Journal of Biosciences

The methyltransferase EZH2 (Enhancer of Zest Homolog 2) and the demethylase KDM6B (Lysine Demethylase 6B) have been associated with epithelial to mesenchymal transition (EMT) and poor prognosis in various cancers. These enzymes methylate and demethylate H3K27me3 and regulate distinct sets of genes controlling EMT induction, despite having opposite catalytic activities. This could be due to their recruitment or the modulation of their activity by partner proteins on specific loci. This work sought to identify proteins associated with chromatin and interacting with EZH2 or with KDM6B during EMT. To do so, co-immunoprecipitation and mass spectroscopy was used under TGF{beta} (Tumor growth factor {beta}) and TNF (Tumor necrosis factor ) treatment to induce EMT in A549 lung cancer cells. Surprisingly, numerous proteins related to focal adhesions were identified to interact with EZH2 or KDM6B. These proteins are part of a nuclear protein interaction network previously described as nucleo-adhesome. Among these proteins, TGFB1I1 (transforming growth factor induced peptide 1) and CSRP2 (cysteine and glycine rich protein 2) were further confirmed to interact with KDM6B in the nucleus and even more so during EMT. The target genes of these complexes were then sought by knocking down KDM6B, TGFB1I1 or CSRP2. Three genes (coding Integrin alpha 5, Laminin y2 and Matrix Metalloproteinase 9) were confirmed to be regulated by KDM6B, TGFB1I1 and CSRP2. These findings may have clinical relevance, as immunohistochemistry analyses performed on a cohort of lung cancer patients revealed increased nuclear localization of TGFB1I1 and CSRP2 in cells undergoing EMT.

Background: Brain banks usually provide small tissue blocks fixed by immersion in neutral-buffered formalin (NBF). While still underexploited for research, gross anatomy laboratories could provide full brains fixed by perfusion with solutions better suited for gross anatomy dissection. However, the chemicals in these solutions might have a different impact on histology protocols for cell quantification than in NBF-fixed brains. The main goal of this study is to compare the effects on the number and size of labeled neurons of the primary motor cortex (PMC) of mouse brains fixed with three different solutions: (1) NBF, typical of brain banks, (2) a saturated salt solution (SSS), and (3) an alcohol-formaldehyde solution (AFS), both used in human anatomy laboratories. Methods: 27 C57BL/6J mouse brains were perfused with the NBF (N=9), SSS (N=9) or AFS (N=9), then cut in 40-m slices and processed with immunohistochemistry to target neurons. Various quantitative variables were assessed manually and automatically on photomicrographs of 3 regions of interest (ROIs) of the PMC per specimen, namely the total and individual neuronal profile areas, number and diameters. The effects of the three fixatives on these variables were compared using ANOVA or Kruskal-Wallis, depending on the distribution. For measures on individual cells, a generalized linear mixed model was applied. Dice coefficients and correlations were applied to evaluate the agreement of the manual and automatic methods. Results: There was no significant difference between the brains fixed by the three fixatives for the total and individual cell areas, the total cell count and the cell diameters. The values obtained from manual and automatic measures had an overall good agreement (Dice coefficients > 0.79). Conclusion: It was found that the SSS and AFS had similar impacts on the quantitative variables in the tissue as the NBF. These results are promising for neuroscientists interested in using brains from anatomy laboratories for quantitative research on neurons from the PMC.

Exposure to chemical cues released by predator or pathogen can evoke anxiety or fear responses in prey/host animals such as fight, flight or freeze both at behavioral and molecular levels. Freezing is a fundamental anxiety response when fighting or fleeing arent feasible. Despite the potential relevance of freezing as a stress-coping mechanism, its behavioral and molecular underpinnings are not understood yet. At molecular level danger cues are perceived by chemosensory receptors expressed in sensory neurons which may further regulate the animals behavioral responses(Ye et al., 2024){Citation}. 2-nonanone (2-NA) is one of the principal volatile organic compounds secreted by many pathogenic bacteria infecting Caenorhabditis elegans as well as humans and may signal danger to worms. Here, we show that olfactory exposure to threat-associated cue 2-NA induces a reversible fear-like freezing response characterized by immobility and halted feeding in C. elegans. With the application of in silico and behavioral approaches we showed that 2-NA is one of the ligands for an olfactory G-protein Coupled Receptor (GPCR) STR-211 and RNAi knockdown of the receptor leads to a defect in 2-NA induced avoidance behavior in worms. We next discovered that STR-211 is required for immediate behavioral changes in C. elegans during freezing response against 2-NA. The study proposes an environment relevant animal model to mimic human anxiety and fear-like behavior, along with the identification of one of the olfactory GPCRs mediating this behavior. The model may help in understanding the neuromolecular basis of freezing response in human anxiety, contributing towards treatment of mental health disorders.

Glycosylation of proteins is arguably the most diverse post-translational modification that is altered in almost all cancer types, which has been demonstrated to play a crucial role in creating an immunosuppressive microenvironment that promotes immune tolerance and evasion. However, the biosynthesis of N-linked glycan is mediated by a series of enzymatic reactions catalysed by glycosyltransferases and glycosidases in a template-independent manner, hindering our understanding of specific structure-function relationships and roles of specific glycans on specific proteins. Here we use HLA class I-negative cell line K562, a known reference target for NK-mediated cytolysis, to establish a model investigating how cell surface glycan dynamics influence its susceptibility to cytolysis mediated by NK-92 cells. Treatment of K562 cells with kifunensine, swainsonine, 2F-peracetyl-fucose, or 3Fax-peracetyl Neu5Ac, inhibitors of N-linked glycan processing, resulted in drastic alterations in cell surface carbohydrate phenotype, as could be shown by flow cytometric analysis of the lectiNbinding properties of the cells. Despite these clear changes in carbohydrate phenotype, only K562 cells treated with either kifunensine or 3Fax-peracetyl Neu5Ac exhibited higher susceptibility to the cytolysis medidated by NK-92 cells accompanied with an increased CD107a expression by NK-92 cells. Although K562 cells overexpressing gene MGAT3 exhibited a decreased NK-susceptibility, we further found that this decrease was not exclusively determined by the overexpression of gene MGAT3 product bisecting {beta}1,4-GlcNAc, because the treatment of 3Fax-peracetyl Neu5Ac reversed the resistance of K562 cell against NK-92 cell in despite of expressing higher levels of bisecting {beta}1,4-GlcNAc. Expressing HLA-G on cell surface as extravillous trophoblast did not change the NK-susceptibility of K562 cells, despite evidence that HLA-G molecules expressed by K562 cells can bind to inhibitory receptor ILT2 expressed on NK-92 cell surface. These findings suggest that the level of terminal sialylation, outweighing other components in N-linked glycan, determines the NK-susceptibility of K562 cell, offering a new strategy to weaken the resistance of cancer cells so that the immune system can maximise the elimination.

Blue light using Flavin (BLUF) proteins are microbial photoreceptors that are involved in various physiological responses. Their occurrence and biochemical properties in fungi remain poorly understood. Here, we investigated a putative BLUF photoreceptor from the corn-smut fungus Mycosarcoma maydis (MmBLUF). Domain analysis, multiple sequence alignment of BLUF core regions, and structural modelling indicated conserved canonical BLUF fold and flavin-pocket residues. However, when heterologously expressed, UV-visible and fluorescence spectroscopy revealed different spectral behaviour than canonical BLUF protein. Further, we tested the role of extended N-terminus in modulation of chromophore binding by expressing N-terminus truncated protein variants. Our results suggest that the unusual spectral behaviour is not linked to the truncation construct (extended N-terminus), which also showed similar spectral features, indicating that the extended N-terminus is unlikely to account for an unusual photodynamics characteristics. Our findings support MmBLUF as a structurally conserved putative fungal BLUF-like photoreceptor with different photochemical properties. Further studies are required to establish its chromophore identity, photocycle and function of this unusual BLUF-like domain from fungal system.

Antibodies generally exhibit high specificity for their cognate epitopes, but structural and physicochemical similarities between distinct epitopes can enable an antibody to recognize different antigens, resulting in cross-reactivity. This property can be exploited for antibody repurposing. To identify epitopes that share such similarities, both sequence- and structure-based approaches can be employed. In this context, 3D Zernike descriptors provide a compact representation of protein surface geometry as numerical feature vectors, enabling quantitative comparisons independently of structural alignment and orientation. Thus, this study aimed to evaluate the application of 3D Zernike descriptors for the structural clustering of antibodies and epitopes and to explore their use in antibody repurposing for the recognition of new targets. To this end, antibody binding sites previously associated with recognition of similar epitopes were analyzed at different structural levels, considering the CDRs, CDRH3, and complete paratopes. Surface similarity was subsequently quantified by calculating the Euclidean distance between their corresponding 3D Zernike feature vectors. Performance was benchmarked against SPACE2. Additionally, different distance thresholds were evaluated based on their ability to recover antibody pairs recognizing the same epitope. The paratope-based approach provided the best balance between the number of identified pairs and precision at a distance threshold of 2.7, whereas epitope clustering showed robust performance up to a distance of 3.0. At these thresholds, the 3D Zernike descriptors identified a greater number of functional pairs than SPACE2 while maintaining comparable precision and identifying complementary sets of antibody pairs.. BTaken together, these findings support the use of 3D Zernike descriptors for structural clustering of antibodies and epitopes and for guiding antibody repurposing G, a highly lethal zoonotic pathogen. Structural screening identified three antibodies with epitopes similar to the NiV target that also showed a consistent binding preference for the target epitope in molecular docking assays. Notably, one candidate, originally directed against a SARS-CoV-2 epitope, formed a stable complex with the NiV epitope, remaining within the 5 [A] RMSD threshold during heated molecular dynamics simulations and emerging as a potential cross-reactive candidate.These results support the use of this computational framework for biopharmaceutical discovery against emerging targets. Taken together, these findings support the use of 3D Zernike descriptors for structural clustering of antibodies and epitopes and for guiding antibody repurposing.

The lack of effective anti-JEV therapy possesses significant challenge to control JEV. {beta}-catenin, a key mediator of Wnt signaling pathway regulates different viral replication and host immune responses. However, its role in JEV infection remains to be elucidated. Thus, the current study focused on evaluating iCRT-14, a specific {beta}-catenin inhibitor, against JEV. Treatment with iCRT-14 following JEV infection resulted efficient reduction in viral progeny release, viral RNA and protein levels in Huh7 and HEK293T cells. Further, active and total {beta}-catenin, Cyclin D-1 and GSK3-{beta}, the other key pathway players were also modulated in infected and inhibitor treated cells. Moreover, iCRT-14 showed an IC of 4.56 in Huh7 cell and maximal inhibition at the early stages of the JEV life cycle. Interestingly, the overexpression of {beta}-catenin in both the cells and siRNA-mediated {beta}-catenin knockdown (in Huh7 cells) significantly abrogated JEV replication, as evidenced by decreased viral titers, viral protein expression, and viral as well as total RNA levels. Moreover, the reduction in extracellular (84%) and intracellular (60%) viral titers following iCRT-14 treatment highlights its role in impairing JEV infection. Further, in silico molecular docking and co-immunoprecipitation studies demonstrated interactions between {beta}-catenin and the JEV NS5 and E proteins. Collectively, these findings suggest that optimum level of {beta}-catenin is required for efficient JEV infection, highlighting its potential as a target for designing host-directed control strategies to regulate viral infection.

Transfection of cells with DNA plasmids typically involves the uptake of lipoparticles by endocytosis, followed by the inefficient escape of these particles from endosomes into the cytoplasm. We found that the expression of transfected plasmids was reduced in cells depleted of either SEPTIN 9 or proteins in the endosomal sorting complexes required for transport (ESCRT) pathway. The reduction in plasmid expression could not be fully explained by effects on endocytosis. SEPTIN 9 depletion appeared to reduce the acidification of plasmid-containing compartments, suggesting that it primarily affects the pH-sensitive escape of plasmids from endosomes. Depletion of the ESCRT proteins VPS36 or ALIX resulted in especially dramatic reductions in transfected plasmid expression, which were accompanied by reduced colocalization between the transfected DNA and CHMP4, an ESCRT protein important for endosomal membrane remodeling during intraluminal vesicle formation. Finally, transfected plasmid DNA was strongly colocalized with LC3B, suggesting that the default pathway for transfected material is autophagy.

Background: Pulmonary arterial hypertension (PAH) is a serious disease with poor prognosis, especially in infants or preterm babies and there is still no optimal treatment for this disease. Noradrenalin (NE) is a vasoactive mediator which is released by sympathetic ganglion. According to previous studies, NE/1-adrenoreceptors is not only in regulating normal physiologic responses, but also in the pathogenesis of PAH. However, the mechanisms of NE in PAH are not fully understood. Methods: Human PASMC (PASMC) was used in this study. Cell viability assay and Wound healing assay were used to evaluate the proliferation and migration of PASMC. Immunoprecipitation and western blots analysis were used to investigate the mechanisms which involved in NE-induced PASMC proliferation. Results: We investigated that NE could induce human PASMC proliferation and migration. Furthermore, we first find that endothelin 1 (ET-1) signaling pathway plays an important role in NE-induced PASMC proliferation. ET1 is a critical molecular which is known for regulating cell growth and migration. We investigated that NE could increase NE-1 secretion, further enhancing ET-1 bind to its receptors. For further clarifying the downstream signals in NE/ET-1 induced PASMC proliferation, we detected the phosphorylation and expression levels of ERK and JNK. Conclusions: By combining the results from ours and previous studies, we believed that JNK/c-jun pathway may play an important role in NE-induced PASMC proliferation. Key Words: Noradrenaline; Pulmonary Arterial Hypertension; Pulmonary Artery Smooth Muscle Cells; Endothelin-1; JNK/c-Jun Signaling.

Long noncoding RNAs encode for microproteins that regulate cellular functions. Small regulatory peptide of amino acid response (SPAR) is a microprotein in the lysosome that responds to amino acid availability of the cell. In this study, we investigated the interactions between SPAR and SLC38A9, a lysosomal amino acid transporter and receptor involved in the mechanistic target of rapamycin 1 (mTORC1) pathway. We found that SPAR binds SLC38A9 and inhibits arginine transport in SLC38A9. Moreover, the downstream recruitment of Rag GTPases is also inhibited when SPAR is present in SLC38A9 liposomes. Docking model shows potential interactions between SPAR and SLC38A9. Together, these findings reveal the mechanism of mTORC1 inhibition through microprotein SPAR and illustrates the power of non long coding RNAs in altering cellular functions. Statement of SignificanceMicroproteins encoded from long noncoding RNAs are emerging as critical regulators of many pathways. This study investigates a novel mechanism of SPAR microprotein that directly regulates the mechanistic target of rapamycin complex1 (mTORC1) signaling pathway through the lysosomal amino acid transporter SLC38A9. SPAR blocks both arginine transport and the downstream recruitment of Rag GTPases. These findings provide critical results in how SPAR controls cellular amino acid availability, while broadly highlighting the powerful regulatory mechanism of microproteins in cellular processes.

Production of aberrant glycans by cancer cells constitutes a key immunosuppressive strategy to avoid destruction by immune cells. Although sialic acid-containing glycans are known to dampen the activation of lymphocytes, including Natural Killer (NK) cells, the role of fucose-containing glycans remains poorly characterized. In this work, we explored the role of Lewis X (LeX) in cancer cell-NK cell interactions. We induced ectopic expression of FUT9, an 1-3/4-fucosyltransferase, in two colorectal cancer cell lines and showed this enzyme only synthesized LeX structures but not sialyl-LeX. FUT9 introduction was not associated with altered MHC class I surface expression, nor with CD2 (which has been proposed as a receptor for LeX) binding to cancer cells. By inhibiting fucosylation we could demonstrate that CD2 binding was furthermore independent of surface fucosylated glycans in three independent cell lines. Lastly, FUT9/LeX had a limited role in cancer cell destruction and expression of activation markers by NK cells. Overall, our study suggests that, unlike sialylated glycans, 1-3/4-fucosylated glycans have limited impact on cancer cell evasion of NK cell-mediated destruction.

THAP9 is a transposable element-derived gene which encodes a protein that is homologous to the active Drosophila P-element transposase (DmTNP). Both THAP9 and DmTNP possess a C-terminal domain (CTD) which is functionally uncharacterized. Sequence and structural analysis suggest that the THAP9-CTD has a novel fold which is only found in THAP9 homologs. To explore the evolutionary history and characteristics of this novel domain, exhaustive phylogenetic analysis (using MSA, structure prediction, MSTA-based clustering) was performed. THAP9-CTD homologs were more widely distributed throughout the animal kingdom in comparison to DmTNP-CTD homologs which were restricted to arthropods. Moreover, the THAP9-CTD homologs were more conserved, especially among mammals and birds and their average length increased in a class-specific manner. Comparison with the DmTNP-CTD homologs demonstrates that although their respective CTDs may have evolved independently, they both surprisingly share similar secondary structure elements consisting of three conserved helical regions made of hydrophobic residues that are predicted to make up a conserved core. The role of the respective CTDs were further investigated by creating truncation mutants lacking the CTD. Interestingly both THAP9 and DmTNP truncation mutants are still capable of DNA excision and integration suggesting that their respective CTDs are not essential for DNA transposition. Moreover, CTD truncation favours DNA integration in THAP9: this suggests that CTD acquisition during evolution may have led to THAP9 domestication as observed in other transposable element-derived genes like Rag1 and piggybac, which have similar terminal regulatory domains.

The marine bacterium Vibrio alginolyticus and the food poisoning bacterium V. parahaemolyticus possess two types of flagella in one cell: proton-driven lateral flagella (Laf) extending from the periphery of the cell body, and sodium ion-driven polar flagella (Pof) extending from a cell pole. For swimming in seawater they use polar flagella, suppressing the expression of lateral flagella. When they attach to the body surface of fish or intestinal tract, lateral flagella are induced, allowing it to crawl along the surface or through mucus. The dynamometer hypothesis, which proposes that polar flagella sense rotation and control the expression of lateral flagellar genes, has been widely accepted. However, how rotation is sensed and how expression is controlled remains unclear. Although swarming has recently been analyzed by physical, biological, or biochemical perspectives, it remains unclear how this motility is controlled, or which substances and conditions are necessary for swarming ability. In this study, we discovered that adding gelatin to agar medium promotes swarming on the agar surface by the lateral flagella of Vibrio. Our data suggested that surfactants or viscous polysaccharides secreted extracellularly are important for promoting swarming on the agar surface and we identified that swarming is likely to be driven by S (social)-motility, in which bacteria move by interacting with each other, and A (adventure)-motility, in which bacteria move by interacting with the agar surface. Our study provides clues that help clarify the mechanism of bacterial swarming IMPORTANCEWe discovered that adding gelatin to hard agar medium promoted swarming on agar surfaces by the lateral flagella of Vibrio cells. The surfactants or viscous polysaccharides secreted extracellularly seem to be important for swarming ability on agar surfaces. We proposed that the swarming is thought to occur through S(social)-motility, where cells move by interacting with cell bodies each other, and A(adventure)-motility, where cells move by interacting with the agar surface and cell body. The present study should provide the clues to clarify the mechanism of bacterial swarming and how to move in a viscous environment.

Motivation Protein language models (PLMs) have emerged as powerful tools for sequence-based prediction of protein function, yet systematic benchmarks comparing frozen embeddings, fine-tuning strategies like Low-Rank Adaptation (LoRA) and classical machine learning (ML) remain limited. We benchmarked four ML strategies: ML using amino acid descriptors (SL-AAFeat), ML using frozen embeddings from 20 PLMs across various pooling strategies (SL-Embed), full model fine-tuning (FT-Full) and LoRA-based fine-tuning (FT-LoRA). Performance was evaluated on the in-house VHH phage display dataset (VHH) for binding affinity prediction and the TAPE fluorescence dataset (FLS and FLS10) for mutational effect prediction. Results Model performance depended strongly on the dataset and adaptation strategy. Max pooling consistently improved embedding-based models, while amino acid descriptors remained competitive under specific datasets and resource constraints. Fine-tuning generally provided the highest predictive performance, but the advantage is not universal. Hyperparameter optimization significantly enhanced FT-LoRA, enabling it to outperform FT-Full on the VHH dataset with less than 10% model parameter adaptation. In contrast, FT-Full achieved the best performance on FLS and FLS10. Several medium-sized PLMs performed comparably to larger models, highlighting favorable performance-efficiency trade-offs. Overall, this paper presents a thorough review of PLM utilization strategies and practical recommendations for selecting suitable strategies based on dataset characteristics and available computational resources. Availability The source code used in this manuscript is available in a Zenodo repository at https://doi.org/10.5281/zenodo.21466255.

Bacterial melanin are macromolecules found in nature that provide a wide range of biological functions, including pigmentation, resistance to radiation, scavenging of free radicals, thermoregulation and protected from oxidative stress and harmful heavy metals. The melanin is crucial for pathogenesis and bacterial survival in a variety of circumstances, and they can also influence how bacteria interact with other organisms. Usually, bacteria produce the melanin is either black or brown colour. The produced melanin has excellent properties like antimicrobial, antioxidant, photoprotective and antibiofilm. This is a report on Corynebacterium amycolatum melanin-producing bacteria isolated from the marine sediment of Thiruvanmiyur beach in Tamil Nadu, India. Corynebacterium amycolatum was screened using tyrosine basal broth (TBB), and UV-visible spectroscopy, FTIR, and SEM were used to analyse the extracted melanin. The non-pathogenic nature of the Cornynebacterium amycolatum strain was verified through antibiotic sensitivity profiling. The cosmetic potential was evaluated using antioxidant and SPF assays. Corynebacterium amycolatum predominantly uses the DOPA pathway for melanin production, was confirmed using kojic acid inhibitor study. The in vitro studies on mouse fibroblast cell line (L929) and in vivo studies on zebra fish embryos shows non-cytotoxicity using this melanin, even in lower concentration confirms its potential to use in cosmetic formulation. This research aims to demonstrate that bacterial melanin is safe for the environment and has qualities that make it safer and more effective in cosmetics.

Investigating the factors that stabilize biological communities is a central topic in ecology. Response diversity, defined as variation in species responses to environmental change, has been proposed as a key mechanism underlying the biodiversity-ecosystem functional stability (BEFS) relationship, whereby greater species diversity enhances ecological stability. Previous studies have shown that response diversity promotes ecological stability by generating asynchronous population fluctuations and the resulting compensatory dynamics. Although several metrics have been proposed to quantify response diversity, they do not explicitly consider the presence of insensitive species whose performance is unaffected by current environmental conditions. To examine how insensitive species influence response diversity, species persistence, and ecological stability, we conducted numerical simulations of a generalized Lotka-Volterra model under environmental forcing. We first confirmed that increasing variation among sensitive species increased the response diversity index and stabilized community dynamics. We then examined a scenario in which response diversity depended solely on the proportion of sensitive and insensitive species, assuming that all sensitive species responded identically to environmental change. Under this assumption, the response diversity index was maximized when sensitive and insensitive species occurred in equal proportions, whereas increasing the number of sensitive species monotonically destabilized community dynamics. Consequently, the relationship between response diversity and community stability depended on how response diversity was generated, such that higher response diversity could even be associated with lower community stability. These findings demonstrate that overlooking environmentally insensitive species can obscure the mechanisms linking response diversity and ecological stability. More broadly, our results reveal that response diversity comprises at least two distinct biological components--species sensitivity and response variation among sensitive species--that can have contrasting consequences for community stability. We therefore highlight the need to quantify sensitive species empirically and to develop response diversity metrics that distinguish these components. Author SummaryUnderstanding why some communities remain stable despite environmental change is a longstanding goal in ecology. Response diversity, which refers to differences in how species respond to environmental change, has been proposed as a key mechanism explaining why greater biodiversity (species richness) can promote ecological stability. Because species respond differently to changing environments, declines in some species can be compensated by increases in others, helping to stabilize community dynamics. However, previous studies have rarely considered species that are insensitive to current environmental changes. Using a mathematical model, we show that response diversity can arise from two distinct biological components--the number of sensitive species and variation in their responses--and that these components can have contrasting effects on ecological stability. When response diversity reflects variation among sensitive species, greater response diversity stabilizes community dynamics, as expected. In contrast, when response diversity changes only because of the proportions of sensitive and insensitive species, higher response diversity can be associated with lower community stability. Our findings highlight the importance of quantifying the number of sensitive species and developing response diversity metrics that distinguish species sensitivity from variation in responses among sensitive species.

Injuries to individual cells happen frequently as a result of physiological and environmental stresses during their normal daily functions that can lead to a ruptured cell cortex (plasma membrane and underlying cortical cytoskeleton). The capacity of cells to rapidly repair general daily injuries, as well as ones resulting from trauma, infection, or diseases/cancer, is essential for their survival. While we know the general cell biological outline of the highly-conserved physiological events taking place during cell wound repair, our knowledge of the molecular mechanisms governing the repair process is still fairly limited, due in large part to the lack of information regarding the molecules, machineries, and pathways involved. Here, we performed a genetic screen of 1322 fluorescent-tagged proteins to identify cell wound repair components that are recruited upon laser wounding or whose expression is lost and/or altered upon laser wounding. We identified 129 proteins that are recruited to wounds during the cell repair process through high resolution spatio-temporal expression analyses of these gene fusions in conjunction with a fluorescent actin reporter. Strikingly, we find that many members of the Rab family GTPases are recruited to wounds where, in addition to their well-known roles in intracellular membrane trafficking, they are affecting actin cytoskeletal organization and dynamics during the repair process. These studies are allowing us to define the earliest acting proteins, as well as those required at specific steps in the repair process based on their recruitment patterns and the precise timing of their recruitment to wounds. Thus, our imaging-based screen is providing us with a global view of the repair processes, as well as a large number of genes/gene families that provide new entry points for examining specific steps in the cell wound repair process. Author SummaryCells in our bodies get injured every day from normal activity, environmental stress, infection, or disease. To survive, they must quickly repair these injuries and restore normal function. While some molecules have been identified as key players of cell wound repair, many of the molecules involved and their roles remain unknown. In this study, we identified new molecules that are involved in different steps of cell wound repair. Using laser-induced injury in the Drosophila model, we examined 1322 proteins and observed their spatial and temporal dynamics in a cell after injury. From the 1322 proteins examined, we identified 129 proteins recruited to distinct regions around the damage site during cell wound repair, suggesting roles in specific steps of the repair process. Interestingly, a subset of these proteins are Rab family GTPase members, highlighting new roles for these proteins in regulating actin dynamics. By identifying new candidate repair molecules, we provide a foundation for understanding how cells maintain their integrity and how repair processes may be influenced by factors such as wound size, infection, aging, and disease.

Valproic acid/sodium valproate (VPA) is a widely prescribed anticonvulsant and has also been used against certain tumor cells. It is a potent modulator of gene expression. Its ability to induce apoptosis has been well documented in HeLa cells. However, another form of cell death - mitotic catastrophe - has not yet been explored in VPA-treated HeLa cells. Here, we investigated the effects of VPA treatment on mitotic catastrophe characteristics, including morphological features and their frequencies, fluorescence intensity signals of caspase-2 and p53, and the expression and abundance of DNMT1 and DNMT3B. An increased frequency of mitotic catastrophe was observed not only morphologically, but also through enhanced induction of caspase-2, involvement of p53, at least under more drastic VPA treatment, but without a decrease in DNMT1 or DNMT3B levels. Additionally, enhancement of mitotic catastrophe coincided with a reduction in mitotic chromosome abnormalities. Increased DNMT3B expression following VPA action, may be favored by previously reported chromatin decondensation induced by this drug. Enhanced CpG methylation of specific DNA sites could thus be promoted. In conclusion, VPA was shown to trigger metabolic pathways linked to different forms of cell death in HeLa cells, supporting its oncosuppressive potential.

Mutations in p53 and its 12 isoforms can alter its functions. As N-terminally truncated isoforms of p53 (delta40p53, delta133p53, and delta160p53) participate in tetramer formation, they are important regulators of cancer fate. Although delta40p53- and delta133p53-mediated regulation of cancer is well reported, the mechanism underlying delta160p53 production and its functional role remains unclear. We investigated the internal ribosomal entry site (IRES)-mediated translation of {Delta}160p53 and its role in cancer regulation. As differential synthesis of delta160p53 was observed under different stress conditions, IRES-mediated translation of this isoform was demonstrated using bicistronic luciferase constructs. No cryptic promoters or splicing sites were detected in the IRES sequence. Cell death and late apoptosis were significantly decreased, while proliferation, the number of cells in the S phase, and drug resistance were induced by delta160p53. Furthermore, delta160p53 did not induce p53-responsive promoters. RNA sequencing analysis of delta160p53 overexpression showed similar results, along with the inhibition of other tumor suppressor genes. Overall, our results provide insights into IRES-mediated translation of delta160p53, which can be considered a novel target for cancer treatment.

HIV transcription is characterized by its stochastic nature, which plays a pivotal role in determining the fate of a provirus--active replication or latent infection--and is therefore a critical determinant of the HIV latency establishment. Building upon our previous work, we identified a unique phenotype of stochastic HIV transcription in a Jurkat T cell clonal model harboring a single lentiviral-based vector, herein referred to as the HIV transcription-sensitized model. A defining feature of this cellular model is that the turnover of HIV transcription shows elevated frequency--a phenomenon designated phenotypic bifurcation--suggesting that, under certain conditions, the regulation of stochastic HIV transcription can be a pure epigenetic phenomenon. In continuation of and to further substantiate this premise, the present study characterizes the contributions of epigenetic regulation of CpG methylation, methionine metabolism that coordinates cell cycle events, and HIV antisense transcription to this phenomenon. This work adds direct causal evidence to the hypothesis that a potential lag prior to the entry of the G2 phase in the bifurcated state of low HIV transcription may serve as one of the underlying mechanisms that lead to the high CpG methylation level compared with that measured in the state of high HIV transcription, contributing to the cyclical turnover of phenotypic bifurcation of HIV transcription.