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Mutations in DNA damage repair (DDR) genes lead to genomic instability, driving a range of degenerative syndromes. In addition to promoting mutation accumulation, unrepaired DNA damage can leak into the cytosol and activate innate immune-sensing pathways, particularly the cGAS-STING axis. However, the extent to which cGAS causally contributes to organismal pathology in DDR syndromes in vivo remains unresolved. Here, we genetically model ataxia telangiectasia (A-T) and Bloom syndrome in the short-lived turquoise killifish (Nothobranchius furzeri) and demonstrate that genetic disruption of cgas in the A-T model partially ameliorates germline failure, hepatic senescence, and cerebellar neuroinflammation. Unexpectedly, cgas loss also reversed cellular hallmarks of genome instability, including reduced micronuclei, improved telomere integrity, and restored H3K9me3-marked heterochromatin landscape, consistent with STING-independent nuclear functions of cGAS that influence DNA repair and chromatin. Together, these data identify cGAS as a context-dependent amplifier of DDR pathology acting through canonical inflammatory signaling and noncanonical nuclear mechanisms that shape genome stability. Accordingly, our findings support pharmacological cGAS inhibition as a potential strategy for DDR syndromes in settings of chronic DNA damage while highlighting that cgas loss in an otherwise naive background exacerbates pathology and genomic instability, underscoring its essential role in normal physiology.

Addiction continues to be heavily stigmatized due to lingering misconceptions that frame substance use as a moral failing rather than a treatable neurobiological disorder. This stigma, when internalized, intensifies shame and obstructs recovery. Awareness Integration Theory, a multidisciplinary therapeutic model, addresses these internalized beliefs by identifying and integrating fragmented aspects of the self across thoughts, emotions, behaviors, and promotes self-awareness, emotional regulation, and cognitive restructuring, critical components in reducing shame and fostering resilience. This paper explores the interplay between self-perception, shame, resilience, and biological predisposition in addiction recovery. Research shows that shame-prone individuals are more likely to relapse, while guilt-prone individuals demonstrate greater capacity for change. Resilience, cultivated through self-forgiveness, social support, and neuroplastic interventions, mitigates the impact of shame. Genetic variants and epigenetic modifications influence reward deficiency syndrome, increasing vulnerability to addiction. The Genetic Addiction Risk Severity test can identify at-risk individuals, enabling precision-targeted interventions. Awareness Integration Therapy's integrative framework complements genomic and neurobiological insights by fostering self-acceptance, enhancing insight into unconscious belief systems, and motivating purposeful action. Neuroimaging studies support the role of resilience-based practices, including those embedded in Alcoholics Anonymous, in promoting dopamine homeostasis and neural recovery. In conclusion, stigma reduction must advance alongside personalized medicine. Integrating Awareness Integration Therapy with genetic screening, trauma-informed care, and psychoeducation offers a comprehensive, compassionate approach. Reframing addiction as a brain-based, treatable condition empowers clients and families, facilitating sustainable recovery grounded in science and self-awareness.

Protein-protein interaction (PPI) is central to all cellular processes and play critical roles in both normal physiology and disease pathogenesis. In this brief guide, we outline the fundamental principles and widely used methods for PPI detection, focusing on 3 key techniques: immunoprecipitation, in vitro pull-down assays, and proximity ligation assay. We also discuss common experimental challenges and provide practical optimization strategies to improve reliability and reproducibility. This resource is designed to aid researchers in molecular and cellular biology, signal transduction, and animal model studies with essential knowledge for selecting and applying PPI detection methods.

Poly(ADP-ribose) (PAR) is a nucleic acid-like heterogeneous polymer in nature. Recently, it was found to engage in liquid-liquid phase separation (LLPS), generating condensates as an emerging class of subcellular structures with pivotal functions in response to stimuli. As a post-translational modification catalyzed by PAR polymerases (PARPs), PAR is known to modulate many key events in cells. However, its involvement in biomolecular condensation remains elusive. Through an imaging-based screening of small molecules with diverse biological activities, we here discovered that PAR undergoes LLPS upon inhibiting proteasome in different types of cells, resulting in co-condensation of PAR with proteasome and ubiquitin chains in nucleus. This unprecedented co-condensation is dependent on PARP2 not PARP1 and requires K6-linked ubiquitylation. PAR is shown for the first time to directly interact with ubiquitin chains. Notably, stalled DNA replication forks arose from proteasome inhibition are co-localized with PAR-proteasome-ubiquitin chain condensates. By attenuating replication and stabilizing stalled replication forks, PAR-proteasome-ubiquitin chain condensates sustain genomic integrity under proteasomal stress. This work demonstrates a self-protective mechanism in stressed cells and provides fundamental understanding of PAR condensation in cell biology.

Pancreatic beta cells contain insulin secretory granules (ISGs), organelles where proinsulin is converted into insulin. As ISGs mature, they undergo extensive biophysical remodeling, producing a spectrum of subpopulations with heterogeneous molecular and spatial characteristics. However, systematic methods to define ISG subpopulations remain underdeveloped. To address this gap in knowledge, we employed soft X-ray tomography (SXT), which can quantitatively measure the biochemical density of ISGs within whole beta cells. Using unsupervised clustering, we classified subpopulations based on molecular density, size, and spatial positioning. Across different insulin secretory stimuli, we observed shifts toward mature and releasable subtypes, demonstrating that exogenous signals can dynamically remodel ISG subpopulation distributions. We extended this methodology to primary beta cells characterized using volume electron microscopy (vEM). Integrating subpopulations from SXT and vEM uncovered insights inaccessible by a single method in isolation. This strategy establishes a framework for defining therapeutic approaches aimed at enriching physiologically beneficial ISG subpopulations.

This commentary discusses the prognostic relevance of leukocyte telomere length and paraoxonase-1 activity in small-cell lung cancer (SCLC) patients undergoing chemotherapy. It emphasizes the importance of integrating telomere biology and oxidative stress assessment in prognostic modeling. The discussion also considers the modifying effects of lifestyle, treatment regimens, and genetic background, advocating for research that combines clinical, biochemical, and molecular data to enhance prognostication in SCLC.

Melanoma, while less prevalent than other skin cancers, remains the most lethal and aggressive type, posing significant treatment challenges. Photodynamic therapy (PDT) offers a promising, less invasive alternative to conventional therapies. In this study, we explored the potential of methylene violet 3RAX (MV), a phenazine-family photosensitizer, for PDT applications through in vitro assays and Langmuir monolayer studies, focusing on its interactions with cell lipid extract membranes derived from two melanoma lineages, A375 and SH-4. Our results demonstrate that MV is non-cytotoxic in the absence of light irradiation but exhibits concentration-dependent cytotoxicity upon photoactivation. Flow cytometry confirmed late apoptosis as the dominant cell death pathway under irradiation. Langmuir isotherms revealed that MV adsorbs onto anionic head groups of the lipid monolayers, particularly interacting with phosphate groups, promoting molecular organization. Upon irradiation, significant material loss to the subphase was observed, suggesting photooxidative interactions with lipid tail unsaturations, leading to hydroperoxidation, chain cleavage, and membrane destabilization. These findings highlight MV dual role as an effective photosensitizer and a molecular probe for membrane interactions, providing new insights into its mechanisms of action in PDT.

Circular RNAs (circRNAs) are emerging regulators in cancer biology, yet the mechanisms underlying their biogenesis remain incompletely defined. RBM10, a splicing regulator frequently mutated in lung adenocarcinoma (LUAD), modulates RNA processing, but its involvement in circRNA regulation has not yet been addressed. Transcriptomic profiling of RBM10-restored LUAD cells, followed by RT-qPCR validation, identified circHIPK3 and circSMARCA5 as consistently RBM10-dependent circRNAs. Subcellular fractionation confirmed nuclear confinement of RBM10 and cytoplasmic enrichment of circRNAs, supporting a nuclear role for RBM10 in circRNA biogenesis. PAR-CLIP and RNA pulldown assays demonstrated direct RBM10 binding to intronic flanking regions of these circRNAs. Using a splicing reporter assay, we found that RBM10 binding to the 3' flanking region promotes exon skipping and circularization more efficiently than 5' binding, revealing a position-dependent mechanism controlling circRNA output. Analysis of RBM10 point mutants showed impaired regulation of circHIPK3 and circSMARCA5, linking defective exon skipping to disrupted circRNA formation. Functionally, modulation of circHIPK3 and circSMARCA5 phenocopied RBM10 restoration in mutant LUAD cell lines and rescued the tumorigenic phenotype driven by RBM10 loss. In two independent LUAD cohorts, circHIPK3 was consistently downregulated, particularly in RBM10-mutant tumors, and strongly correlated with RBM10 expression. Proteomic analyses further identified RBM10–SF3B1 interaction as a key upstream event governing circHIPK3 biogenesis. Together, these findings uncover a previously unrecognized mechanism through which RBM10 exerts tumor-suppressive functions via circRNA regulation and highlight circHIPK3 as a promising biomarker and potential therapeutic target in RBM10-deficient LUAD. The online version contains supplementary material available at 10.1186/s40364-026-00891-6. [Image: see text]

A class of organic, naturally occurring compounds known as phytohormones affects physiological functions in plants at trace levels. Phytohormones play important roles in plant growth, development, nutrient transport, and survival by regulating plant responses to abiotic stress and form the basis of ability of plants for adapting to changing conditions. To carry out some specific and distinct functions, cells of complex organisms implement gene regulation dynamics according to their cell types, including during the synthesis and control of various plant hormones. Rather than the traditional approaches for phytohormone quantification, single-cell RNA sequencing (scRNA-seq) and/or single-nucleus RNA sequencing (snRNA-seq) technologies that enable high-resolution mapping of cellular heterogeneity at the transcriptomic level, provide a novel approach for the phytohormone signaling. Here, we detailed the methodology of single cell/nucleus technologies to investigate underlying gene regulatory networks of plant hormone biology.

One of the most eye-catching events in cell biology is the condensation of chromosomes. During mitosis, the diffuse interphase chromatin rearranges into compact, rod-shaped chromosomes that can be precisely segregated between the daughter cells. This drastic reorganization of the DNA relies on condensin I and II complexes, large ring-shaped ATPases that extrude and stabilize loops of DNA. Since condensin II is always present in the nucleous its activity is repressed by the protein MCPH1 during interphase. But what are the molecular mechanisms regulating the activation of condensin II? New evidence suggests that this activation depends on the interaction between the condensin II subunit CAP-G2 with the centromeric protein M18BP1. Both the repressor and the activator bind on the same subunit of condensin II and their alternative binding is regulated by phosphorylation, which acts as the signal to trigger DNA condensation by condensin II.

Immune protection against coronavirus disease 2019 (COVID-19) relies, along with cellular immunity, on anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies. We studied the effect of IgG avidity, the average antibody binding strength, on anti-SARS-CoV-2 neutralizing antibodies (nAbs), often considered the hallmark of effective immunity. Prior studies estimating the significance of avidity for nAb-mediated immunity have been complicated by the fact that not only the quality but also the quantity of antibodies impacts the results. Here we provide means for quantifying the impact of IgG avidity on neutralization, irrespective of antibody titer. We introduce for anti-SARS-CoV-2 spike protein (S) and nucleoprotein (N) antibodies, IgG avidity assays shown to be unaffected by the IgG concentration. Hospitalized (n = 14) and nonhospitalized (n = 14) COVID-19 patients and vaccinees (n = 20) of early 2020 were assayed for Wuhan S-IgM, S-IgA, S-IgG, and S-IgG avidity; Wuhan N-IgG and N-IgG avidity; and Wuhan, Beta, and Delta nAbs, to identify the factors contributing to neutralization efficiency. N-IgG avidity was superior to S-avidity in pinpointing the time of SARS-CoV-2 primary infection. Both Wuhan nAb and Delta nAb correlated, expectedly, with Wuhan S-IgG level (P < .0001 each). Wuhan S-IgG avidity intensified homologous (Wuhan; P = .001) but not significantly heterologous (Delta; P = .053) neutralization. Accordingly, along with postinfection time, the average neutralization efficiency of S-IgG molecules increased while their concentration decreased. Quantitatively, doubling of Wuhan S-IgG avidity, at constant S-IgG quantity, augmented Wuhan neutralization 1.58- to 1.68-fold. Comprehensive serological profiles of early SARS-CoV-2 primary infections and immunizations provided a model showing that the antiviral neutralization potency is enhanced by anti-spike IgG avidity. The methodology presented is applicable widely beyond COVID-19.

Chimeric antigen receptor (CAR) T-cell (CAR-T) therapy is a transformative modality in cancer immunotherapy that employs genetically engineered T-cells to eliminate malignant cells selectively. Its efficacy and limitations are governed by cytokine- and growth factor-mediated signaling networks that shape T-cell activation, proliferation, differentiation, and persistence. This review traces the molecular evolution of CAR-T architecture across generations, highlighting how synthetic modulation of cytokine and co-stimulatory pathways enhances potency while reducing exhaustion and toxicity. We discuss strategies that incorporate cytokine engineering, metabolic reprogramming, and logic-gated activation to counteract the immunosuppressive tumor microenvironment. Recent technological advances-such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-based cytokine pathway editing, induced pluripotent stem cell (iPSC)-derived "off-the-shelf" CAR-T platforms, and extracellular vesicle (EV)-mediated cytokine delivery-are reshaping adoptive immunotherapy. Framing CAR-T development through the lens of cytokine and growth factor biology, we outline how integrating these pathways enables safer, more durable, and scalable next-generation therapies for hematologic and solid tumors.

The pancreatic &#x3b2;-cell contains several functional subpopulations of insulin secretory granules (ISGs). These subpopulations vary in maturity, age, and secretory capacity. Differences in protein and lipid composition of ISGs are correlated with disease but require further study to understand how ISG remodeling regulates normal biology. Due to limitations in traditional separation methods, the extent of these subpopulations, any overlap between them, and how they are affected by insulinotropic signals have not been determined. In this work, we adapted direct current insulator-based dielectrophoresis (DC-iDEP) to separate ISGs isolated from INS-1E cells, an immortalized rat insulinoma cell line model, according to their electrokinetic mobility ratio (EKMr). We were able to separate ISG subpopulations from unstimulated cells to determine a baseline distribution and identify characteristic profiles for immature, young, and old ISGs. We then analyzed distributions of subpopulations from cells stimulated with insulin secretion signals known to induce biophysical remodeling and maturation. We found significant changes in each subpopulation studied in response to stimulation, consistent with the increases in maturation, crystallization, and changes in size reported in the literature. This work provides new insights into how the cell controls ISG remodeling and may drive future development of more effective therapies.

Abelson-1 (ABL-1) is a nonreceptor tyrosine kinase that plays essential roles in various cellular processes, including proliferation, survival, differentiation and its kinase activity is tightly regulated. The dysregulated ABL-1 kinase activity is linked to disease pathogenesis like Chronic Myeloid Leukemia (CML), where the BCR::ABL-1 fusion oncoprotein drives oncogenic signaling. Due to its central role in CML pathogenesis, understanding the structure of ABL-1 is crucial for the effective management of the disease and drug development studies. This study focuses on optimizing the expression, purification and crystallization of the recombinant human ABL-1 kinase domain for its structural analysis via X-ray crystallography and structure-based drug screening applications. The human ABL-1 kinase domain, fused with a SUMO-tag, was expressed in Escherichia coli Rosetta2 BL21 using the pET28(a)+ expression vector. The ABL-1 aggregates seen under native culture conditions were successfully solubilized by the mild ionic detergent sarkosyl. After obtaining soluble expression of the protein, Ni-NTA affinity chromatography was performed and high yield of purified ABL-1 was obtained. The 6X-His-SUMO-tag of purified ABL1 was cleaved by ULP1 protease. The recombinant ABL-1 was subsequently used in crystallization trials to enlighten structural features of ABL-1 that could guide the development of novel therapeutics and drug screening platforms targeting ABL-1 in CML.

Cancer cells face a hostile microenvironment characterized by hypoxia, nutrient deprivation, endoplasmic reticulum (ER) stress, and oxidative imbalance. To cope with these challenges, they activate an interconnected network of adaptive pathways including autophagy, the unfolded protein response, metabolic reprogramming, and the integrated stress response., which promote cell survival, therapy resistance, immune evasion, and metastasis. CRISPR-based functional genomics has emerged as a powerful strategy to systematically dissect these stress-adaptive networks, enabling the identification of key regulators and vulnerabilities across diverse contexts. In this review, we first summarize tumor progression in major stress conditions and then highlight how CRISPR screening strategies ranging from genome-wide loss-of-function studies to single-cell and combinatorial platforms, are unraveling critical stress regulators. We further discuss emerging tools, model systems, and translational perspectives, underscoring how the integration of CRISPR technologies with multi-omics, artificial intelligence, and advanced preclinical models is reshaping our understanding of cancer stress biology and guiding the development of novel therapeutic strategies. Finally, we addressed how these novel dissection technologies influence translational opportunities, specifically in the context of combining stress-pathway modulators with immunotherapy and targeted therapy drugs.

Across biological subdisciplines, the last decade has seen an explosion of high-dimensional datasets. At the ICTS workshop &#x2018;Unifying Theories in High-Dimensional Biophysics&#x2019;, we discussed whether this high dimensionality poses a challenge or an opportunity for theoretically describing, understanding and predicting biological systems. We discussed methods, models and frameworks that can be used for this purpose. This Comment summarizes our discussions from the perspectives of individual participants.

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Since 2000, rates of suicide and opioid overdose have sharply increased. Approximately one-third of individuals with major depressive disorder (MDD) experience treatment-resistant depression (TRD), highlighting the urgent need for novel therapeutic approaches. This review synthesizes pivotal preclinical and clinical findings on low-dose ketamine's rapid antidepressant effects and examines proposed mechanisms underlying its therapeutic action. This is a narrative review of key contributions in the literature addressing ketamine's fast-acting antidepressant properties. Low-dose ketamine rapidly alleviates depressive symptoms, including in refractory depression. Despite multiple hypotheses supported by preliminary data, there is no consensus regarding its definitive mechanism of action. Proposed mechanisms include modulation of dopamine signaling via epigenetic neuroadaptation, interactions with D1/D2 receptor systems, optogenetic activation of D1 pathways, and alterations in D2/D3 receptor availability. Elucidating ketamine's mechanism of action may inform the development of next-generation psychoplastogens that promote neural plasticity in TRD and unipolar MDD. However, ketamine's psychoactive properties and abuse potential, along with concerns regarding misuse and diversion, underscore the need for enhanced clinical oversight and regulatory frameworks. A recent editorial in the British Medical Journal (BMJ) highlighted a dramatic rise in non-prescribed ketamine use in the UK, with over 3,600 individuals seeking treatment for ketamine addiction in 2023&#x2013;2024, an eightfold increase compared to a decade earlier. This trend is not isolated to the UK; misuse has surged across Europe and the United States. It is worth noting that as early as 2015, the BMJ cautioned against overstating ketamine&#x2019;s antidepressant benefits and warned of its methodological flaws and risks of misuse, warnings that appear to have been largely overlooked (Zhang & Ho, 2015). Once confined to occasional recreational use in nightlife settings, ketamine is now frequently self-administered at home, often daily, by individuals attempting to manage symptoms of anxiety, depression, PTSD, or chronic stress without medical oversight.Despite its therapeutic promise at controlled doses, ketamine carries well-established risks. A 2016 publication by one of the present authors outlined these concerns early on. The BMJ editorial affirms ketamine-associated urotoxicity, which can manifest as urinary incontinence, severe bladder pain (commonly referred to as &#x201c;K-cramps&#x201d;), and reduced bladder capacity, sometimes necessitating surgical intervention, including bladder removal. The editorial also reported new adverse outcomes from chronic use, including cognitive deficits, affective disturbances (e.g., anxiety, depression, psychosis), gastrointestinal complications, and renal injury. Ceban et&#xa0;al. (CNS Drugs, 2021) reported that while most ketamine- and esketamine-related adverse effects are mild and transient, clinicians should remain vigilant regarding potential long-term toxicity and abuse liability. Ng et&#xa0;al. (Psychopharmacology, 2021) further demonstrated that chronic or high-frequency ketamine exposure may lead to urothelial inflammation and bladder dysfunction, highlighting the importance of monitoring genitourinary symptoms during treatment (Ceban et&#xa0;al., 2021; Ng et&#xa0;al., 2021). Though ketamine-related fatalities remain comparatively low, the numbers are steadily rising. The drug remains accessible and inexpensive, often costing less than cocaine. DEA data show a sharp increase in trafficking activity, with ketamine seizures increasing by 349% between 2017 and 2022.Medically, ketamine has long been used as an anesthetic and, at lower doses, is prescribed for chronic pain, suicidality, and treatment-resistant depression. In the U.S., intranasal esketamine is FDA-approved for depression. However, findings from earlier research by Dr. Linda Cottler (University of Florida) and Dr. Joseph Palamar (NYU) raise concerns. Palamar&#x2019;s group documented a consistent increase in nonmedical ketamine use and exposures between 2006 and 2019. Cottler&#x2019;s team, using U.S. National Poison Data System records from 2019 to 2021, noted rising poisonings involving ketamine, despite its relatively low prevalence at the time. Alarmingly, nearly 40% of those cases involved intentional misuse or suicide attempts. Palamar&#x2019;s 2025 follow-up study confirmed that this trend has continued through 2023.The study by Zhang et&#xa0;al. (2017) found that between 2000 and 2015, North American print media coverage of ketamine shifted significantly, from predominantly emphasizing its abuse potential to increasingly portraying it as a promising antidepressant treatment. From 2008 to 2015, articles were notably more likely to highlight ketamine&#x2019;s efficacy for treatment-resistant depression and its superiority over conventional antidepressants, suggesting that evolving media narratives may have contributed to its growing popularity and off-label clinical use (Zhang et&#xa0;al., 2017).Polysubstance use is also a concern. Ketamine is often co-ingested with benzodiazepines, alcohol, opioids, stimulants like cocaine, and club drugs including MDMA, LSD, and GHB. This pattern of co-use dramatically raises the risk of overdose and death, particularly when injected. Cottler and colleagues stressed that ketamine poisonings rarely occur in isolation but are part of a larger polysubstance crisis. Ketamine is also prescribed off-label for pain conditions such as Complex Regional Pain Syndrome (CRPS), where transient benefits may be tied to NMDA receptor blockade. These alternative applications, while promising in some cases, highlight unresolved questions about the drug&#x2019;s durability, mechanism of action, and potential for misuse across both psychiatric and pain-related domains.

Bilacunaria microcarpa, a traditionally consumed yet underexplored species of the Apiaceae family, was evaluated for its neuroprotective potential in an in vitro Parkinson's disease model induced by 1-methyl-4-phenylpyridinium (MPP&#x207a;). Differentiated SH-SY5Y neuronal cells were co-treated with MPP&#x207a; and aqueous extracts derived from the plant's flowers, stems, and leaves. Cell viability was assessed using the MTT assay, while nuclear morphology was examined via Hoechst 33258 staining. Enzymatic activities of AChE and caspase-3 were analyzed to investigate cholinergic and apoptotic responses, respectively. The antioxidant and oxidant status of the samples was determined by measuring the total antioxidant status and total oxidant levels. Chemical profiling analysis by HPLC-DAD identified chlorogenic acid as the predominant compound across all plant parts. The extracts demonstrated substantial enhancement in cell viability and were non-cytotoxic in fibroblast cultures. Moreover, all sample extracts caused a statistically significant reduction in caspase&#x2009;-&#x2009;3 activity (p&#x2009;<&#x2009;0.05). Furthermore, in silico blood-brain barrier permeability predictions indicated that some phytochemicals present in the extracts, such as resveratrol, o-coumaric acid, hydroxybenzoic acid, and vanillin, have the potential to permeate the blood-brain barrier. These outcomes indicate that Bilacunaria microcarpa exhibits considerable potential as a neuroprotective agent, warranting further exploration as a candidate for the development of therapeutic interventions for Parkinson's disease.