Biology Direct

Cyanobacteria utilize type IV pili for many behavioural responses, such as phototaxis, aggregation, floating, and DNA uptake. Type IV pilus-dependent functions are regulated by the nucleotide second messengers, c-di-GMP and cAMP. In this study, we investigated the role of a recently identified c-di-GMP receptor (CdgR) in cyanobacteria that harbours a ComFB domain. ComFB-domain proteins are widespread in cyanobacteria and are also present in heterotrophic bacteria. We demonstrated that the CdgR homolog from the cyanobacterium Synechocystis sp. PCC 6803, a model organism for studying type IV pilus-dependent functions, specifically binds to c-di-GMP. Genetic and phenotypic analyses revealed that Synechocystis CdgR is involved in phototactic motility and natural competence. Inactivation of cdgR resulted in altered expression of specific sets of minor pilins, which are essential for motility or natural competence. We identified interactions between CdgR and the CRP-family transcription factors, SyCRP1 and SyCRP2. Disruption of these CdgR-SyCRP1 and CdgR/SyCRP2 complexes is initiated by elevated c-di-GMP levels. Moreover, the assembly and stability of these complexes are influenced by other cyclic nucleotides, such as cAMP and c-di-AMP. These observed interactions imply a complex regulatory mechanism by which CdgR influences gene expression in response to cyclic nucleotide messenger signalling, particularly c-di-GMP. The present findings highlight the importance of CdgR in c-di-GMP signalling and its role in regulating type IV pilus-dependent functions in Synechocystis. The modulation of the expression of specific minor pilin genes by CdgR, through interactions with the transcription factors SyCRP1 and SyCRP2, contributes to the establishment of multiple type IV pilus functions and adaptive behaviours of cyanobacteria.

The unicellular green alga Chlamydomonas reinhardtii provides a tractable model for investigating how carbon availability influences metabolic organization and cell-cycle control in photosynthetic eukaryotes. Its capacity for autotrophic (light, CO2), mixotrophic (light, CO2, acetate), and heterotrophic (acetate, dark) growth enables systematic analysis of trophic-state-dependent regulation. We performed comparative transcriptomic analyses of strain 21gr grown under these three regimes at 30 {degrees}C. Mixotrophy resulted in the highest biomass accumulation and was associated with earlier cell-cycle commitment compared with autotrophy, whereas heterotrophy displayed delayed commitment and reduced growth. Transcriptomic profiling revealed coordinated upregulation of central carbon metabolic pathways under mixotrophy, including photorespiration, glycolysis, the oxidative pentose phosphate pathway, and tricarboxylic acid cycle functions, consistent with enhanced carbon flux and biosynthetic capacity. In contrast, heterotrophy preferentially induced acetate assimilation and glyoxylate cycle genes and was accompanied by elevated expression of cell-cycle regulators, including the CDK-inhibitory kinase WEE1. Together, these findings indicate that trophic mode modulates the coupling between carbon metabolism and cell-cycle progression, with mixotrophy supporting integrated metabolic and proliferative activity, whereas heterotrophy is associated with delayed cell-cycle timing and transcriptional signatures of metabolic adjustment.

Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in elderly individuals for which no effective treatments are currently available. The photoreceptor loss in dry AMD is secondary to the demise of the retinal pigment epithelium (RPE) cells. The accumulation of extracellular deposits, known as drusen, resulting in part from deficient lysosomal and autophagosomal degradation, is a key feature of dry AMD pathogenesis. Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway that maintains proteostasis by targeting specific cytosolic proteins for lysosomal translocation and degradation. LAMP2A (lysosome-associated membrane protein 2A) functions as the key lysosomal receptor required for CMA. Using Lamp2a knockout mouse, we show that selective CMA dysfunction recapitulates AMD-like pathologies, including sub-RPE lipid and protein deposits, RPE atrophy, Bruchs membrane thickening, and impaired autophagic activity. Furthermore, we identify large-conductance Ca{superscript 2}-activated K (BK) channels as a therapeutic target for restoring autophagic activity. Mechanistically, pharmacological activation of BK channels with the small-molecule agonist GLA-1-1 enhances macroautophagy and stimulates autophagic flux by promoting autophagosome-lysosome fusion. Importantly, oral administration of GLA-1-1 in markedly attenuates structural, functional, and molecular retinal abnormalities in Lamp2a-deficient mice, suggesting that pharmacological activation of macroautophagy through facilitating autophagosome-lysosome fusion can partially compensate for CMA deficiency. Together, these findings demonstrate that pharmacological activation of macroautophagy can ameliorate the retinal phenotype resulting from CMA dysfunction and support BK channel activation by GLA-1-1 as a promising therapeutic strategy for dry AMD.

Chromosome spatial organization plays critical roles in transcriptional regulation and DNA protection. In cyanobacteria--oxygenic photosynthetic bacteria that experience dramatic fluctuations in light intensity--chromosome reorganization may facilitate rapid transcriptional reprogramming and protect DNA from photodamage. However, direct observation of chromosome organization in these polyploid organisms has remained technically challenging, leaving light-dependent chromosomal responses unexplored. Here we show that local chromosome organization in Synechocystis sp. PCC 6803 is reorganized in response to high-light stress. We established fluorescence in situ hybridization (FISH) methods for this model cyanobacterium carrying multi-copy genomes, together with a computational pipeline for optimal same-genome-copy probe pairing. Under standard conditions, spatial distance between paired signals increased with genomic distance (slope {beta} = 0.972 nm/kbp, R{superscript 2} = 0.12), demonstrating that linear genome organization is reflected in three-dimensional chromosome structure at the 25-124 kbp scale. This genomic-spatial distance relationship substantially weakened under high-light conditions ({beta} = 0.450 nm/kbp, R{superscript 2} = 0.02), indicating that local chromosome organization is disrupted by elevated light intensity. Same-color nearest-neighbor distances further revealed that the spatial distribution of genome copies differed between conditions, independently supporting condition-dependent chromosome reorganization. Hi-C analysis corroborated these findings, revealing reduced short-range interactions within the 10-100 kbp genomic range under high-light conditions. Our integrative single-cell and population-level approach provides a framework for investigating how environmental signals modulate higher-order chromosome structure in polyploid bacteria.

DNA methylation plays a central role in the regulation of gene expression. In plants, methylation occurs in the CG, CHG and CHH contexts, via distinct DNA methyltransferases including MET1, CMT3 and the RNA-directed DNA Methylation (RdDM) pathway via DRM2. In interspecific hybrids, these epigenetic mechanisms are confronted to a mixed small RNA population and two subgenomes harbouring specific methylation patterns, therefore generating unique expression profiles. The aim of this work was to understand these regulations by analysing gene expression, DNA methylation and small RNAs in a Citrus hybrid resulting from the cross between C. reticulata (mandarin) and C. australasica (finger lime). Haplotype-resolved subgenomes assembly identified hundreds of allele-specifically expressed genes. Asymmetric reprogramming of methylation was observed, in particular an increase in CHH in C. australasica haplotype. Surprisingly, CHH methylation, usually associated with gene silencing, was correlated here with increased expression, but also 24nt small RNA populations at their promoter regions. Similar analyses of the parental lines and other citrus species suggest the correlation between CHH methylation-enriched promoter and high expression level is not due to the hybridization, but seem to be generally true for all citrus. These observations suggest that, in citrus fruit, RdDM could activate transcription. This work also provides a full pipeline to analyse the expression profiles and DNA methylation in complex hybrids, which could be crucial for anticipating varieties resistant to diseases and the current threats affecting citriculture such as the Huanglongbing disease.

In 2016, the glycolytic Entner-Doudoroff (ED) pathway was reported in cyanobacteria and plants (1). The claim was based on the biochemical characterization of its key enzyme the 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase EDA (ED aldolase), on protein sequence alignments, physiological data from cyanobacterial mutants, and the in vivo detection of an ED pathway specific metabolite (1). However, two enzymes 6-phoshogluconate (6PG) dehydratase (EDD) and EDA are unique to this route. A recent study suggests that EDD (Slr0452) from Synechocystis sp. PCC 6803 most likely encodes an enzyme involved exclusively in amino acid synthesis, indicating that a complete ED pathway would be missing (2). To answer the presence or absence of the ED pathway in Synechocystis, we conducted extended biochemical and physiological studies, revisited old data and resolved contradictions. These investigations reveal that Synechocystis lacks both an ED pathway and a glucose dehydrogenase/glucokinase (GDH/GK) bypass but contains a promiscuous aldolase EDA. EDA prefers KDPG as substrate but also decarboxylates oxaloacetate (OAA) and cleaves 2-keto-4-hydroxyglutarate (KHG). Synthesis of KDPG from pyruvate and glyceraldehyde 3-phosphate (GAP) is catalyzed with very low efficiency. These in vitro data suggest that EDA might be involved in the phosphoenolpyruvate (PEP)-pyruvate-OAA node and proline catabolism, which requires further clarification. The previous misconception was based on missing enzymatic characterizations, the oversight of a secondary mutation in a deletion strain, and an outdated view on carbohydrate fluxes. We conclude with a list of lessons and provide a solid foundation for future investigations into the role of EDA in cyanobacteria and other photoautotrophs. Significance statementThis study provides a retrospective on why, for many years, it was mistakenly assumed that the glycolytic Enter-Doudoroff (ED) pathway exists in the cyanobacterium Synechocystis sp. PCC 6803. It shows that the first enzyme of this pathway, ED dehydratase EDD, is absent, while the second enzyme, 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase EDA, is present but is promiscuous, cleaving KDPG in addition to 2-keto-4-hydroxyglutarate (KHG) and decarboxylating oxaloacetate (OAA) in vitro. Finally, valuable lessons are drawn from prior misconceptions and experimental limitations. This study provides a solid foundation for future studies on the role of the ED aldolase in absence of the ED pathway in cyanobacteria and other photoautotrophs.

Virulent pathogens commonly circulate in wildlife populations without causing mass mortality; the triggers of die-offs remain poorly understood. Prevailing frameworks emphasize individual host susceptibility, yet experimental manipulations of susceptibility factors often fail to predict population-level outcomes. We tracked ranavirus epizootics across 40 wood frog breeding ponds over three years, comparing lagged viral state variables against abiotic and host predictors at each epizootic stage. Lagged viral state--environmental DNA concentration and infection prevalence--outperformed abiotic and host predictors of transmission, intensification, and viral accumulation. Infected hosts shed virus into the water column throughout epizootics, but the reciprocal pathway, environmental virus driving new and more severe infections, activated only at the transition to die-off, consistent with a self-reinforcing feedback. The rate of viral accumulation discriminated die-offs, while no static pond or host feature was predictive, reframing mass mortality as an emergent property of pathogen accumulation in shared environments rather than of individual host susceptibility.

O_LIAquatic algae are key primary producers in the Arctic and Antarctic, yet how cold-water species respond to environmental change is poorly understood. The Polar Regions are increasingly exposed to frequent heat waves, leading to declining ice cover, increased light availability, and decreasing salinity in polar waters. We compared three phylogenetically related but geographically distant polar Chlamydomonas species to test how habitat history shapes algal responses to light, salinity, and temperature stress. C_LIO_LIWe assessed the growth, morphology, and photochemistry of psychrophilic Chlamydomonas acclimated to native-like (lower light, higher salinity) and climate-shifted conditions (higher light, lower salinity). Next, we exposed acclimated cultures to a lethal heat shock and observed how acclimation affects algal temperature stress resilience. C_LIO_LIAll three species acclimated to climate-shifted conditions grew rapidly but showed the greatest sensitivity to temperature stress, with rapid loss of viability and photosynthetic efficiency. In contrast, slow-growing cultures acclimated to native-like conditions exhibited significantly greater resilience to temperature stress. C_LIO_LIOur work is the first to directly link light and salinity acclimation with temperature resilience in psychrophilic algae, suggesting that fast-growing polar green algae may be particularly vulnerable to increasingly frequent heat waves, with major implications for primary productivity in polar environments. C_LI

Bacteria evolve antibiotic resistance through various mechanisms, including horizontal gene transfer, sequence-altering mutations, and tandem duplications/amplifications in genomic regions that increase the dosage of resistance determinants. This phenomenon, known as gene duplication-amplification (GDA), is a transient and reversible genetic mechanism that allows bacteria to rapidly adapt to antibiotic stress. Due to the unstable nature of amplification, the quantitative relationship between gene copy number, resistance levels, and the associated physiological costs remains poorly defined. Here, we constructed strains with fixed GDA copy numbers in a clinical Enterobacter cloacae strain. Using growth curve and time-kill assays, we show that ceftazidime (CAZ) resistance scales with GDA copy number, but the benefit under selection comes with a growth cost in the absence of antibiotic. Notably, we find that while GDA provides a survival advantage under CAZ pressure, it simultaneously increases susceptibility to rapid killing at supra-MIC concentrations. Our analysis further illustrates that survival is jointly shaped by gene dosage, antibiotic concentration, and exposure time. Together, these results clarify that GDA-mediated resistance is highly context dependent, shaped by trade-offs between resistance benefits and physiological costs.

Human immunodeficiency virus (HIV) infection promotes considerable bioenergetic, spatially heterogenous strain to the brain that is incompletely ameliorated through viral suppression afforded by antiretroviral therapy (ART). Disrupted homeostasis of brain lipids after HIV in humans or simian immunodeficiency virus (SIV) infection in rhesus macaques occurs due to elevated energetic demands, neuroinflammation, reactive oxygen species, and barrier leakiness. Brain lipids are particularly vulnerable to HIV-associated dysregulation due to their high abundance, unique composition, and specialized functional roles. Using rhesus macaques exposed to SIV and ART (tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and dolutegravir (DTG), we investigated the spatial distribution and abundance of lipids across brain regions and metabolically relevant peripheral tissues using mass spectrometry imaging. When comparing lipid abundance, individual lipids representing a multitude of species were more varied across tissues than by treatment condition. Further, we discerned either solely SIV infection or ART outweighed one another in altering phospholipids in different tissues Presence of ART had a greater influence on phospholipid homeostasis in the temporal cortex and hippocampus than in the midbrain, possibly due to differences in penetrance and turnover of ART across brain regions. Overall, these data demonstrate ART robustly increased phospholipids across brain regions while SIV infection had a varied impact depending on the brain region. These findings inform the need to further evaluate the neurologic consequences that may result in the brain due to disrupted lipid homeostasis across ART regimens.

Small regulatory RNAs (sRNAs) are major post-transcriptional regulators in bacteria and, together with transcriptional regulators such as the two-component systems (TCSs), participate in the rapid adaptation of these microorganisms to changing environments. Several examples of paralogous sRNAs with overlapping functions have been reported, that could in theory integrate different environmental cues. Consistent with this idea, we have identified the acid-responsive RstB-RstA two-component system, important for virulence of multiple bacterial species, as a specific multicopy activator of the Escherichia coli OmrB sRNA, but not of the paralogous sRNA OmrA. Further characterization of this regulation unexpectedly revealed the asr-ydgU operon, itself a target of RstB-RstA, as a dual modulator of this TCS via two opposite effects. First, the 27 aminoacids YdgU small protein exerts a negative feedback by directly interacting with RstB and, second, Asr in contrast mediates a positive feedback on RstB-RstA activity via a not completely elucidated mechanism. These results provide a new example of retro-control of a TCS, here RstB-RstA, by one of its direct targets. They further highlight the major role of small proteins in controlling TCS activity and ydgU was thus renamed samT, for Small Acid-responsive Modulator of the RstB-RstA TCS.

There is growing interest in understanding how hormonal signaling pathways contribute to the pathophysiology of mood disorders, based on the premise that fluctuations in sex hormones influence mood, a relationship particularly evident in conditions such as premenstrual dysphoric disorder, prenatal depression, postpartum depression, and perimenopausal depression. Estrogen receptor alpha (ER) is predominantly localized in the nucleus, but can also be associated with the cell membrane, thus mediating a broad range of genomic and non-genomic effects through distinct intracellular pathways. By employing a combination of computational simulations and in vitro biochemical and cell-based assays, we systematically evaluated the potential binding and functional interactions of antidepressant compounds with ER. Our results provide compelling evidence that antidepressants may not only affect classical monoaminergic targets but also modulate hormone receptor activity, particularly that of ER. These findings are consistent with the hypothesis that ER plays an important role in mood regulation and highlight it as a potential therapeutic target. Moreover, this work raises the possibility that the clinical efficacy of certain antidepressants may, at least in part, derive from their capacity to influence estrogen receptor-mediated signaling. Significance statementClinical observations suggest a link between female sex hormones and mood, highlighted by the higher prevalence of depression in women and increased vulnerability to depression during hormonal fluctuations. Here, we report that structurally diverse conventional and rapid-acting antidepressants directly interact with estrogen receptor alpha (ER). This interaction is associated with rapid intracellular signaling in cellular models. These findings indicate that, alongside their conventional targets, antidepressants may also engage sex steroid receptor components and signaling. This work broadens our basic understanding of antidepressant pharmacology at the cellular level, offering an additional perspective that may inform future research into the biological mechanisms of mood disorders and suggest a framework for developing targeted therapies for hormone-associated depressive disorders.

Fitness increase, impacted by genetic and environmental traits, requires evolutionary changes in genomes or ecological changes in habitats. Whether and how habitat reconstruction can compensate for the genetic changes remains unclear. The present study offers an experimental comparison of bacterial fitness increase via evolutionary and ecological strategies to verify that habitat reconstruction has the potential to avert genetic restriction, challenging the genetic-only view of adaptation. Six finely tuned media with different combinations and five evolved lineages with different mutations, both of which allowed equivalent increases in bacterial growth rates, were achieved through active machine learning and experimental evolution, respectively. Transcriptome changes accompanied by fitness increase were limited but divergent among the six fitted media, compared to changes that were broad but similar among the five fitted genomes. The universal transcriptome reorganization in fitted genomes and media was likely driven by surrounding metabolisms, indicating escape routes for genetic changes that increase fitness through habitat reconstruction.

Trade-offs are central to biodiversity because they prevent the emergence of dominant phenotypes by limiting the simultaneous optimization of multiple fitness components. Yet trade-offs are often difficult to detect empirically when variation in overall performance produces positive correlations that mask underlying constraints. Here we use Pareto fronts--boundaries that capture optimal trade-off solutions--to test for evolutionary constraints on niche-determining traits in phytoplankton, including minimum nutrient requirements, thermal breadth, salt tolerance, and population growth rates. Using experimentally evolved Chlamydomonas reinhardtii populations subjected to nutrient and salt stress, we detected widespread Pareto fronts limiting the joint optimization of growth rate and niche-determining traits, thereby restricting the emergence of multivariate stress tolerance. Importantly, Pareto fronts revealed trade-offs even when underlying trait correlations were positive. We found that the structure of trait covariation behind Pareto fronts strongly predicted evolutionary outcomes: populations moved toward Pareto-optimal phenotypes primarily when trait correlations were neutral or positive, whereas negative trait correlations were associated with limited evolutionary optimization. Extending this framework across phytoplankton diversity, we compiled niche-determining traits for 299 phytoplankton taxa. At a macroevolutionary scale, we detected significant Pareto fronts constraining the evolution of niche-determining traits in phytoplankton. These fronts, however, did not always recapitulate the structure of trade-offs evident among C. reinhardtii populations, suggesting that forces that dictate microevolutionary outcomes, such as genetic correlations, can be resolved across macroevolutionary time. Together, our results highlight that evolutionary trajectories may differ across scales, but that fundamental limits on multivariate trait optimization persist across phytoplankton. Significance StatementTrade-offs among biological traits are central to evolutionary theory but often prove difficult to detect empirically. Here, we apply Pareto fronts--a framework borrowed from economics and engineering--to detect and reveal trade-offs among key niche-determining traits in phytoplankton. By combining experimental evolution in the laboratory with a synthesis of ecological traits across 299 taxa, we demonstrate widespread limits on the simultaneous optimization of growth rate, nutrient competition, salt tolerance, and thermal breadth. Importantly, Pareto fronts reveal trade-offs even when conventional correlation-based approaches fail, uncovering evolutionary constraints that remain hidden in trait correlations. These results show that trade-offs shape phenotypic variation across both micro- and macroevolutionary scales and impose fundamental limits on phytoplankton responses to multiple environmental stressors.

Down syndrome (DS) results from trisomy for human chromosome 21 and is the most frequent genetic cause of intellectual disability. No effective treatments currently exist that improve neurodevelopment and cognition. Atypical brain development in individuals with DS is apparent before birth, which suggests that the optimal time to begin administration of therapies is prenatally. Human neural progenitor cell (NPC) cultures provide a tractable in vitro model system to examine the effects of trisomy 21 (T21) on neurodevelopment and to measure the effects of pharmacological interventions. Here we report the results of preclinical studies evaluating 24 candidate therapies. RNA-Seq analyses found that euploid and T21 NPCs showed different transcriptomic responses to five candidate pharmacotherapies. The Rho-associated coiled-coil kinase (ROCK) inhibitor fasudil increased proliferation of T21 NPCs, reduced expression of inflammatory pathway genes in T21 NPCs, and reduced markers of inflammation in LPS-stimulated microglia model systems. These results demonstrate that fasudil can alter multiple T21-associated abnormalities in a beneficial manner, suggesting that fasudil warrants further study as a candidate prenatal pharmacotherapy for DS.

Favoured by global changes, freshwater cyanobacterial harmful blooms generate major ecological, economical and public health challenges. Microcystis, one of the most widespread cyanobacterial genera, grows within a phycosphere where specialised interactions with its microbiome occur, and are suspected to influence bloom appearance and its potential toxicity. Using a combination of metagenomic, metabolomic and metabolic modelling, we characterised the phycospheres of twelve Microcystis strains isolated from a French pond. The distribution of metabolic reactions within Microcystis was consistent with their genospecies, whereas the metabolic landscape at the community level diverged from cyanobacterial phylogeny indicating functional decoupling between cyanobacteria and their associated microbiomes. Phycosphere-associated bacteria substantially expand the metabolic repertoire of the system, while maintaining functional redundancy within and across communities. On the other hand, metabolomic profiles were largely driven by cyanobacterial metabolic outputs. Metabolic modelling, together with the identification of toxic specialised metabolites produced by specific biosynthetic gene clusters, further highlighted differences in metabolic potential among phycospheres. Together, these findings deepen the understanding of Microcystis phycosphere functioning, demonstrate the value of multi-omics systems biology approaches, and underscore the ecological relevance of interspecies and inter-phycosphere metabolic interactions as a structuring process in bloom-associated microbiomes.

Photosynthetic electron transport is mediated by several protein supercomplexes that are spatially arranged in the thylakoid membranes of chloroplasts. The chloroplast NADH dehydrogenase-like (NDH) complex is part of the photosynthetic alternative electron transport (AET) chain, which reduces the plastoquinone (PQ) pool using reduced ferredoxin as a substrate. This NDH complex is associated with photosystem I (PSI) and mediates a portion of AET in stroma lamellae, whereas photosystem II (PSII) is concentrated in grana stacks. This study presents the findings regarding post-illumination chlorophyll fluorescence increase (PIFI), a protein crucial for regulating AET via the NDH pathway. A marked increase in NDH activity and a reduction in the PQ pool in the dark were observed in PIFI-deficient mutant strains (g-pifi) generated by genome editing. Blue native PAGE analysis indicated that PIFI was associated with the NDH-PSI supercomplex in the wild type, and the NDH complex was dissociated from PSI in the g-pifi mutants. Additionally, the g-pifi mutants exhibited a decrease in the maximum quantum yield of PSII (Fv/Fm). Notably, Fv/Fm was restored in a double mutant harboring both g-pifi and NDH-deficient pnsl1 mutations, demonstrating that deregulated NDH activity in g-pifi causes downregulation of PSII efficiency. However, the lower Fv/Fm was not observed in a mutant lacking thioredoxin m4 (trxm4), which showed deregulated NDH activity but maintained the NDH-PSI supercomplex. These data suggest that PIFI stabilizes the NDH-PSI supercomplex and maintains the spatial localization of PQ reduction via AET in thylakoid membranes, which is essential for the proper functioning of PSII.

BackgroundRising global temperatures and eutrophication are increasing the intensity and frequency of cyanobacterial harmful algal blooms that release toxins including microcystin-LR (MC-LR). MC-LR inhibits protein phosphatases in the human liver and brain, but its accumulation in the placenta is unclear. Placental transporter expression varies across pregnancy and is influenced by physiological cues, such as low oxygen concentrations which activate HIF1A, and trophoblast cell fusion forming syncytiotrophoblasts that engage CREB-driven transcription. This study examined whether MC-LR accumulates in placental cells, which transporters mediate uptake, and how these transporters are regulated by HIF1A and CREB. MethodsIntracellular accumulation of MC-LR (0.1-10 {micro}M, 3 hour) was measured in human cytotrophoblasts (JAR, BeWo) and extravillous trophoblasts (HTR-8/SVneo) by western blotting for MC-LR-adducted proteins. Organic anion transporting polypeptide (OATP) involvement was tested using cyclosporin A (10 {micro}M), an OATP inhibitor, before exposure to the OATP substrate or MC-LR. Cells were also cultured under 3%, 8%, or 20% O2 to induce hypoxic responses or treated with forskolin (a potent intracellular cAMP inducer) to stimulate cell fusion before MC-LR exposure. ResultsMC-LR accumulated in all three placenta cell lines in a concentration-dependent manner. Cyclosporin A reduced MC-LR uptake by 57% in JAR cells, confirming OATP-mediated transport. Low O2 increased OATP4A1 expression and function but reduced protein phosphatase expression, decreasing MC-LR-bound proteins by 52-72%. Forskolin increased OATP4A1 expression and enhanced MC-LR uptake >2.5-fold. ConclusionMC-LR enters placental trophoblasts via active OATP transport, likely OATP4A1, and uptake increases under hypoxia and trophoblast fusion.

Xylella fastidiosa is a xylem-limited phytopathogen bacterium responsible for severe diseases in numerous plant species of major agricultural importance. Despite its economic impact, its metabolism remains poorly characterized due to the bacteriums fastidious growth and the limited availability of defined culture media. In this work, we reconstructed the first pangenome-based genome-scale metabolic model for X. fastidiosa, integrating the conserved metabolic capabilities of 18 strains representing five described subspecies. The resulting core metabolic model, Xfcore, was manually curated and used to investigate the metabolic potential of the species. Model simulations predict minimal nutritional requirements that guide the formulation of defined media capable of supporting biofilm formation in vitro. Analysis of the metabolic network also suggests an undescribed metabolic pathway that enables growth on acetate as a sole carbon source. Furthermore, the model predicts that X. fastidiosa could overproduce polyamines, compounds previously associated with virulence in other phytopathogens. Experimental analyses confirm the production and secretion of polyamines in several X. fastidiosa strains, providing the first in vitro evidence of polyamine production in this pathogen. These findings suggest that polyamine biosynthesis may represent an uncharacterized virulence factor in X. fastidiosa, potentially contributing to bacterial protection against host-induced oxidative stress. Overall, the Xfcore model provides a systems-level framework to explore the metabolism of X. fastidiosa, generate testable hypotheses about its physiology and virulence, and establish a basis for future strain-specific reconstructions and host-pathogen metabolic interaction studies.

Knock-out mutants are often used to study gene function by disrupting a specific gene and comparing the mutant to a wild-type strain. Reliable interpretation, however, requires that the two strains differ only by the intended mutation and that the observed phenotype is caused specifically by the deleted gene. In the highly polyploid cyanobacterium Synechocystis sp. PCC 6803, this is particularly challenging because incomplete segregation can mask genetic heterogeneity or secondary suppressor mutations. The genetic variation among laboratory wild-type lines can further confound phenotypic analyses. We show that these challenges can be addressed by routine strain validation via whole-genome sequencing (WGS). To this end, we developed and tested user friendly workflows for short-read (Illumina), long-read (Oxford Nanopore Technologies; ONT), and hybrid data, providing standardized quality control, variant calling, and structural variant detection. We benchmarked their performance in detecting single-nucleotide polymorphisms (SNPs), small indels, and structural variants using simulated datasets across different coverages and mixed populations. Applying the workflows to three Synechocystis sp. PCC 6803 wild-type lines revealed multiple sequence and structural differences relative to the reference genome, including previously undescribed genetic variants, underscoring the importance of documenting the strain background and the value of long-read sequencing. Characterization of two independent 6-phosphogluconate dehydrogenase (gnd) knock-out mutants and their complemented strains highlighted how a failed rescue can reveal a phenotype unrelated to the intended knock-out. An automated literature analysis revealed that only a minority of the investigated Synechocystis studies that used knock-out mutants included complementation as a control (39%), whereas this practice is more common in studies involving Escherichia coli (63%) and Saccharomyces cerevisiae (55%). Based on these results, we propose a practical guide for standardizing knock-out phenotyping in Synechocystis PCC 6803. Combined with accessible workflows for routine whole-genome validation, this framework aims to support more robust and reproducible knock-out studies in the future.