The interplay between chromatin structure and phase-separating proteins is an emerging topic in cell biology with implications for understanding disease states. Here, we investigate the functional relationship between bromodomain protein 4 (BRD4) and chromatin architecture. By combining molecular dynamics simulations with live-cell imaging, we demonstrate that BRD4, when mutated at specific N-terminus sites, significantly impacts the organization and dynamics of chromatin nanodomains, known as nucleosome clutches. Our findings reveal that a constitutively phosphorylated mutant of BRD4 condenses nucleosome clutches, while treatment with (+)-JQ1 increases the diffusion dynamics of single nucleosomes and decondenses nucleosome clutches. Simultaneously, we demonstrate that BRD4 mutations can alter localization of BRD4 to chromatin as well as modify single nucleosome dynamics. These results suggest that both chromatin binding and phase separation of BRD4 could co-regulate the nanoscale chromatin architecture and the chromatin microenvironment. Our observations shed light on the nuanced regulation of chromatin structure by BRD4, offering insights into its role in maintaining the nuclear architecture and transcriptional activity.
Abstract Immune evasion and immunosuppression are important hallmarks of human malignancies. Astragaloside IV (AST) is one of the effective ingredients in Astragalus, which has been confirmed to enhance antitumor immunity. However, the functions and underlying mechanism of AST on oral squamous cell carcinoma (OSCC) tumorigenesis remain undetermined. Our present work tried to test whether and how AST inhibited OSCC immune evasion and ameliorated CD8+ T cell-mediated antitumor response in immune microenvironment. The results of the present work indicated that AST repressed OSCC cells’ proliferation and migration in dosage-dependent manner. In a co-culture system analysis of CD8+ T and OSCC cells, AST enhanced the antitumor activity of CD8+ T cells to impair the OSCC immune evasion. Moreover, AST also repressed the lactate secretion and extracellular acidification. Furthermore, excess lactate accumulation triggered the PD-L1 enrichment on OSCC cells in acidic microenvironment. Mechanistically, AST targeted MCT1 to degrade its mRNA stability, thereby mitigating the extracellular acidification and inhibiting the escape of OSCC from CD8+ T cells’ killing. This study indicates that AST could ameliorate the acidic microenvironment in OSCC to improve CD8+ T cell-mediated antitumor immune response. Our finding might offer novel insights for the anti-tumor effect of AST and provide a potential therapeutic strategy for OSCC.
Abstract The seminal work by Shi et al. unveils a novel cytoprotective mechanism in intervertebral disc degeneration, whereby USP5‐rich apoptotic extracellular vesicles (ApoEVs) stabilize the transcription factor E2F1 to suppress apoptosis and enhance DNA repair in nucleus pulposus cells. While this study elegantly reframes the reparative potential of ApoEVs, its conceptual and translational premises invite critical scrutiny. This letter highlights several overlooked challenges. We argue that the contextual duality of E2F1—a well‐established regulator of both cell survival and senescence—poses a considerable therapeutic hazard, as sustained E2F1 stabilization may inadvertently trigger a senescent phenotype. Moreover, the generalizability of this mechanism remains uncertain: ApoEV cargo and functionality are highly dependent on the apoptotic stimulus, and vesicles derived from etoposide‐induced apoptosis may not mirror those produced under pathophysiological disc conditions. Crucially, the proposed pathway—from vesicle internalization and endosomal escape of luminal USP5 to its nuclear trafficking and engagement with E2F1—represents a mechanistic black box requiring rigorous validation. Resolving these issues is essential to translating this compelling discovery into a safe and effective therapeutic strategy.
Peng Gao, Schrodinger Cenatus, Nathalie Henley
et al.
Abstract The role of tubular epithelial cells (TEC) senescence in the progression from acute kidney injury (AKI) to chronic kidney disease (CKD) remains debated due to the complexity of senescent cell populations and their pro-survival mechanisms. To directly assess the contribution of TEC senescence to AKI-to-CKD progression, we employed an aristolochic acid nephropathy (AAN) mouse model. Here, we demonstrated that AAI-induced DNA damage specifically drives TEC senescence during AKI-to-CKD progression. Concomitant with the emergence of senescence, immunofluorescence staining revealed the expression of anti-apoptotic proteins, including BCL-2, BCL-xL, and MCL-1, within KIM1⁺ tubules—a marker of tubular injury. To further characterize these senescent cells, we integrated this model with snRNA-Seq data and identified a distinct population of KIM1+ senescent TEC exhibiting resistance to apoptosis through upregulation of pro-survival proteins such as MCL-1, BCL-2, and BCL-xL. To evaluate the therapeutic potential of targeting these pathways, we treated AAN mice with the MCL-1-specific inhibitor UMI-77 and the senolytic ABT-263 (targeting BCL-2/BCL-xL) during both the acute and late phases. Interestingly, only UMI-77 administration during the acute phase effectively reduced tubular senescence and mitigated fibrosis. In contrast, late-phase treatment had only marginal benefits. Notably, ABT-263 failed to eliminate senescent cells and instead exacerbated fibrosis, suggesting that while senescent TEC relies on pro-survival mechanisms to evade apoptosis, their dependency on specific anti-apoptotic proteins varies. Our study provides a high-resolution molecular framework for understanding TEC senescence and identifies MCL-1 inhibition as a precise and effective therapeutic strategy to prevent AKI-to-CKD progression, with early intervention being critical for therapeutic success.
Suresh Sivakumar, Sonja Lieber, Raimund Dietze
et al.
Abstract Background High expression of basal cell adhesion molecule (BCAM) is a hallmark of ovarian cancer (OC) progression. BCAM facilitates transcoelomic dissemination by promoting mesothelial cell clearance at peritoneal attachment sites of tumor cell spheroids. We investigated how BCAM mediates this effect and potentially drives other pro-metastatic functions. Methods The impact of BCAM on the tumor cell secretome and the mesothelial cell phenotype was analyzed by affinity proteomics, bulk and single-cell RNA sequencing, life-cell and multiphoton microscopy, biochemical and functional in vitro assays as well as a murine tumor model. BCAM manipulation involved ectopic overexpression, inducible expression and treatment with soluble BCAM. Results All forms of BCAM enhanced the secretion of cytokines that impact cell motility, mesenchymal differentiation and angiogenesis, including AREG, CXCL family members, FGF2, TGFB2, and VEGF. Notably, their levels in OC ascites were correlated with BCAM expression, and recombinant BCAM-induced cytokines triggered mesothelial-mesenchymal transition (MMT). Mesothelial cells undergoing MMT exhibited enhanced motility away from attaching tumor spheroids, leading to mesothelial clearance at spheroid attachment sites. BCAM-mediated MMT-associated transcriptional changes were also observed in subpopulations of omental mesothelial cells from OC patients, and were associated with poor survival. Consistent with the secretome data, BCAM induced endothelial tube formation in vitro and markedly promoted tumor angiogenesis in a mouse model. Conclusion We have identified previously unknown functions of the BCAM-induced secretome potentially impacting distinct stages of OC metastasis. While BCAM’s impact on MMT may facilitate initiation of micrometastases, neo-angiogenesis is essential for tumor growth. Taken together with the observed clinical adverse association, our findings underscore the potential of BCAM as a therapeutic target.
Sergio Antonio Oropeza-de Lara, Idalia Garza-Veloz, Bertha Berthaud-González
et al.
Endometrial cancer (EC) is a significant cause of cancer-related deaths in women. MicroRNAs (miRs) play a role in cancer development, acting as oncogenes or tumor suppressors. This study evaluated the diagnostic potential of hsa-miR-185-5p and hsa-miR-191-5p in EC and their correlation with clinical and histopathological features. A cross-sectional study analyzed formalin-fixed, paraffin-embedded tissue samples from 59 patients: 18 with EC, 21 with endometrial hyperplasia (EH), 17 with normal endometrium (NE), and 3 with endometrial polyps (EPs). Quantitative reverse transcription-polymerase chain reaction and TaqMan probes were used for miR expression analysis. The Shapiro–Wilk test was used to analyze the normal distribution of the data. Subsequently, parametric or non-parametric tests were used to evaluate the associations between the expression levels of each miR and clinical parameters. Both miRs were underexpressed in some precursor and malignant lesions compared to certain NE subtypes and benign lesions. Specifically, hsa-miR-185-5p showed underexpression in grade 3 EC compared to some NE and EH subtypes (FC: −57.9 to −8.5, <i>p</i> < 0.05), and hsa-miR-191-5p was underexpressed in EH and EC compared to secretory endometrium and EPs (FC: −4.2 to −32.8, <i>p</i> < 0.05). <i>SETD1B</i>, <i>TJP1</i>, and <i>MSI1</i> were common predicted target genes. In conclusion, hsa-miR-185-5p and hsa-miR-191-5p are underexpressed in EC tissues, correlating with histopathological grades, highlighting their potential as diagnostic biomarkers and their role as tumor suppressors in EC.
Loukia Touramanidou, Sonam Gurung, Claudiu A. Cozmescu
et al.
Recently approved adeno-associated viral (AAV) vectors for liver monogenic diseases haemophilia A and B are exemplifying the success of liver-directed viral gene therapy. In parallel, additional gene therapy strategies are rapidly emerging to overcome some inherent AAV limitations, such as the non-persistence of the episomal transgene in the rapidly growing liver and immune response. Viral integrating vectors such as in vivo lentiviral gene therapy and non-viral vectors such as lipid nanoparticles encapsulating mRNA (LNP-mRNA) are rapidly being developed, currently at the preclinical and clinical stages, respectively. Macrophages are the first effector cells of the innate immune response triggered by gene therapy vectors. Macrophage uptake and activation following administration of viral gene therapy and LNP have been reported. In this study, we assessed the biodistribution of AAV, lentiviral, and LNP-mRNA gene therapy following the depletion of tissue macrophages by clodronate pre-treatment in neonatal and juvenile mice. Both neonatal and adult clodronate-treated mice showed a significant increase in lentiviral-transduced hepatocytes. In contrast, clodronate pre-treatment did not modify hepatocyte transduction mediated by hepatotropic AAV8 but reduced LNP-mRNA transfection in neonatal and juvenile animals. These results highlight the importance of age-specific responses in the liver and will have translational applications for gene therapy programs.
Objective To compare the costs and effects of three sampling strategies for human papillomavirus (HPV) primary screening.Design Cost-consequence analysis from a health system perspective using a deterministic decision tree model.Setting England.Participants A cohort of 10 000 women aged 25–65 years eligible for the National Health Service Cervical Screening Programme (NHSCSP).Methods The model was based on the NHSCSP HPV primary screening pathway and adapted for self-sampling. It used a 3-year cycle: routine screening (year 1) and recall screening (years 2/3). Parameter inputs were informed using published studies, NHSCSP reports and input from experts and manufacturers. Costs were from 2020/2021, British pound sterling (£).Interventions Three sampling strategies were implemented: (1) routine clinician-collected cervical sample, (2) self-collected first-void (FV) urine, (3) self-collected vaginal swab. The hypothetical self-sampling strategies involved mailing women a sampling kit.Main outcome measures Primary outcomes: overall costs (for all screening steps to colposcopy), number of complete screens and cost per complete screen. Secondary outcomes: number of women screened, number of women lost to follow-up, cost per colposcopy and total screening costs for a plausible range of uptake scenarios.Results In the base case, the average cost per complete screen was £56.81 for clinician-collected cervical sampling, £38.57 for FV urine self-sampling and £40.37 for vaginal self-sampling. In deterministic sensitivity analysis, the variables most affecting the average cost per screen were the cost of sample collection for clinician-collected sampling and the cost of laboratory HPV testing for the self-sampling strategies. Scaled to consider routine screening in England, if uptake in non-attenders increased by 15% and 50% of current screeners converted to self-sampling, the NHSCSP would save £19.2 million (FV urine) or £16.5 million (vaginal) per year.Conclusion Self-sampling could provide a less costly alternative to clinician-collected sampling for routine HPV primary screening and offers opportunities to expand the reach of cervical screening to under-screened women.
INTRODUCTION: With cellular lipid storage varying, the balance between lipid intake and lipid degradation was a must to keep healthy and determined the level of lipid droplets. Although lipid droplets accumulation had been well demonstrated in adipocytes, gene expression profiling and gene function during adipogenesis and osteoblastogenesis remain unknown. MATERIAL AND METHODS: Here, this work profiled gene transcriptional landscapes of lipid droplets formation during adipogenesis from human mesenchymal stem cells (hMSCs) using RNA-Seq technique. By using RNA interference (RNAi) we investigated the function of candidate genes during adipogenesis and osteoblastogenesis using Oil Red/Alizarin Red/alkaline phosphatase (ALPL) staining and qRT-PCR (quantitative real-time PCR). RESULTS: Eleven differentially up-regulated genes associated with lipid droplets formation were identified at 3, 5, 7, 14, 21, and 28 days during adipogenesis. Unexpectedly, APOB per se inhibiting adipogenesis weakened osteoblastogenesis and METTL7A facilitating adipogenesis negligibly inhibited osteoblastogenesis according to the phenotypic characterization of adipocytes and osteoblasts and transcriptional condition of biomarkers through lentivirus transfection assays. CONCLUSIONS: The establishment of the gene transcriptional profiling of lipid droplets formation would provide the molecular switches of hMSCs cell fate determination and the study targets for fat metabolic diseases.
Abstract Homeobox A5 (HOXA5) is a transcription factor in mammalian and can regulate cell differentiation, proliferation, and apoptosis as well as tumorigenesis. However, little is known on whether and how HOXA5 can regulate the malignant behaviors of cholangiocarcinoma. The methylation levels of HOXA5 were evaluated by methylation microarray and bisulfite sequencing PCR. HOXA5 expression in tissue samples was examined by immunohistochemistry and Western blot. The proliferation of tumor cells was assessed by CCK-8, EdU, and nude mouse tumorigenicity assays. The invasion, apoptosis and cell cycling of tumor cells were evaluated by Wound healing assay and flow cytometry. The interaction between HOXA5 and the MXD1 promoter was examined by CUT & Tag assay, luciferase reporter assay and chromatin immunoprecipitation. Hypermethylation in the HOXA5 promoter down-regulated HOXA5 expression in extrahepatic cholangiocarcinoma (ECCA) tissues, which was correlated with worse overall survival. HOXA5 overexpression significantly inhibited the proliferation and tumor growth. HOXA5 overexpression enhanced MXD1 expression by directly binding to the MXD1 promoter in ECCA cells. MXD1 overexpression inhibited the proliferation and tumor growth while MXD1 silencing abrogated the HOXA5-mediated proliferation inhibition. HOXA5 overexpression increased p53 protein expression in an MXD1-dependent manner. HOXA5 and MXD1 acted as tumor suppressors to inhibit the mitosis of ECCA cells by enhancing the p53 signaling. Our findings may uncover molecular mechanisms by which the HOXA5/MXD1 axis regulates the progression of ECCA, suggesting that the HOXA5/MXD1 may be therapeutic targets for ECCA.
Matteo Ghisa, Angelo Bellumat, Manuela De Bona
et al.
The diagnostic approach to the biliary tree disorders can be challenging, especially for biliary strictures. Albeit the great diagnostic impact of endoscopic retrograde cholangiopancreatography (ERCP) which allows one to obtain fluoroscopic imaging and tissue sampling through brush cytology and/or forceps biopsy, a considerable proportion of cases remain indeterminate, leading to the risk of under/over treated patients. In the last two decades, several endoscopic techniques have been introduced in clinical practice, shrinking cases of uncertainties and improving diagnostic accuracy. The aim of this review is to discuss recent advances and emerging technologies applied to the management of biliary tree disorders through peroral endoscopy procedures.
S. E. Romanov, D. A. Kalashnikova, P. P. Laktionov
The correct deployment of genetic programs for development and differentiation relies on finely coordinated regulation of specific gene sets. Genomic regulatory elements play an exceptional role in this process. There are few types of gene regulatory elements, including promoters, enhancers, insulators and silencers. Alterations of gene regulatory elements may cause various pathologies, including cancer, congenital disorders and autoimmune diseases. The development of high-throughput genomic assays has made it possible to significantly accelerate the accumulation of information about the characteristic epigenetic properties of regulatory elements. In combination with high-throughput studies focused on the genome-wide distribution of epigenetic marks, regulatory proteins and the spatial structure of chromatin, this significantly expands the understanding of the principles of epigenetic regulation of genes and allows potential regulatory elements to be searched for in silico. However, common experimental approaches used to study the local characteristics of chromatin have a number of technical limitations that may reduce the reliability of computational identification of genomic regulatory sequences. Taking into account the variability of the functions of epigenetic determinants and complex multicomponent regulation of genomic elements activity, their functional verification is often required. A plethora of methods have been developed to study the functional role of regulatory elements on the genome scale. Common experimental approaches for in silico identification of regulatory elements and their inherent technical limitations will be described. The present review is focused on original high-throughput methods of enhancer activity reporter analysis that are currently used to validate predicted regulatory elements and to perform de novo searches. The methods described allow assessing the functional role of the nucleotide sequence of a regulatory element, to determine its exact boundaries and to assess the influence of the local state of chromatin on the activity of enhancers and gene expression. These approaches have contributed substantially to the understanding of the fundamental principles of gene regulation.
Geert A. Martens, Geert Stangé, Lorenzo Piemonti
et al.
Ongoing beta cell death in type 1 diabetes (T1D) can be detected using biomarkers selectively discharged by dying beta cells into plasma. microRNA-375 (miR-375) ranks among the top biomarkers based on studies in animal models and human islet transplantation. Our objective was to identify additional microRNAs that are co-released with miR-375 proportionate to the amount of beta cell destruction. RT-PCR profiling of 733 microRNAs in a discovery cohort of T1D patients 1 h before/after islet transplantation indicated increased plasma levels of 22 microRNAs. Sub-selection for beta cell selectivity resulted in 15 microRNAs that were subjected to double-blinded multicenter analysis. This led to the identification of eight microRNAs that were consistently increased during early graft destruction: besides miR-375, these included miR-132/204/410/200a/429/125b, microRNAs with known function and enrichment in beta cells. Their potential clinical translation was investigated in a third independent cohort of 46 transplant patients by correlating post-transplant microRNA levels to C-peptide levels 2 months later. Only miR-375 and miR-132 had prognostic potential for graft outcome, and none of the newly identified microRNAs outperformed miR-375 in multiple regression. In conclusion, this study reveals multiple beta cell-enriched microRNAs that are co-released with miR-375 and can be used as complementary biomarkers of beta cell death.
Seyyedeh Tahereh Nabavi, Farah Farahani, Masoud Sheidai
et al.
Ziziphus jujuba (jujube) of buckthorn family (Rhamnaceae) is an important medicinal crop plant cultivated in different provinces of Iran. It has also wild populations in some geographical areas. We carried out population genetic study on 8 populations of cultivated versus wild jujuba by using ISSR molecular markers to produce data on population genetic structure, gene flow, and genetic variability in the studied populations. We also aimed to investigate genetic differentiation between wild and cultivated plants and identify the potential gene pools of this medicinal plant species. The studied populations had a moderate genetic variability and were grouped in two major groups by PCoA plot. AMOVA revealed significant genetic difference among these cultivars. Mantel test showed significant correlation between genetic distance and geographical distance in the studied populations. PCoA analysis showed genetic differentiation between wild and cultivated plants within each province. STRUCTURE analysis identified two potential gene pools for jujube cultivars. Data obtained may be used in genetic conservation and future breeding programs of this medicinal plant species in the country.
Antonello Nicolini,1 Bruna Grecchi,2 Maura Ferrari-Bravo,3 Cornelius Barlascini4 1Respiratory Diseases Unit, Hospital of Sestri Levante, Sestri Levante, Italy; 2Rehabilitation Unit, ASL4 Chiavarese, Chiavari, Italy; 3Statistics Unit, ASL4 Chiavarese, Chiavari, Italy; 4Health Medicine Unit, Hospital of Sestri Levante, Sestri Levante, Italy Purpose: Chest physiotherapy is an important tool in the treatment of COPD. Intrapulmonary percussive ventilation (IPV) and high-frequency chest wall oscillation (HFCWO) are techniques designed to create a global percussion of the lung which removes secretions and probably clears the peripheral bronchial tree. We tested the hypothesis that adding IPV or HFCWO to the best pharmacological therapy (PT) may provide additional clinical benefit over chest physiotherapy in patients with severe COPD. Methods: Sixty patients were randomized into three groups (20 patients in each group): IPV group (treated with PT and IPV), PT group with (treated with PT and HFCWO), and control group (treated with PT alone). Primary outcome measures included results on the dyspnea scale (modified Medical Research Council) and Breathlessness, Cough, and Sputum scale (BCSS), as well as an evaluation of daily life activity (COPD Assessment Test [CAT]). Secondary outcome measures were pulmonary function testing, arterial blood gas analysis, and hematological examinations. Moreover, sputum cell counts were performed at the beginning and at the end of the study. Results: Patients in both the IPV group and the HFCWO group showed a significant improvement in the tests of dyspnea and daily life activity evaluations (modified Medical Research Council scale, BCSS, and CAT) compared to the control group, as well as in pulmonary function tests (forced vital capacity, forced expiratory volume in 1 second, forced expiratory volume in 1 second/forced vital capacity%, total lung capacity, residual volume, diffusing lung capacity monoxide, maximal inspiratory pressure, maximal expiratory pressure) and arterial blood gas values. However, in the group comparison analysis for the same variables between IPV group and HFCWO group, we observed a significant improvement in the IPV group maximal inspiratory pressure, maximal expiratory pressure, BCSS, and CAT. Similar results were observed in changes of sputum cytology with reduction of inflammatory cells (neutrophils and macrophages). Conclusion: The two techniques improved daily life activities and lung function in patients with severe COPD. IPV demonstrated a significantly greater effectiveness in improving some pulmonary function tests linked to the small bronchial airways obstruction and respiratory muscle strength and scores on health status assessment scales (BCSS and CAT) as well as a reduction of sputum inflammatory cells compared with HFCWO. Keywords: severe COPD, intrapulmonary percussive ventilation, high-frequency chest wall oscillation, daily life activity
Mariana Pavelski, Daniele Von Kruger Amaral, Giovana Paladino Vieira
et al.
There is a high incidence of bronchitis and asthma cases in veterinary medicine. Thoracic radiographs and bronchoalveolar lavage (BAL) are commonly performed for definitive diagnosis in dogs and cats with suspected bronchitis and asthma. It is believed that a combination of diagnostic tools is the best choice to achieve a diagnosis. The aim of this study was to evaluate the efficacy of thoracic radiographs and BAL in the diagnosis of chronic bronchial disease (CBD) in dogs and cats and whether there is any specific radiographic finding that could influence the indication for bronchoalveolar lavage. It was performed a cross-sectional, prospective, observational study including forty client-owned dogs and cats with lower respiratory tract signs and positive radiographic opacities that were evaluated with BAL followed by cytology and culture. The radiographic results compared with BAL culture showed a sensitivity of 38%, specificity of 95% and accuracy of 65% in detecting patients with pneumonia associated with chronic bronchial disease. Thoracic radiographs were effective in diagnosing 65% of the patients, radiographs plus BAL cytology diagnosed 75% of patients and the combination of radiographs, BAL cytology and culture diagnosed 95% of the patients with chronic bronchial disease. In conclusion, the combination of radiographic examination with BAL followed by cytological and microbiological analyses increases diagnostic success in CBD.