Journal of Biosciences

The IFN-I (type I interferon) signaling pathway is the first line of defence against foreign pathogens. Stringent control of signalling pathways is necessary to maintain host immune responses and homeostasis. However, the underlying mechanism for its tight regulation is yet completely understood. In this study, we demonstrated that the TRIM family protein tripartite motif-containing 52 (TRIM52) is a novel negative regulator of IFN-{beta} production. Ectopically expressed TRIM52 markedly inhibited the activation of the IFN-{beta} promoter by ectopic expression of cGAS/STING, RIG-IN, or TRIF, MAVS, STING, and TBK1 but not by IRF3/5D, indicating that TRIM52 targets TBK1. TRIM52 also significantly inhibited the IFN-{beta}, ISG54, and ISG56 production, the dimerization of IRF3 and the nuclear localization of IRF3-YFP induced by ectopic expression of TBK1. Co-immunoprecipitation experiment revealed that TRIM52 specifically interacted with TBK1. Furthermore, the TBK1 protein, but not its mRNA, decreased considerably with increasing expression of TRIM52, and TRIM52 did not decrease the expression of the cGAS, STING, or IRF3 proteins. In addition, proteasome inhibitor MG-132 blocked the reduced TBK1 induced by TRIM52, indicating that TRIM52 caused TBK1 degradation via the proteasome pathway. Co-IP and ubiquitination assays demonstrated that TRIM52 promotion of K48-linked ubiquitination of TBK1, which depends on its E3 ubiquitin ligase. Collectively, our findings identify a previously unrecognized role of TRIM52 in regulating the IFN-I signalling pathway through targeting TBK1 for polyubiquitination and degradation.

Lymphocyte-extracellular matrix (ECM) interactions occur intermittently throughout the lymphocytes life cycle. Alterations in blood insulin levels following feeding modulates naive lymphocyte trafficking and adhesion to fibronectin via a pathway involving insulin-like growth factor-1 receptor (IGF-1R), phospholipase C gamma 1 (PLC-{gamma}1) and {beta}2 integrin activation. Lymphocytes exert traction forces, on the ECM during the process of extravasation. While these forces are essential for several homeostatic processes, the role of insulin in modulating lymphocyte-derived traction forces upon ECM adhesion is unknown. The aim of the current study was to investigate the effect of insulin on the traction generated by lymphocytes when adhered onto a fibronectin-coated substrate. Jurkat T-cells were placed on a fibronectin layer (50{micro}g/ml, 100{micro}m thickness) coated on polyacrylamide gels of stiffness 400Pa with red fluorescence beads as fiduciary markers. The cellular force generated by Jurkat T-cells was mapped using traction force microscopy. To elucidate the role of PLC-{gamma}1 in cellular force generation, the traction of Jurkat T-cells lacking PLC-{gamma}1, as well as those of a knockout cell where PLC-{gamma}1 was restored were quantified and compared with wild-type Jurkat T-cells. Lack of PLC-{gamma}1 attenuated adhesion when compared to wild-type Jurkat T-cells. Additionally, the traction force generated by each cell type decreased with increasing concentration of extracellular calcium. Treatment of adherent Jurkat T-cells with insulin increased traction in lower extracellular calcium condition while a dip was observed when a high extracellular calcium was present, in comparison to the untreated cells. However, the effect of insulin treatment was lost in the case of Jurkat T-cells lacking PLC-{gamma}1. Together these results indicate that insulin regulates traction force generated by adherent Jurkat T-cells via a process involving PLC-{gamma}1, in a calcium dependent manner.

Hypoxia and anoxia are known to suppress cell proliferation due to an increase in replication stress and activation of DNA damage checkpoints. Embryos of the annual killifish Austrofundulus limnaeus show a strong tolerance to extended anoxic exposure, indicating an improved genomic stability under oxygen starvation. Here we investigate the cell cycle regulation of the anoxia tolerant killifish embryonic cell line PSU-AL-WS40NE during anoxic exposure. Live cell imaging confirms continued cell proliferation of WS40NE cells for the first 24 hours of anoxic exposure with minimal cell death. Fluorescent imaging shows that cells begin to accumulate in G1 after the first day in anoxia with a pronounced and rapid entry into the S phase upon reoxygenation. Pharmacological inhibition tests show that this response appears to be reliant more on ATR signaling then ATM, suggesting that increased {gamma}H2AX levels are driven by increased replication stress instead of DNA damage. This conclusion is further supported by an apparent lack of induction of a G2 checkpoint in these cells suggesting that DNA damage during anoxic replication is minimal. Maintaining cellular proliferation during initial exposure to anoxia and accumulating cells in the G1 phase for extended anoxic exposure is likely one way that embryos of the annual killifish are able to survive prolonged anoxia and provides insight into mechanisms that enable cells to proliferate under metabolic stress.

Predicting protein-ligand interactions in the modern drug discovery has revolved from the involvement of artificial intelligence and structural bioinformatics using Graph Neural Networks (GNNs). The limited explainability of GNN models presents an important encumbrance in biomedical research, but it has achieved a high degree of accuracy in determining and identifying binding affinity and active compounds, as evidenced by [1] [2] [3] [4]. Here this research focuses on the interpretation of protein-ligand interactions at a molecular level, a rapidly developing area within Graph Neural Networks (GNNs). Now days modern study handling techniques such as visualization techniques, attention mechanism and model-based feature ascription by model to boost, and make robust and decrease false predictions on binding. Along with some approaches include like graph pooling strategies, message-passing optimization, self-supervised learning, transfer learning and contrastive learning are rapidly utilized to enhance the representative learnings. Furthermore, integration of molecular docking simulations, hybrid deep learning architectures and protein language model gives more reliable & biological predictions of protein-ligand interactions. That focuses on given process that identifies key ligand atoms and binding residues, as well as physicochemical factors influencing affinity, through chemical thought processes. Here this research work identified the challenges of developing biologically significant explanations, transparency, and the corollary dataset biases on interpretability. The research work conducted an in-depth investigation into the consolidation of protein language models to establish more reliable pathways for future research, examining hybrid architectures, transparent and energy-efficient GNNs, and scientifically grounded AI models for drug discovery. My research work highlights that XGNNs establishes a connection between Deep Learning and Biochemical expertise with increased confidence, which will enhance the accuracy of predictive models and computational models.

LIM kinases, LIMK1 and LIMK2, play a crucial role in cytoskeleton dynamics. They are involved in many physiological processes but also in several pathologies such as cancer, neuronal diseases and neurofibromatosis. Although LIM kinases appear as promising therapeutic targets, they remain undruggable. A better understanding of their activity and regulation is thus required to better design efficient targeted therapies. Here, we have shown the impact of a single amino acid on LIMK activity on cofilin, their main substrate in actin filament remodelling. We demonstrated that Y632 and Y630, for LIMK1 and LIMK2 respectively, mediate LIMK dimerization, resulting in their transphosphorylation. This process seems to be a prerequisite for their canonical phosphorylation on their respective T508 and T505 residues within the activation loop. These Tyrosine are not phosphorylated, their aromatic nature is rather critical to ensure proper LIMK activity on cofilin. These results bring new insights into LIMK molecular features.

Small molecule modifiers whence bound, allosterically, will alter the binding of a macromolecule to one- or more-cognate substrates/partners via conformational and non-conformational changes. Although allostery is inferred directly from empirical data, the mathematical basis of these models, constraints deployed and choice of parameter(s) are not clear. Here, we present and characterize a discrete-to-continuous mathematical model for ensemble distributions of a ligand-interacting macromolecular species across milieux-dependent conformational states and examine its role in the genesis and progression of cooperative binding. The premise, of our model, is a set of occupancy matrices (sparse, binary, strictly delocalized) which can be partitioned by a probability-based hyperparameter into mutually exclusive proper subsets of occupancy matrices with identical multinomial probabilities. Since each subset is canonical with a constituent occupancy matrix, it is characterized by a unique multinomial probability. The inner product of combinatorial pairs of all mutually exclusive subsets of occupancy matrices, with an expression for the summed transitional probabilities (finite differences between unique multinomial probabilities), is the differentiable matrix of strictly positive real-valued numbers for the system of ensemble distributions. Whilst the harmonic mean is presented as a generic solution for a system of ensemble distributions, the row-wise definite integral for each column is the finite union of open intervals (contiguous, strictly monotone) which in tandem with a set of interval-specific and bounded transitional probabilities constitutes a piecewise smooth curve (path-connected-, closed- and compact-set). Our discrete-to-continuous model is phenomenological and able to recapitulate the basic tenets of cooperative binding whilst offering insights into the genesis and progression of the same.

How cells balance growth (cell size) and division (cell number) requires a complex interplay between response to external signals including growth factors, nutrient availability and metabolic cues, along with regulation of the cell cycle. The protein Tuberin (gene TSC2) is a critical regulator of these decisions. In a complex with the protein Hamartin, Tuberin functions as a negative regulator of the Target of Rapamycin (mTOR) pathway, preventing excessive growth under unfavorable conditions. However, how this growth pathway connects to decisions to progress through the G2 phase of the cell cycle and permit cell division is still unclear. In this study, we show that post-translational modification of Tuberin by the Extracellular Signal-Regulated Kinase (ERK) pathway abrogates binding between Tuberin and the mitotic cyclin, Cyclin B1. This causes an increase in mitotic cells, due to an unregulated G2/M transition, increasing the proliferation rate. Our work shows a novel role of Tuberin in cell cycle regulation by growth and mitogenic factors independent of mTOR regulation.

Crown-of-thorns sea star (CoTS), Acanthaster cf. solaris, outbreaks are a major cause of hard coral cover decline across the west Pacific, threatening coral reefs. Coral taxa vary in susceptibility to CoTS predation from preferred (Acropora spp.) to non-preferred (Porites spp.), yet the mechanisms underlying these differences are poorly understood. We investigated coral defenses during an ongoing CoTS outbreak in Mo'orea, French Polynesia by examining gene expression (including putative toxin genes) in healthy and actively predated colonies of a preferred (Acropora hyacinthus) and a non-preferred (Porites sp.) coral prey species. During predation, A. hyacinthus exhibited molecular signatures of cellular stress responses involving oxidative stress signalling, inflammation, and tissue proteolysis. In contrast, Porites sp. showed enrichment of genes involved in mitochondrial metabolic adjustment and aerobic metabolism, suggesting metabolic compensation to maintain cellular function. Furthermore, A. hyacinthus demonstrated a reactive defense behaviour by differentially expressing toxins (e.g., kunitz-type neurotoxins) while Porites sp. employed constitutive expression of all putative toxins regardless of active predation, suggesting a proactive defense strategy. Together, these findings suggest that preferred and non-preferred coral prey exhibit fundamentally different molecular and defensive strategies during CoTS predation, shedding light on the evolutionary arms race between corals and their predators.

Ergosterol has multiple functions in filamentous fungi and yeasts, although only a part of the functions seems to be understood. An antifungal peptide, theonellamide A (TNM-A) induces drastic morphological changes in fission yeast cells by targeting plasma membrane ergosterol. TNM-A induces overproduction and ectopic accumulation of cell wall glucan at both growing tips and septum through a yet unknown mechanism. Here we show that TNM-A treatment causes accumulation of 1,3-{beta}-glucan at cell-polarity sites, not by increased activity of 1,3-{beta}-glucan synthase, but by an increased, persistent localization of the glucan synthase enzymes. Screening based on subcellular localization of proteins at periphery or polarity sites suggested the involvement of the Rho family GTPase Cdc42. In agreement, TNM-A induced both activation of Cdc42 and enhancement of membrane trafficking of glucan synthase enzymes. In conclusion, our chemical genetics analyses using TNM-A suggest that membrane ergosterol regulates the activity of Cdc42, which further regulates the localization of glucan synthases and cell wall biosynthesis. Highlights (four sentences)- Thenoellamide A (TNM-A) induces an ectopic overproduction of cell wall glucan. - TNM-A treatment causes increased, persistent localization of glucan synthases at the cell tips and septum. - TNM-A activates Cdc42 and upregulates membrane trafficking of glucan synthases. - Ergosterol is involved in proper activation/inactivation of Cdc42.

Behcets disease (BD) is a systemic inflammatory disease. It is considered as an autoinflammatory disease triggered by innate immunity rather than adaptive immunity. Human leukocyte antigen-B51 (HLA-B51) is the strongest genetic factor associated with BD. This study investigated how HLA class 1 molecules interact with innate immune cells and induce cytokine secretion. For this purpose, 293T cells transfected with a plasmid encoding HLA-B51 were cultured with natural killer (NK) cells obtained from healthy human donors. Within 24 h, the concentrations of interleukin-4 (IL-4), IL-8, and interferon-{gamma} (IFN-{gamma}) in the medium increased, indicating that NK cells secreted cytokines without undergoing cellular expansion for cytolysis. NK cells stimulated by nonself HLA-B51 produced IFN-{gamma} levels comparable to those produced by NK cells stimulated by self HLA-B51. NK cells carrying HLA-B51 were accurately recognized by overexpressing HLA-B51 on 293T cells. Moreover, ample intracellular IFN-{gamma} levels were detected in NK cells after stimulation with phorbol 12-myristate-13-acetate (PMA) plus ionomycin. KLRK1 (CD314)-positive cells mainly primarily accounted for IFN-{gamma}-producing cells, whereas KLRK1-negative cells did not. In contrast, both NCR1 (CD335)-positive and -negative cells contributed to IFN-{gamma} production. We next investigated whether HLA-B51 on the surface of 293T cells stimulates KLRK1 as a ligand causing IFN-{gamma} secretion. In masking experiments using anti-KLRK1 antibodies, NK cells with high levels of cell surface KLRK1 decreased the production of IFN-{gamma}. Conversely, human NK cell line KHYG1 cells also produced IFN-{gamma} in culture with 293T cells, but did not increase IFN-{gamma} through HLA-B51 stimulation. The mRNA expression of the signal adaptor protein HCST (DAP10) in KHYG1 cells was lower than that in NK cells, whereas the relative expression of IL-2RA in KHYG1 cells was higher than that in NK cells. These findings suggest that HLA-B51 can interact with KLRK1 on the NK cells inducing IFN-{gamma} secretion, whereas IL-2 signals outweigh HLA-51 stimulation in KHYG1 cells.

Fv-HSP72 is a rapid cell-penetrating human heat shock protein for the treatment of traumatic organ injuries. We have shown this re-engineered protein (HSP72) is capable of crossing the blood brain barrier (BBB) of rats suffering a controlled cortical impact (CCI) and remains in brain tissue for up to 12 hours; long after clearance from the cortex of uninjured rats. Peptide sequences unique to Fv-HSP72 allow for its differential detection from endogenous HSP72. Male Sprague-Dawley rats were divided into 10 groups of n=10 with those animals receiving a CCI subjected to a unilateral cortical contusion simulating a moderate to severe brain injury using an electronically controlled pneumatic impact device. Control groups were either uninjured (Sham), injured (TBI Only), or injured and given buffer (TBI+Vehicle). Rats treated with one of three Fv-HSP72 variants were dosed at 10 or 30mg/kg 15m post-impact, then sacrificed 48 hours later. Cortical tissues were extracted from the ipsilateral and contralateral hemispheres for biomarker analysis. Here we report results of our drug inhibiting neurodegeneration based on five biomarkers (NF-L, pNF-H, pTau [T181, T231, S396]). These results were statistically significant, especially for one of the Fv-HSP72 variants, when comparing differences both between treatment groups and within groups (i.e. when comparing ipsi-vs. contralateral hemispheres). Significant inhibition of oxidative stress (3-NT) and inflammatory (IL-6) biomarkers were also observed (both p<0.0001). With similar results obtained for a blast injury model being published elsewhere, the analyses suggest Fv-HSP72 is neuroprotective following a direct impact brain injury. One sentence summaryThis study describes the effectiveness of a biologic agent, Fv-HSP72, in significantly inhibiting neuronal tissue damage in the brain when administered after a direct cortical impact.

Cerebral Palsy (CP) is the most common motor disability in childhood, and the most frequent comorbidity is pain. Rabbit kits subjected to prenatal hypoxia-ischemia (HI) exhibit allodynia and an expansion of nociceptive afferents in the lumbar spinal cord at postnatal day (P5). In this study, we examined how HI alters the development of multiple sensory modalities and its effect on psychosocial measures and C-fiber distribution in the spinal cord. To do this, we performed an HI surgery to occlude blood flow to fetal New Zealand White rabbits for 40 minutes, or a sham surgery. We performed von Frey, Hargreaves, and a cold allodynia test at P1, P5, P11, and P18. Additionally, we performed open field, a two-texture preference test, and immunofluorescence assays at P18. HI kits exhibit altered development and allodynia in von Frey and Hargreaves and minor decreased sensitivity in cold allodynia. HI kits spend less time on the aversive side of the two-texture preference apparatus and more time in the center of an open field but a higher ratio of that time immobile. This is accompanied by changes in the distribution of C-fibers in the dorsal horn of the cervical and lumbar spinal cord. A principal components analysis revealed altered nociception and psychosocial changes are important for differentiating between control and HI kits but not distribution of C-fibers. Overall, HI rabbits kits exhibit altered sensory development, allodynia, anxiety-like behavior, and changes to the distribution of nociceptive afferents in the dorsal horn of the spinal cord.

The heterotrimeric G-protein transducin (Gt) is among the key proteins mediating phototransduction in rod and cone cells of the vertebrate retina. Even though this protein has been extensively characterized in mammals, little is known about its expression patterns in migratory songbirds. Here we characterised Gt expression in the European robin, a night-migratory songbird known for its light-dependent magnetoreception. The mechanism underlying magnetoreception is not fully understood, but one well-supported hypothesis involves a radical-pair formation in the blue light receptor cryptochrome type 4a. The - and {gamma}-subunits of cone specific transducin have been identified as possible interaction partners of cryptochrome 4a. Therefore, we analysed the expression patterns of various G-protein subunits in bird photoreceptors. Specifically, we combined single cell RNA sequencing and immunohistochemistry, and tested for protein interaction by pulldown, co-immunoprecipitation, and NanoBiT luminescence assays. We show that genes for G-protein subunits GNB1 and GNB3 (coding for Gt{beta}1 and Gt{beta}3, respectively) are predominantly expressed in rods and cones. Among {gamma}-subunits, GNGT2 (coding for Gt{gamma}T2) was the principal isoform in cones, whereas GNG11 (coding for Gt{gamma}11) was associated with rods. In contrast, we did not detect GNG10 (coding for Gt{gamma}10) expression in either photoreceptor type. Interaction assays demonstrated that all three {beta}{gamma} combinations; {beta}{gamma}T2, {beta}{gamma}10, and {beta}{gamma}11, can associate in vitro. These findings indicate that {beta}{gamma} dimer formation in vivo is likely constrained by the photoreceptor-specific expression of the respective subunits. Furthermore, the absence of GNG10 expression in rods and cones does not support a role of this {gamma}-subunit in photoreceptor-based magnetoreception.

Nucleobindin 1 (NUCB1) is a multifunctional conserved protein located in Golgi luminal, nucleus, extracellular and cytosolic pools. NUCB1 is multidomain protein comprised of a signal peptide, a DNA-binding domain, a leucine zipper and Ca2+ -binding domain. The multiple domains and localization of NUCB1 potentiates its interactions with various partners, such as DNA, Gi3 protein, cyclooxygenase 2, LRP10 and RNA suggests its importance in the regulation of many cellular events. We revealed that NUCB1 contains three RNA-binding regions and able to interact with two RNA fragments. It was suggested possible variants of the participation of NUCB1 in the interaction of the two partially complementary RNAs. The RNA-binding properties of the NUCB1 were also confirmed in vivo experiments.

B-cell leukemia/lymphoma 11B (BCL11B), despite its name, is a key regulator of T-cell development, specification, and T-cell malignancies. BCL11B contains a bipartite DNA binding domain composed of two C2H2 zinc finger arrays: low-affinity ZF2-3 and high affinity ZF4-6. These arrays function as homotypic modules that recognize similar six-nucleotide motifs, TG(O_SCPLOWNC_SCPLOW)CC(O_SCPLOWCC_SCPLOWO_SCPCAP/C_SCPCAPO_SCPLOWTC_SCPLOWO_SCPCAP/C_SCPCAPO_SCPLOWAC_SCPLOW), as seven of the eight DNA base-contacting residues are conserved between them. The most conserved interactions involve GG dinucleotides, contacted by arginine and lysine residues at key base-interacting positions in ZF3 and ZF5. The two ZF arrays are connected by a long [~]300-residue linker that provides flexibility in how the arrays engage DNA, allowing ZF2-3 and ZF4-6 binding to the same or opposite strands with variable orientation, spacing and positioning along the DNA. This extended linker is enriched in serine/threonine, acidic residues (aspartate/glutamate), and structural residues (glycine/proline), providing additional layers of transcriptional regulation possibly through post-translational modification, electrostatic modulation, and/or condensate formation. We also examined six missense mutations in base-interacting residues, that are associated with neurodevelopmental disorders. Substitutions replacing bulky, positively charged arginine or lysine with smaller or hydrophobic residues likely reduce DNA-binding affinity and/or specificity, whereas substitutions between asparagine and lysine may alter base recognition preferences.

Microtubules are cytoskeletal structures composed of polymers of /{beta}-tubulin heterodimers. They play a central role in cell division and motility by a stochastic process of alternating polymerization and depolymerization episodes (dynamic instability) that can be modulated by phosphorylation. Protein kinase C and cyclin-dependent kinase 1 are known to phosphorylate Ser165 of -tubulin (:Ser165) and Ser172 of {beta}-tubulin, ({beta}:Ser172), respectively. Using all-atom molecular dynamics simulations of 6-mer {beta}-tubulin systems modeled on the cryo-EM structure of a microtubule (PDB 3J6E), the impact of phosphorylation at each site is explored in terms of secondary structures (:helix H8/loop T7 segment and {beta}:loops T3/T5) that lie at the inter-dimer cleft near the E-site {beta}:GTP. If properly aligned, :Glu254 (helix H8) hydrolyzes {beta}:GTP to GDP thereby triggering the transition from a polymerizing to a depolymerizing microtubule. -Tubulin phosphorylated at :Ser165 displaces helix H8 (:Glu254/:Gln256) and loop T5 towards the {gamma}-phosphate of {beta}:GTP. This movement coincides with a shift of the {beta}:GTP nucleotide by 4.5-5.5 [A], stabilization of the {gamma}P of {beta}:GTP by additional H-bonding and weakened inter-dimer interactions. In a phosphorylated {beta}:Ser172 system, loop T5 is displaced toward {beta}:GTP and coincides with stabilization of inter-dimer interactions. Therefore, phosphorylation of either - or {beta}-tubulin generates a distinct profile of intramolecular rearrangements that remodel the inter-dimer cleft and modulate dynamic instability. These profiles may provide a useful reference for screening mutations identified in tumor genomes.

Understanding the drivers of biodiversity in the worlds rivers, which are known to be hyperdiverse relative to their coverage area, has been an enduring goal in ecology. While regression-based empirical studies have identified a suite of environmental factors that are correlated with riverine fish biodiversity, these insights are often system-specific and inconsistent across regions. In contrast, a more limited body of studies have suggested that network connectivity of rivers affects fish biodiversity by limiting dispersal, and basin shape may modulate these relationships. The few theoretical papers that have explored basin morphology effects have tended to use extreme network shapes and inconsistent methods, thus limiting general insights. Here, we build on these results to demonstrate that river basin morphology, as measured by log aspect ratio, can alter both network connectivity and biodiversity in simulated, all-else-equal scenarios. First, we quantify variation in log aspect ratio across the worlds 100 largest rivers and use this empirical range of shape variation to guide synthetic experiments. In particular, we use Optimal Channel Networks (OCNs) constrained to basins with log aspect ratios within realistic range to study how shape alters connectivity profiles when node number and basin area are held constant. By coupling OCNs with dendritic neutral models, we demonstrate that variation in aspect ratio and concomitant changes in connectivity lead to substantial changes in simulated biodiversity. Finally, we use Earth Movers Distance to establish that basin-shape-induced changes in node-level connectivity distributions are predictive of transformations in node-level distributions of and {beta} diversity. Overall, elongated basins such as the Mekong River feature lower species richness (-diversity), higher turnover ({beta}-diversity), and less variable distributions of both quantities relative to a square reference basin. Furthermore, approximately one third of the worlds largest rivers are elongated enough to potentially feature statistically-detectable, shape-mediated variation in connectivity and biodiversity.

Dengue virus (DENV) infection generates a significant health burden throughout the world, and there are no clinically approved antiviral drugs, as of now. The virus also depends on lipid metabolism in the host to conduct effective replication and this makes lipid-directed compounds promising as therapeutic options. We assessed the antiviral effect of lauric acid, a 12-carbon medium-chain fatty acid, against DENV serotype 2 (DV2) in the presence of a stable cell line, DV2-replicon, expressing all the non-structural proteins (NS1-NS5) and a luciferase reporter. Active viral replication in replicon cells was established by morphological examination and immunofluorescence of cells. The MTT assay was used to determine the cytotoxicity of lauric acid revealing the LD50 of 2.52 uM, so higher concentrations were toxic as the effect of the drug is dose-related. The antiviral effect was tested through replicon inhibition (luciferase) assay which showed an incredible inhibition of viral RNA replication with a IC50 of 1.70 uM and this is equivalent to antiviral mycophenolphycic acid. The cytopathic effects, as well as a decrease in the activity of luciferase, proved the presence of viral translation and replication inhibition within the process of the treatment of the lauric acid. These results propose that lauric acid has cytotoxic and antiviral dual effect and can be a possible inhibitor of DENV replication. The toxicity needs to be reduced and future research is necessary to explain its molecular pathway and also to come up with the best delivery methods. IMPORTANCEDengue virus (DENV) remains a significant health challenge to the world since there are no effective antiviral agents. This work will recognize lauric acid as a possible dengue virus replication inhibitor in a model of a DV2 replicon, exhibiting antiviral action that is similar to that of mycophenolic acid. These results support lipid-directed compounds as potential dengue antiviral targets, but more research is needed to minimize toxicity and better understand the molecular mechanism of action.

O_LIIn this study, we demonstrate that Benzo[a]pyrene (B[a]P) induces keratinocyte senescence and p21Cip1-dependent keratinocyte differentiation. Atmospheric and environmental pollution are known to induce senescence and promote terminal differentiation in human primary keratinocytes, thus driving skin aging. However, much is still unknown about the underlying molecular mechanisms. We observed that B[a]P, a common atmospheric pollutant, induced senescence in primary keratinocytes in both two-dimensional and three-dimensional (reconstructed human epidermis) culture. This was accompanied by signs of DNA damage in B[a]P-treated cells. B[a]P-treated cells also underwent accelerated late-stage terminal differentiation, indicated by increased IVL and FLG expression from 48 to 96 hours post-exposure. While pharmacological and genetic attenuation of p21Cip1 did not rescue cellular senescence, it prevented the expression of IVL and FLG, suggesting that the late-stage terminal differentiation induced by B[a]P exposure was p21-dependent. Our data thus suggest a key role for the p21Cip1 in the keratinocyte response to pollution-induced damage, where p21Cip1 induces terminal differentiation to maintain skin barrier homeostasis. C_LI

Cholera, caused by Vibrio cholerae, continues to pose a serious global public health challenge, with its impact worsened by rising antibiotic resistance associated with bacterial biofilm formation. This study reveals the role of the hypothetical protein (HP) TYC33605.1 in cyclic-di-GMP (c-di-GMP)-mediated biofilm regulation and identifies natural inhibitors that disrupt this mechanism. Functional annotation revealed TYC33605.1 as a membrane-associated diguanylate cyclase (DGC) with GGDEF and sensory domains, critical for c-di-GMP synthesis and biofilm persistence. Homology modelling and molecular dynamics (MD) simulations validated its stable 3D structure (C-score: -1.22, Ramachandran favoured regions: 91.1%) and dynamic behaviour (average RMSD: 8.55 [A]). Virtual molecular docking screening of 1,092 natural compounds identified Luteolin (CID 5280445) and Sativanone (CID 13886678) as top candidates, exhibiting strong binding affinities (-9.1 and -9.0 kcal/mol, respectively) and forming hydrogen bonds, {pi}-cation, and hydrophobic interactions with key residues (Glu293, Arg364, Ala176). MD simulations (100 ns) confirmed complex stability, with Luteolin and Sativanone showing lower RMSD fluctuations (7.78 [A] and 8.13 [A]) compared to the control and apoprotein. The ADME/Tox profiles highlighted favourable pharmacodynamics (PD), pharmacokinetics (PK), high gastrointestinal absorption, no hepatotoxicity, and drug-likeness (Lipinski compliance). Principal component, probability density function, and binding free energy analyses underscore ligand-induced conformational stability. This study proposes the molecular characterisation of the HP and the bioactive compounds Luteolin and Sativanone as promising inhibitors targeting TYC33605.1, offering a novel strategy to combat biofilm-mediated antibiotic resistance and a framework for analogous antimicrobial discovery in Vibrio cholerae.