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Scavenger Receptor Class B Member 2 (SCARB2) is an integral lysosomal membrane protein essential for lysosomal integrity and autophagy regulation. The aim of this study was to investigate the functional impact of SCARB2 overexpression on chemotherapy response, reactive oxygen species (ROS) production and proteomic composition of human ovarian adenocarcinoma cells A2780. To induce SCARB2 overexpression, A2780 cells were transfected using a PiggyBac vector system. Two clones with the highest SCARB2 expression (L and V) were selected for further analyses. Differences in chemosensitivity were assessed using the MTS assay. Proteomic analysis was used to identify differentially expressed proteins and enriched pathways. We also performed flow cytometry to investigate changes in ROS production and lysosomal activity. Lysosomal distribution was assessed using LAMP1 immunofluorescence staining followed by confocal microscopy, and total cholesterol levels were determined using an enzymatic colorimetric assay. Both clones showed increased sensitivity to cisplatin compared to the control group. In contrast, clone V showed resistance to doxorubicin and no significant differences were observed for gemcitabine, except for a transient sensitizing effect when low concentrations used. Elevated ROS levels were detected in untreated clones, and after doxorubicin exposition. Proteomic analysis showed significant changes in lysosome-associated proteins, with consistent enrichment of the lysosomal pathway across all experimental comparisons. Immunofluorescence analysis of LAMP1 and LysoTracker staining demonstrated altered lysosomal distribution and activity in SCARB2-overexpressing clones. In addition, both SCARB2-overexpressing clones exhibited significantly reduced total cholesterol levels compared with control cells. This study broadens our understanding of SCARB2 in ovarian cancer. SCARB2 overexpression induces extensive lysosomal reprogramming in A2780 ovarian cancer cells and modulates chemotherapy response.

Pseudomonas aeruginosa is an opportunistic pathogen that has been reported to cause a variety of nosocomial infections, partly due to its high levels of antimicrobial resistance. With limited genomic surveillance at hand, knowledge about high-risk clones and epidemiological trends in Africa has been hindered. This systematic review and meta-analysis aimed to investigate the clonal diversity of P. aeruginosa in Africa, focusing on the prevalence and geographic spread of globally recognized high-risk clones. To synthesize molecular evidence on P. aeruginosa circulating in Africa, to describe clonal diversity, estimate the prevalence of high-risk clones, and their antimicrobial resistance characteristics. A literature search was conducted across PubMed, Web of Science, African Journal Online, and Scopus to retrieve studies mainly on the phenotypic and genotypic characteristics of P. aeruginosa in Africa. This study did not limit the literature by publication date. A random-effects meta-analysis model was used to estimate pooled resistance rates, and subgroup analyses were performed for specific antibiotic classes and high-risk clones (HRCs) This systematic review and meta-analysis was registered in PROSPERO database (ID: CRD420251071203). From the initial search, 794 records were obtained, out of which 28 studies from 13 African countries met the inclusion criteria. The pooled resistance rate across all antibiotic classes was 54.4% (95% CI: 45.6-63.0), with resistance reaching 100% for tetracyclines and glycylcyclines. While non-HRCs were more prevalent (67.54%, 95% CI: 53.79-80.01), the prevalence of the HRCs was also substantial at 32.46% (95% CI: 19.99-46.21). The eBURST analysis revealed 127 sequence types in 30 clonal complexes. Egypt and Algeria contributed the highest diversity and frequency of HRCs of P. aeruginosa, with ST244 (47.84%, 95% CI 20.99-75.31) being the most prevalent HRC, followed by ST773 at 31.48%. The widespread detection of HRCs such as ST244 and ST773 in most regions in Africa highlights a critical need to enhance genomic surveillance and implement systematic monitoring of antimicrobial resistance (AMR).

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease driven by uncensored B and T cell autoreactivity. Understanding this pathogenic process has been hampered by lack of studies of secondary lymphoid organs in human SLE. Using minimally invasive lymph node fine needle aspirates (LN-FNAs), we profiled tissue-resident immune cells from 59 SLE patients and 34 healthy controls through high-dimensional 43-color flow cytometry, antigen-specific tetramer probing, and sc-RNA sequencing with paired VH/VL repertoire analysis. Our findings reveal hyperactive lymph node immunity in SLE characterized by spontaneous germinal center (GC) activation, plasma cell accumulation enriched in mature CD19 - and CD138 + antibody-secreting cells, and increased frequencies of both GC-T FH and PD-1 + CXCR5 - T extra-follicular helper cells. SLE lymph nodes harbored large oligoclonal B cell families with altered isotype usage, dominated by IgG1 and IgG4. Critically, self-reactive 9G4 + and Ro60 + B cells showed defective tolerance checkpoint control, accumulating in activated naïve, GC, and plasma cell compartments with distinctive PD-1 + Tox + expression absent in viral-specific responses. Single-cell repertoire analysis revealed VH 4-34 clones in SLE B GC and B PC , that in contrast to HD, had not experienced clonal redemption. Instead, SLE VH 4-34 clones displayed low somatic hypermutation and preserved the AVY hydrophobic patch associated with autoreactivity. Monoclonal antibody testing confirmed that unmutated AVY + VH 4-34 clones retained polyreactivity against naïve B cells, apoptotic cells, and multiple self-antigens. Together, these results define "clonal damnation" as a key mechanism in SLE whereby autoreactive VH 4-34 clones of pathogenic potential escape tolerance checkpoints, expand in germinal centers, and differentiate into tissue plasma cells while preserving germline-encoded self-reactivity. Combined, our study defines critical mechanisms of tolerance breakdown in lupus pathogenesis.

Giardia lamblia, a protozoan parasite that causes diarrhea in humans, contains two centrins implicated in cellular morphology and cell division. To identify proteins interacting with each G. lamblia centrin (GlCent), we performed a yeast two-hybrid assay. The coding sequences of the two centrins, GlCent1 (GL50803_6744) and GlCent2 (GL50803_104685), were cloned into pGBKT7 as bait constructs. A G. lamblia cDNA library, driven by pGADT7, was transformed into yeast strains expressing each bait protein. Initial screening on selective media yielded 17 and 23 candidate clones for GlCent1 and GlCent2, respectively. After retransformation and interaction strength analysis using growth assays, 10 GlCent1-interacting clones and 5 GlCent2-interacting clones were selected. Their interactions were validated using co-immunoprecipitation and immunofluorescence assays in transgenic Giardia cells co-expressing hemagglutinin-tagged centrins and myc-tagged candidate proteins. The results showed that ubiquitin protein ligase 1 (GL50803_9779) and preimplantation protein 3 (GL50803_3417) were GlCent1-binding proteins, whereas two hypothetical proteins (GL50803_4239 and _27739) interacted with GlCent2. Proteins interacting with the two centrins transiently co-localized during cell division. These findings suggest that GlCent1 and GlCent2 might participate in cell division by associating with distinct partner proteins in Giardia.

Clonal hematopoiesis of indeterminate potential (CHIP) is a recognized risk factor for hematologic malignancies, but its contribution to different types of solid cancers remains incompletely defined. Here, we performed a systematic, gene-specific analysis of CHIP across 19 common solid cancer types using two large population-based cohorts, the UK Biobank and All of Us with Cox proportional hazards models and nested case-control logistic models. We demonstrate that the relationship between CHIP and solid tumors is highly cancer-type specific, with lung cancer exhibiting the strongest association. In lung cancer, this association is largely driven by ASXL1-mutant clones. Specifically, high variant allele fraction (high-VAF) ASXL1 conferring a significantly increased risk (hazard ratio = 3.2), and the associations remained robust after adjustment for age, sex, body mass index (BMI), smoking status, and genetic ancestry. Notably, ASXL1 CHIP was substantially enriched among smokers, and its association with lung cancer risk was restricted to ever-smokers, highlighting a key interaction between CHIP and environmental exposure. The enrichment of ASXL1 CHIP in lung cancer was further validated in two independent cancer-only cohorts, including MSK-IMPACT and TCGA. In addition, rare germline variant association analysis revealed that germline variation in ASXL1 had the strongest association with lung cancer susceptibility among all solid tumors. Collectively, our findings support a model in which smoking-associated expansion of ASXL1-mutant clones contributes to lung cancer development and suggest that gene-specific CHIP metrics may enhance risk stratification and early detection strategies.

Persistent Human Papillomavirus (HPV) infection causes 84% of cervical cancers (CC) worldwide. The current gold standard, Pap smear, has a high false-negative rate, highlighting the need for a specific and sensitive molecular method for early diagnosis. T cell receptor (TCR)-like antibodies offer a promising platform for cancer immunodiagnostics and immunotherapy. Using the phage display technique, a monoclonal TCR-like antibody (16 Ab) against HPV 16 oncoprotein E7 for human leukocyte antigen (HLA)-A2 was produced in this study. 16 Ab was selected from a single-domain antibody (sDAb) library via biopanning, an affinity-selection method. Once the clone of 16 Ab was selected, it was sequenced, and the result was analyzed using IMGT/V-Quest and VBase2 databases before proceeding with 16 Ab protein expression and purification. The purified 16 Ab was evaluated for its binding capability towards its target peptide-major histocompatibility complex (pMHC) by Western blot and Enzyme-Linked immunosorbent assay (ELISA). A target pMHC complex was generated and panned against a phage display library. Highly enriched antibody phages from the 3rd biopanning round were chosen for phage ELISA analyses. Of the nine clones selected and sequenced, only two had proper sequences, and 16 Ab was chosen for the downstream process because it shows higher specificity for the target pMHC complex than the other clones. The dot blot showed that the soluble protein from 16 Ab was successfully expressed. The binding of 16 Ab towards the target pMHC complex (OD405 nm: 1.06) significantly differs from the non-target pMHC complex (OD405 nm: 0.09). This study demonstrates the potential of 16 Ab as a companion diagnostic to reduce the rate of false-negative Pap smears. Further research is needed to assess its therapeutic potential, expand coverage to all HPV variants and HLA types, and evaluate its performance in cervical cancer cell lines before clinical testing.

Class-switch recombination (CSR) allows B cells to produce antibodies with distinct effector functions, but its dynamics during a primary human response remain poorly understood. We sampled COVID-19-naive healthy volunteers every other day during the first 3 weeks after SARS-CoV-2 vaccination, combining bulk and single-cell B cell receptor repertoires, single-cell transcriptomics, immunophenotyping, and IGHC sterile transcript analysis. Vaccine-specific B cells show sterile transcription across all IGHC genes up to IGHG2, contradicting the prevailing idea of single-gene sterile transcription. Clonal tracking confirms that sequential CSR exists: e.g., IGHG3 to IGHG1 and IGHG1 to IGHA1 and IGHG2, with sparse switching beyond IGHG2. VDJ gene usage associates with specific isotype subclasses and differential CSR timing. CSR and somatic hypermutation are temporally decoupled, with antigen-specific clones remaining hypomutated up to 10 weeks post-immunization. These findings complement textbook models of CSR and inform strategies for vaccines requiring switching to key isotypes such as IgG1 or IgA2.

Hematological patients are at higher risk of severe SARS-CoV-2 infection and exhibit impaired vaccine responses due to disease and therapy. Treatments like Rituximab are known to compromise immune function, reducing the generation of protective antibodies. In the CORSA trial, we evaluated humoral and cellular responses to messenger RNA vaccines against SARS-CoV-2 in 77 patients with hematological malignancies receiving active treatment and in matched healthy controls. Peripheral blood samples were processed to assess Spike-specific immunoglobulin G titers, T-cell responses by enzyme-linked immunospot assay, and T-cell receptor repertoire sequencing from baseline to six months after vaccination to determine clonal breadth and depth of Spike-specific T-cell receptor clones. Seroconversion occurred in only 8% and 21% of patients after the first and second dose, compared to 93% and 100% of healthy controls. Despite poor antibody responses, 69% of seronegative patients showed measurable T-cell activity, suggesting some level of vaccine-induced protection. Spike-specific TCR repertoire analysis in lymphoma patients (LP) and HC revealed significantly broader clonal breadth (p = 0.0013) and higher clonal depth (p = 0.0265) in HC at day 50 versus baseline, while blunted diversification in LP (p = 0.0407). Lymphoma patients showed significantly weaker Spike-specific clonal responses and reduced diversity compared with healthy controls, supporting the association with increased patient vulnerability.

Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are leading causes of urinary tract infections (UTIs). In Morocco, carbapenem resistance data are scarce and largely hospital-based. We investigated carbapenem-resistant E. coli and K. pneumoniae isolates from community settings in Morocco. In this prospective multicentre study, we analysed 1296 non-duplicate E. coli and K. pneumoniae isolates recovered from outpatients with community-acquired UTIs (CA-UTIs) across 12 Moroccan cities (January 2022-December 2023). Antimicrobial susceptibility testing was performed following EUCAST guidelines. All phenotypically carbapenem-resistant isolates (n = 54, 13 E. coli, et 41 K. pneumoniae) were subjected to whole-genome sequencing (WGS) using Illumina technology to identify resistance determinants, mobile genetic elements and clonal lineages. E. coli predominated [n = 1000, 77.2% (95% CI: 74.8-79.4)] while K. pneumoniae accounted for 22.8% [n = 296 (95% CI: 20.6-25.2)]. Carbapenem resistance rates were significantly higher in K. pneumoniae than in E. coli [ertapenem: 21.1% (95% CI: 16.6-26.4) versus 2.4% (95% CI: 1.6-3.6); imipenem: 15.0% (CI: 11.2-19.6) versus 1.9% (95% CI: 1.2-3.0)].WGS revealed a high clonal diversity [eight sequence types (ST) in E. coli and 12 in K. pneumonnie]. Carbapenemase-mediated resistance was predominant in K. pneumoniae (notably bla NDM-14, bla NDM-7, bla NDM-5 and bla OXA-48). By contrast, E. coli primarily exhibited efflux and permeability defect mechanisms with sporadic carbapenemase-encoding gene carriage (bla NDM or bla OXA-48). The K. pneumoniae international high-risk clone ST147 producing NDM-14 was dominant (n = 23 isolates), followed by ST307 (n = 4), whereas ST410 was the most frequently identified among E. coli (n = 5). Carbapenem-resistant E. coli and K. pneumoniae in community UTIs in Morocco, are driven by multiple clones. These findings underscore the urgent need to expand community-based AMR surveillance beyond hospital settings.

The hospital environment is increasingly recognized as a critical reservoir for antimicrobial-resistant (AMR) bacteria. In sub-Saharan Africa, maternity wards represent high-risk settings where environmental contamination poses a direct threat to vulnerable mothers and neonates. Despite this, there is a significant lack of data integrating phenotypic resistance with whole-genome sequencing (WGS) to understand antimicrobial resistance (AMR) in these settings. This study characterized the AMR patterns and genomic features of ESBL-producing Escherichia coli and Klebsiella spp. isolated from maternity ward surfaces in Yaounde, Cameroon. A cross-sectional environmental study was conducted across four maternity wards. Isolates were identified via standard microbiological methods, and antimicrobial susceptibility testing against 13 antibiotics was performed following European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2024 guidelines. Short-read WGS was utilized to identify sequence types (STs), plasmid incompatibility groups, antibiotic resistance genes (ARGs), and virulence factors. Plasmid-ARG association networks were constructed to visualize resistance dynamics. Nineteen ESBL-producing Enterobacterales were identified, comprising 15 E. coli and four Klebsiella spp. isolates. High levels of multidrug resistance were observed against ciprofloxacin, penicillins, and third-generation cephalosporins. While the isolates remained sensitive to colistin and imipenem, alarming resistance to meropenem was detected. Genomic analysis revealed the presence of globally disseminated high-risk lineages, including E. coli ST131, ST1193, and ST410, alongside K. pneumoniae ST1324 and K. quasipneumoniae ST489. Critical resistance determinants, including ESBLs, AmpC enzymes, and carbapenemases (blaNDM, blaOXA-48-like), were associated with epidemic plasmids such as IncF, IncA/C2, and IncL/M. Additionally, the isolates harboured virulence factors characteristic of extraintestinal pathogenic Enterobacterales. The widespread presence of high-risk carbapenemase-producing clones on maternity ward surfaces identifies the hospital environment as a significant AMR reservoir in Yaounde. These findings highlight the urgent need for reinforced infection prevention and control (IPC) measures, robust antimicrobial stewardship, and the integration of genomic surveillance to safeguard highly susceptible maternal and neonatal populations from life-threatening infections.

Caenorhabditis elegans is a powerful model for studying gene function and disease pathogenesis. While RNA interference effectively suppresses gene expression, CRISPR activation (CRISPRa) provides a robust tool for upregulating endogenous gene expression in C. elegans. Compared with traditional injection-based methods, feeding-based CRISPRa, similar to RNA interference, is easy, cost-effective and efficient. However, the traditional cloning workflows remain a bottleneck for high-throughput gene screening. Here, we present a pooled, one-step dual gRNA cloning protocol that enables rapid and efficient construction of gRNA expression vectors for CRISPRa. In a test case, by designing 55-mer and 54-mer primers, each containing one gRNA, we amplified and pooled 126 gRNA inserts in a single reaction pipeline. The 126 gRNA clones were completed in three pooled rounds, achieving 42%-56% coverage for each round, with remaining clones processed individually. This protocol dramatically reduces time, labor, and reagent consumption, while increasing scalability and maintaining reproducibility. It is particularly well suited for high-throughput screening of gene libraries or pathways and supports downstream applications such as phenotypic screening and lifespan analysis. This work advances CRISPRa-based functional genomics in C. elegans by providing a practical tool for large-scale gene activation studies.

In South Korea, OXA-48-like carbapenemase-producing Enterobacterales (OXA-48-like-CPE) remain uncommon. We investigated an OXA-48-like-CPE outbreak in a Korean hospital between January and May 2025. We conducted integrated epidemiological and genomic investigations using contact tracing, environmental sampling, and a case-control study. Whole-genome sequencing (WGS) was performed on 23 isolates for phylogenetic analysis, with long-read sequencing of the index isolate to characterize plasmids. Thirty-eight hospital-linked cases of OXA-48-like-CPE (36 Klebsiella pneumoniae, 1 K. pneumoniae and E. coli, and 1 E. coli) were identified across 12 hospital wards, primarily affecting the haematology unit. The outbreak originated from a single outside case admitted to a haemato-oncology unit. Despite contact precautions, five secondary cases were linked to the index patient. The spread was further amplified by inter-ward patient transfers, with a medical unit serving as a transmission hub, facilitating cross-departmental spread. Despite detection mainly via surveillance cultures, 10 (26.3%) patients, predominantly from haematology, developed bacteremia. WGS confirmed clonal dissemination of OXA-181-producing Klebsiella pneumoniae sequence type 16 (OXA-181 KPN ST16), which originated from the index case and harbored the KL51 capsule and ybt10/ICEKp virulence determinants. blaOXA-181 was carried on a conserved conjugative plasmid containing multiple replicons (ColKP3, IncX3, IncFII(K), IncFIB(K)). Intensified infection prevention and control (IPC) measures, including weekly surveillance of high-risk patients, successfully contained the outbreak within 4 months. This outbreak highlights the potential for rapid hospital-wide dissemination of high-risk clones and underscores the critical role of integrated genomic-epidemiological investigations and intensified IPC measures in effective outbreak containment.

Three-dimensional (3D) cancer models, notably patient-derived organoids (PDOs), address the critical limitations of traditional preclinical systems, including two-dimensional (2D) monolayer cultures and patient-derived xenografts (PDXs), by better recapitulating physiological tumor architecture and patient-specific heterogeneity, thereby revolutionizing oncology research. We chart the complementary technological landscape, from high-fidelity PDOs and scalable spheroids to engineered systems that reconstruct the tumor microenvironment (TME) via co-culture, organ-on-a-chip, and 3D bioprinting. Beyond foundational biology, these tools are driving functional precision medicine, where PDO avatars predict clinical drug response, and accelerating drug discovery through physiologically relevant screening. A core focus is their unparalleled utility in modeling therapy resistance, enabling the induction and multi-omic dissection of resistant clones, deconstructing stroma-mediated protection, and testing rational combination therapies to overcome relapse. Despite challenges in standardization and complete TME integration, the convergence of 3D models with single-cell omics, CRISPR screening, and artificial intelligence heralds a new era of predictive oncology. Ultimately, rigorous validation and clinical translation of these models promise to bridge the gap between bench and bedside, enabling truly personalized and effective cancer therapies.

Reducing tumor cell oxygen consumption has emerged as a promising strategy to counter hypoxia-induced radioresistance in solid tumors. Previously, we found that OXPHOS inhibition using PEGylated mitochondria-targeted atovaquone (Mito-PEG-ATO) and mitochondria-targeted tamoxifen (MitoTam) alleviated hypoxia in tumor spheroids. Here, we investigated the underlying metabolic and redox-related mechanisms-of-action and examined whether mitochondria-targeted OXPHOS inhibition enhances radiotherapy (RT)-induced DNA damage. The metabolic and redox-related effects of Mito-PEG-ATO and MitoTam alone or combined with RT were examined in B16OVA, MOC1.3D5 and MC38 cells or clones containing an HRE-eGFP-ODD construct. Viability was assessed using a CCK-8 assay on 2D cells. ROS levels were measured after treatment with Mito-PEG-ATO and MitoTam for 24 h with CellRox green and fluorescence monitoring via IncuCyte Zoom. Antioxidant capacity was measured after OXPHOS inhibition relative to Trolox, an analogue of Vitamin E used as antioxidant standard, and mitochondrial membrane potential (MMP) was examined using MitoTracker Orange. Intracellular ATP and metabolic dehydrogenase activity was assessed in tumor spheroids via CellTiter-Glo and CCK-8 assays. Viability, MMP, ATP levels and metabolic activity were measured at 4 h and 24 h post-RT. DNA damage was quantified by γH2AX immunofluorescence in spheroids. Tumor hypoxia following treatment with Mito-PEG-ATO was determined with immunohistochemistry in MOC1.3D5 tumor-bearing mice. Mito-PEG-ATO and MitoTam reduced cell viability independent of hypoxia and increased ROS production, which was most pronounced for Mito-PEG-ATO, while no effect was observed on antioxidant capacity. Mitochondrial function was impaired by OXPHOS inhibition, shown by MMP disruption, ATP depletion and reduced metabolic activity, with no further impairment upon combination with RT. However, combining Mito-PEG-ATO or MitoTam with RT increased DNA damage compared to either treatment alone. Treatment with Mito-PEG-ATO in MOC1.3D5 tumor-bearing mice did not alleviate hypoxia or alter lactate levels. Mito-PEG-ATO and MitoTam increase ROS production and disrupt mitochondrial function, enhancing RT-induced DNA damage and potentially improving RT efficacy. However, mice treated with Mito-PEG-ATO did not show a reduction in tumor hypoxia.

The thermo-acidophilic unicellular algal class Cyanidiophyceae diverged from other red algae soon after primary chloroplast acquisition. Cyanidiophyceae possess extremely simple genomes (8.7-17.8 Mb; approximately 4,800-7,800 genes), and the cell-wall-less, genetically tractable strain Cyanidioschyzon merolae 10D has served as a model organism. However, its unknown sexual life cycle has limited its utility in studies of evolution and genetics. Here, we show that in the genera Cyanidioschyzon, Cyanidiococcus, and Cyanidium, the cell-walled diploid form, exclusively observed in nature, produces a cell-wall-less haploid form when the culture pH is lowered, and both proliferate asexually. Cyanidioschyzon merolae 10D is a haploid clone that forms a cell-walled diploid through mating with other haploid clones. In addition, we generated high-quality genomic resources with phase-specific transcriptomes, identified a compact candidate mating-type region, and developed genetic manipulation systems using the cell-wall-less haploids of these genera. We further uncovered phase-specific distributions of histone H3 lysine 27 trimethylation linked to haploid- and diploid-specific gene expression, including transcription factors involved in differentiation associated with sexual reproduction in plants. Additionally, biparental inheritance of organelle DNA occurs following isogamous mating of haploid cells but resolves into uniparental inheritance during diploid proliferation. These advances position Cyanidiophyceae as a powerful model lineage for studying early Archaeplastida evolution, the shared mechanisms of photosynthetic eukaryotes, and environmental adaptation.

Innate immune cells constitute the majority of the tumor microenvironment (TME) and mediate anti-tumor immunity and immunotherapy responses. While single-cell T and B cell receptor sequencing have revealed insights into the clonal dynamics of adaptive immunity, the lack of analogous tools has precluded similar analysis of innate immune cells. Here, we describe a method leveraging somatic mitochondrial DNA (mtDNA) mutations to reconstruct clonal lineage relationships between cells in native human tissues. By jointly profiling single-cell chromatin accessibility and mtDNA variants, we resolve clonal dynamics of 218,715 cells from matched tumors, tissues, and blood from patients with lung and ovarian cancers. Clonal tracing reveals that TME-resident myeloid subsets, including macrophages and type 3 dendritic cells (DC3), are clonally related to circulating and tissue-infiltrating monocytes. We further identify distinct DC-biased and macrophage-biased clones, whose circulating monocyte precursors exhibit distinct epigenetic profiles, suggesting intratumoral myeloid differentiation fate may be peripherally programmed before TME infiltration.

the RhD antigen is highly immunogenic, and poses a significant clinical risk of hemolytic transfusion reactions (HTRs), rendering it a critical factor in safe blood transfusion. Conventional immunization strategies relying on full-length RhD protein are limited by restricted antigen availability, high production costs, and ethical concerns associated with in vivo antibody generation. To address these challenges, we evaluated an in vitro immunization platform using human peripheral blood mononuclear cells (PBMCs) stimulated with either full length RhD protein or a designed synthetic RhD peptide, aiming to establish a rapid, ethical, and reproducible biotechnological alternative for in vitro antibody production. PBMCs from O-negative blood units were isolated by Ficoll density gradient, and treated with L-leucine methyl ester to deplete immunosuppressive cells. PBMCs were stimulated with full length RhD protein, synthetic peptide, or peptide conjugated to Keyhole Limpet Hemocyanin (KLH). Conjugation efficiency was confirmed via ELISA and spectrophotometry (280/412 nm). Cultures were supplemented with IL-4, IFN-γ, and conditioned medium, and then incubated at 37°C for one week. Antibody production in culture supernatants was quantified by ELISA. Peptide-KLH induced higher antibody levels (84.87±13.6 ng/mL) than free peptide (52.4±9.8 ng/mL), while RhD protein elicited (108.7±10.4 ng/mL). Combined stimulation with peptide-KLH and RhD protein further enhanced antibody levels (157.9±23.6 ng/mL). Two-step immunization significantly enhanced responses (P=0.006). The combination of IL-4 and IFN-γ yielded the highest cytokine-driven enhancement. Synthetic RhD peptide, especially when conjugated to KLH, provides an attractive and convenient alternative to full-length protein for in vitro antibody production. This approach has practical applications in biotechnology, including the generation of antibodies and antibody producing clones, reducing reliance on in vivo immunization.

Cancer originates from a single ancestor cell that acquires driver mutations, which confer a selective growth advantage, followed by the acquisition of additional driver mutations by descendant cells. Recently, it has become evident that somatic mutations accumulate even in normal cells with aging; exposure to environmental factors such as alcohol, smoking, and ultraviolet radiation increases this mutation rate. Clones harboring cancer driver mutations expand even in normal tissues with age, leading to tissue remodeling known as somatic mosaicism. Chronic inflammatory diseases can also influence this remodeling, and identification of disease-specific driver mutations can help elucidate the pathogenesis of such diseases. Phylogenetic analysis of cancer and surrounding tissues, including normal cells, has revealed the early evolutionary history of carcinogenesis. In addition, accumulating evidence that somatic mosaicism in the blood affects various diseases-including atherosclerosis and chronic liver disease-highlights the potential role of somatic mosaicism in non-neoplastic disease pathogenesis. This article summarizes recent updates on somatic mosaicism in normal tissues.