Cell DNA Research Articles

LIG1 is a synthetic lethal target in BRCA1 mutant cancers.

Synthetic lethality approaches in BRCA1/2-mutated cancers have focused on poly(ADP-ribose) polymerase (PARP) inhibitors, which are subject to high rates of innate or acquired resistance in patients. Here, we used CRISPR/Cas9-based screening to identify DNA Ligase I (LIG1) as a novel target for synthetic lethality in BRCA1-mutated cancers. Publicly available data supported LIG1 hyperdependence of BRCA1-mutant cells across a variety of breast and ovarian cancer cell lines. We used CRISPRn, CRISPRi, RNAi, and protein degradation to confirm the lethal effect of LIG1 inactivation at the DNA, RNA, and protein level in BRCA1-mutant cells in vitro. LIG1 inactivation resulted in viability loss across multiple BRCA1-mutated cell lines, whereas no effect was observed in BRCA1/2 wild-type cell lines, demonstrating target selectivity for the BRCA1-mutant context. On-target nature of the phenotype was demonstrated through rescue of viability with exogenous wild-type LIG1 cDNA. Next, we demonstrated a concentration-dependent relationship of LIG1 protein expression and BRCA1 mutant cell viability using a titratable, degradable LIG1 fusion protein. BRCA1 mutant viability required LIG1 catalytic activity, as catalytically dead mutant LIG1K568A failed to rescue viability loss caused by endogenous LIG1 depletion. LIG1 perturbation produced proportional increases in PAR staining in BRCA1 mutant cells, indicating a mechanism consistent with the function of LIG1 in sealing ssDNA nicks. Finally, we confirmed LIG1 hyperdependence in vivo using a xenograft model in which LIG1 loss resulted in tumor stasis in all mice. Our cumulative findings demonstrate that LIG1 is a promising synthetic lethal target for development in patients with BRCA1 mutant cancers.

Evaluating H2BC9 as a potential diagnostic and prognostic biomarker in head and neck squamous cell carcinoma

BackgroundHistone H2B is highly expressed in many types of cancers and is involved in cancer development. H2B clustered histone 9 (H2BC9), a member of the H2B family, plays critical roles in gene expression regulation, chromosome structure, DNA repair stability, and cell cycle regulation. However, the diagnostic and prognostic value of H2BC9 in head and neck squamous cell carcinoma (HNSCC) remains unclear. This study aimed to evaluate the potential diagnostic and prognostic value of H2BC9 in HNSCC and investigate its biological role using bioinformatics.MethodsThe expression pattern and diagnostic value of H2BC9 in HNSCC were explored using UCSC Xena and GEO database. H2BC9 expression was validated using the Human Protein Atlas database, qRT-PCR, and western blotting. Prognostic value was assessed using Kaplan–Meier curves, Cox regression analysis, and a nomogram. Drug sensitivity was predicted using the R package pRRophetic, and molecular interactions were analyzed using the DepMap database. The impact of H2BC9 on HNSCC cells was further investigated through in vitro experiments.ResultsH2BC9 was markedly upregulated in HNSCC cell lines and tissues. High expression of H2BC9 was correlated with advanced-stage disease and poor prognosis. KEGG analysis linked H2BC9 to cell cycle regulation and DNA replication. H2BC9 expression influenced the drug sensitivity of paclitaxel, docetaxel, cisplatin, and 5-fluorouracil. Key molecules, such as TONSL, PITX2, NOTCH1, and H2BC10, were positively correlated with H2BC9 expression. Silencing H2BC9 suppressed cell proliferation, induced G2/M cell cycle arrest, and enhanced apoptosis and DNA damage in HNSCC cells.ConclusionWe demonstrated that H2BC9 expression may be associated with HNSCC development and prognosis. These findings may provide a potential therapeutic target for HNSCC.

Propidium Monoazide is Unreliable for Quantitative Live-Dead Molecular Assays.

Propidium monoazide (PMA) is a dye that distinguishes between live and dead cells in molecular assays like the Polymerase Chain Reaction (PCR). It works by cross-linking to the DNA of cells that have compromised membranes or extracellular DNA upon photoactivation, making the DNA inaccessible for amplification. Currently, PMA is used to detect viable pathogens and alleviate systemic bias in the microbiome analysis of samples using 16S rRNA gene sequencing. In these applications, treated samples consist of different amounts of dead bacteria and a range of bacterial strains, variables that can affect the performance of PMA and lead to inconsistent findings across various research studies. To evaluate the effectiveness of PMA, we used a sensitive qPCR assay and post-treatment sample concentration to determine PMA cross-linkage and activity accurately under varying sample conditions. We report that PMA is unreliable for viability assays when the concentration and composition of the bacterial mixtures are unknown. PMA is suitable only for qualitatively assessing viability in samples containing a known number of dead microbes or extracellular DNA.

Investigations on the effects of in vitro exposure of mouse ovaries to withaferin A, a new candidate for chemotherapy.

This study aimed to investigate, in vitro, the toxicity of WTA on ovarian follicles. Initially, a cytotoxicity assay was conducted using tumor and non-tumor cell lines to determine the IC50of the WTA and validate its antitumor activity. Mouse ovaries were cultured in vitro (IVC) for 6 days in the presence of 1% dimethyl sulfoxide (DMSO), doxorubicin at 0.3µg/ml (DXR), or WTA at 0.6µM or 6.0µM. DXR or WTA were added to the IVC medium once (1DXR, 1WTA0.6, 1WTA6.0) or three times (3DXR, 3WTA0.6, 3WTA6.0). After the IVC, the ovarian stroma, follicular morphology and development, cell proliferation, senescence, DNA damage, and apoptosis were assessed. The degeneration rate in 3DXR and WTA6.0 (1x and 3x) was higher (p < 0.05) compared to the DMSO group. 1DXR and 3WTA0.6 reduced (p < 0.05) the percentage of primordial follicles and increased (p < 0.05) the number of developing follicles compared to the control (CTR) and DMSO groups. An increase (p < 0.05) in lipofuscin granules was observed with DXR and WTA at both concentrations and exposure frequencies compared to the CTR. In the presence of 3WTA0.6, staining for cleaved caspase-3 was more pronounced (p < 0.05). Additionally, 3WTA0.6, 1WTA6.0, and 3DXR increased (p < 0.05) DNA fragmentation in the stroma compared to the CTR and DMSO groups. We conclude that, like chemotherapy agents used for cancer treatment, WTA induces severe cytotoxic effects on ovarian follicles and stroma, especially at high concentrations and exposure frequencies.

RIOK1: A Novel Oncogenic Driver in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common and highly lethal cancers worldwide. RIO kinase 1 (RIOK1), a protein kinase/ATPase that plays a key role in regulating translation and ribosome assembly, is associated with a variety of malignant tumors. However, the role of RIOK1 in HCC remains largely unknown. Changes in RIOK1 expression in HCC and patient prognosis were evaluated using HCC tissues and public databases. The functional role of RIOK1 in HCC was analyzed by RTCA assay, clonogenic assay, and flow cytometry invitro, and by mouse tumor xenograft model invivo. Potential mechanism studies were performed using multi-omics analysis, public database screening, and qRT-PCR assay. In this study, we found that RIOK1 was elevated in HCC tissues and correlated with poor prognosis. Functional assays demonstrated that RIOK1 knockdown suppressed HCC cell proliferation, survival, and tumor growth invivo, while RIOK1 overexpression enhanced these oncogenic phenotypes. Meanwhile, RIOK1 knockdown affected cell cycle progression and the expression of cyclin A2 and cyclin B1. Furthermore, integrated transcriptomic and proteomic analysis revealed that RIOK1 may promote HCC cell proliferation by affecting the cell cycle and DNA repair pathways. Moreover, we identified five potential effectors regulated by RIOK1: PMS1, SPDL1, RAD18, BARD1, and SMARCA5, which were highly expressed in HCC tissues and negatively correlated with the overall survival of HCC patients. Our findings suggest that RIOK1 is a novel oncogenic driver that may serve as a potential diagnostic and therapeutic target for HCC.

Retinol-Binding Protein 4 as a Biomarker in Cancer: Insights from a Pan-Cancer Analysis of Expression, Immune Infiltration, and Methylation

Background: Retinol-binding protein 4 (RBP4) is primarily recognized for its role in retinoid transport, but has recently been implicated in cancer progression and prognosis. However, a comprehensive pan-cancer analysis of RBP4’s expression, prognostic significance, and functional associations across various cancers is lacking. Methods: We conducted a pan-cancer analysis of RBP4 using data from public databases. RBP4 expression levels were examined in 33 tumor types, and correlations with clinical outcomes, immune cell infiltration, DNA methylation, and gene mutations were assessed. Enrichment analyses of RBP4 and its co-expressed genes were performed to explore associated biological pathways. Additionally, in vitro experiments were conducted to assess the effects of RBP4 on cell migration and proliferation. Results: RBP4 showed differential expression between tumor and normal tissues, with downregulation in 21 cancer types and upregulation in 6. High expression levels of RBP4 were associated with poor overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) in specific cancers, notably in BRCA, HNSC, and STAD, whereas it was a favorable prognostic factor in cancers such as KIRP and MESO. RBP4 expression was also associated with immune cell infiltration, particularly with CD4+ Th2 cells and immune checkpoint genes. DNA methylation analysis suggested that the methylation of RBP4 may play a role in its regulatory mechanisms across cancer types. Enrichment analyses revealed that RBP4 and its co-expressed genes are involved in metabolism-related pathways and immune regulation. Functional assays indicated that RBP4 knockdown promoted tumor cell migration and proliferation. Conclusions: This study provides a comprehensive pan-cancer analysis of RBP4, identifying its prognostic potential and possible involvement in tumor immunity and metabolism. Our findings suggest that RBP4 could serve as a novel biomarker and therapeutic target in cancer, although further experimental studies are required to elucidate its precise mechanisms in specific cancer types.

Identification of glycogen synthase kinase 3alpha/beta as a host factor required for hepatitis B virus transcription using high-throughput screening.

Hepatitis B virus (HBV) leads to severe liver diseases, such as cirrhosis and hepatocellular carcinoma. Identification of host factors that regulate HBV replication can provide new therapeutic targets. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV entry receptor has enabled the establishment of hepatic cell lines for analyzing HBV infection and propagation. Using this new system, studies aimed at identifying host factors that regulate HBV propagation have increased. We established an HBV-based-reporter gene expression system that mimics HBV replication from transcription to virus egress. Using this approach, we screened 1,827 Food and Drug Administration-approved compounds and identified glycogen synthase kinase 3 (GSK3)alpha/beta inhibitors, including AZD1080, CHIR-98014, CHIR-98021, BIO, and AZD2858, as anti-HBV compounds. These compounds suppressed hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) production in HBV-infected human primary hepatocytes. Proteome analysis revealed that GSK3alpha/beta phosphorylated forkhead box K1/2 (FOXK1/2)s. A double-knockout of FOXK1/2 in HBV-infected HepG2-NTCP cells reduced HBeAg and HBsAg production. The rescue of FOXK2 expression, but not FOXK1 expression, in FOXK1/2-double-knockout cells restored HBeAg and HBsAg production. Importantly, phosphorylation of FOXK2 at Ser 424 is required for GSK3alpha/beta-mediated HBeAg and HBsAg production. We observed the binding of FOXK2 to HBV DNA in HepG2-NTCP cells. Our recombinant HBV-based screening system enables the discovery of new targets. Using our approach, we identified GSK3 inhibitors as potential anti-HBV agents.

Retention mechanism in slalom chromatography: Perspectives on the characterization of large DNA and RNA biopolymers in cell and gene therapy.

Significant progress has been made in the last two decades in producing small (<2μm), high-purity, and low-adsorption particles, columns and system hardware, for ultra-high pressure liquid chromatography (UHPLC). Simultaneously, the recent rapid expansion of cell and gene therapies for treating diseases necessitates novel analytical technologies for analyzing large (>2 kbp) plasmid double-stranded (ds) DNA (which encodes for the in vitro transcription (IVT) of single-stranded (ss) mRNA therapeutics) and dsRNAs (related to IVT production impurities) biopolymers. In this context, slalom chromatography (SC), a retention mode co-discovered in 1988, is being revitalized using the most advanced column technologies for improved determination of the critical quality attributes (CQAs) of such new therapeutics. In this review, we first recall non-exhaustively the main currently available analytical techniques (enzyme-linked immunosorbent assay (ELISA), agarose gel electrophoresis (AGE), pulse field gel electrophoresis (PFGE), capillary gel electrophoresis (CGE), mass photometry (MP), anion-exchange chromatography (AEX), ion-pairing reversed-phase liquid chromatography (IP-RPLC), hydrophobic interaction chromatography (HIC), size-exclusion chromatography (SEC), hydrodynamic chromatography (HDC), highly converging flow ultra-filtration (HCF-UF), asymmetrical flow field-flow fractionation (AF4), mass spectrometry (MS), and atomic force microscopy (AFM)) for analyzing mixtures containing large nucleic acid biopolymers, while assessing their strengths and weaknesses. We then focus comprehensively on the SC technique, report on its past applications since its birth, and review in detail the history and evolution of the proposed retention mechanisms accounting for the observations made in SC. This includes and emphasizes the latest physico-chemical insights (shear rates in packed HPLC columns, entropic elasticity and relaxation of dsDNA, dsRNA, and mRNA biopolymers) governing the retention behavior of such biopolymers in SC. Finally, based on the recent advancements in understanding the fundamentals of retention in SC, we provide some perspectives and recent proof-of-concept for the analytical characterization by SC of large dsDNAs (plasmid digests, polymerase chain reaction (PCR) verification), the separation of supercoiled/circular and linear dsDNAs (plasmid linearization), the isolation and quantification of large dsRNAs impurities present in mRNA samples produced by IVT, and the differentiation between dsRNA conformers.

Optimized protocol for single-cell isolation and alkaline comet assay to detect DNA damage in cells of Drosophila wing imaginal discs.

Reduced expression of nucleolar genes induces stress and DNA damage. Here, we present a protocol to analyze DNA fragmentation at the single-cell level in Drosophila imaginal discs using an optimized alkaline comet assay. We describe steps for larvae development, tissue disaggregation, and single-cell dissociation. We then detail procedures for cell lysis, electrophoresis, and DNA visualization. This approach provides insights into the molecular consequences of nucleolar stress induction and its role in the DNA damage response pathways invivo.

PLK3 weakens antioxidant defense and inhibits proliferation of porcine Leydig cells under oxidative stress

Aging is characterized by cellular degeneration and impaired physiological functions, leading to a decline in male sexual desire and reproductive capacity. Oxidative stress (OS) lead to testicular aging by impairing the male reproductive system, but the potential mechanisms remain unclear. In the present study, the functional status of testicular tissues from young and aged boars was compared, and the transcriptional responses of Leydig cells (LCs) to hydrogen peroxide (H2O2)-induced senescence were explored, revealing the role of OS in promoting aging of the male reproductive system. 601 differentially expressed genes (DEGs) associated with OS, cell cycle regulation, and intracellular processes were identified. These DEGs were significantly enriched in critical aging pathways, including the p53 signaling pathway, autophagy, and cellular senescence. Protein-protein interaction (PPI) network analysis unveiled 15 key genes related to cell cycle and DNA replication, with polo-like kinase 3 (PLK3) exhibiting increased expression under OS. In vitro, PLK3 knockdown significantly enhanced the viability and antioxidant capacity of LCs under OS. This study deepens our understanding of how LCs respond to OS and provides new therapeutic targets for enhancing cellular resistance to oxidative damage and promoting tissue health.

Short-term exposure of 2.4 GHz electromagnetic radiation on cellular ROS generation and apoptosis in SH-SY5Y cell line and impact on developing chick embryo brain tissue.

Electromagnetic radiation (EMR) from wireless technology and mobile phones, operates at various frequencies. The present study analyses the major impact of short-term exposure to 2.4GHz frequency EMR, using the two model systems chick embryos and SH-SY5Y cell lines. We hypothesized that exposure to this frequency would induce oxidative stress and apoptosis in neurons. Chick embryos were exposed continuously to 2.4GHz EMR for 4h each day over a 5-day period, and comparisons were made with a control group. At the end of the exposure, brain tissues were dissected for histopathological analysis, antioxidant assays, and reactive oxygen species (ROS) detection. Additionally, SH-SY5Y cells were exposed to 2.4GHz EMR to assess cell viability, DNA damage, and apoptosis. Our results showed that exposure to 2.4GHz EMR induces oxidative stress in both chick embryos and the SH-SY5Y cells, though no significant tissue-level impact was observed. In SH-SY5Y cells, ROS production increased after 4h of exposure, accompanied by moderate DNA damage and early markers of apoptosis, such as upregulation of the Bax gene. Furthermore, we observed that antioxidants, such as NAC and Mito-TEMPO, helped mitigate the cytotoxic effects of EMR in both the study models. In conclusion, short-term exposure (4h) to 2.4GHz EMR induced moderate cellular and molecular changes, primarily oxidative stress. The oxidative stress was reduced by antioxidants, which suggests potential benefits in preventing EMR-induced cytotoxicity. Extended exposure to EMR beyond 4h may pose adverse health risks to humans, endorsing further investigation.

Unveiling cell-type-specific microRNA networks through alternative polyadenylation in glioblastoma

BackgroundGlioblastoma multiforme (GBM) is characterized by its cellular complexity, with a microenvironment consisting of diverse cell types, including oligodendrocyte precursor cells (OPCs) and neoplastic CD133 + radial glia-like cells. This study focuses on exploring the distinct cellular transitions in GBM, emphasizing the role of alternative polyadenylation (APA) in modulating microRNA-binding and post-transcriptional regulation.ResultsOur research identified unique APA profiles that signify the transitional phases between neoplastic cells and OPCs, underscoring the importance of APA in cellular identity and transformation in GBM. A significant finding was the disconnection between differential APA events and gene expression alterations, indicating that APA operates as an independent regulatory mechanism. We also highlighted the specific genes in neoplastic cells and OPCs that lose microRNA-binding sites due to APA, which are crucial for maintaining stem cell characteristics and DNA repair, respectively. The constructed networks of microRNA-transcription factor-target genes provide insights into the cellular mechanisms influencing cancer cell survival and therapeutic resistance.ConclusionsThis study elucidates the APA-driven regulatory framework within GBM, spotlighting its influence on cell state transitions and microRNA network dynamics. Our comprehensive analysis using single-cell RNA sequencing data to investigate the microRNA-binding sites altered by APA profiles offers a robust foundation for future research, presenting a novel approach to understanding and potentially targeting the complex molecular interplay in GBM.

PI3K/mTOR dual inhibitor GSK458 and arsenic trioxide exert synergistic anti-tumor effects against ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC), particularly advanced or recurrent settings, is generally resistant to platinum-based chemotherapy, warranting novel therapeutic strategies. Mutations in the phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin kinase (PI3K/AKT/mTOR) pathway are frequently reported in OCCC. Therefore, we hypothesized that the PI3K/mTOR dual inhibitor, GSK458, and arsenic trioxide may exert synergistic anti-tumor effects on OCCC. We investigated the effects of GSK458, arsenic trioxide, and the combination of GSK458 and arsenic trioxide on cell viability, colony formation, and apoptosis in seven OCCC cells. Mechanistically, transcriptomic differences were assessed among the groups. Additionally, their anti-tumor effects were evaluated on the three-dimensional cultures of OCCC patient-derived xenografts as well as in vivo. Low-dose combination of GSK458 and arsenic trioxide exerted synergistic anti-tumor effects in vitro. Viability of the three-dimensional OCCC patient-derived xenograft cultures treated with the combination of GSK458 and arsenic trioxide decreased to 23.8% of that of the control. RNA sequencing revealed that the mechanism was associated with cell cycle and DNA damage repair. The combination of GSK458 and arsenic trioxide synergistically inhibited the PI3K/AKT/mTOR pathway and angiogenesis and increased apoptosis. Compared to any monotherapy, the combination treatment significantly suppressed tumor growth in vivo, thereby enhancing survival. Overall, our findings highlight the potential of the novel combination of GSK458 and arsenic trioxide combination for OCCC treatment.

12-Hydroxy-Lauric Acid Tethered Self-Assembled Heterochiral Diphenylalanine-Based Mechanoresponsive and Proteolytically Stable Hydrogel: A Dual Player for Handling Cancer and Bacterial Challenges.

Deciphering the most promising strategy for the evolution of cancer patient management remains a multifaceted, challenging affair to date. Additionally, such approaches often lead to microbial infections as side effects, probably due to the compromised immunity of the patients undergoing such treatment. Distinctly, this work delineates a rational combinatorial strategy harnessing stereogenic harmony in the diphenylalanine fragment, tethering it to an amphiphile 12-hydroxy-lauric acid at the N-terminus (compounds I-III) such that a potential therapeutic could be extracted out from the series. Aligned to the goal, the cytotoxic properties and cell viability of the compounds were investigated using two distinct cell lines: MCF-7 (human breast cancer cell) and HEK 293 (human embryonic kidney). Our rigorous investigations revealed that compounds I-III exhibited substantial cytotoxic impact on the MCF7 cell line. But from a pool of three constructs, compound III (12-hydroxy-lauric acid -d-Phe-l-Phe-OH) showed better selectivity toward cancerous MCF7 over normal HEK 293 in comparison to others, backed by computational calculations. Henceforth, it was fished out from the series and used for its elaborate anticancer activities using cell reactive oxygen species generation, DNA fragmentation, and caspase-dependent gene expression employing extrinsic and intrinsic apoptotic factors as well as inflammatory biomarkers, namely, TNF-α and IL1-β. We anticipated that compound III, possessing mechanoresponsiveness and a nanofibrillar network, could be administered in patients with injections because of its shear-thinning properties. Moreover, the optimum partition coefficient of compound III might have allowed the scaffold to penetrate the cellular membranes and form a dilactate complex (compound VI) when exposed to the accumulated lactates/lactic acids, a common phenomenon observed within the hypoxic cancerous tumor cores, in accordance with the Warburg mechanism, thereby evading the cytotoxicity within normal cells. Besides, the supramolecular β-sheets of compound III manifest substantial antimicrobial efficacy against the common pathogens, two Gram-positive bacteria, S. aureus and B. subtilis, two Gram-negative bacteria, E. coli and P. aeruginosa, and a fungus, C. albicans, along with proteolytic stability and high mechanical strength at physiological pH. Overall, we speculate that the discovery of these multifunctional bioinspired materials holds future promise as preferential therapeutics for the remediation of immune-susceptible cancer patients, afflicted by microbial infections arising alone or as side effects of chemotherapeutic medications.

The impact of obesity on mitochondrial dysfunction during pregnancy.

Mitochondria play a central role in nutrient metabolism, besides being responsible for the production of adenosine triphosphate (ATP), the main source of cellular energy. However, the ATP production process is associated with the generation of reactive oxygen species (ROS), which excessive accumulation can cause mitochondrial dysfunction. This dysfunction, in turn, causes the accumulation of fatty acids in the adipose tissue, triggering a local inflammatory process that can evolve into systemic inflammation. In women with obesity, an increase in lipid levels in the placental environment is observed. The high presence of fatty acids compromises the structural integrity and mitochondrial membrane, culminating in the release of ROS. This process damages the DNA of placental cells and causes an inflammatory state, affecting metabolic efficiency. This vicious cycle is characterized by defects in mitochondrial ATP production, which can lead to lipid accumulation and inflammation. In pregnant women with obesity, these mitochondrial changes play a determining role in pregnancy outcomes. Hence, the objective of this study was to search the literature to review the impact of mitochondrial dysfunction in the maternal obesity.

Identification of MORF4L1 as an endogenous substrate of CRBN and its potential role as a therapeutic target in cancer

The ubiquitin-proteasome system (UPS) is essential for cellular homeostasis, regulating the degradation of proteins involved in key processes such as cell cycle, apoptosis, and DNA repair. Dysregulation of the UPS is implicated in hepatocellular carcinoma (HCC), contributing to tumor progression and therapeutic resistance. The cereblon (CRBN) E3 ubiquitin ligase complex is a crucial component of the UPS, particularly in modulating protein degradation in response to small-molecule modulators like thalidomide. However, the endogenous substrates of CRBN in solid tumors like HCC remain poorly characterized. Here, we identify MORF4L1, a member of the MRG family involved in chromatin remodeling and DNA damage response, as a substrate of CRBN. Using proteomic analysis, co-immunoprecipitation, and structural modeling, we demonstrate that CRBN promotes MORF4L1 degradation under physiological conditions, which is further enhanced by the modulator CC-885. Importantly, MORF4L1 is upregulated in multiple cancers, including HCC, suggesting a broader role in tumorigenesis. Our findings reveal MORF4L1 as a physiological CRBN substrate and highlight the therapeutic potential of targeting CRBN substrates in cancer.

Non-thermal plasma as promising anti-cancer therapy against bladder cancer by inducing DNA damage and cell cycle arrest

Bladder cancer often recurs, necessitating innovative treatments to reduce recurrence. We investigated non-thermal plasma’s potential as a novel anti-cancer therapy, focusing on plasma-activated solution (PAS), created by exposing saline to non-thermal plasma. Our study aims to elucidate the biological effects of PAS on bladder cancer cell lines in vitro, as well as the combination with mitomycin C (MMC), using clinically relevant settings. PAS treatment exerts a potent cytotoxic effect through the production of intracellular reactive oxygen species, resulting in DNA damage and subsequent induction of G1 cell cycle arrest/senescence. This is induced via upregulation of cell cycle checkpoint signalling and DNA damage repair pathways using LC-M/MS-based phospho-proteomics. Importantly, combining PAS with MMC reveals a synergistic effect (Combination Index of 0.59–0.67), suggesting the potential of utilizing PAS in combination therapies. Our findings demonstrate PAS’s mode of action and suggest its potential as a promising treatment for bladder cancer, warranting further clinical studies.

Casein Kinase I Protein Hrr25 Is Required for Pin4 Phosphorylation and Mediates Cell Wall Integrity Signaling in Saccharomyces cerevisiae.

Casein kinase I protein Hrr25 plays important roles in many cellular processes, including autophagy, vesicular trafficking, ribosome biogenesis, mitochondrial biogenesis, and the DNA damage response in Saccharomyces cerevisiae. Pin4 is a multi-phosphorylated protein that has been reported to be involved in the cell wall integrity (CWI) pathway and DNA damage response. Pin4 was reported to interact with Hrr25 in yeast two-hybrid and large-scale pulldown assays. Co-immunoprecipitation and yeast two-hybrid assays were utilized to confirm whether Pin4 and Hrr25 interact and to determine how they interact. Genetic interaction analysis was conducted to examine whether hrr25 mutations form synthetic growth defects with mutations in genes involved in CWI signaling. Immunoblotting was used to determine whether Hrr25 phosphorylates Pin4. We show that Hrr25 interacts with Pin4 and is required for Pin4 phosphorylation. pin4 mutations result in synthetic slow-growth phenotypes with mutations in genes encoding Bck1 and Slt2, two of the protein kinases in the MAP kinase cascade that regulates CWI in the budding yeast. We show that hrr25 mutations result in similar phenotypes to pin4 mutations. Hrr25 consists of an N-terminal kinase domain, a middle region, and a C-terminal proline/glutamine-rich domain. The function of the C-terminal P/Q-rich domain of Hrr25 has been elusive. We found that the C-terminal region of Hrr25 is required both for Pin4 interaction and CWI. Our data suggest that Hrr25 is implicated in cell wall integrity signaling via its association with Pin4.

Mechanism of RPA phosphocode priming and tuning by Cdk1/Wee1 signaling circuit.

Replication protein A (RPA) is a heterotrimeric single-strand DNA binding protein that is integral to DNA metabolism. Segregation of RPA functions in response to DNA damage is fine-tuned by hyperphosphorylation of the RPA32 subunit that is dependent on Cyclin-dependent kinase (Cdk)-mediated priming phosphorylation at the Ser-23 and Ser-29 sites. However, the mechanism of priming-driven hyperphosphorylation of RPA remains unresolved. Furthermore, the modulation of cell cycle progression by the RPA-Cdk axis is not clearly understood. Here, we uncover that the RPA70 subunit is also phosphorylated by Cdk1 at Thr-191. This modification is crucial for the G2 to M phase transition. This function is enacted through reciprocal regulation of Cdk1 activity through a feedback circuit espoused by stabilization of Wee1 kinase. The Thr-191 phosphosite on RPA70 is also crucial for priming hyperphosphorylation of RPA32 in response to DNA damage. Structurally, phosphorylation by Cdk1 primes RPA by reconfiguring the domains to release the N-terminus of RPA32 and the two protein-interaction domains that markedly enhances the efficiency of multisite phosphorylation by other kinases. Our findings establish a unique phosphocode-dependent feedback mechanism between RPA and RPA-regulating kinases that is fine-tuned to enact bipartite functions in cell cycle progression and DNA damage response.

Fabrication of nanoparticles with precisely controllable plasmonic properties as tools for biomedical applications.

Metal nanoparticles are established tools for biomedical applications due to their unique optical properties, primarily attributed to localized surface plasmon resonances. They show distinct optical characteristics, such as high extinction cross-sections and resonances at specific wavelengths, which are tunable across the wavelength spectrum by modifying the nanoparticle geometry. These attributes make metal nanoparticles highly valuable for sensing and imaging in biology and medicine. However, their widespread adoption is hindered due to challenges in consistent and accurate nanoparticle fabrication and functionality as well as due to nanotoxicological concerns, including cell damage, DNA damage, and unregulated cell signaling. In this study, we present a fabrication approach using nanoimprint lithography in combination with thin film deposition which yields highly homogenous nanoparticles in size, shape and optical properties with standard deviations of the main geometry parameters of less than 5% batch-to-batch variation. The measured optical properties closely match performed simulations, indicating that pre-experimental modelling can effectively guide the design of nanoparticles with tailored optical properties. Our approach also enables nanoparticle transfer to solution. Particularly, we show that the surface coating with a PEG polymer shell ensures stable dispersions in buffer solutions and complex cell media for at least 7 days. Furthermore, our in vitro experiments demonstrate that these nanoparticles are internalized by cells via endocytosis, exhibit good biocompatibility, and show minor cytotoxicity, as evidenced by high cell viability. In the future, our high-precision nanoparticle fabrication method together with tunable surface plasmon resonance and reduced nanotoxicity will offer the possibility to replace conventional nanomaterials for biomedical applications that make use of an optical response at precise wavelengths. This includes the use of the nanoparticles as contrast agents for imaging, as probes for targeted photothermal cancer therapy, as carriers for controlled drug delivery, or as probes for sensing applications based on optical detection principles.