Biology Direct

In the next years, several space missions will search for evidence of life on exoplanets, focusing on robust biosignatures associated with oxygenic photosynthesis, including atmospheric oxygen accumulation and the Vegetation Red-Edge in surface reflectance spectra. Many potentially habitable rocky exoplanets orbit M-dwarf stars, whose spectral energy distribution may challenge oxygenic photosynthesis. Differently from the Sun, M-dwarf stars emit predominantly far-red (700- 750 nm) and infrared (750-1000 nm) light, and relatively little visible (400-700 nm) radiation, which constitutes photosynthetically active radiation. Some organisms have been found to photosynthesize under such spectrum but less efficiently than under solar light, as their photosynthetic apparatus evolved to harvest visible light emitted by the Sun. Around M-dwarfs, such different irradiation might have selected adaptations optimized for harvesting far-red / infra-red light. On Earth, similar selection can be found in Acaryochloris marina strains, constitutively presenting high chlorophyll d content in photosystem II & I, with in vivo absorption peaks beyond 700 nm. Here we tested the Moss Beach strain under a simulated M-dwarf spectrum and a simulated primeval atmosphere - anoxic and enriched in carbon dioxide. Results underline how this permanently red-shifted photosynthetic apparatus does not require acclimation to the stellar spectrum and enables for a strong growth and oxygen production, higher than under simulated solar light. Moreover, cells reflectance spectrum highlights a shift of the canonical red-edge toward longer wavelengths, resulting in a Chl d-near-infrared edge, suggesting a similar metabolism on exoplanets orbiting M-dwarfs could successfully produce both a gaseous biosignature and a characteristic surface biosignature. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/719884v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@7f91bdorg.highwire.dtl.DTLVardef@1391bdborg.highwire.dtl.DTLVardef@53f7b4org.highwire.dtl.DTLVardef@ab59fa_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Liistro, E. (2026) https://BioRender.com/j2de4ay

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.

Under natural growth conditions, plants are not usually exposed to the high-energy ultraviolet C range (UV-C, 100-280 nm) of the solar spectrum, as this is absorbed by the ozone layer. However, low doses of UV-C radiation can trigger stress responses in plants. Nevertheless, it is not yet fully understood how UV-C light affects plant development at the hormonal level. Here we show that a single one-min UV-C light pulse (20 W/m2) alters gibberellin (GA) homeostasis in Arabidopsis in two phases: initially, the level of GA12 - a key precursor of the final part of gibberellin biosynthesis - is reduced. Consistent with this, the transcript levels of the CPS, KS and KAO2 genes, which encode enzymes involved in the initial parts of gibberellin biosynthesis, decrease. The level of the plant hormone GA4 also decreases initially, probably due to the reduced GA12 precursor levels. However, in a second phase, the endogenous GA4 levels rise in UV-C treated plants relative to control plants. This increase leads to an early onset of flowering, as well as increased growth and fertility, in UV-C-treated Arabidopsis plants. The GA signalling mutant gdella does not exibit wild-type phenotypic responses to UV-C treatment, indicating that GA signalling is essential for the UV-C response. To further narrow down the responsible steps in the GA-signalling pathway, we tested the kao1 and kao2 mutants, which are both impaired in early gibberellin biosynthesis. Neither mutant displays phenotypic responses to the UV-C treatment, indicating that both genes are required for mediating the UV-C response. In contrast, the quintuple 2-oxidase mutant C19--2oxqM exhibits responses to UV-C treatment similar to the wild-type, suggesting that the five catabolic 2-oxidases that act on C19-GAs play a negligible role in regulation GA-hormone levels for growth and development in this case. HighlightUV-C pulse triggers biphasic gibberellin dynamics, delaying early development but ultimately enhancing growth and fertility in Arabidopsis thaliana.

Chromosome spatial organization plays critical roles in transcriptional regulation and DNA protection. In cyanobacteria--photosynthetic bacteria that experience dramatic fluctuations in light intensity--chromosome reorganization could facilitate rapid transcriptional reprogramming and protect DNA from photodamage. However, chromosome organization in these polyploid organisms has remained technically challenging to observe, leaving light-dependent responses unexplored. Here, we show that higher-order chromosome organization in Synechocystis sp. PCC 6803 is associated with light intensity, revealing a previously unrecognized light-dependent adaptation in cyanobacteria. We established fluorescence in situ hybridization (FISH) methods for this model cyanobacterium carrying multi-copy genomes, together with a computational pipeline to assign paired FISH signals to individual genome copies. The slope relating genomic and spatial distance was steeper under standard conditions ({beta} = 0.972 nm/kbp, R{superscript 2} = 0.12) than under high-light conditions ({beta} = 0.450 nm/kbp, R{superscript 2} = 0.02), indicating that local chromosome organization is substantially disrupted by elevated light intensity. The spatial distribution of the multiple genome copies also differed between conditions, independently supporting condition-dependent chromosome reorganization. Hi-C analysis corroborated these findings, revealing reduced chromosomal interactions within the 10-100 kbp range under high-light conditions. Together, these results demonstrate that light intensity is a previously unrecognized determinant of higher-order chromosome organization in a photosynthetic bacterium.

BackgroundTraumatic brain injury (TBI) together with non-cerebral injuries characterizes the TBI-polytrauma (P-TBI) constellation, which is associated with acute neurological deterioration, delirium and unfavourable prognosis. It is hypothesized that systemic inflammatory mediators my enhances the focal, cerebral neuroimmune reaction with overall detrimental consequences, in particular in terms of acute microglial reactivity. MethodsWe explored the role of the Complement factor 3 (C3) and of the TLR-co receptor cluster of differentiation (CD14) in a murine polytrauma model that involves a mild TBI together with femur fracture, blunt thorax trauma and resuscitated haemorrhagic shock, making use of mice genetically lacking either C3, CD14 or both. ResultsWe show that P-TBI results in a rapid (4h) and brain-wide induction of inflammatory cytokines, although with distinct profiles (TNF and CCL2 having brain-wide involvement and IL-1{beta} restricted to ipsilateral cortex and striatum). TNF and CCL2 mRNA as well as protein synthesis were upregulated in microglia upon P-TBI in cortex, hippocampus and striatum which was fully abolished in the C3-/-CD14-/-animals. The analysis of single-KO animals revealed that induction of TNF and CCL2 was prevented in animals lacking C3, but not CD14, in the contralateral cortex and striatum, with an abolishment in hippocampus in mice lacking both C3 and CD14. In the cortical area of focal lesion neither C3 nor CD14 affected the induction of pro-inflammatory cytokines. ConclusionThus, C3 and CD14 are dispensable for the acute cytokine response to P-TBI in the site of injury but play differential roles across the cortex, hippocampus and striatum for the induction of cytokines in the non-injured parenchyma and in particular in microglia. Thus, interventions on C3 (mainly) and/or CD14 may reduce the encephalopathy risk associated with P-TBI but not the acute response in the injury site, where additional DAMP signalling may offer redundant activation pathways.

Ischemic stroke triggers a cascade of molecular and cellular processes leading to fibrotic scar formation, entailing activation of brain platelet-derived growth factor receptor (PDGFR){beta}+ cells. Kruppel-like factor (KLF)4 plays an important role in regulating the activation of peripheral PDGFR{beta}+ perivascular cells in response to hypoxia/ischemia. Herein, we aimed to characterize the spatiotemporal responses of brain PDGFR{beta}+ cells while assessing the contribution of KLF4. This was achieved using transgenic mice that enable tracking or conditionally depleting KLF4 in PDGFR{beta}+ cells, which were subjected to experimental ischemic stroke. Next, we employed point pattern analysis (PPA) and topological data analysis (TDA) to quantitatively characterize cell phenotypic changes and spatial distribution over injury progression after ischemic stroke. We show that brain PDGFR{beta}+ cells rapidly become reactive and early localize to regions prone to irreversible damage. We report the emergence of parenchymal PDGFR{beta}+ cells, which cannot be causally linked to proliferation or vascular detachment. Moreover, our analysis reveals that KLF4 is barely expressed in brain PDGFR{beta}+ cells under normal conditions, and that its expression is slightly induced in reactive cells in the injured brain. Notably, specific attenuation of KLF4 induced expression in PDGFR{beta}+ cells does not affect cell reactivity and spatiotemporal distribution, nor scar formation and injury severity. These observations suggest that in contrast with the periphery, KLF4 is not implicated in regulating the responses of brain PDGFR{beta}+ cells. Our results indicate that the reactivity of brain PDGFR{beta}+ cells after stroke is spatiotemporally diverse, evolve over injury progression, and is distinct from peripheral perivascular cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/712632v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1149c62org.highwire.dtl.DTLVardef@26edaaorg.highwire.dtl.DTLVardef@1bd3d35org.highwire.dtl.DTLVardef@fd8030_HPS_FORMAT_FIGEXP M_FIG C_FIG

Formaldehyde (FA) is an environmentally abundant and endogenously produced aldehyde that has been shown to cause DNA damage, mutagenesis, and carcinogenesis. Several studies have demonstrated that FA induces guanine mutations resulting in a mutation signature like SBS40. In this work, we demonstrate that replication defects generating single-stranded DNA (ssDNA) caused by the downregulation of the major replicative polymerases results in elevated FA mutagenesis. We found that loss of Mrc1 (CLASPIN) resulted in a high accumulation of ssDNA and FA mutagenesis, and that these phenotypes were not dependent on Mrc1s checkpoint activity. Loss of DNA-protein crosslink repair results in elevated FA sensitivity with no alteration to mutagenesis, likely due to the inability of the fork to bypass unprocessed protein adducts. Finally, we show that FA-induced mutagenesis is dependent on Pol {zeta}-mediated translesion synthesis, while deficiencies in the template switching pathway do not alter error-free bypass of FA adducts. Overall, our work points towards replication-associated ssDNA as a major substrate for FA-induced damage and elucidates the pathways that function to prevent FA mutagenesis at replication forks.

Translation reinitiation (REI) is one of the most important gene-specific regulatory mechanisms by which eukaryotic cells influence expression of main translons, for example during highly conserved integrated stress response (ISR). In S. cerevisiae, expression of the key stress response gene, GCN4, is controlled by an intricate interplay among four short upstream translons (uTranslons, formerly uORFs), resulting in high or low levels of REI at GCN4 depending on the growth conditions. Under nutrient rich conditions, GCN4 expression is repressed, but upon amino acid starvation, it is derepressed, despite of a general translational shut down. Capitalizing on our screening reporter system, we identified three new factors influencing efficiency of REI after translation of GCN4 uTranslons: Rai1p (an RNA quality control and processing factor), and Ssz1p and Zuo1p (members of the Ribosome Associated Complex [RAC]). Importantly, we showed that depletion of these factors deregulated derepression of Gcn4p synthesis under starvation. Furthermore, we found that similar to RAC, Rai1p associates with 40S subunits and actively translating ribosomes. We also explored interactomes of these proteins. Collectively, we present three previously unknown factors that co-regulate stress response to amino acid starvation in the budding yeast by unique mechanisms.

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.

The circadian clock is a highly conserved evolutionary advantage which allows organisms to anticipate regular changes in daily environmental conditions. Clocks from fungi to mammals rely on a transcription-translation feedback loop (TTFL) mechanism. Phosphorylation is understood to be a critical regulatory step for maintaining the period of the circadian clock and feedback loop closure. The role of kinases in the Neurospora clock has been examined extensively; however, phosphatases have not been systematically interrogated. By re-examining the Neurospora genome using current informatic tools we identified the 30 genes previously identified as encoding protein phosphatases as well as 13 novel genes, and we assessed the function of the core circadian clock in 39 non-essential phosphatases using a real-time luciferase reporter. We observed both period lengthening and shortening effects, which are not restricted to a single phosphatase family or fold. All but one deletion mutant maintained a rhythmic core clock. In addition, we observed a new temperature compensation defect in the previously studied knockout of phosphatase pph-4, the result of nutritional growth conditions.

Hepatocytes undergo extensive proliferation to facilitate liver repair after injury, yet early adaptive changes prior to proliferation remain unclear. Here, we report that during early acetaminophen (APAP)-induced liver injury, hepatocytes exhibit transient proliferation suppression, most pronounced in mid-zone hepatocytes due to zonal APAP metabolism. Using spatial transcriptomics (ST), immunohistochemistry, and functional studies, we identified a unique mid-zone stress-response program. Central to this adaptation is the Atf4-Chop axis, which actively suppresses proliferation via the cell cycle inhibitor Btg2, prioritizing cytoprotection over cell division. This transient arrest is a critical survival strategy: halting energy-intensive proliferation during peak injury allows mid-zone hepatocytes to redirect resources towards protection, enhancing their survival in early APAP-induced liver injury. Thus, Atf4-Chop-mediated quiescence preserves a hepatocyte reservoir necessary for subsequent regenerative proliferation and effective repair. Our findings reveal a key adaptive trade-off in mid-zone hepatocytes where transient proliferation arrest promotes early survival to enable repair.

The sigma-1 receptor (S1R) is an endoplasmic reticulum transmembrane protein implicated in a wide range of physiological and pathological processes, including neurodegeneration, cancer, and pain modulation. Although X-ray crystallography has revealed S1R as a trimeric assembly with a distinctive triangular architecture, the dynamic behavior of this oligomeric state and its modulation by ligands and membrane composition remain poorly understood. In particular, agonists and antagonists have been experimentally proved to differentially regulate S1R oligomerization although the underlying molecular mechanisms are still obscure. Here, we present the first atomistic molecular dynamics study of trimeric S1R embedded in a physiologically relevant lipid environment. Using a total of 12 {micro}s of simulation time, we investigate the impact of membrane composition, with a specific focus on cholesterol, as well as the conformational response of S1R to pharmacologically distinct ligands: the agonist (+)-pentazocine and the antagonist haloperidol. Our simulations reveal how ligands can alter S1R interprotomer interaction through a mechanism involving the {beta}6-strand of the protein and in particular W136, data that correlate with experimentally observed differences in S1R oligomerization. These findings provide new molecular-level insights into S1R regulation and establish a framework for rationalizing the distinct functional outcomes induced by agonists and antagonists.

Advances in NASAs astrobiology program have demonstrated the feasibility of cultivating plants in space and in analog extraterrestrial habitats. In addition to abiotic stressors, plants grown in terrestrial and space-like environments are challenged by both phytopathogens and opportunistic human pathogens, with implications for plant productivity and human health. The persistence of human-associated pathogens in spacecraft and space stations raises significant concerns regarding food safety. The molecular, biochemical, and signaling mechanisms governing stomatal development and function under microgravity remain poorly understood. We employed an experimental system incorporating human pathogen Salmonella enterica and lettuce microgreens exposed to simulated microgravity through two-dimensional clinorotation to investigate plant innate immunity and stomatal development and function. We further evaluated four lettuce cultivars to determine whether genetic variation impacts these factors under simulated microgravity conditions. Our findings indicate that simulated microgravity significantly influences stomatal development and function, as evidenced by an increase in stomatal density and variable changes to stomatal aperture. Notably, cultivar-dependent variation in stomatal traits and responses to Salmonella enterica was observed under microgravity conditions. Although increased stomatal density was hypothesized to enhance pathogen ingression, internalization was more strongly predicted by cultivar selection and simulated microgravity; simulated microgravity increased ingression, with red pigmented cultivars having less pathogen than green cultivars. These results suggest that targeted selection of cultivars with favorable physiological traits may improve food safety and the viability of crop production systems in space environments. They also suggest that development and function of stomata may change in spaceflight conditions.

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.

RNA helicases encoded by positive-strand RNA viruses are essential for genome replication, yet the specific biological functions and mechanochemical basis underlying these functions remain poorly defined. Progress has been limited by the difficulty of resolving individual catalytic steps under single-turnover conditions, which are often experimentally inaccessible for viral enzymes. Alphaviruses replicate within membrane-bound spherules that may alter local metabolite concentrations, raising the possibility that the enzymatic properties of alphaviral proteins differ from those of viruses with greater cytosolic exposure. Here, we present a kinetic and binding analysis of full-length non-structural protein 2 (nsP2) from Chikungunya virus, a multifunctional superfamily 1B NTPase and RNA helicase. Purified nsP2 binds nucleoside triphosphates with high affinity, exhibiting equilibrium dissociation constants in the single digit micromolar range. This property enabled single-turnover, pre-steady-state, and isotope-trapping experiments that are rarely feasible for viral helicases. These analyses identified two sequential conformational-change steps required for nucleotide hydrolysis. Molecular dynamics simulations suggest tightening of the RecA1 and RecA2 domains upon ATP binding followed by compaction of the enzyme mediated by interactions between the 1B subdomain and RecA2 domain. Product inhibition patterns support random release of ADP and inorganic phosphate, with relative binding affinities indicating that ADP dissociates first. The reaction is irreversible. Although nsP2 binds RNA tightly, strand separation under single-turnover conditions is too slow to represent ATP-driven unwinding, instead likely reflecting formation of an unwinding-competent nsP2-RNA complex. Together, these findings establish a quantitative framework for nsP2 function and provide a roadmap for mechanistic studies of alphaviral helicases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/723793v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@13899a1org.highwire.dtl.DTLVardef@ee1aadorg.highwire.dtl.DTLVardef@1991e1org.highwire.dtl.DTLVardef@b877f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

The hippocampus is essential for memory consolidation, a process mediated by high-frequency oscillations known as ripples during non-rapid eye movement (NREM) sleep. Ramelteon, a selective MT1/MT2 receptor agonist, has been reported to possess cognitive-enhancing properties; however, its impact on the fine-scale dynamics of hippocampal ripples remains unclear. We performed chronic local field potential recordings from the dorsal hippocampus and prefrontal cortex in mice. Following the intraperitoneal administration of either vehicle or ramelteon, we evaluated sleep architecture and characterized ripple properties, including occurrence rate, amplitude, instantaneous frequency, and duration during NREM sleep. Ramelteon administration significantly increased NREM sleep occupancy. Notably, we found that ramelteon significantly enhanced both the occurrence rate and amplitude of hippocampal ripples compared to the control group. While a slight increase in intra-ripple frequency was observed, other structural features, such as ripple duration and asymmetry index, remained unaffected. Our findings demonstrate that ramelteon facilitates hippocampal ripple dynamics by increasing their occurrence and synchrony during NREM sleep. Given the critical role of ripples in memory consolidation, these neurophysiological changes may underlie the procognitive effects of ramelteon. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=55 SRC="FIGDIR/small/723673v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@c798c7org.highwire.dtl.DTLVardef@1ff616eorg.highwire.dtl.DTLVardef@1557dc8org.highwire.dtl.DTLVardef@1b4e89e_HPS_FORMAT_FIGEXP M_FIG C_FIG