory and synaptic plasticity, highlighting its relevance in memory reconsolidation processes.
Parkinson's disease (PD) is a progressive neurodegenerative disorder in which dopaminergic dysfunction is associated with oxidative stress, chronic neuroinflammation, and endoplasmic reticulum (ER)-stress-related signaling. ER-resident neurotrophic factors, cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF), have emerged as potential modulators of neuronal stress responses. Silybin, a flavonolignan derived from S. marianum, has antioxidant, anti-inflammatory, and neuroprotective effects in experimental PD models, although the molecular pathways underlying these effects remain incompletely defined. In this study, we evaluated whether silybin was associated with changes in CDNF/MANF levels, unfolded protein response-related markers, inflammatory mediators, and antioxidant enzyme activities in a subchronic MPTP mouse model. Silybin improved survival, reduced motor impairment, and partially preserved tyrosine hydroxylase content in the nigrostriatal pathway. Also, silybin selectively increased CDNF levels, whereas MANF remained unchanged. In parallel, silybin modulated PERK/eIF2α/ATF4- and IRE1α/XBP1-associated markers, lowered total NF-κB p65 and pro-inflammatory cytokine levels, and normalized several endogenous antioxidant enzyme activities. Correlation analyses identified CDNF as a prominent correlate of the protective phenotype, and in silico docking and molecular dynamics analyses further suggested that silybin may interact with PERK and IRE1α domains, providing a structural hypothesis compatible with the experimental findings. Overall, these results indicate that silybin modulates convergent changes in stress-, inflammatory-, and redox-related pathways in the MPTP model, together with selective CDNF upregulation and improved behavioral outcomes.
Aporocotyle is the second richest genus of fish blood flukes within the family Aporocotylidae. These cosmopolitan trematodes infect the heart, bulbus arteriosus, and blood vessels of marine fishes belonging to the orders Gadiformes, Ophidiiformes, Perciformes, Pleuronectiformes, and Scorpaeniformes. In this study, three species of Aporocotyle, i.e. A. argentinensis, A. mariachristinae, and A. ymakara, from teleost fishes collected in the San Matías Gulf, Patagonia, Argentina, are morphologically and genetically characterized. Re-examination of these blood flukes provided new detailed drawings, measurements, photomicrographs, confocal and scanning electron microscopy (SEM) images. Aporocotyle argentinensis is redescribed based on type-, voucher- and newly collected material from the bulbus arteriosus, heart and gill blood vessels of the Argentine hake Merluccius hubbsi from Argentina. We generated partial sequences for the large subunit of the nuclear ribosomal RNA (28S rRNA) and the mitochondrial cytochrome c oxidase subunit 1 (cox1) genes for A. argentinensis and A. ymakara and used these sequences to investigate the phylogenetic relationships of these blood flukes. Our morphological analyses highlighted the taxonomic importance of the distribution of tegumental spines, the extension of the posterior caeca, and the presence of a genital atrium to circumscribe and classify species of Aporocotyle. Phylogenetic analyses recovered A. mariachristinae and A. ymakara as closely related taxa and placed the new sequences of A. argentinensis in a clade together with previously published sequences of isolates identified as A. argentinensis from three hake species (Merluccius hubbsi, M. gayi and M. australis) from Argentina, Chile, Peru and the Falkland Islands. This study provides a comprehensive review of the diversity of Aporocotyle in the southwest Atlantic, contributing to a better understanding of the systematics and evolutionary history of these blood flukes.
Pesticide exposure during pregnancy, particularly to organophosphates and carbamates, has been associated with oxidative stress imbalance and adverse maternal and neonatal outcomes. This study aimed to evaluate cholinesterase activity and oxidative stress biomarkers in maternal peripheral blood and umbilical cord blood from mother and newborn pairs residing in an agricultural region of southern Brazil with environmental pesticide exposure. A total of 98 third-trimester pregnant women were recruited. Biomarkers of oxidative stress (MDA, GST, protein carbonyls, and 8-OHGua) and cholinesterase activity (AChE and BChE) were assessed in maternal and cord blood at delivery. Sociodemographic, clinical, and behavioral data were also collected. AChE activity was significantly reduced in cord blood (0.305 ± 0.15 μmol/min/mg) compared to maternal blood (0.612 ± 0.27 μmol/min/mg; p < 0.0001), with inhibition observed in 95% of neonatal samples. Elevated 8-OHGua levels were detected in 87% of maternal and 90% of neonatal samples. GST activity was significantly reduced in 65% of cord samples (p = 0.0001). Exploratory MCA analyses identified association patterns between biomarkers and maternal or neonatal variables. In cord blood, low birth weight showed the highest contribution in Dimension 3 (32.6%), followed by normal GST activity (20.9%). However, no significant associations were observed between biomarkers and major perinatal outcomes. The findings suggest relevant biochemical alterations in the maternal-newborn dyad, involving cholinesterase inhibition and oxidative imbalance. However, the absence of direct pesticide quantification requires cautious interpretation of the findings.
Down syndrome (DS) is linked to increased risks of metabolic, gastrointestinal, and neurodegenerative disorders. Early alterations in the gut microbiota have potential long-term health impacts. This report appears to be the first study to stratify DS participants by age to explore early-life microbiota changes. We conducted a cross-sectional analysis of gut microbiota in children, adolescents, and adults with DS, compared with a control group, using Illumina iSeq100 sequencing of the V4 polymorphic region of the 16S rRNA gene. Children with DS exhibited lower microbial diversity and a higher abundance of genera such as Sutterella and Enterococcus. These differences lessened in older groups, indicating a convergence with control profiles. Early alterations in gut microbiota in DS may contribute to metabolic and neurodegenerative risks, emphasizing the need for early interventions to potentially improve long-term health outcomes.
Urban ecological reserves in Buenos Aires city (Argentina) were intended as spaces for outdoor recreation, preservation of natural environments and protection of native species. Reserva Ecológica Costanera Sur (RECS) and Reserva Ecológica Ciudad Universitaria Costanera Norte (RECUCN) are located on the right bank of the Río de la Plata estuary. In view of the repeated appearance of non-native land snails and slugs in Argentina during the last four decades, we wanted to know to what extent the terrestrial gastropod faunas of these two reserves were composed of native or non-native species. As RECS is larger than RECUCN, we also tested the hypothesis that abundance, species richness and diversity would be higher in the former than in the latter. Fifty-two and 25 sites were sampled for terrestrial snails and slugs in RECS and RECUCN, respectively. Land gastropod species richness was similar in RECS (14 species) and RECUCN (12 species). Non-native gastropods were much more diverse and overwhelmingly more abundant than native ones. The terrestrial gastropod fauna is dominated by non-native snails and slugs (98.4% non-native individuals vs. 1.6% native in RECS, 97.5% vs. 2.5% in RECUCN), most of them never previously reported from these reserves. More than 78% of the individuals found in RECUCN belonged to the non-native snail Vallonia pulchella, which was the species that most contributed to the dissimilarity between reserves. This study shows that so far, urban ecological reserves in Buenos Aires city are sites where mainly exotic rather than native terrestrial molluscs are being preserved. The dominance of nonnative species in these urban ecological reserves endorses the pervasive advance of synanthropic species in urban land snail assemblages already documented for other biogeographic regions.
Hypervirulent Klebsiella pneumoniae (hvKp) is an emerging threat due to its capacity to cause severe community-acquired infections and its increasing convergence with multidrug resistance. However, functional assessment of hvKp virulence still relies largely on mammalian infection models, which are costly, ethically constrained, and poorly suited for scalable early-stage screening. Here, we establish a proof-of-concept cell-based biosensing framework using the social amoeba Dictyostelium discoideum to detect capsule-associated virulence phenotypes in the model hvKp strain SGH10. Using SGH10 and a scarless capsule-null ΔwcaJ mutant, we show that loss of capsule production strongly reduces mucoviscosity, zebrafish lethality, resistance to amoebal predation, inhibition of social development, and impairment of amoebal motility. These host behavioral responses were quantified through complementary readouts, including predation plaque expansion, multicellular development, fluorescence microscopy, live-cell tracking, and exploratory multivariate integration. Compared with SGH10, the ΔwcaJ mutant produced phenotypes resembling those of the avirulent control strain KpGe, supporting capsule production as a major driver of phagocytosis resistance and host-response disruption in this model. Rather than providing a broadly validated classifier for hvKp, our results define a calibrated experimental framework in which D. discoideum functions as a living phenotypic sensor for capsule-dependent virulence traits. This platform offers a low-cost, genetically tractable, and imaging-compatible system for dissecting hvKp host interactions and for guiding future validation of scalable virulence biosensing across larger and genetically diverse K. pneumoniae collections.
Trait-based approaches are increasingly used in ecology to understand biodiversity responses to environmental changes. However, information on diatom traits, particularly in temporary aquatic systems, remains limited. Here, we present DIATPOOL, the first database focused on diatom traits from disconnected pools. DIATPOOL includes 17 morphological, ecological, and physiological traits, derived from literature and expert knowledge, including size, shape, apical constriction, dispersal capacity, ecological guilds, colony formation, or habitat tolerance. The database also includes an initial inventory of diatom species, their relative abundances, and environmental characteristics of disconnected pools sampled across the Iberian Peninsula. DIATPOOL provides an open and standardized resource to explore diatom biodiversity and functional traits in temporary and intermittent aquatic systems, offering a valuable framework for ecological, taxonomic, and bioassessment studies in these transitional habitats.
Somatic embryogenesis (SE) is a biological process in which somatic cells undergo molecular reprogramming, leading to embryo formation. SE is considered the most efficient plant propagation system, supporting breeding programs that use genetic engineering/editing and omics-based research. However, its success depends on a complex interaction between internal and external factors, including biomolecules released by explants into the culture medium. The use of nurse cell cultures, established decades ago, exemplifies this process. Nevertheless, few studies have characterised the secreted molecules involved or investigated their role in SE efficiency. This review compiles current knowledge on molecules released during SE, focusing on secreted proteins and metabolites. In addition, the involvement of extracellular vesicles (EVs) as modulators of SE response is hypothesised, emphasising their capacity to mediate targeted communication during the early stages of embryogenic reprogramming. EVs transport regulatory biomolecules and may participate in coordinating the cellular responses triggered by SE-inducing stimuli, as supported by the recurrent identification of EVs-associated proteins linked to stress perception and developmental signalling. Understanding how these extracellular factors interface with intracellular pathways could advance efforts to optimise SE and broaden its biotechnological applications.
This study investigates whether salinity affects the upper thermal tolerance (CTmax) of the euryhaline mummichog (Fundulus heteroclitus). Mummichogs were acclimated to seawater (38 ppt, SW) or freshwater (<1 ppt, FW) at 18.5°C (Tacc), then transferred to the opposite salinity. CTmax and physiological profiles were assessed on days 0, 1, 3, 6, and 13 post-transfer. CTmax was consistently lower in FW than in SW, regardless of prior acclimation. Compared to the Tacc controls, CTmax exposure reduced plasma ion levels in FW-transferred fish and increased them in SW-transferred fish. In both salinities, CTmax exposure raised plasma cortisol, glucose, lactate, and hematocrit. Second-order Akaike Information Criterion (AICc) modeling identified the Salinity/Acclimation time interaction as the most parsimonious predictor of CTmax. Furthermore, multivariate analysis reveals that potential underlying mechanisms of thermal shock diverged by environment. In FW, CTmax progressively declined over time, constrained by a direct metabolic tax-the recruitment of high-overhead, residual (ouabain-insensitive) ATPase activity required to maintain ionic stability-alongside a unique stress-erythropoiesis response. Conversely, in SW, thermal performance improved over time and was tightly coupled to osmolality, glucose and cortisol. Additionally, lactate accumulation was a shared response across salinities. We conclude that the synergy of ion-poor environments and rising temperatures creates a unique performance trade-off that imposes a permanent thermal penalty, potentially increasing the climate vulnerability of estuarine populations beyond current ecological forecasts.
Wine quality assessment is the most time-consuming and labor-intensive task in grapevine breeding. Breeding programs worldwide would benefit by accelerating this process, regardless of their primary objectives, such as climate adaptation or disease resistance, because wine quality ultimately determines consumer acceptance. In this study, an F1 mapping population derived from 'Calardis Musqué' × 'Villard Blanc', segregating for perceived wine quality, was comprehensively characterized for key aroma-relevant metabolites and sensory attributes. A total of 25,284 sensory evaluations were conducted on 1,635 micro-vinified wine samples, which were obtained from 147 genotypes grown at two locations over six vintages. A multivariate Bayesian multilevel modeling approach was applied to disentangle the effects of genotype, environment, and individual taster subjectivity on eight sensory attributes and the total quality score (TQS). Quantitative trait locus (QTL) analysis based on a dense genetic map was performed using the modeled sensory phenotypes as well as quantification data of volatile monoterpenes acquired with a targeted SIDA-SPE-GC-MS. This integrative approach identified overlapping genetic loci linking aroma-active metabolites with their sensory quality attributes. Two major pleiotropic QTL clusters associated with wine quality were identified. The first, located on chromosome 2, simultaneously controlled all major quantified monoterpenes (e.g., linalool: LOD 10.1, PVE 19.7%) and is co-localized with QTLs for the most discriminating sensory attributes, including "floral" (LOD 14.8, PVE 14.3%), "fruity" (LOD 14.4, PVE 17.2%), and "tropical fruit" (LOD 13.1, PVE 16.3%). A second major QTL cluster on chromosome 11 harbored strong QTLs for terpene concentrations as well as quality-related aroma attributes (e.g., floral: LOD 11.1, PVE 10.9%). In addition, QTLs for TQS and the attribute "off-flavor" were identified on several chromosomes, indicating further genomic targets relevant for breeding on wine quality. This integrated workflow combines sensory phenotyping with genotype × environment modeling and estimation of taster bias for subsequent genomic analysis. The approach represents a significant advancement in grapevine quality assessment and provides a framework to accelerate the breeding of new grapevine cultivars that are agronomically valuable, consumer-accepted, and both ecologically and economically sustainable.
To preserve proteome homeostasis and survive at higher-than-optimal temperatures, organisms have evolved the conserved heat-shock (HS) response (HSR), characterised by increased expression of specific chaperone-encoding genes. In the human blood, malaria parasites are frequently exposed to elevated temperatures associated with host fever episodes. The protective HSR of Plasmodium falciparum, the parasite that produces the vast majority of malaria clinical cases and deaths, is regulated by the transcription factor AP2-HS. Here, we systematically investigated the conditions that trigger the AP2-HS-dependent HSR and found that even mild HS conditions that do not compromise parasite viability can activate this response. As in other organisms, activation of the HSR in P. falciparum is rapid, as it was observed after a HS of only 10 min. Dihydroartemisinin (DHA), a drug that produces general proteome damage, also triggered the HSR, indicating that activation of the malarial HSR is not restricted to thermal stress. The AP2-HS-dependent HSR can be activated in all asexual blood stages, with the exception of very young rings, but not in intermediate or mature gametocyte stages. Accordingly, these gametocyte stages are highly sensitive to HS. Since mature gametocytes are the only stage that can mediate human-to-mosquito transmission, these results suggest that malaria patients with high fever may become transiently non-infectious.
Cancer progression involves not only uncontrolled proliferation but also the strategic entry of tumour cells into reversible (quiescent) or irreversible (senescent) states of cell cycle arrest (G0). These states can give rise to rare persister-like cancer cells that survive hostile tumour microenvironment conditions, facilitating drug resistance, metastasis and disease relapse. Despite their importance, identifying and understanding the mechanisms regulating these cell populations remains challenging. We leveraged single-cell and spatially profiled primary breast tumours to quantify G0 arrest and proliferation decisions in cancer cells, revealing molecular and spatial features associated with proliferation-G0 dynamics. We uncovered a G0 persister-like state with reduced copy number alteration burden and hallmarks of dormancy, characterised by transcriptional reprogramming of stress response pathways and increased epithelial-mesenchymal plasticity. Spatial analyses revealed distinct ecological niches: G0 cells inhabited protective niches with complement pathway activity proximal to CXCL10+ macrophages and myofibroblastic cancer-associated fibroblasts (CAFs), whereas proliferative zones were associated with CLEC9A+ dendritic cells and PERK signalling, with distinct drug sensitivities. Our findings highlight key principles underpinning G0-proliferation dynamics and niche specialisation in breast cancer, offering novel insights into the spatial drivers of tumour heterogeneity and evolution.
Adolescence is a vulnerable window in which stress and alcohol exposure can induce long-lasting neuroimmune, endocrine, and cardiac molecular alterations. The fractalkine axis (CX3CL1/CX3CR1) regulates neuron-microglia signaling and inflammatory responses, but its role in linking adolescent stress/alcohol exposure with adult anxiety-like behavior and cardiac biomarker/molecular signatures remains unclear. To test whether genetic or pharmacological disruption of CX3CR1 modifies adult anxiety-like behavior, circulating biomarkers, and cardiac transcriptional responses after adolescent stress and/or alcohol exposure. Male and female C57BL/6J wild-type (WT) and CX3CR1 knockout (KO) mice were exposed to restraint stress (90 min) and/or alcohol (2 g/kg, 14 days) during adolescence. In adulthood, elevated plus maze (EPM) behavior was assessed; plasma cTnI/cTnT, CX3CL1, ACTH, and corticosterone were quantified; and cardiac mRNA expression of chemokines, inflammatory receptor/NF-κB-related genes, glucocorticoid/mineralocorticoid receptor-related genes, and RAAS-related transcripts was profiled. WT mice received the CX3CR1 antagonist AZD8797 (20 mg/kg, i.p.) for behavioral validation. CX3CR1 loss or blockade was associated with increased anxiety index and reduced open-arm exploration under selected exposure conditions. CX3CR1 KO mice showed higher plasma cTnI/cTnT, CX3CL1, and ACTH, and lower corticosterone, stress and alcohol were also associated with lower corticosterone. In cardiac tissue, alcohol exposure, mainly under the no-stress condition, increased Cx3cl1, Ccl2, Ackr3, and selected RAAS-related transcripts, whereas CX3CR1 deficiency was associated with broadly lower expression across chemokine, NF-κB-related, mineralocorticoid receptor-related, and RAAS-related targets. Correlation analyses indicated a chemokine/Cxcl12-anchored inflammatory-RAAS transcriptional architecture in WT mice that was attenuated and shifted toward glucocorticoid/mineralocorticoid receptor-NF-κB/IκBα-RAAS-related associations in CX3CR1 KO mice. These findings suggest that CX3CR1 signaling contributes to the long-term coordination of behavioral stress-related responses, HPA-axis activity, circulating cardiac injury biomarkers, and cardiac inflammatory/RAAS-related transcriptional programs after adolescent stress and alcohol exposure. Because cardiac function and histopathology were not assessed, these results should be interpreted as evidence of cardiac biomarker and molecular vulnerability rather than definitive myocardial pathology.
Biological dinitrogen (N2) fixation sustains productivity in oligotrophic oceans and is now also thought to contribute substantially to the nitrogen supply in the warming Arctic. Here we demonstrate significant N2 fixation by particle-associated diazotrophs in subsurface waters of the Barents Sea. Comparing our findings with subtropical studies reveals particle-associated non-cyanobacterial diazotrophs as the primary N2 fixers in subsurface Arctic waters of the Barents Sea, contrasting with diverse communities in warmer regions. As the Arctic shifts towards oligotrophication, understanding the magnitude and controls of particle-associated N2 fixation will provide critical insights into future nitrogen supply required to sustain productivity in the rapidly changing Arctic Ocean. However, particle-associated N2 fixation may be a distinctive feature of the Barents Sea, where in contrast to other Arctic shelves the seasonal and long-term trends in nitrogen dynamics are heterogeneously determined by changes in the external Atlantic Water supply, sea-ice extent, and terrestrial inputs. In this context, the role of particle-associated N2 fixation across the wider Arctic Ocean will require further investigation.
Immunoglobulin binding protein (BiP) is a chaperone protein that plays crucial roles in protein folding and transport by binding unfolded proteins in the substrate binding domain (SBD), an interaction allosterically linked to the nucleotide occupancy of the nucleotide binding domain (NBD). BiP also forms oligomers that influence its activity, particularly in binding polypeptide clients. Using single-molecule approaches and mechanical and enzymatic activity assays, BiP monomer stability and oligomerization were analyzed both alone and in response to nucleotides and peptides. We find that the formation of BiP dimers exhibits biphasic behavior as a function of BiP concentration, suggesting concentration-dependent changes in dimer dissociation constants. At low BiP concentrations, dimers were disrupted by peptide substrate and adenosine triphosphate (ATP) but remained unaffected by ADP or Adenosine 5'-O-(3-thio)triphosphate (ATPγS). At high concentrations, dimers are unaffected by peptides, but their assembly is inhibited by ATP and ATPγS to the same degree. These results suggest the formation of two distinct BiP dimers exhibiting unique binding affinities, kinetics, and potentially structures. We propose the existence of a high-affinity dimer binding site within the SBD of BiP, while the formation of a low-affinity dimer involves interactions between the lid and NBD of each protomer. Our findings demonstrate the importance of considering single-molecule characteristics when interpreting bulk studies on protein function and regulation.
Vitamin D signaling produces context-dependent transcriptional responses, but how gene-level variability relates to recurrent pathway-level programs across perturbational models remains incompletely defined. To systematically characterize these responses, we analyzed publicly available LINCS L1000 perturbational transcriptomic data comprising 258 replicate-supported 24-h signatures across five LINCS cell-line models and seven vitamin D-related compounds. Global transcriptomic analysis indicated that cellular context contributes slightly more to transcriptional variability than compound identity and dose. At the gene level, we defined a direction-specific consensus transcriptional core composed of 42 upregulated and 35 downregulated genes, which was used to derive an operational transcriptional activity metric (core_score). Despite strong context-dependent variability at the gene level, pathway-level analysis revealed recurrent functional themes involving metabolic regulation, stress response, proteostatic control, and proliferative programs. Dose-related analyses showed aggregate dose-ordered core_score trends in several cellular contexts, but these associations should be interpreted as dataset-level patterns rather than compound-specific pharmacodynamic curves. Transcript-level assessment of selected VDR/RXR-axis genes showed small or context-dependent changes in VDR, CYP24A1, CYP27B1, and RXR isoform transcripts, indicating that transcript abundance of these selected components alone does not explain downstream response heterogeneity. Overall, this study provides a reproducible, hypothesis-generating framework for distinguishing shared and context-dependent vitamin D-related transcriptional programs in the analyzed LINCS subset.
Adolescence is a period of several brain changes, making it especially vulnerable to external influences. Abuse of psychoactive drugs, such as caffeine (CAFF), generates changes in cognitive functions. The main pharmacological targets of CAFF are mainly the A1R and A2AR adenosine receptors, which can regulate GABA homeostasis. We therefore evaluated the influence of CAFF intake upon GABA uptake and release in the frontal cortex (FC) of adolescent Swiss mice and addressed the underlying mechanism. Mice were treated for 5 days with a subcutaneous injection of CAFF (10, 20, and 40 mg/kg) every 24 h, and the FC dissected out at 1 h after the last injection for measurement of [3H]-GABA uptake and release, cAMP accumulation, and density levels of GAT-1, A1R, A2AR, PKA, and PKC. Calcium (Ca2+) imaging was performed on primary neuronal cultures treated with CAFF (200 μM). CAFF increased [3H]-GABA uptake at all doses studied, an effect reversed by incubation with the selective GAT-1 uptake inhibitor, NO-711 (10 μM). At 20 and 40 mg/kg, CAFF also increased [3H]-GABA release. CAFF also increased A1R, but not A2AR levels. The influence of CAFF involves pPKC activity since CAFF enhanced the pPKC/PKC ratio, while the PKC-inhibitor Gö 6983 (100 nM) reversed the facilitatory action of CAFF upon GABA transport and prevented the CAFF-induced increase in the frequency of Ca2+ transients in neuronal cell cultures. We conclude that CAFF alters GABAergic homeostasis in the FC, increasing GABA transport through PKC-activity modulation.