Biomedical subjects

Li Zhang

Publications and source records attributed to Li Zhang.

At least 37 records · Page 2Linked to original sources

PubMed2026

Extreme Temperature and Incident Diabetes Risk Among Middle-Aged and Older Adults in China: A National Longitudinal Cohort Study.

BACKGROUND AND AIMS: The metabolic consequences of extreme temperature exposure in nondiabetic populations remain poorly understood. This study aimed to examine associations between heatwave and coldwave exposure and incident diabetes mellitus (DM) and impaired glucose tolerance (IGT) in middle-aged and older Chinese adults. METHODS: A total of 1803 China Health and Retirement Longitudinal Study participants aged ≥45 years with normoglycemia at baseline were followed from Wave 1 (2011) to Wave 3 (2015). Eighteen extreme-temperature indicators were derived from city-level fifth-generation European Centre for Medium-Range Weather Forecasts atmospheric reanalysis data. Outcomes were classified according to American Diabetes Association criteria. Generalized linear mixed-effects models (GLMMs) were pooled across five imputed datasets, with Bonferroni correction for multiple comparisons (α = 0.0028) and sensitivity analysis adjusting for individual follow-up duration. RESULTS: All nine heatwave indicators showed odds ratios (ORs) < 1.0 for DM and IGT. HT9 (≥97.5th percentile, ≥4 consecutive days) was the sole Bonferroni-significant result: OR = 0.845 (95% confidence interval [CI]: 0.802-0.890). Coldwave indicators showed no consistent associations. Age significantly modified the HT9 effect (P-interaction = 0.004): adults aged 65-84 showed a stronger inverse association (OR = 0.616) than those aged < 65 (OR = 0.921). CONCLUSION: Prolonged heatwave exposure was consistently associated with reduced diabetes risk, with pronounced age heterogeneity. Replication in larger prospective studies is warranted.

Humans↗

PubMed2026

Histone demethylase PHF2 drives olanzapine-induced dyslipidemia via epigenomic rewiring of hepatic lipogenic genes.

Olanzapine, an atypical antipsychotic agent, is widely used in treating psychotic disorders, yet its metabolic side effects remain a clinical concern. Emerging evidence suggests that dynamic alterations in histone methylation are implicated in olanzapine-induced hepatic lipid metabolic disorders. PHF2, a JmjC family histone demethylase mediating H3K9me2 demethylation, functions as a transcriptional repressor by regulating downstream targets. To elucidate PHF2's role in this process, we utilized an olanzapine-induced dyslipidemia rat model. ChIP-qPCR analysis demonstrated a significant reduction in dimethylated histone H3 lysine 9 (H3K9me2) on the promoters of lipogenic genes (Fasn, Acc1, Scd1) in the liver, accompanied by elevated nuclear expression of PHF2 in olanzapine-treated rats. Co-immunoprecipitation (Co-IP) assays revealed a physical interaction between PHF2 and ChREBP, a glucose-responsive lipogenic transcription factor. Olanzapine was found to enhance the formation of this complex. Overexpression of PHF2 led to upregulated protein levels of FASN/ACC1 and intracellular lipid accumulation, whereas knockdown of PHF2 using siRNA attenuated these effects. Notably, the upregulation of FASN/ACC1 expression induced by olanzapine was markedly diminished in PHF2-deficient AML12 cells via ChREBP-PHF2-mediated H3K9me2 demethylation. Additionally, olanzapine inhibited the nuclear translocation of FOXA2, a PHF2 transcriptional regulator, thereby augmenting PHF2 expression. These findings uncover a novel epigenetic mechanism underlying olanzapine-induced dyslipidemia, positioning the FOXA2-PHF2-ChREBP axis as a potential therapeutic target through modulation of hepatic histone methylation.

Animals↗

PubMed2026

Aneuploidy selects for the acquisition of driver genes in breast cancer.

Chromosome instability is highly prevalent in cancer and drives large-scale chromosomal imbalances, known as aneuploidies1-4. How aneuploidy contributes to tumorigenesis remains difficult to study due to the vast numbers of genes affected. Here we established a CRISPR knockout- and activation-linked assay (CRISPR-KOALA), enabling high-throughput bidirectional genetic screens in immunocompetent mouse models of cancer. We developed a compendium of the ten most frequent human chromosome-arm-level alterations in basal-like breast cancer (BLBC), a disease type that is driven by large copy-number alterations (CNAs)5-8. Using CRISPR-KOALA, we screened the mouse orthologues of 3,752 genes on these arms and identified 90 cancer driver genes, the function of the vast majority of which is unknown. These genes drive distinct signalling pathways including MAPK, HIPPO and WNT, reflecting the high degree of BLBC heterogeneity. Manipulating the identified cancer driver genes overcomes the need for CNAs in Trp53-mutant BLBC mouse models. Mechanistically, we identify that PLGRKT is a potent oncogene that lies on chromosome 9p and show that its tumour-promoting activity is associated with highly stress-resistant mitochondria and an increased ability to detoxify reactive oxygen species. Together, our findings reveal that arm-level CNAs can function to select specific driver genes to promote heterogeneous biological processes.

Animals↗

PubMed2026

GT-Mamba: a Topology-Aware Graph-State space model for robust and interpretable epigenetic age prediction.

MOTIVATION: Current epigenetic clocks face a trade-off between predictive accuracy and biological interpretability, often relying on dataset-specific correction to generalize across cohorts. We propose GT-Mamba, a novel architecture that integrates a Structure-Aware Graph Transformer with the Mamba state space model. This design captures CpG topological correlations and genome-wide long-range dependencies. RESULTS: GT-Mamba demonstrates strong out-of-the-box robustness across heterogeneous independent validation cohorts, achieving a weighted average MAE of 4.43 years. Notably, it effectively generalizes to EPIC 850k arrays despite partial feature missingness, and maintains consistent performance across homologous age distribution shifts (MAE 2.94 years in a young cohort). Ablation studies confirm that graph topology contributes to improved robustness against noise. Mechanistic analysis suggests that the model captures methylation patterns associated with both developmental and functional processes. AVAILABILITY: Source code and pre-trained models are freely available at https://github.com/NENUBioCompute/GT-Mamba and archived on Zenodo (DOI: 10.5281/zenodo.19703155).

Epigenesis, Genetic↗

PubMed2026

Mitochondrial genomes of Dactylogyrus wunderi (Monopisthocotyla: Dactylogyridae): structural features, codon usage patterns, and phylogenetic implications.

BACKGROUND: Codon usage bias (CUB) is a common phenomenon reported among many species and genes, but its unique characteristics in the mitochondrial genome of class Monopisthocotyla remain unknown. METHODS: The complete mitochondrial genome of Dactylogyrus wunderi was sequenced and characterized, and the mitochondrial genome compositions and CUB of six Dactylogyrus species and 35 Monopisthocotyla species were analyzed using bioinformatics methods. RESULTS: The mitochondrial genome of D. wunderi is a typical circular structure in length of 14,920 bp. The A + T contents of the six Dactylogyrus species (58.4% ± 5.7%) were significantly lower than that of Monopisthocotyla species (71.0% ± 5.80%, p < 0.01). Neutrality plot analysis showed slopes of 0.3136 and 0.389 in the six Dactylogyrus and the 35 Monopisthocotyla species, respectively. Furthermore, 98.3% and 77.4% of the genes in the six Dactylogyrus and the Monopisthocotyla species, respectively, had effective number of codons (ENC) higher than 35, but 23.3% and 0.5% genes of ENC ratio ranged from - 0.05 to 0.05 in the six Dactylogyrus and Monopisthocotyla species. Phylogenetic analysis revealed that, within the context of the sampled taxa, the families of Monopisthocotyla were monophyletic groups, except for Ancyrocephalidae. CONCLUSIONS: The nucleotide composition had AT base bias in Monopisthocotyla, and natural selection was the main factor affecting CUB in the mitochondrial genomes of Monopisthocotyla species. These results provided insights into the factors affecting CUB in Monopisthocotyla species and deepened our insight of phylogeny, evolution, and codon usage of Monopisthocotyla.

Genome, Mitochondrial↗

PubMed2026

Nucleolin promotes neuropathic pain by increasing chromatin accessibility at the Ccl2 promoter in primary sensory neurons.

Nerve injury-induced transcriptional alterations in primary sensory neurons of the dorsal root ganglion (DRG) constitute a key molecular basis for the development of neuropathic pain. Nucleolin (NCL), a highly conserved multifunctional nucleolar protein, regulates gene transcription. Here, we identify that NCL is expressed exclusively in the nuclei of DRG neurons. Peripheral nerve injury time-dependently upregulates Ncl mRNA and NCL protein levels in injured DRG neurons. Blocking this upregulation through DRG microinjection of the adeno-associated virus 9 (AAV9) expressing an shRNA targeting Ncl attenuates nerve injury-induced increases of C-C motif chemokine ligand 2 (CCL2) mRNA and its protein in injured DRG and alleviates the development and maintenance of mechanical, heat and cold hypersensitivities. Conversely, mimicking DRG NCL upregulation through DRG microinjection of AAV9 carrying the full-length Ncl coding sequence increases Ccl2 mRNA and CCL2 protein levels in microinjected DRGs and produces neuropathic pain-like symptoms in the absence of nerve injury. Mechanistically, peripheral nerve injury increases NCL occupancy at the Ccl2 promoter and enhances chromatin accessibility at this locus, resulting in elevated CCL2 expression in injured DRG neurons, which is reversed by NCL knockdown. Given that Ncl mRNA is co-expressed with Ccl2 mRNA in individual DRG neurons, our findings suggest that NCL upregulation in the DRG contributes to neuropathic pain likely by increasing chromatin accessibility at the Ccl2 promoter in primary sensory neurons.

Animals↗

PubMed2026

Characterization of small intestinal neuroendocrine tumorlets.

Small intestine neuroendocrine tumors (SI-NETs) frequently present as multifocal primaries. We commonly observe microscopic lesions in the superficial layer of the small intestine of SI-NET patients. We aimed to define them as small intestinal neuroendocrine tumorlets (SINTs) and explore their clinical and biological significance. Twenty multifocal and twenty unifocal SI-NETs patients who received resection at a single institution were retrospectively reviewed. Four hundred and forty six archived pathological slides were examined for microscopic lesions located in the lamina propria, muscularis mucosa, and superficial submucosa. Clinicopathological associations and progression-free survival were analyzed. Previously published genomic data were re-analyzed. SINTs were identified in 50% of multifocal and 30% of unifocal SI-NET patients. Median SINT size was 95 μm, with a median distance of 2.2 mm from the nearest mass. Compared to the 'true unifocal' group (unifocal without SINT), the 'multifocal-spectrum' group (multifocal or unifocal with SINT) had higher BMI (median: 27.6 vs 22.8, P = 0.0060), higher rates of perineural invasion (OR: 5.5, P = 0.044), larger mesenteric mass (median: 2.6 vs 1.6 cm, P = 0.034), and more advanced pT stage (pT3 or pT4, OR: 7.2, P = 0.018). Genomic re-analysis suggested that 13% of cells in multifocal primary tumors could share clonal origins, possibly indicating clonal spread via SINTs. SINTs may serve as a new biomarker for multifocal spectrum with local aggressiveness. The actual frequency of multifocal SI-NET may be higher than currently recognized in clinical practice. Further studies are needed to validate their prognostic and biological significance.

Humans↗

PubMed2026

Identification of cryosensitive niches and a targetable FOS/AP‑1 program in the human ovarian cortex by single‑cell and spatial transcriptomics.

BACKGROUND: The ovary is a vital and dynamic reproductive organ. Ovarian tissue cryopreservation (OTC) plays a vital role in preserving female fertility. However, the cellular subtypes most susceptible to cryoinjury and the molecular mechanisms underlying cryopreservation-associated damage remain poorly understood. This study aimed to identify cell populations vulnerable to freezing-thawing and to elucidate the key transcriptomic alterations and signaling pathways associated with ovarian cryoinjury at the single-cell and spatial levels. METHODS: Ovarian cortical tissues from patients undergoing three gender reassignment surgery (GRS) were divided into fresh and vitrification-rapid warming groups. Following collagenase IV digestion, 10x Genomics single-cell RNA-seq was used for dissociated ovarian cell suspensions (27,185 fresh and 25,480 frozen-thawed cells). Eight major cell clusters were identified. Additionally, 110 oocytes (66 fresh, 44 vitrification-rapid warming) were isolated and analyzed using the Smart-seq2 platform. Spatial transcriptomics was performed via BGI Stereo-seq. Molecular validation was performed via β-galactosidase staining, immunofluorescence, and qRT-PCR. RESULTS: Cryopreservation significantly altered the activity of pathways related to focal adhesion, oxidative stress, and apoptosis, particularly in stromal and perivascular cells. The number of FOS-positive perivascular cells was notably increased after vitrification-rapid warming, whereas the number of PTGDS-positive stromal cells decreased. Oocyte analysis revealed that cryopreservation primarily disrupted pathways involved in the cell cycle and meiosis, although the damage was not irreversible, supporting the relative safety of long-term cryostorage. Spatial transcriptomics and functional validation further confirmed the rapid and robust activation of the FOS/AP-1 pathway after vitrification-rapid warming, particularly in perivascular and granulosa cells. Treatment with T-5224 (a FOS/AP-1 inhibitor) significantly rescued the morphology and function of cultured frozen-thawed ovaries. CONCLUSIONS: Stromal and perivascular cells are the main cell types that are sensitive to ovarian cryopreservation. The FOS/AP-1 pathway is markedly activated after, suggesting the exacerbation of metabolic impairment. In oocytes within the ovarian cortex, the cell cycle and meiosis-related physiological processes were the primary processes affected.

Female↗

PubMed2026

Widespread Molecular Imprints in the Serum Proteome of COVID-19 Convalescents Uncovering Immune System Sequelae.

Post-COVID-19 sequelae have become an emerging global health issue, but the mechanisms for the sustained susceptibility of convalescents to the sequelae remain poorly understood. Here we report the use of a restricted open-search approach to explore the molecular imprints of SARS-CoV-2 infection left on the proteome of 412 COVID-19 patients and convalescences. A total of 827 non-standard amino acid variations, chemically modified residues as well as post-translational modifications, termed non-coded amino acids (ncAAs), are found spreading over 29,814 sites in patient's serum proteins. Markedly, widespread ncAAs are induced and sustainedly imprinted on the serum proteome predominately perturbing the immunoglobulin-mediated immune response, complement activation and coagulation regulation even 12 months after recovery. Sustained amino acid variations and chemical modifications are found in the complementary‑determining regions (CDRs) of the variable region of immunoglobulin contributing to the interactions between the emerging antibody and antigens; durable chemical amino acid modifications found in the hyper ncAA-modified regions of the constant region of immunoglobulin important for the interaction with the complement and regulatory receptors. In the complement system, inducible ncAAs are memorized in the components essential for the complement activation, amplification cascades and membrane attack processes. Thus, the workflow described in this study can be used to identify the molecular imprints of viral infection at the proteomic scale, particularly the specific antibodies and the immune targets left in COVID-19 patients and convalescents.

Humans↗

PubMed2026

Ivonescimab plus chemotherapy versus placebo plus chemotherapy in patients with advanced EGFR-mutated non-small-cell lung cancer after disease progression on EGFR tyrosine kinase inhibitor therapy (HARMONi): a multicentre, randomised, double-blind, phase 3 trial.

BACKGROUND: Ivonescimab has shown clinical efficacy in non-small-cell lung cancer (NSCLC). We aimed to assess the efficacy and safety of ivonescimab plus chemotherapy versus placebo plus chemotherapy in patients with advanced EGFR-mutated NSCLC whose disease progressed after third-generation EGFR tyrosine kinase inhibitor (TKI) therapy. METHODS: HARMONi is a randomised, placebo-controlled, double-blind, phase 3 trial done at 114 cancer centres and hospitals across Asia, Europe, and North America. Eligible patients were aged at least 18 years (upper limit: 75 years in Asia) with stage IIIB/IIIC or IV non-squamous EGFR-mutated NSCLC, disease progression after treatment with a third-generation EGFR-TKI, and an Eastern Cooperative Oncology Group performance status score of 0 or 1. Patients were randomly assigned (1:1) via a centralised interactive voice response system or interactive web response system to receive ivonescimab (20 mg/kg) or placebo plus pemetrexed (500 mg/m2) and carboplatin (target area under the curve 5 mg/mL per min) intravenously every 3 weeks. Randomisation was stratified by brain metastases status at enrolment and geographical region. The primary endpoints were progression-free survival by blinded independent radiology review committee and overall survival in the intention-to-treat population. Safety was assessed in patients who received at least one dose of trial treatment. This study is registered with ClinicalTrials.gov (NCT06396065), has completed enrolment, and is ongoing for treatment and follow-up. FINDINGS: From Jan 25, 2022, to Oct 1, 2024, 660 individuals were screened for eligibility; of these, 438 were enrolled and randomly assigned to receive ivonescimab plus chemotherapy or placebo plus chemotherapy (219 per group). Of enrolled patients, 257 (59%) were female and 181 (41%) were male; 306 (70%) reported race as Asian, and 105 (24%) as White. At a median follow-up of 22·3 months (95% CI 21·5-23·0), 275 progression or death events had occurred in 345 patients (129 events among 172 patients in the ivonescimab plus chemotherapy group and 146 events among 173 patients in the placebo plus chemotherapy group). Median progression-free survival was 6·8 months (95% CI 5·7-7·1) in the ivonescimab plus chemotherapy group versus 4·4 months (4·1-5·5) in the placebo plus chemotherapy group (hazard ratio [HR] 0·52; 95% CI 0·41-0·66; p<0·0001). At a median follow-up of 29·7 months (95% CI 27·7-31·0), 262 deaths occurred in 438 patients (122 in the ivonescimab plus chemotherapy group and 140 in the placebo plus chemotherapy group). Median overall survival was 16·8 months (14·3-19·0) in the ivonescimab plus chemotherapy group versus 14·0 months (12·8-15·7) in the placebo plus chemotherapy group (HR 0·79; 0·62-1·01). The most common grade 3-4 treatment-related adverse events in the ivonescimab plus chemotherapy versus the placebo plus chemotherapy group were decreased neutrophil count (42 [19%] of 218 vs 36 [17%] of 218), decreased white blood cell count (28 [13%] vs 24 [11%]), decreased platelet count (27 [12%] vs 14 [6%]), and anaemia (22 [10%] vs 27 [12%]). Serious treatment-related adverse events occurred in 61 (28%) patients in the ivonescimab plus chemotherapy group and 33 (15%) patients in the placebo plus chemotherapy group. Treatment-related adverse events led to death in four patients (disease progression, multiple organ dysfunction syndrome, and hepatic failure, each in one patient; gastrointestinal haemorrhage and pulmonary embolism in one patient) in the ivonescimab plus chemotherapy group and five patients (pneumonitis, myocardial infarction, cerebrovascular accident, cognitive disorder, and embolic stroke, each in one patient) in the placebo plus chemotherapy group. INTERPRETATION: Ivonescimab plus chemotherapy showed a clinically meaningful and statistically significant progression-free survival benefit in patients with EGFR-mutated NSCLC after progression on EGFR-TKI therapy. The clinical benefit and lack of new safety signals of ivonescimab with chemotherapy support the potential for the combination as a new treatment option in this patient population. FUNDING: Summit Therapeutics.

Humans↗

PubMed2025

A randomized pilot study of HPV16 L2E7E6 fusion protein vaccination site post-treatment for HPV16+ cervical cancer.

OBJECTIVE: Since anti-tumor immunity is enhanced by vaccination of mice adjacent to human papillomavirus type 16 (HPV16+) tumors, we examined whether HPV16 L2E7E6 fusion protein (TA-CIN) vaccination in the thigh of HPV16+ cervical cancer patients would be more immunogenic than their arm. METHODS: HPV16+ cervical cancer (stage IB1-IVA) patients, who had completed standard-of-care treatment within the past year and absent evidence of disease (NED), were enrolled in a pilot study (NCT02405221). Participants were randomized 1:1 to receive three 100 μg TA-CIN monthly intramuscular immunizations either in the arm or thigh and followed for two years for safety (CTCAEv4.0), immune response, and recurrence. RESULTS: Fifteen patients were enrolled (median age 44, range 35-83 years); one patient experienced a non-vaccine-related adverse event after one vaccination and withdrew. Treatment-related adverse events (n = 8) were grade 1, primarily at the injection site, and self-resolved. No recurrence was observed. TA-CIN-specific antibody titers tended to be higher in thigh-vaccinated patients. Bulk TCRseq revealed significant increases in expanded and de novo T cell clones following thigh-vaccination compared with the arm. No correlation with prior treatment modality was observed. E6-, E7-, and L2-specific TCR clones expanded, although L2-specific T cell responses were predominant. One month post-vaccination, scRNAseq revealed significant expansion of MAIT and cytotoxic CD8+ T cells, and both expanded and novel TCR clonotypes were identified in the latter. CONCLUSIONS: Thigh or arm vaccination with TA-CIN was well tolerated, but the former elicited higher CD8 T cell and antibody responses in HPV16+ cervical cancer patients with NED after primary therapy.

Humans↗

PubMed2025

Oncogenic roles of young human de novo genes and their potential as neoantigens in cancer immunotherapy.

Young human de novo genes, recently emerging from non-coding regions, are expected to contribute to human-specific traits and diseases. However, systematic explorations of this connection have been lacking. Here, we report 37 recently originated de novo genes in humans, with their evolution and characteristics defined within an updated genomic context. The expression of these genes is significantly upregulated and temporospatially expanded in tumors, partially associated with extrachromosomal DNA amplification. Depletion of 57.1% of these genes suppresses tumor cell proliferation, underscoring their roles in tumorigenesis. As a proof of concept, we developed mRNA vaccines expressing ELFN1-AS1 and TYMSOS-young genes specifically expressed during early development but reactivated exclusively in tumors. In humanized mice, these vaccines triggered specific T cell activation and inhibited tumor growth. The antigens derived from these genes are immunogenic and capable of eliciting antigen-specific T cell activation in colorectal cancer patients. These findings underscore young human de novo genes as neoantigens in cancer immunotherapy.

Humans↗

PubMed2025

Exploring biosynthetic potential of the endophytic Penicillium turbatum BLH34 using whole-genome sequence analysis and molecular networking.

An in-depth genomic and metabolomic investigation was conducted on the endophytic fungus Penicillium turbatum BLH34, isolated from Macleaya cordata. Hybrid sequencing (Illumina-Nanopore) generated a high-quality 27.9 Mb genome (GC 48.6%) encoding 9798 proteins, with functional annotation linking 5350 genes to the NCBI non-redundant database and 3404 to KEGG pathways. AntiSMASH analysis uncovered 35 biosynthetic gene clusters (BGCs), 23 of which lacked homology to known pathways, highlighting BLH34's potential for novel metabolite discovery. Molecular networking (GNPS) and LC-MS/MS identified 19 specialised metabolites, including antimicrobial polyketides. Bioassays demonstrated potent inhibition against Staphylococcus aureus (36 mm), Bacillus subtilis (28 mm) and Escherichia coli (24 mm), underscoring its pharmaceutical relevance.

Penicillium↗

PubMed2025

N6-methyladenosine modification of the subgroup J avian leukosis viral RNAs attenuates host innate immunity via MDA5 signaling.

Subgroup J avian leukosis virus (ALV-J), a retrovirus, elicits immunosuppression and persistent infections in chickens. Although it is widely acknowledged that ALV-J can evade the host's innate immune defenses, the mechanisms behind this immune evasion remain elusive. N6-methyladenosine (m6A), the most prevalent internal RNA modification, plays a role in innate immune evasion. Our research identified ALV-J as an inefficient stimulator of innate immunity in vitro and in vivo, with its genomic RNA featuring m6A modifications predominantly in the envelope protein (Env) region and 3' untranslated region (3'UTR). To elucidate the functional consequences of m6A modification, we subsequently generated m6A-deficient ALV-J through its culturing in the DF-1 overexpressing fat mass and obesity-associated protein (FTO) cells. The m6A-deficient ALV-J virus, or its RNAs significantly enhanced IFN-β production compared to the wild-type (wt) ALV-J, suggesting a pivotal regulatory function of m6A modifications in modulating innate immune response. Mechanistically, the m6A modification of the ALV-J genomic RNA directly impacted its recognition by MDA5, weakening its binding and ubiquitination and attenuating IFN-β activation. Moreover, m6A-deficient ALV-J, created by inducing mutations in m6A sites within Env and 3'UTR, exhibited reduced replication capacity and elevated IFN-β expression in host cells. Importantly, this phenomenon was abolished in MDA5-knockout DF-1 cells, further demonstrating the core role of MDA5. These data demonstrate that m6A modification of ALV-J genomic RNA dampens the host's innate immune response through MDA5 signaling pathway.

Animals↗

PubMed2025

Development and validation of a deep learning model based on cascade mask regional convolutional neural network to noninvasively and accurately identify human round spermatids.

INTRODUCTION: The difficulty of identifying human round spermatids (hRSs) has impeded applications of the human round spermatid injection (ROSI) technique. RSs can be accurately screened through flow cytometric analysis utilizing the Hoechst fluorescence profile reflecting DNA, but this method is not suitable for isolating hRSs due to the toxicity associated with Hoechst staining. OBJECTIVE: To evaluate the capacity of a deep learning model grounded in a cascade mask region-based convolutional neural network (R-CNN) for the noninvasive and accurate identification of hRSs. METHODS: In this study, we presented the development and validation of a deep learning model for identifying hRSs through the analysis of 3457 optical light microscope images of sorted hRSs obtained via flow cytometric analysis. The model's accuracy and specificity were evaluated by calculating the mean average precision (mAP). Furthermore, a double-blind experiment was conducted to access the reliability of the proposed model in accurately identifying hRSs. It detected the expression of protamine (PRM1) and/or peanut lectin (PNA), which are established markers for RSs. RESULTS: Our deep learning-based model demonstrated a high precision, achieving a mAP of over 0.80 for isolating hRSs in test datasets. The expression of PRM1 and/or PNA was observed in all cells noninvasively selected by our AI model during an independent double-blind test. This phenomenon confirmed the accuracy and effectiveness of the proposed model. The model's capability for noninvasive and accurate isolation of hRSs among spermatogenic cells highlighted its robustness and generalizability for clinical applications. CONCLUSION: The deep learning AI model based on a cascade R-CNN has the ability to accurately identify hRSs among spermatogenic cells. The application of this noninvasive method, which requires no additional procedures in clinical practice, is able to facilitate the widespread implementation of ROSI technique. Therefore, it can provide patients with spermatogenic arrest the opportunity to become biological fathers.

Humans↗

PubMed2025

Evolutionary fingerprints of epithelial-to-mesenchymal transition.

Mesenchymal plasticity has been extensively described in advanced epithelial cancers; however, its functional role in malignant progression is controversial1-5. The function of epithelial-to-mesenchymal transition (EMT) and cell plasticity in tumour heterogeneity and clonal evolution is poorly understood. Here we clarify the contribution of EMT to malignant progression in pancreatic cancer. We used somatic mosaic genome engineering technologies to trace and ablate malignant mesenchymal lineages along the EMT continuum. The experimental evidence clarifies the essential contribution of mesenchymal lineages to pancreatic cancer evolution. Spatial genomic analysis, single-cell transcriptomic and epigenomic profiling of EMT clarifies its contribution to the emergence of genomic instability, including events of chromothripsis. Genetic ablation of mesenchymal lineages robustly abolished these mutational processes and evolutionary patterns, as confirmed by cross-species analysis of pancreatic and other human solid tumours. Mechanistically, we identified that malignant cells with mesenchymal features display increased chromatin accessibility, particularly in the pericentromeric and centromeric regions, in turn resulting in delayed mitosis and catastrophic cell division. Thus, EMT favours the emergence of genomic-unstable, highly fit tumour cells, which strongly supports the concept of cell-state-restricted patterns of evolution, whereby cancer cell speciation is propagated to progeny within restricted functional compartments. Restraining the evolutionary routes through ablation of clones capable of mesenchymal plasticity, and extinction of the derived lineages, halts the malignant potential of one of the most aggressive forms of human cancer.

Animals↗

PubMed2025

Characterization of group I introns in generating circular RNAs as vaccines.

Circular RNAs are an increasingly important class of RNA molecules that can be engineered as RNA vaccines and therapeutics. Here, we screened eight different group I introns for their ability to circularize and delineated different features that are important for their function. First, we identified the Scytalidium dimidiatum group I intron as causing minimal innate immune activation inside cells, underscoring its potential to serve as an effective RNA vaccine without triggering unwanted reactogenicity. Additionally, mechanistic RNA structure analysis was used to identify the P9 domain as important for circularization, showing that swapping sequences can restore pairing to improve the circularization of poor circularizers. We also determined the diversity of sequence requirements for the exon 1 and exon 2 (E1 and E2) domains of different group I introns and engineered a S1 tag within the domains for positive purification of circular RNAs. In addition, this flexibility in E1 and E2 enables substitution with less immunostimulatory sequences to enhance protein production. Our work deepens the understanding of the properties of group I introns, expands the panel of introns that can be used, and improves the manufacturing process to generate circular RNAs for vaccines and therapeutics.

RNA, Circular↗

PubMed2025

Construction and In Vitro and In Vivo Analysis of Coxsackievirus B4 Reporter Viruses: Attenuated Virulence but Highly Efficient for Antiviral Drug Screening and Evaluation.

Coxsackievirus B4 (CVB4) is an enterovirus with one of the highest mortality rates following infection, yet research on it remains limited. To enhance the efficiency of CVB4 research, we developed the rCVB4-EGFP and rCVB4-NanoLuc reporter viruses. The replication kinetics of these reporter viruses in SH-SY5Y and HeLa cells were essentially consistent with those of the wild-type CVB4. A strong correlation was observed between the fluorescence and bioluminescence signals of rCVB4-EGFP and rCVB4-NanoLuc and viral titers at specific times postinfection. When evaluating the anti-CVB4 drug fluoxetine using these reporter viruses, the half-maximal effective concentrations derived from fluorescence signals, bioluminescence signal intensities, and viral genome copies were consistent. In In Vivo drug evaluations, because CVB4 can infect various tissues and organs, the bioluminescence signal of rCVB4-NanoLuc effectively demonstrated the antiviral effects of drugs, offering significant advantages over traditional tissue viral titer analysis. The reporter viruses exhibited reduced virulence compared with wild-type CVB4 both In Vitro, in SH-SY5Y and HeLa cells, and In Vivo, in ICR suckling mice. Although this reduced virulence may limit their application for studying pathogenic mechanisms, these reporter viruses can serve as highly efficient tools for high-throughput screening and evaluation of anti-CVB4 drugs, vaccines, and neutralizing antibodies.

Humans↗