Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer (BC) with higher incidence rates in India. Ionizing radiation (IR) is a key component of TNBC treatment regimens. However, overall suboptimal response during the course of multiple repeat radiotherapy with eventual attainment of radioresistance remains a major challenge for this oncologic treatment modality. To overcome this limitation, it is important to understand molecular signatures that drive cells to transform into non-responsive populations against radiation therapy. Here, we demonstrate a constitutively high basal level of autophagy in TNBC cells, which might contribute to their relatively lower sensitivity to radiation-induced cell death. We also observed an elevated level of p62 in TNBC cells, which is regulated independently of autophagic flux. Concurrent upregulation of p62 and basal level of autophagy were found to be the driving forces in acquiring radioresistance in breast cancer cells. Ectopic expression of p62 enabled breast cancer cells to proliferate rapidly with enhanced migration potential. Reciprocally, posttranscriptional or pharmacological inhibition of p62 prevented the proliferation and migration potential of TNBC cells. Our high-throughput next-generation sequencing (NGS) data revealed VCAM-1 as a key mediator in p62-driven cell proliferation and acquired radioresistance. Likewise, impairment of autophagic flux, either by pharmacological inhibitors (CQ and bafA1) or by genetic deletion of ATG5, led to reversal of radioresistance. Collectively, our data highlight that an elevated basal level of autophagy with co-induction of p62 de novo protein synthesis confers radioresistance in TNBC.

This study presents the first fabrication of Arbortristoside-A (Arbor-A)-loaded PLGA nanoconstructs (Arbor-A-PNC) for targeted intramacrophagic delivery intended to enhance the therapeutic efficacy of Arbor-A in the treatment of visceral leishmaniasis (VL). VL treatment remains a formidable health challenge due to the parasites' intramacrophagic persistence, drug-induced toxicities, emerging drug resistance, and no vaccines in hand. Arbor-A, a bioactive iridoid glycoside naturally occurring in Nyctanthes arbor-tristis Linn., has shown potent leishmanicidal activity by inhibiting trypanothione reductase, a validated drug-target enzyme in Leishmania parasites, leading to redox homeostasis disruption, elevated ROS, cell membrane damage, and apoptotic-led parasitic death. Despite encouraging anti-VL potential, its further investigation remained unexplored, attributed to poor aqueous solubility posing a challenge in developing Arbor-A formulations. To address this and maximise Arbor-A-Leishmania parasite contact, this work introduces macrophage-targeted Arbor-A-PNC that have been systematically optimised through a Box-Behnken statistical design approach. The optimised nanoconstructs exhibited a spherical geometry, optimal size (Z(av) = 166.25 +/- 2.90 nm), monomodal dispersion (PDI = 0.111 +/- 0.008), -11.75 +/- 0.07 mV zeta-potential, 65.91 +/- 12.47% Arbor-A entrapment and biphasic, sustained release governed by quasi-Fickian (n < 0.43) diffusion kinetics. Mechanistic insights gained using flow cytometry and fluorescence imaging revealed its efficient intramacrophagic uptake, while cytotoxicity assays demonstrated toxicity reduction of free Arbor-A towards macrophages in vitro. Notably, the ex vivo infection assays showed its significant 9.97-fold higher efficacy compared to native Arbor-A against Leishmania donovani amastigotes. The study introduces a pioneering approach for the intramacrophagic delivery of plant-derived antileishmanial iridoid glycoside based on a nanotherapeutic platform, positioning nanoformulated Arbor-A as a prospective option to existing VL chemotherapeutics that are increasingly undermined by the emergence of drug resistance and systemic toxicities.

Air pollution-induced emphysema is accompanied by changes in pulmonary vasculature, leading to pulmonary hypertension (PH) and ultimately heart failure. Pyrroloquinoline Quinone (PQQ), a potent antioxidant with cardio-protective properties, upregulates mitochondrial biogenesis and functions. Previously, we have shown that PQQ protects against PH; however, the effect of PQQ on emphysema and the mitochondrial dysfunction due to air pollution still remains unexplored. In our study, we unraveled the effect of PQQ on Ultrafine carbon particle (UFCP) induced emphysema and PH. In the in vitro studies, human lung adenocarcinoma epithelial cells (A549 cells) were exposed to UFCP (50 μg/ml) and PQQ (100 μM) for 24 h, and following this, the redox state and mitochondrial health of the cells were examined. For the in vivo study, SD rats were administered UFCP (100 μg/dose, three times a week, intranasally) and PQQ (2 mg/kg, oral/day) for four weeks. Plethysmography, 2-D Echo, and invasive blood pressure measurement were used to study pulmonary, hemodynamic, and cardiac functions, and metabolic changes were studied by untargeted metabolomics of the lungs. PQQ treatment improved mitochondrial structure, dynamics, and biogenesis and reduced oxidative stress in UFCP-exposed A549 cells. PQQ significantly improved pulmonary functions, inflammation, structure, and muscularization of vessels in UFCP-exposed rats (#p < 0.01). Metabolomics study showed improved metabolism in the lungs of PQQ-treated rats. Further, PQQ significantly reduced right ventricular pressure (RVP) and hypertrophy (RVH) in UFCP-exposed rats (#p < 0.05). Our findings suggest that improving mitochondrial functions by PQQ preserves alveolar integrity and prevents pulmonary hypertension, and it can be a promising prophylactic, especially for pollution-ridden settings.

Intriguingly, 13-mer frog-peptide, temporin L (TempL) contains 50% aromatic residues within its first eight residues. Considering the role of aromatic residues in self-assembly of peptides and the potential of such peptides in biomedical applications, we envisaged to identify new short self-assembling peptides from the amino-terminus of TempL and characterize their structural and biological properties. Thus, starting from the eighth to the first residue of TempL, we synthesized five 4 to 8 residue peptides (T-4mer to T-8mer). Different ultrastructural studies suggested nano-spherical/nano-fibrillar structures of these peptides. Remarkably, T-6mer, T-7mer and T-8mer exhibited polyproline type-II circular dichroism spectra of collagen-like triple-helical structure and sigmoidal melting curves like that we observed with rat-tail type-I collagen. Amazingly, the T-8mer peptide at 1.5% (w/v) forms hydrogel within an hour indicating its ability to form supramolecular assembly, saturated with water. We further studied collagen-mimetic nature of these TempL-derived peptides. HepG2 cells showed significant adhesions onto the coatings of T-6mer, T-7mer, T-8mer peptides and rat-tail type-I collagen which got compromised when these cells were pre-treated with antibody of collagen receptor, integrin α2β1. Interestingly, following the adhesions onto the surface of these TempL-derived peptides, cytoskeletal organization was induced in HepG2 cells like that observed in the presence of a collagen protein. Overall, the current results demonstrated the dissection of a frog-peptide, TempL with revelation of collagen-mimetic peptides from its aromatic-residue rich amino-terminus.

ABSTRACT The influenza virus poses a significant global health threat due to its continuous evolution, immune evasion, and zoonotic spillover. The rise of drug resistance, reduced susceptibility to existing antiviral medications, and the limited effectiveness of annual vaccines underscore the need for new antiviral strategies. To infect, the influenza virus binds to sialic acid (SA)-containing molecules on host cell membranes through hemagglutinin (HA). Blocking this interaction represents a promising antiviral approach. Herein, we report that SA containing plasma membrane-derived vesicles (PMV) efficiently inhibits in vitro Influenza A virus (IAV) infection. Using orthogonal methods, we demonstrate that PMV derived from A549, MDCK, and HEK cells competitively bind to H1N1 (WSN) and H3N2 (X-31) IAV strains, block entry and infection in human respiratory epithelial cells in a dose-dependent manner, without causing significant toxicity. When the size of the vesicles was reduced through extrusion, the antiviral activity was enhanced, and this was found to be correlated with a size-dependent increase in hemagglutination inhibition and reduced IAV internalisation. Plasma membrane-derived vesicles may serve as a novel antiviral strategy against influenza virus infections due to their simple production method and conserved SA binding site on HA.

SK (SK), a secondary plant metabolite from Lithospermum erythrorhizon, is an inducer of oxidative stress and a DNA Topoisomerase inhibitor with promising anticancer properties. However, the underlying mechanisms, especially the involvement of autophagy in cancer cell death, are poorly understood. Here, we report a novel mechanism of action that activates a noncanonical, Beclin1-independent but ATG5-dependent autophagy pathway triggered by oxidative stress in two distinct subtypes of triple-negative breast cancer (TNBC) cell lines: mesenchymal stem cell-like MDA-MB-231 and basal-like-1 MDA-MB-468. We observed that this noncanonical autophagy pathway specifically targets and degrades nuclear material by nucleophagy. Electron microscopy analysis of both cell lines revealed distinct nuclear alterations, including envelope-limited chromatin sheets (ELCS), nuclear buds, and micronuclei after SK treatment. Furthermore, numerous autophagosomes and lysosomes were found in close proximity to the nuclear membrane, suggesting the occurrence of nucleophagy. The localization of γ-H2AX in nuclear buds and micronuclei observed by confocal microscopy indicated cytosolic leakage of damaged DNA. Additionally, Western blot analysis confirmed the role of the cGAS-STING pathway, which is essential for detecting damaged DNA in the cytosol. Inner nuclear membrane protein Lamin B1 was found to interact with LC3II and was subsequently degraded through the nucleophagy pathway. Knockout of ATG5 using CRISPR-Cas9 reduced autophagy, while Beclin1 knockdown did not reduce LC3II conversion, indicating that the process follows a noncanonical autophagy pathway that is dependent on ATG5 and independent of Beclin1. SK induces oxidative stress, leading to mitochondrial depolarization and DNA damage accumulation, which subsequently triggers autophagy and ultimately causes apoptotic cell death. Treatment with the ROS scavenger N-acetylcysteine (NAC) reduced nuclear stress, mitochondrial dysfunction, autophagy, and cell death, emphasizing the role of oxidative stress in SK-induced cell death. MDA-MB-468 cells exhibited greater sensitivity to SK-induced nuclear stress and cell death compared to MDA-MB-231 cells. Taken together, we demonstrate that SK exerts its anticancer effects in TNBC cells through the generation of oxidative stress and noncanonical autophagy, thus highlighting SK's potential for targeted anticancer therapeutics.

BACKGROUND:Lower levels of Qki were reported in human and mouse-failing hearts, implicating its involvement in cardiac diseases. However, the molecular and functional effects of its downregulation in adult myocardium remain largely unknown. OBJECTIVE:We aim to uncover the effects of Qki knockdown in adult hearts. METHODS & RESULTS:Here we show that AAV9-mediated knockdown of Qki by shRNAs in the hearts of adult BALB/c mice led to cardiac malfunction, atrophy, apoptosis, heart failure, and death within two weeks. Global transcriptomic analysis of Qki knockdown hearts revealed significant dysregulation of 996 alternative splicing events upon Qki knockdown. Mechanistically, we discovered that loss of Qki promotes the exclusion of the third exon of Morf4l2, leading to higher expression of exon three excluded variant (Morf4l2Δex3). Like rodents, the RNA-seq dataset from 108 human hearts revealed a lower splice junction count of MORF4L2 exon three in hearts with low levels of QKI compared to subjects with higher QKI levels. Specific knockdown of Morf4l2Δex3 rescues Qki knockdown-induced cardiac cachexia and improves cardiac function. Moreover, Morf4l2Δex3 was increased in the colon cancer-induced cardiac cachexia mouse model, and its inhibition prevented cardiac cachexia and improved cardiac function. Mechanistically, exon three of Morf4l2 lies in the 5'UTR, and its exclusion leads to higher expression of MORF4L2 upon Qki knockdown due to the lack of a G2-quadruplex. Importantly, MORF4L2 protein sequence and localization were not affected by alternative splicing as exon three lies in the 5'UTR. We found that MORF4L2 is a chromatin-bound protein and regulates H3K27ac. CONCLUSION:Qki knockdown in the adult heart leads to cardiac cachexia due to the alteration of Morf4l2 splicing. Inhibition of Morf4l2Δex3 inhibits cancer-induced cardiac cachexia, demonstrating it as a potential therapeutic target.

Background The functional impact of m6A modifications on RNA is governed by reader proteins that read the m6A marks and process the RNA accordingly. Recent studies have highlighted the importance of m6A reader proteins in the heart. However, the function of a reader protein Ythdf3 in the heart remains completely unknown. Objective We aim to uncover the function of Ythdf3 in the hearts. Methods& Results Here, we show that Ythdf3 is indispensable for the heart, and its knockdown leads to cardiac cachexia evident by cardiomyocyte atrophy, death, and less dense myofibrils visualized by electron microscopy. We also found downregulation of Ythdf3 in response to doxorubicin, and its overexpression rescues doxorubicin-induced cardiomyocyte atrophy and death. Contrary to primarily cytoplasmic localization, we found that Ythdf3 localizes in the cardiomyocyte nucleus. Furthermore, using co-immunoprecipitation coupled with LC-MS/MS, we show that Ythdf3 interacts with splicing proteins like DDX5 and HNRNPU and regulates alternative splicing in the heart. Mechanistically, we found that Ythdf3 regulates the splicing of CaMKIIδ, and its knockdown leads to an increase in CaMKIIδA and CaMKIIδC isoforms while a decrease in CaMKIIδ9 isoform. Conclusion Collectively, Ythdf3 knockdown in the adult heart leads to cardiac cachexia due to the alteration of the splicing program. ### Competing Interest Statement The authors have declared no competing interest. * RBPs : RNA-binding proteins AAV : Adeno-associated Viruses TUNEL : Terminal deoxynucleotidyl transferase dUTP nick end labeling TEM : Transmission Electron Microscope FDR : False Discovery Rate PSI : Percent Splicing Index mRNA : Messenger RNA DAPI : 4′,6-diamidino-2-phenylindole AS : Alternative Splicing SE : Skipped Exon LC-MS/MS : Liquid Chromatography with tandem mass spectrometry CaMKII : calcium/calmodulin-dependent protein kinase II m6A : N6-methyladenosine PBS : Phosphate buffer saline cTnT : cardiac Troponin T

The understanding of pyroptotic cell death and its mechanistic insights in neutrophils remains less explored and ambiguous. This study analyzes neutrophil pyroptotic responses under different conditions and dissects distinct pyroptosis-associated events, including IL-1β release and cell death. We decipher the signalling pathways underlying these responses and distinguish them from other cell death mechanisms. Interestingly, we observe IL-1β release and enhanced survival in neutrophils in response to LPS-primed ATP-treatment, in contrast to a combination of IL-1β release with cell death in macrophages. While LPS-primed nigericin-treated neutrophils exhibit IL-1β release and cell death, characterized by nuclear rounding, cell swelling/ ballooning, plasma membrane pore formation, and subsequent cell rupture, confirming the occurrence of pyroptotic cell death. While nigericin alone triggers cytokine uncoupled pyroptosis. Intriguingly, these phenomena do not induce under other death programs, including apoptosis, NETosis. This provides opportunity to unravel the regulation of these pyroptotic responses in neutrophils. Data observed reveal the role of NLRP3 and context-dependent caspase −1 & 11 in IL-1β secretion. While, DNA damage and caspase-7 & 9 regulate pyroptotic death. Intriguingly, forced DNA damage by ATM kinase inhibitor mitigated inflammasome activation and IL-1β release, while spur death. Furthermore, this study identifies perinuclear actomyosin forces driving nuclear rounding and pyroptotic death. Both murine neutrophils exposed to bacteria and human neutrophils treated with nigericin exhibit these pyroptotic processes, highlighting their broad relevance. This study depicts decision whether to secrete cytokines or undergo pyroptotic cell death emphasizing the regulatory role of DNA damage-cytoskeletal axis beyond inflammasome activation. Moreover, LPS and bacteria induced acute lung injury in mice displays nuclear rounding presenting pyroptotic neutrophils and enhanced NLRP3, Caspase-11 activation, and IL-1β release. Together, this study defines intriguing crosstalk of NLRP3, caspases, DNA damage, and actomyosin forces that orchestrate divergent inflammatory fates, potentially leading to opportunities for targeted strategies for combating inflammation. HIGHLIGHTS ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. Department of Science and Technology, ECR/2022/001339 Council of Scientific and Industrial Research, https://ror.org/021wm7p51, FBR070302 [1]: pending:yes

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease that currently has no cure. Fibroblast-like synoviocytes (FLS), present in the RA synovium, play a pivotal role in RA pathogenesis. Notably, FLS in the RA patients (RA-FLS) exhibit characteristics similar to cancer cells, like enhanced migration, invasiveness, uncontrolled proliferation, resistance to apoptosis, and metabolic reprogramming. RA-FLS invasiveness is linked to radiographic joint damage in the patients, whereas inhibiting the FLS migration mitigates disease pathology. However, the molecular mechanisms underlying the migration and invasion capabilities of RA-FLS are not entirely understood. In this work, we have explored the function of mitochondrial calcium uniporter (MCU) and calcium signaling in FLS invasion. Our findings demonstrate a positive correlation between MCU expression and RA disease score. Interestingly, mitochondrial size was reduced, and peripheral localization was more pronounced in the RA-FLS when compared to the control FLS. Mitochondrial calcium import inhibition in the FLS by specific MCU inhibitor, Ruthenium-360 restored these altered mitochondrial dynamics and reduced the invasive phenotype. Through unbiased transcriptome analysis, we identified that MCU-mediated calcium signaling in RA-FLS leads to the enriched actin cytoskeleton and focal adhesion pathways responsible for the invasion phenotype, which can be effectively suppressed by inhibiting MCU. Additionally, we found that mitochondrial transport facilitator Miro1 binds to MCU in a calcium-dependent manner and regulates MCU-mediated mitochondrial dynamics and RA-FLS invasion. Experiments utilizing mice xenograft model demonstrated that MCU silencing diminishes the migration of RA-FLS toward the sites of inflammation in the immunocompromised SCID mice. Altogether, our findings highlight MCU as a promising therapeutic target to inhibit RA-FLS migration and RA progression.

The unabated increase in antimicrobial resistance has underlined the importance of identifying novel drug combinations which eliminate infections more potently and likely reduce the emergence of resistance. In this context, we have identified Vancomycin and many β-lactams as being potently active against drug-resistant Mycobacterium tuberculosis and non-tuberculous mycobacteria including M. abscessus, emerging as pathogens of concern owing to their inherent drug resistance profile. In this study, we have identified combinations of Vancomycin, a glycopeptide and β-lactams, especially Ceftriaxone, Ceftazidime and Meropenem, in the presence or absence of Sulbactam, a β-lactamase inhibitor, as possessing potent antimicrobial activity against several drug-resistant mycobacterial strains. The combination of Vancomycin and β-lactams exhibited potent bactericidal activity and reduced the bacterial load better than either drug alone. The molecular basis of synergy was mediated by increase in permeability of mycobacterial cell as demonstrated by ethidium bromide assay. In the murine model of mycobacterial infection, synergistic combination of Vancomycin and β-lactams outperformed clinically utilized drugs including Isoniazid, Rifampicin and Ethambutol against M. tuberculosis and Amikacin, Clarithromycin against M. abscessus. The combinations caused a significant reduction in bacterial load in various organs in M. tuberculosis and M. abscessus infected mice. Thus, the synergistic combination of Vancomycin and β-lactams could potentially be utilized for treatment of recalcitrant mycobacterial infection especially those caused due to drug-resistant pathogens. ### Competing Interest Statement The authors have declared no competing interest.

Ormeloxifene (ORM) is a nonsteroidal selective estrogen receptor modulator (SERM), developed by the CSIR-Central Drug Research Institute that is approved as an oral contraceptive. However, it has also shown promising anti-cancer activity, especially in breast cancer. Here, we have investigated the anti-cancer effect of ORM on colon cancer cells and show that its antiproliferative activity is mediated through mitochondrial fission and autophagy-associated cell death. We observed that ORM treatment led to an elevation in autophagy markers like LC3II, Beclin1, and Atg7. Autophagy induction and LC3II turnover were monitored by immunofluorescence staining and confocal microscopy. Transmission electron microscopy results confirmed the formation of autophagosomes and autophagolysosomes. Autophagic flux was confirmed by the increased expression of LC3II in cells co-treated with BafilomycinA1(autophagy inhibitor) and ORM. This was further corroborated using tandem mRFP-GFP-LC3 (tfLC3) transfection in DLD-1 cells. Interestingly, we observed that inhibition of autophagy reduced the apoptotic cell population, suggesting pro-death autophagy. ORM treatment caused notable ultrastructural alterations indicative of cellular stress. Notably, ORM triggered the generation of mitochondrial ROS, associated with increased levels of mitochondrial fission and a decrease in mitochondrial fusion proteins. Changes in mitochondrial dynamics were observed under the TEM, which included reduced mitochondrial size and increased mitochondrial number. Inhibition of mitochondrial fission resulted in enhanced cell survival and a concomitant decrease in the autophagic markers, implying that ORM-induced autophagy depends on mitochondrial fission. Taken together, our findings bring to light a novel mechanism where Ormeloxifene targets mitochondrial dynamics to promote autophagy-associated cell death in colon cancer cells.

Spiniferin is a 13-mer scorpion-origin antimicrobial peptide having poor antimicrobial activity. To augment Spiniferin's antimicrobial activity, we enhanced its net positive charge by replacing a glutamic acid residue with an arginine residue toward its amino terminus. We envisaged that a cation-π interaction could be introduced between this arginine residue and the tryptophan residue located near the middle of Spiniferin. This cation-π interaction could promote stronger interaction of the peptide with a negatively charged bacterial membranes, resulting in its increased antimicrobial activity. Though glutamic acid-to-arginine substitution [Spiniferin-(E4R)] enhanced both the antimicrobial and toxic properties of Spiniferin, the same replacement with a d-arginine residue [Spiniferin-(E4dR)] significantly enhanced its antimicrobial activity against selected Gram-negative/positive bacteria and a MRSA strain while maintaining low hemolytic/cytotoxic properties. Interestingly, Spiniferin-(E4dR) analogs, with its aromatic-tryptophan residue substituted with an aromatic phenylalanine or an aliphatic valine residue, and its d-arginine residue replaced with a d-lysine residue, showed much lesser antibacterial activity than Spiniferin-(E4dR) or Spiniferin-(E4R). The results indicated a crucial role of the tryptophan and l-/d-arginine combination in augmenting the antimicrobial activity of Spiniferin analogs, Spiniferin-(E4R) and Spiniferin-(E4dR). Spiniferin-(E4dR) showed bactericidal properties against selected Gram-positive/negative bacteria. It permeabilized bacterial membranes and induced damages in bacterial membrane organization, suggesting that the bacterial plasma membrane is its target for exhibiting antimicrobial activity. Further, Spiniferin-(E4dR) in the intravenous route demonstrated the survival of E. coli ATCC 25922-infected mice and the clearance of bacteria from the visceral organs of these mice. Computational studies showed the requisite distance between the arginine's cationic side chain and the π-electron site of the tryptophan residue for a possible intramolecular cation-π interaction in Spiniferin-(E4dR)/Spiniferin-(E4R).

Neutrophils play a critical role in various pathophysiological conditions. However, their therapeutic targeting has been challenging due to the associated risk of infections. Therefore, identifying disease-associated neutrophil populations is essential for targeted strategies. This study identifies a distinct neutrophil subpopulation, CD11b+Ly6G+Sca-1+ neutrophils expressing stem cell antigen-1 (Sca-1). Sca-1pos neutrophils, enriched in the liver, are mature, long lived, and proinflammatory. They display enhanced effector functions, including superoxide production, degranulation, NETosis, and T cell proliferation. Sca-1 is not merely a marker but actively contributes to these functions. Intriguingly, inflammatory and metabolic cues drive the emergence of Sca-1pos neutrophils from conventional neutrophils and progenitors. Models of acute and chronic inflammatory conditions such as peritonitis and non-alcoholic steatohepatitis demonstrate increased Sca-1pos neutrophils. Pharmacological interventions reduce their enhanced functional activity and associated inflammatory outcomes. Collectively, this work reveals a distinct inflammatory neutrophil subpopulation and provides a potential avenue for selectively targeting neutrophil subpopulations in inflammatory diseases.

Human positive coactivator 4 (PC4) is a highly abundant non-histone chromatin protein involved in diverse cellular processes, including transcription regulation, genome organization, autophagy, B-cell differentiation, neurogenesis, DNA repair, etc. Most PC4 is phosphorylated in cells, which interacts with core histones and the linker histone H1 to confer the compact heterochromatin state of the genome. Knocking down PC4 at both cellular and organismal levels leads to significant chromatin decondensation, altered epigenetic landscape, enhanced autophagy, and increased DNA damage susceptibility. Here, we report that besides p300, PC4 also gets acetylated by DNA repair, facilitating lysine acetyltransferase KAT5 (Tip60) at a specific lysine residue (PC4K80) when the cells are subjected to DNA damage. The vulnerability of DNA in PC4 devoid cells was substantially reduced by reintroducing wild-type PC4 to the cells but not the mutant PC4 (PC4K80R), defective in KAT5-mediated acetylation. High-resolution microscopy techniques, including transmission electron microscopy and atomic force microscopy, are employed to visualize chromatin structural changes in response to DNA damage and repair in a Tip60-mediated PC4 acetylation-dependent manner. Presumably, KAT5-mediated acetylation of PC4 at K80 residue facilitates access to the damaged DNA by altering chromatin structures at damage sites, thus promoting DNA repair. This process could be highly significant both in cancer and in aging.

AIMS:Our aim was to evaluate the antileishmanial potential of Shikonin (SKN), a natural topoisomerase inhibitor derived from Lithospermum erythrorhizon, against Leishmania donovani and to elucidate its mechanism of action through physiological, ultrastructural, and in silico studies. METHODS AND RESULTS:SKN inhibits the in vitro proliferation of both parasite forms, with IC50 values of 3 μM for promastigotes and 0.75 μM for intracellular amastigotes. Using a combination of biophysical and biochemical methods, we found that SKN exerts its cytotoxic effect by generating oxidative stress, mitochondrial dysfunction, and nuclear damage, leading to apoptotic cell death in the parasites. Ultrastructural analysis revealed morphological alterations and apoptotic features in treated parasites, which included mitochondrial swelling and organelle damage, nuclear pyknosis, and pronounced nuclear membrane swelling in the parasites. Since the unique bi-subunit topoisomerase of these parasites has structural differences from the human counterpart and is considered a potential drug target, we performed in-silico molecular docking studies to determine the binding affinity of SKN with topoisomerase 1B (TOP1B). SKN exhibits a stronger binding affinity with L. donovani 1B than the standard topoisomerase inhibitor, camptothecin, forming favorable interactions at the catalytic site. It also shows a lesser binding affinity with human TOP1B than camptothecin. CONCLUSIONS:Targeting the Leishmania parasite Topoisomerase with the natural naphthoquinone SKN inhibits parasite proliferation. SKN generates oxidative stress, leading to mitochondrial dysfunction, elevation of cytosolic calcium, and nuclear damage resulting in apoptotic cell death.

Intricately modulated by a spectrum of proteins, Chromatin structure governs gene expression and cellular homeostasis. In Caenorhabditis elegans , critical components like the TATA-binding protein tbp-1 play pivotal roles in orchestrating chromatin dynamics. While the function of many of the interacting partners of tbp-1 is well-understood, our study brings into focus a lesser-known entity, T13F2.2, an unexplored tbp-1 interacting protein and a putative RNA polymerase II transcriptional coactivator. Employing reverse genetics, we found that RNAi-induced depletion of T13F2.2 resulted in pronounced disruptions to nuclear architecture, evidenced by nuclear staining and transmission electron microscopy. Accompanying these structural anomalies, we observed increased autophagy, pointing to cellular stress and a hyperacetylation of the core histones, suggesting potential chromatin decompaction. Notably, multifaceted functional alterations, upon the partial knockdown of the T13F2.2, culminated in a substantial reduction in the worm’s lifespan. Intriguingly, interventions such as administering ROS scavengers and autophagy modulators offered a reprieve from this life-shortening effect. Transcriptomic analysis upon T13F2.2 knockdown revealed upregulation of genes related to autophagy and chromatin remodelling, alongside downregulation of genes involved in longevity pathways and oxidative stress response. This study, thus, not only puts forward the functional implication of an uncharacterized gene in C. elegans biology, but also further emphasizes the role of chromatin organization in aging at the organismal level. ### Competing Interest Statement The authors have declared no competing interest. A Nazir acknowledges funding received from CSIR-CDRI vide projects UNCIDAN and NISTHA (MLP 2030)., TKK acknowledges JNCASR and J C Bose Fellowship, DST, Government of India.