Immune‑regulatory dysfunction caused by human papillomavirus (HPV) infection can lead to severe condyloma acuminate (CA), and the treatment is challenged by obstinacy and recurrence. Hyperthermia treatment stimulates the immune response by raising the body temperature locally to fight pathogens. Hyperthermia treatment possesses advantages of low recurrence rate and good efficacy in curing viral warts. However, the exact modulatory mechanism of hyperthermia treatment on immune response remains to be addressed. HPV 16 pseudovirus (HPV.PSV)‑infected HaCaT cells were established to mimic clinical HPV infection. Flow cytometry showed higher major histocompatibility complex class I (MHC‑I) expression in HPV.PSV infected cells. HPV.PSV‑infected cells, CaSki cells and CA tissue demonstrated upregulated MHC‑Ⅰ expression following 44˚C water bath incubation, as detected by flow cytometry and IHC. 4D‑FastDIA quantitative proteomics was used to analyze differential protein expression in CaSki cells following 37‑ or 44˚C incubation. High mobility group box (HMGB)1 was upregulated following 44˚C incubation, as shown by ELISA and western blotting. By applying HMGB1 knockdown cell lines established by small interfering RNA, the present study demonstrated HMGB1 was essential to MHC‑Ⅰ expression, which was detected by flow cytometry and the recombinant HMGB1 protein addition test. Western blotting and ELISA demonstrated that hyperthermia increased heat shock protein family A member 6 (HSPA6) expression and JNK phosphorylation, which resulted in greater secretion of HMGB1. Hyperthermia treatment facilitated HSPA6‑modulated JNK phosphorylation, which lead to HMGB1 secretion and enhanced the expression of MHC‑Ⅰ in HPV‑infected epithelial cells, as well as strengthened the host immune regulation and recognition.

Actinic keratosis is a precancerous skin lesion caused by long-term sun exposure, and may progress to cutaneous squamous cell carcinoma. Bowen disease is a squamous cell carcinoma in situ of the epidermis. Differential diagnosis of actinic keratosis, Bowen disease and cutaneous squamous cell carcinoma, as well as early diagnosis of cutaneous squamous cell carcinoma, has always been research hotspots. This review summarizes biomarkers related to the malignant progression of actinic keratosis, with a view to providing a reference for early clinical diagnosis.

Acute myeloid leukemia (AML) is a hematologic malignancy, and lymphangiogenesis can affect the proliferation, invasion, and other biological behaviors of leukemia cells. This study explored lymphangiogenesis-associated mechanisms in AML. AML datasets were downloaded from public databases. Differential expression analysis, univariate Cox regression, and machine learning were used to identify prognostic lymphangiogenesis-related genes (LYMRGs) and build a risk model. Prognostic analyses included enrichment pathway, genetic mutation, immune microenvironment, and drug sensitivity analyses. Dataset GSE116256 explored LYMRG expression in key cells; GSE142698 and RT-qPCR verified prognostic LYMRG expression. A 6-LYMRG (ANGPT1, HGF, MAPK8, PCNA, TBL1XR1, TLR4) risk model was the optimal prognostic signature. Moreover, pathways like cytokine-cytokine receptor interaction and immune cells such as macrophages were found to be associated with risk stratification in AML patients. Mutational patterns differed between different risk AML patients. High-risk AML patients showed greater sensitivity to UMI.77, vorinostat, BI.2536, tozasertib, daporinad, carmustine, MIM1, and WEHI.539. Furthermore, significant changes in prognostic LYMRG expression were observed during key cell (including progenitor cells, monocyte-derived dendritic cells, and erythroblasts) differentiation. Importantly, GSE142698 and RT-qPCR confirmed that HGF, MAPK8, PCNA, and TBL1XR1 were abundantly expressed, while TLR4 showed low expression in AML patients. This study identified ANGPT1, HGF, MAPK8, PCNA, TBL1XR1, and TLR4 as the key prognostic indicators for AML. The lymphangiogenesis-associated risk model provided an efficient tool for predicting patient survival and might facilitate the development of personalized treatment strategies for AML.

BACKGROUND:The mechanisms of chemotherapy sensitivity and toxicity are complex. Metabolomics can better reflect the status of anticancer drugs, tumors, and hosts simultaneously. METHODS:Mice were implanted with human gastric cancer cells through subcutaneous xenografting, and then treated with the PF (platinum-fluorouracil) regimen, with saline serving as the control. Tumor growth was monitored by measuring tumor volume, and body weight was recorded on Days 0, 2, 4, 6, and 8. Kidney damage was assessed using H&E staining. To analyze differential responses, PF-treated mice were grouped separately according to chemotherapy sensitivity (high/medium/low via tumor response) and toxicity (high/medium/low via body weight changes). Serum metabolomics was evaluated using Mass Spectrometry. RESULTS:Platinum-Fluorouracil (PF) chemotherapy significantly reduced tumor weight in mice, although it also induced notable body weight loss and renal toxicity compared to controls. Serum metabolomic analysis revealed significant differences between PF and control groups, involving metabolites like deoxymethylmycin and dehydrocorticosterone, associated with AMPK and cortisol synthesis/secretion pathways. Further comparisons highlighted: (1) High- vs. lowsensitivity subgroups differed significantly in metabolites, such as palmitoyl-CoA and indoleacetic acid (linked to AGE-RAGE, insulin resistance, and AMPK pathways). (2) High- vs. lowtoxicity subgroups displayed significant metabolic differences, including methylguanosine and methylcytidine (implicated in ferroptosis, ether lipid, and fatty acid metabolism pathways). CONCLUSION:The PF regimen effectively inhibits the growth of subcutaneous tumors in nude mice, while causing varying levels of sensitivity and toxicity in tumor chemotherapy. These observed effects of sensitivity and toxicity are linked to underlying metabolic mechanisms.

Psoriasis is a common immune-mediated skin disease driven largely by interleukin-17A (IL-17A). Although IL-17A plays a key role in disease pathogenesis, the underlying mechanisms remain incompletely understood. Through bioinformatic analysis, we discovered that proline/arginine-rich end leucine-rich repeat protein (PRELP) expression is upregulated in skin lesions from psoriasis patients following treatment with IL-17A inhibitors (secukinumab, ixekizumab, and brodalumab), despite being significantly downregulated in lesional compared to nonlesional skin at baseline. Experimental assays confirmed that IL-17A suppressed PRELP expression in keratinocytes, consistent with its reduced expression in lesional tissues from both murine models and human patients. Functionally, PRELP suppressed keratinocyte proliferation, promoted apoptosis, and attenuated activation of the NF-κB and MAPK pathways, along with downstream proinflammatory cytokine and chemokine production. By downregulating IL6 in keratinocytes, PRELP further attenuated local IL-17A production via IL6 modulation, suggesting a break in the feed-forward loop of psoriatic inflammation. Intradermal administration of AAV-K14-PRELP ameliorated psoriasis-like findings in mice, including erythema, scaling, epidermal hyperplasia, and Th17 cell infiltration. Mechanistically, IL-17A suppressed PRELP transcription by activating STAT3, which directly binds to the PRELP promoter as a transcriptional repressor. Collectively, our findings identify PRELP as a negative regulator of IL-17A signaling in psoriasis, acting through keratinocyte dysregulation and modulation of Th17 cells. Therapeutic strategies aimed at enhancing PRELP expression may represent a novel approach for treating psoriasis and other Th17-driven inflammatory diseases.

Neonatal hypoxic-ischemic encephalopathy (NHIE) is a leading cause of morbidity and mortality in term infants. The anesthetic dexmedetomidine (Dex) has been shown to reduce brain damage. In this study, hypoxia-ischemia (HI) in neonatal rats caused significant cerebral infarction, neurological deficits, learning and cognitive impairments, inflammatory responses, and microglia polarization. Dex treatment mitigated HI-induced brain injury in rats. Lipopolysaccharide (LPS) increased inflammation in BV2 cells, elevated M1 polarization markers, and raised the proportion of M1 cells. Dex reduced inflammation and M1 polarization in BV2 cells. Rbm47 was identified as a target of Dex, being downregulated in NHIE rat brain tissues and upregulated by Dex. Rbm47 co-localized with microglia and was decreased as the microglia marker Iba-1 increased. Adenovirus-mediated overexpression of Rbm47 alleviated brain injury in NHIE rats and reduced microglial inflammation and M1 activation, both in vitro and in vivo. Conversely, knockdown of Rbm47 hindered the protective effects of Dex against BV2 cell inflammation and M1 polarization. This study indicates that Rbm47 mediates the protective effects of Dex against NHIE brain injury.

Diabetic retinopathy (DR), the leading cause of adult blindness, has its risk increased by excessive endoplasmic reticulum stress (ERS). Nuclear receptor subfamily 2 group f member 2 (NR2F2) is an orphan nuclear receptor with essential roles in angiogenesis. However, its roles in DR remain unknown. Notably, NR2F2 protein expression was upregulated in streptozotocin-induced diabetic mice retina and the retinal endothelial cells characterized by endothelial marker CD31. Retinal NR2F2 expression was knocked down in diabetic mice using adeno-associated virus serotype 2. NR2F2 knockdown increased retinal thickness, decreased acellular capillary formation and FITC-labeled dextran leakage, and increased tight junction (TJ) protein ZO-1 expression in vivo. Additionally, NR2F2 knockdown inhibited reactive oxygen species generation and the ERS marker CHOP expression in vivo. In high glucose (HG)-induced human retinal microvascular endothelial cell (HRMEC) monolayers, NR2F2 knockdown inhibited FITC-dextran flux, increased transendothelial electrical resistance, reduced VEGFA secretion and restored expression and continuous distribution of TJ proteins. NR2F2 knockdown also reduced ERS markers expression and inhibited ERS-related signaling pathways in vitro. mRNA-seq analysis in NR2F2-knockdown HRMECs exposed to HG showed that NR2F2 inhibition upregulated CYB5R2 mRNA level. NR2F2 was bound directly to the CYB5R2 promoter for transcriptional repression in HRMECs. CYB5R2 overexpression inhibited ERS-related protein expressions and barrier dysfunction in HRMEC monolayers, and CYB5R2 knockdown reversed the endothelial protective effect of NR2F2 inhibition. In conclusion, NR2F2 knockdown attenuates vascular dysfunction by alleviating ERS in retinal microvascular endothelial cells and alleviates disease progression in DR mice. This may be achieved by reducing CYB5R2 transcriptional repression.

Background:Locally advanced gastric/gastroesophageal junction (G/GEJ) adenocarcinoma faces high recurrence risks despite radical surgery. Perioperative chemotherapy (e.g., FLOT regimen) improves survival but has limited pathological complete response (pCR) rates and significant toxicity. Immunotherapy and anti-angiogenic agents show promise in advanced G/GEJ cancer. This trial evaluates fruquintinib (a VEGFR-1/2/3 inhibitor), sintilimab (PD-1 inhibitor), and SOX (oxaliplatin+S-1) as perioperative therapy for resectable locally advanced G/GEJ adenocarcinoma. Methods:This prospective, single-arm, phase II trial (N = 25) enrolls treatment-naïve adults (18-75 years) with histologically confirmed, resectable cT3-4aN+M0 G/GEJ adenocarcinoma (AJCC 8th edition). Patients receive 3 cycles of neoadjuvant therapy:Fruquintinib:4 mg orally, days 1-14 (21-day cycle). S-1: 80-120 mg orally twice daily (based on BSA), days 1-14. Oxaliplatin: 130 mg/m² IV, day 1. Sintilimab: 200 mg IV, day 1.Radical gastrectomy with D2 lymphadenectomy follows 4-6 weeks post-neoadjuvant therapy. Adjuvant therapy (3 cycles of sintilimab for pCR patients; 3 cycles of preoperative regimen for non-pCR) starts 4-6 weeks post-surgery. Endpoints: Primary: pCR rate (ypT0/Tis ypN0 per CAP criteria). Secondary: R0 resection rate, major pathological response (MPR, ≤10% residual tumor), 2-year event-free survival (EFS), 2-year overall survival (OS), safety (NCI CTCAE v5.0). Exploratory: Biomarker analysis of tumor microenvironment. Statistical Analysis: Sample size (25 patients) was calculated using Fisher's exact test (one-sided α = 0.05, power = 80%), assuming pCR improvement from 5% (historical control) to 20%. Efficacy analyses use intention-to-treat (ITT) population; safety analyses include patients receiving ≥1 neoadjuvant dose. Discussion:This is the first trial combining fruquintinib, sintilimab, and SOX in perioperative G/GEJ cancer. If successful, it may expand treatment options for locally advanced disease. Limitations include single-arm design and small sample size. Trial Registration:Chinese Clinical Trial Registry (ChiCTR2400084194).

BACKGROUND:Acute liver failure (ALF) is a heterogeneous syndrome with high mortality. Current prognostic models fail to capture pathophysiological heterogeneity, necessitating refined patient stratification and personalized therapeutic strategies. METHODS:We analyzed 2,691 adult patients with ALF from six international critical care databases (MIMIC-IV as discovery cohort, n = 1,227; five validation cohorts: MIMIC-III CareVue, eICU, HiRID, NWICU, and SICdb, n = 1,464). We developed a Multi-algorithm Consensus-based Acute Liver Failure Classification (MCALFC) approach integrating ten clustering algorithms to identify distinct subtypes based on clinical parameters, interventions, comorbidities, and medications. We validated these subtypes across databases, performed survival analyses, conducted SHAP analysis using XGBoost to identify key distinguishing features, and evaluated treatment response heterogeneity. RESULTS:Three robust ALF phenotypes emerged: Subtype 1 (28.3 %) - "critical hemodynamic collapse" with severe cardiovascular instability (MAP 78.9 mmHg, HR 113.5 bpm), highest illness severity (SAPS II: 62.0, SOFA: 12.2), and organ support requirements; Subtype 2 (44.6 %) - "cardiovascular dysfunction" with profound hypotension (MAP 69.1 mmHg) but low heart rate (84.3 bpm); and Subtype 3 (27.0 %) - "hyperacute hepatic necrosis" with preserved hemodynamics (MAP 99.4 mmHg) but severe hepatocellular injury (ALT 1059.8 U/L, AST 1427.3 U/L). Subtypes demonstrated distinct survival trajectories maintained through one-year follow-up (28-day mortality: Subtype 2 HR 0.48, 95 % CI: 0.40-0.59; Subtype 3 HR 0.39, 95 % CI: 0.31-0.50 vs. Subtype 1). Treatment responses varied significantly: epinephrine was harmful in Subtype 1 (OR 1.68) but protective in Subtypes 2 and 3 (OR 0.47, 0.34); dexmedetomidine benefited Subtype 3 (OR 0.26) but harmed Subtype 2 (OR 1.47); renal replacement therapy showed highest risk in Subtype 3 (OR 2.05). CONCLUSION:ALF comprises three distinct phenotypes with unique pathophysiological features, prognostic trajectories, and treatment responses. This phenotypic classification, validated across multiple international databases, provides a framework for precision medicine in ALF management and challenges the current one-size-fits-all treatment paradigm.

Sepsis can trigger systemic inflammation and lead to detrimental effects on several organs, with particular emphasis on the lungs. In sepsis-associated lung injury, macrophages assume a pivotal role, as their overactivation could facilitate the secretion of inflammatory factors and the imbalance of polarization. Hepatocyte nuclear factor 4 alpha (HNF4A) has been reported its potential involvement in the regulation of inflammatory response and macrophage polarization. This study discusses the role and mechanism of HNF4A in sepsis-induced lung damage. HNF4A exhibits a decrease in expression by analyzing the differentially expressed genes in the lungs of septic mice from the Gene Expression Omnibus dataset GSE15379. Then, we established a mouse sepsis model through a cecal ligation and puncture method and observed that the expression of HNF4A was reduced in both lung tissues and alveolar macrophages. To evaluate the function of HNF4A, we overexpressed HNF4A mediated by adenovirus vectors, which were injected into mice. We found that HNF4A overexpression resulted in a higher survival rate in septic mice and an amelioration of pulmonary damage. Meanwhile, HNF4A overexpression mitigated the infiltration of inflammatory cells and impeded the M1 polarization but facilitated the M2 polarization of macrophages in the lung tissues or the alveolar lavage fluid. In vitro, we treated bone marrow-derived macrophages with interleukin-4. Consistent results were obtained that HNF4A overexpression promoted the M2 polarization of macrophages. Mechanistically, we found that HNF4A transcriptionally regulate the expression of nuclear receptor coactivator 2 (NCOA2) through binding to its promoter region. NCOA2 interacted with glucocorticoid receptor (GR). Stabilin 1 (STAB1) was selected as a possible target by transcriptome sequencing analysis. Functional experiments confirmed STAB1 as a downstream target of the HNF4A/NCOA2/GR axis. Overall, this research investigated the potential impact of HNF4A on pulmonary injury in sepsis. It is suggested that one of the regulatory mechanisms involved in this association may be the NCOR2/GR/STAB1 axis.

Spinal cord injury (SCI) represents a significant neurological disorder that profoundly impacts human life. Transplantation of extracellular vesicles (EVs) from human umbilical cord mesenchymal stem cells (hUC-MSCs) has emerged as a promising therapeutic strategy. microRNA (miRNA) containing EVs serve as crucial mediators of intercellular communication, playing vital roles in physiological and pathological processes. Research indicates that EVs from hUC-MSCs could attenuate inflammation and facilitate recovery from SCI. Nevertheless, their application in clinical treatment necessitates further investigation. We are actively pursuing an effective approach to modulate the intensity of the inflammatory response, thereby addressing secondary SCI. Initially, we activated hUC-MSCs with interleukin-4 (IL-4) and subsequently harvested their EVs. We investigated the influences of A-hUC-MSCs-EVs compared to routinely acquired EVs on macrophage polarization phenotypes both in vitro and in vivo. Our results show that EVs originating from A-hUC-MSCs are more effective at promoting macrophage polarization from the M1 phenotype to the M2 phenotype than those derived from hUC-MSCs. Notably, we found that A-hUC-MSCs-derived EVs had a superior impact on motor function recovery in mice with SCI. Importantly, we observed that IL-4 activation significantly upregulated the expression of miR-21-5p within these EVs. More specifically, our data demonstrate that A-hUC-MSCs-EVs depend on miR-21-5p to inhibit the effects of PDCD4 on macrophage polarization. This mechanism regulates inflammatory responses while simultaneously reducing apoptosis. In summary, EVs derived from IL-4 primed hUC-MSCs are enriched with miR-21-5p, which exerts a pivotal influence in shifting macrophage polarization, alleviating inflammatory responses following SCI, and facilitating recovery.

Endothelial barrier dysfunction and the resulting vascular injury are responsible for multiorgan failure in sepsis. Myeloid C-type lectin domain family 5 member A (CLEC5A) is a pattern recognition receptor involved in host defense against infection. Mice lacking CLEC5A were resistant to cecal ligation and puncture (CLP)-induced polymicrobial sepsis and lipopolysaccharide (LPS)-induced endotoxemia, as observed by decreased mortality. Single-cell RNA sequencing revealed transcriptomic heterogeneity of vascular endothelial cells in CLEC5A-deficient lungs following CLP. Endothelial-specific knockdown of CLEC5A improved survival of CLP-challenged mice, which was completely ineffective with reexpression of endothelial CLEC5A. The survival benefits were attributed to alleviated inflammatory storm and vascular leakage. Furthermore, endothelial CLEC5A deficiency protected mice against Escherichia coli-induced pneumonia. In vitro, CLEC5A deletion maintained trans-endothelial electrical resistance, and inhibited adhesion and trans-endothelial migration of monocytes/neutrophils under LPS stimulation. The study unveils the importance of CLEC5A in regulating endothelial barrier function and suggests endothelial CLEC5A as a therapeutic target for pneumonia or sepsis-causing bacterial infection.

BACKGROUND:Serum lactate levels are used to evaluate tissue hypoxia and predict outcomes in cases of sepsis and septic shock. Lactate can participate in a posttranslational modification known as lactylation. Myocardial depression during sepsis and septic shock is common. Here, we investigated the role of lactate in sepsis-induced myocardial depression. METHODS:Septic myocardial depression in rats was induced by lipopolysaccharide administration or cecal ligation and puncture. Lactylation and protein profiles of heart tissues from the control and lipopolysaccharide groups were analyzed using proteomic analysis. Lactylation of the HADHA (trifunctional enzyme subunit alpha) at K166 and K728 was detected in septic heart tissues and lipopolysaccharide-induced cultured cells. Mutation of K166 and K728 HADHA were used to clarify the effects of HADHA lactylation on mitochondrial function, ATP production, energy metabolism, and heart function. Transcriptomic and metabolomic analyses were used to identify differentially expressed genes and differential metabolites in H9c2 (rat cardiomyoblast cell line) cells. RESULTS:We identified 1127 lysine lactylation sites, with 83 differentially lactylated lysine sites. By integrating multifeature hybrid learning and protein language models, we identified lactylation at K166 and K728 of the HADHA as functionally important. We confirmed that lactylation at these sites was influenced by lactate levels and inhibited the HADHA activity, which disturbed mitochondrial function, ATP production, and energy metabolism. This reduction in the contraction force of cardiomyocytes can influence heart function in vitro and in vivo. Furthermore, this study revealed that sirtuin 1 and sirtuin 3 regulated the lactylation of HADHA at K166 and K728. CONCLUSIONS:This study reveals the significant impact of lactylation on cardiomyocyte metabolism. Lactate-induced HADHA lactylation disturbs cardiomyocyte mitochondrial function and metabolism and promotes sepsis-induced cardiac dysfunction. These findings inform the development of new therapeutic targets for sepsis-induced myocardial depression.

To investigate the effect and mechanism of exosomes derived from human amniotic mesenchymal stem cells (hAMSC-Exos) promoting angiogenesis. HAMSC-Exos were isolated using ultracentrifugation and characterized by transmission electron microscopy, NTA, and Western blot. The uptake of hAMSC-Exos by hUVECs was analyzed using PKH-26 labeling, and the effect of hAMSC-Exos on angiogenesis was analyzed in human umbilical vein endothelial cells hUVECs by cell viability assay, Transwell migration assay, Matrigel tube formation assay, and Matrigel plug assays in nude mice. Bioinformatics methods were used to analyze miRNA high-throughput sequencing data of hAMSC-Exos, and RT-qPCR was used to validate the novel miRNAs. HAMSC-Exos with high and low N-194 expression were obtained by transfection, respectively. Target genes were predicted using TargetScan, and the mRNA and protein levels of potential target genes were analyzed by RT-qPCR and Western blot after N-194 mimics transfection. Interaction between miRNAs and target genes was detected using the dual-luciferase reporter assay. Target genes were overexpressed in hUVECs by transfection. The roles of target genes in the influence of N-194 on cell function were determined by analyzing angiogenesis. The extracted hAMSC-Exos showed saucer-shaped under transmission electron microscopy, and the NTA results showed the particle size of 115.6 ± 38.6 nm. The positive expression of CD9, CD63, and CD81 were verified using Western blot. The treatment of hUVECs with hAMSC-Exos significantly increased cell proliferation, migration, and angiogenesis. HAMSC-Exos contained the novel miRNAs N-194, N-314, N-19, N-393, and N-481, and the expression of N-194 was higher. The Exos derived from hAMSCs which were transfected with FAM-N-194 mimics were able to deliver FAM-N-194 mimics to hUVECs. The hAMSC-Exos with high N-194 significantly promoted angiogenesis in hUVECs. N-194 mimics transfection significantly reduced mRNA and protein levels of potential target gene ING5, and N-194 mimics significantly reduced the luciferase activities expressed by wild-type reporter gene vectors for ING5. The ING5 overexpression significantly reduced the angiogenic capacity of hUVECs. ING5 overexpression suppressed the expression of HSP27 and PLCG2. HAMSC-Exos promotes angiogenesis in hUVECs by delivering novel miRNA N-194 which targets ING5.

Breast cancer is characterized by significant molecular heterogeneity; therefore, there are distinct clinical features, treatment modalities, and prognostic outcomes across its various molecular subtypes. In the era of precision medicine, liquid biopsy has emerged as a convenient and minimally invasive technique capable of dynamically representing the comprehensive tumor gene spectrum. This review systematically elaborates the clinical value of liquid biopsy as a breakthrough tool for precision diagnosis and treatment in breast cancer through dynamic detection of key biomarkers, including circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), exosomes, and non-coding RNA (ncRNA). Specific genetic mutations and methylation signatures in ctDNA can be applied to early breast cancer screening, minimal residual disease monitoring, and tracking drug resistance mechanisms. CTCs enumeration (≥1/7.5 mL in early-stage cancer or ≥ 5/7.5 mL in metastatic cancer) and PD-L1 expression levels demonstrate direct correlations with prognostic stratification and the efficacy of immunotherapy. As the specificity and sensitivity of liquid biopsy continue to improve, personalized treatment strategies, informed by biomarker analysis and targeted precision therapies, have unveiled new avenues of hope for patients with breast cancer. However, several challenges persist in the practical application of liquid biopsy. Despite persistent challenges, such as insufficient standardization and difficulties in resolving low-abundance variants, future advancements should focus on multi-omics integration and AI-driven technological breakthroughs to overcome bottlenecks in clinical translation. This review summarizes cutting-edge liquid biopsy technologies for identifying clinically significant molecular biomarkers, focusing on discussing critical challenges in the strategies to advance precision oncology applications for optimized treatment guidance and disease surveillance in breast cancer.

BACKGROUND: Intracellular Ca 2+ cycling governs effective myocardial systolic contraction and diastolic relaxation. SERCA2a (sarco/endoplasmic reticulum Ca 2+ ATPase type 2a), which plays a crucial role in controlling intracellular Ca 2+ signaling and myocardial cell function, is downregulated and inactivated during sepsis-induced heart dysfunction. However, the cause of this dysregulation remains unclear. In this study, we investigated the effect of lysine succinylation in lipopolysaccharide-induced septic heart dysfunction through global succinylome analysis of myocardial tissues from septic rats. METHODS: We conducted a succinylome profiling and developed a protein language model–based framework to prioritize succinylation at a functionally important site, and further analysis revealed crosstalk between ubiquitination and succinylation of SERCA2a. The succinylation of SERCA2a in septic rats or lipopolysaccharide-treated cells were detected by co-immunoprecipitation. Thereafter, a desuccinylated SERCA2a K352R was introduced and its function and stability were determined by Ca 2+ transient and Western blot, respectively. Meanwhile, the effect on SERCA2a K352R on heart function was assessed in vivo by echocardiography and hemodynamics. RESULTS: We identified 10 324 succinylated lysine sites in heart tissues, including 1042 differentially succinylated lysine sites, in response to lipopolysaccharide. SERCA2a was hypersuccinylated in the myocardial tissues of septic rats and lipopolysaccharide-treated cardiomyocytes. Increased ubiquitination level, reduced protein level, and activity of SERCA2a were observed, along with increased succinylation of SERCA2a in vivo and in vitro. K352 was essential for SERCA2a succinylation, which reduced SERCA2a protein level by promoting formation of the K48 ubiquitin chain on SERCA2a and its degradation by proteasomes. Co-immunoprecipitation combined with liquid chromatography–tandem mass spectrometry identified that SIRT2 (sirtuin2), a deacylase, exhibited interaction with SERCA2a. Furthermore, SIRT2 decreased K352 succinylation of SERCA2a, suggesting that SIRT2 may function as a desuccinylase for SERCA2a. CONCLUSIONS: Succinylation of SERCA2a at K352, which was controlled by SIRT2, promotes its ubiquitinoylation and degradation by proteasomes in sepsis-induced heart dysfunction.

BACKGROUND Gastric cancer (GC) is one of the most common malignancies worldwide. Glycolysis has been demonstrated to be pivotal for the carcinogenesis of GC. AIM To develop a glycolysis-based gene signature for prognostic evaluation in GC patients. METHODS Differentially expressed genes correlated with glycolysis were identified in stomach adenocarcinoma data (STAD). A risk score was established through a univariate Cox and least absolute shrinkage and selection operator analysis. The model was evaluated using the area under the receiver operating characteristic curves. RNA-sequencing data from high- and low-glycolysis groups of STAD patients were analyzed using Cibersort algorithm and Spearman correlation to analyze the interaction of immune cell infiltration and glycolysis. Multiomics characteristics in different glycolysis status were also analyzed. RESULTS A five-gene signature comprising syndecan 2, versican, malic enzyme 1, pyruvate carboxylase and SRY-box transcription factor 9 was constructed. Patients were separated to high- or low-glycolysis groups according to risk scores. Overall survival of patients with high glycolysis was poorer. The sensitivity and specificity of the model in prediction of survival of GC patients were also observed by receiver operating characteristic curves. A nomogram including clinicopathological characteristics and the risk score also showed good prediction for 3- and 5-year overall survival. Gene set variation analysis showed that high-glycolysis patients were related to dysregulation of pancreas beta cells and estrogen late pathways, and low-glycolysis patients were related to Myc targets, oxidative phosphorylation, mechanistic target of rapamycin complex 1 signaling and G2M checkpoint pathways. Tumor-infiltrating immune cells and multiomics analysis suggested that the different glycolysis status was significantly correlated with multiple immune cell infiltration. The patients with high glycolysis had lower tumor mutational burden and neoantigen load, higher incidence of microsatellite instability and lower chemosensitivity. High glycolysis status was often found among patients with grade 2/3 cancer or poor prognosis. CONCLUSION The genetic characteristics revealed by glycolysis could predict the prognosis of GC. High glycolysis significantly affects GC phenotype, but the detailed mechanism needs to be further studied.

BACKGROUND Increasing evidence has demonstrated that N6-methyladenosine (m6A) RNA modification plays an essential role in a wide range of pathological conditions. Impaired autophagy is a critical hallmark of acute pancreatitis (AP). AIM To explore the role of the m6A modification of ZKSCAN3 in the regulation of autophagy in AP. METHODS The AP mouse cell model was established by cerulein-treated mouse pancreatic acinar cells (MPC-83), and the results were confirmed by the levels of amylase and inflammatory factors. Autophagy activity was evaluated by specific identification of the autophagy-related microstructure and the expression of autophagy-related genes. ZKSCAN3 and ALKBH5 were knocked down to study the function in AP. A m6A RNA binding protein immunoprecipitation assay was used to study how the m6A modification of ZKSCAN3 mRNA is regulated by ALKBH . RESULTS The increased expression of amylase and inflammatory factors in the supernatant and the accumulation of autophagic vacuoles verified that the AP mouse cell model was established. The downregulation of LAMP2 and upregulation of LC3-II/I and SQSTM1 demonstrated that autophagy was impaired in AP. The expression of ZKSCAN3 was upregulated in AP. Inhibition of ZKSCAN3 increased the expression of LAMP2 and decreased the expression of the inflammatory factors, LC3-II/I and SQSTM1 . Furthermore, ALKBH5 was upregulated in AP. Knockdown of ALKBH5 downregulated ZKSCAN3 expression and restored decreased autophagic flux in AP. Notably, the bioinformatic analysis revealed 23 potential m6A modification sites on ZKSCAN3 mRNA. The m6A modification of ZKSCAN3 mRNA was significantly decreased in AP. Knockdown of ALKBH5 increased the modification of ZKSCAN3 mRNA, which confirmed that ALKBH5 upregulated ZKSCAN3 expression in a m6A-dependent manner. CONCLUSION ALKBH5 inhibits autophagic flux through m6A demethylation of ZKSCAN3 mRNA in AP, thereby aggravating the severity of the disease.