Digitoxin inhibits ICC cell properties via the NF­κB/ST6GAL1 signaling pathway.

Intrahepatic cholangiocarcinoma (ICC) is a type of liver cancer associated with poor prognosis and increased mortality; the limited treatment strategy highlights the urgent need for investigation. Traditional Chinese Medicine (TCM), used alone or in combination with other treatments, can enhance therapeutic efficacy, improve life quality of patients and extend overall survival. In total, two rounds of screening of a TCM library of 2,538 active compounds were conducted using a Cell Counting Kit­8 assay and ICC cell lines. Cell proliferation and migration abilities were assessed through colony formation, 5­ethynyl­2'­deoxyuridine, would healing and Transwell assays. The impact of digitoxin (DT) on signaling pathways was initially investigated using RNA sequencing and further validated using reverse transcription­quantitative PCR, western blotting, lectin blotting and flow cytometry. ICC cells stably overexpressing ST6 ß­galactoside α­2,6­sialyltransferase 1 (ST6GAL1) were generated through lentiviral transfection. It was shown that DT emerged as a highly effective anti­ICC candidate from two rounds high­throughput library screening. DT could inhibit the proliferation and migration of ICC cells by suppressing NF­κB activation and reducing nuclear phosphorylated­NF­κB levels, along with diminishing ST6GAL1 mRNA and protein expression. The aforementioned biological effects and signal pathways of DT could be counteracted by overexpressing ST6GAL1 in ICC cells. In conclusion, DT suppressed ICC cell proliferation and migration by targeting the NF­κB/ST6GAL1 signaling axis. The findings of the present study indicated the promising therapeutic effects of DT in managing ICC, offering new avenues for treatment strategies.

N-glycan signatures identified in the serum from biliary tract cancer patients: Association with clinical diagnosis and prognosis.

BACKGROUND: Changes in the expression of genes related to glycosyltransferases may lead to alterations in N-glycan structure abundance, potentially acting as markers for diagnosis and prognosis in biliary tract cancer (BTC). METHODS: This study was divided into cross-sectional and longitudinal approaches. The cross-sectional study included 316 BTC and 301 non-BTC. Propensity score matching was applied to adjust for sex and age differences between BTC and non-BTC. Univariate and multivariate logistic regression identified independent risk factors for BTC and constructed the BTC-G model. The ROC curve was used to validate the diagnostic performance of BTC-G. Longitudinal follow-up studies included postoperative (N = 50) and immunotherapy (N = 43) follow-up cohorts. Cox regression analysis identified N-glycan structures impacting BTC prognosis postoperative and immunotherapy, with further confirmation through Kaplan-Meier curves. RESULTS: Univariate and multivariate analyses identified Peak3 (OR: 0.790, 95% CI: 0.658-0.949), Peak9 (OR: 1.646, 95% CI: 1.409-1.922), and Peak9p (OR: 2.467, 95% CI: 1.267-4.804) as independent BTC risk factors, leading to the creation of the BTC-G. The ROC curve confirmed that BTC-G performed well in training (AUC: 0.753, 95% CI: 0.703-0.799), validation (AUC: 0.811, 95% CI: 0.740-0.870), and CA19-9 negative cohorts (AUC: 0.717, 95% CI: 0.664-0.767). Cox regression analysis and Kaplan-Meier curves established that Peak12 (HR: 5.578, 95% CI: 1.145-27.170) and Peak11 (HR: 1.104, 95% CI: 0.611-1.994) are independent risk factors for BTC prognosis following surgery and immunotherapy, respectively. CONCLUSIONS: Our NGFP technology supplements BTC diagnostics, distinguishing survival and recurrence subtypes for postoperative and immunotherapy, thereby supporting the development of treatment strategies.

Serum N-glycomic profiling identifies candidate biomarker panels for assessing coronary artery stenosis severity.

Stenosis severity may escalate over the course of coronary artery disease (CAD), increasing the risk of death for the patient. Conventionally, the assessment of stenosis degree relies on invasive coronary angiography (ICA), an invasive examination unsuitable for patients in poor physical condition or those with contrast allergies and one that imposes a psychological burden on patients. Although abnormal serum N-glycan profiles have exhibited robust associations with various cardiovascular diseases, including CAD, their potential in diagnosing CAD stenosis remains to be determined. In this study, we performed a comprehensive analysis of serum N-glycome from 132 patients who underwent ICA and 27 healthy controls using MALDI-TOF-mass spectrometry. The patients who underwent ICA examination were categorized into four groups based on stenosis severity: no/mild/moderate/severe stenosis. Twenty-seven N-glycans were directly quantified, and 47 derived glycan traits were obtained. Notably, among these 74 glycan features, 18 exhibited variations across the study groups. Using a combination of least absolute shrinkage and selection operator and logistic regression analyses, we developed five diagnostic models for recognizing stenosis degree. Our results suggested that alterations in serum N-glycosylation modifications might be valuable for identifying stenosis degree and monitoring disease progression in individuals with CAD. It is expected to offer a noninvasive alternative for those who could not undergo ICA because of various reasons. However, the diagnostic potential of serum N-glycan panels as biomarkers requires multicenter, large cohort validation in the future.

Risks and Clinical Predictors of Hepatocellular Carcinoma in Chinese Populations: A Real-World Study of 10,359 Patients in Six Medical Centers.

Purpose: Early detection of hepatocellular carcinoma (HCC) through surveillance could reduce this cancer-associated mortality. We aimed to develop and validate algorithms using panel serum biomarkers to identify HCC in a real-world multi-center study in China. Patients and Methods: A total of 10,359 eligible subjects, including HCCs and benign liver diseases (BLDs), were recruited from six Chinese medical centers. The three nomograms were built using logistic regression and their sensitivities and specificities were carefully assessed in training and validation cohorts. HCC patients after surgical resection were followed to determine the prognostic values of these algorithms. Prospective surveillance performance was assessed in a cohort of chronic hepatitis B patients during 144 weeks follow-up. Results: Independent risk factors such as alpha-fetoprotein (AFP), lens cuinaris agglutinin-reactive fraction of AFP (AFP-L3), des-gamma-carboxy prothrombin (DCP), albumin (ALB), and total bilirubin (TBIL) obtained from train cohort were used to construct three nomograms (LAD, C-GALAD, and TAGALAD) using logistic regression. In the training and two validation cohorts, their AUCs were all over 0.900, and the higher AUCs appeared in TAGALAD and C-GALAD. Furthermore, the three nomograms could effectively stratify HCC into two groups with different survival and recurrence outcomes in follow-up validation. Notably, TAGALAD could predict HCC up to 48 weeks (AUC: 0.984) and 24 weeks (AUC: 0.900) before clinical diagnosis. Conclusion: The proposed nomograms generated from real-world Chinese populations are effective and easy-to use for HCC surveillance, diagnosis, as well as prognostic evaluation in various clinical scenarios based on data feasibility.

Analysis on dynamic changes of etizolam and its metabolites and exploration of its development prospect using UPLC-Q-exactive-MS.

As one of the most widely abused designer benzodiazepines in the world, etizolam has been found in many cases in many countries. In this study, UPLC-Q-Exactive-MS was used for the first time to establish a dynamic change model of etizolam and its metabolites in rats. Compared with previous studies, the detection sensitivity and reproducibility of the instrument were higher. In the experiment, we optimized the traditional pharmacokinetic model based on Gauss function. According to the significant difference of etizolam in the plasma elimination phase of rats, a new pharmacokinetic model based on Lorentz function was established to describe the dynamic changes of etizolam more rigorously, which made the error effects lower and the accuracy of the pharmacokinetic parameters was improved. At the same time, the pharmacokinetic parameters of etizolam were compared with four other designer benzodiazepines reported in previous studies in rats, and we found the direct reason for the popularity of etizolam in the NPS market and explored the future development of etizolam for the first time. In addition, 21 metabolites were found through rat experiments to effectively detect etizolam abuse for a long time, of which 4 metabolites had the longest detection window and could be used as long-acting metabolites for experiments, which greatly prolongs the detection window and extends the time range in which etizolam was detected in real cases. This study is the first to conduct a systematic and comprehensive study on the metabolism and pharmacokinetics of etizolam and find out the direct reason for the prevalence of etizolam abuse, and we also discuss the development trend of etizolam in the future market of new psychoactive substances, which is beneficial for forensic experts to assess the trend of drug abuse and can provide reference for relevant drug control and drug treatment.

Quercetin, a key active ingredient of Jianpi Zishen Xiehuo Formula, suppresses M1 macrophage polarization and platelet phagocytosis by inhibiting STAT3 activation based on network pharmacology.

Primary immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disease, and abnormal M1 macrophage polarization participates in the pathogenesis of ITP. Jianpi Zishen Xiehuo (JZX) Formula has a good therapeutic effect on ITP. However, its key active ingredients and molecular mechanisms remain unclear. In this study, we explored the key active ingredients and potential targets of JZX in treating ITP using network pharmacology combined with in vitro experimental verification. A total of 157 active ingredients of JZX were identified from public databases, and quercetin was the most important one. One hundred sixty-five intersection targets of active ingredients in JZX, ITP, and macrophage polarization were obtained by Venn diagram. The top three potential targets were signal transducer and activator of transcription 3 (STAT3), protein kinase B (PKB/AKT) 1, and c-JUN through protein-protein interaction analysis. Molecular docking showed that quercetin had strong binding affinities with them all. In vitro experiment, CD16+ monocytes increased in ITP patients compared with healthy controls, which indicated a M1/M2 polarization imbalance in ITP. The expression levels of M1 polarization markers, CD86, CD80, and inducible nitric oxide synthase (iNOS), M1 polarization-associated cytokines, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), and antibody-opsonized platelet phagocytosis significantly increased in THP-1 macrophages stimulated with lipopolysaccharide (LPS). Quercetin markedly inhibited the expressions of M1 markers, decreased the levels of TNF-α and IL-6, and down-regulated the phosphorylated STAT3 (p-STAT3) protein, which confirmed the prediction by network pharmacology and molecular docking. Importantly, quercetin significantly reduced the phagocytosis of antibody opsonised platelet. In conclusion, quercetin suppressed platelet phagocytosis in M1 macrophages via its anti-inflammatory effects and may serve as a potential drug for the treatment of ITP. Quercetin could be a key ingredient for JZX against ITP.

Chinese expert consensus statement on the clinical application of AFP/AFP-L3%/DCP using GALAD and GALAD-like algorithm in HCC.

BACKGROUND: Primary hepatocellular carcinoma (HCC) is one of the most prevalent world-wide malignancies. Half of the newly developed HCC occurs in China. Optimizing the strategies for high-risk surveillance and early diagnosis are pivotal for improving 5-year survival. Constructing the scientific non-invasive detection technologies feasible for medical and healthcare institutions is among the key routes for elevating the efficacies of HCC identification and follow-up. RESULTS: Based on the Chinese and international guidelines, expert consensus statements, literatures and evidence-based clinical practice experiences, this consensus statement puts forward the clinical implications, application subjects, detection techniques and results interpretations of the triple-biomarker (AFP, AFP-L3%, DCP) based GALAD, GALAD like models for liver cancer. CONCLUSIONS: The compile of this consensus statement aims to address and push the reasonable application of the triple-biomarker (AFP, AFP-L3%, DCP) detections thus to maximize the clinical benefits and help improving the high risk surveillance, early diagnosis and prognosis of HCC.

Comprehensive Overview the Role of Glycosylation of Extracellular Vesicles in Cancers.

Extracellular vesicles (EVs) are membranous structures secreted by various cells carrying diverse biomolecules. Recent advancements in EV glycosylation research have underscored their crucial role in cancer. This review provides a global overview of EV glycosylation research, covering aspects such as specialized techniques for isolating and characterizing EV glycosylation, advances on how glycosylation affects the biogenesis and uptake of EVs, and the involvement of EV glycosylation in intracellular protein expression, cellular metastasis, intercellular interactions, and potential applications in immunotherapy. Furthermore, through an extensive literature review, we explore recent advances in EV glycosylation research in the context of cancer, with a focus on lung, colorectal, liver, pancreatic, breast, ovarian, prostate, and melanoma cancers. The primary objective of this review is to provide a comprehensive update for researchers, whether they are seasoned experts in the field of EVs or newcomers, aiding them in exploring new avenues and gaining a deeper understanding of EV glycosylation mechanisms. This heightened comprehension not only enhances researchers' knowledge of the pathogenic mechanisms of EV glycosylation but also paves the way for innovative cancer diagnostic and therapeutic strategies.

Pulmonary fungal infection in a neonate with methylmalonic acidemia: A case report.

BACKGROUND: Methylmalonic acidemia (MMA) is characterized by non-specific symptoms such as vomiting, and feeding difficulties, along with delayed mental and physical development. However, no case of MMA combined with pulmonary fungal infection has been reported yet. CASE SUMMARY: We report the case of a neonate who presented pulmonary fungal infection along with the non-specific features of MMA. Exome sequencing revealed a c.331C>T variant in exon 3 of MMACHC from the father, and a c.658-c.660delAAG variant in exon 4 from the mother, which confirmed the diagnosis of cblC type MMA combined with hyperhomocysteinemia. CONCLUSION: Invasive fungal infection might occur in some infants with MMA. Therefore, early diagnosis is recommended for unexplained pulmonary infection.

Impact of apolipoprotein A1 on tumor immune microenvironment, clinical prognosis and genomic landscape in hepatocellular carcinoma.

Background: Current knowledge on apolipoprotein A1 (APOA1) in hepatocellular carcinoma (HCC) is fragmented and even contradictory. Multi-dimensional analyses are required to comprehensively elucidate its value and underlying mechanism. Methods: We collected 49 RNA-seq datasets, 40 cell line types data and 70 scRNA pan-cancer datasets public available, including 17 HCC datasets (1754 tumor samples), and enrolled 73 pairs of HCC tissue and 516 blood samples independently from our clinics. APOA1 impacting on the HCC tumor microenvironment (TME) was analyzed using intensive data mining. Methylation sequencing, flow cytometry, quantitative PCR, western blot, immunohistochemistry and clinical chemistry assays were conducted for wet experimental investigation. Results: The APOA1 ontology fingerprint indicated that it played various crucial biological roles in HCC, primarily involved in cholesterol efflux. Consistent findings at histology, serology, and clinical follow-up revealed that high APOA1 was a good prognosis indicator of HCC. Hypermethylation in the APOA1 promoter region was found in clinical samples which is in accordance with the reduction of APOA1 in HCC. The cell cycle, DNA replication, mismatch repair pathways, and tumor cell proliferation were less observed in the HCC APOA1high subgroup. The favorable immunoregulatory abilities of APOA1 showed interesting findings: a positive correlation between APOA1 and anti-tumor immune cells (NK, CD8+ T cells) and a negative association with immune cells exerting immunosuppressive effects, including M2 macrophages. Conclusion: This is an integrative multidimensional exploration of APOA1 using bioinformatics and experiments. Both the prognostic value and anti-tumor effects based on APOA1 panoramic exploration in the HCC TME demonstrate a new potential clinical target for HCC assessment and intervention in the future.

Usability of Serum Stanniocalcin-1 as a Prognostic Biochemical Marker of Acute Supratentorial Intracerebral Hemorrhage: A Prospective Cohort Study.

Objective: Stanniocalcin-1 (STC1) may be neuroprotective. This study aimed to evaluate the prognostic role of serum STC1 levels in intracerebral hemorrhage (ICH). Methods: This prospective observational study was assigned in two parts. In the first part, blood samples of 48 patients with ICH were acquired on admission and on days 1, 2, 3, 5, and 7 after ICH, and those of 48 controls were collected at their entry into the study. In the second part, blood samples of 141 patients with ICH were obtained upon admission. Serum STC1 levels were measured, and the National Institutes of Health Stroke Scale (NIHSS), hematoma volume, and poststroke 6-month modified Rankin Scale (mRS) scores were recorded. Dynamic changes in serum STC levels and their correlation with disease severity and prognosis were investigated. Results: Serum STC1 levels were elevated after ICH, peaked on day 1, plateaued on day 2, declined gradually afterwards, and were significantly higher than those in controls. Serum STC1 levels were independently correlated with NIHSS scores, hematoma volume, and the 6-month post-injury mRS scores. Serum STC1 levels, NIHSS scores, and hematoma volume independently predicted a poor prognosis (mRS scores of 3-6). The model integrating serum STC1 levels, NIHSS scores, and hematoma volume was visually displayed using a nomogram and was relatively stable using the Hosmer-Lemeshow test and calibration curve analysis. Under the receiver operating characteristic curve, serum STC1 levels efficiently predicted a poor prognosis and showed similar prognostic ability to NIHSS scores and hematoma volume. The preceding model had significantly higher prognostic capability than NIHSS scores and hematoma volume alone and their combination. Conclusion: Substantial enhancement of serum STC1 levels after ICH, which is strongly correlated with severity, independently distinguished the risk of poor prognosis, assuming that serum STC1, as a prognostic parameter, may be clinically valuable in ICH.

Electrokinetic ion enrichment in asymmetric charged nanochannels.

Artificial bionic nanochannels have attracted wide attention and successfully used in various fields. In this work, a novel nanochannel with asymmetric surface charge is proposed to investigate the ion enrichment effect. The results show that the proposed nanochannel has excellent ion enrichment performance and the obtained ion enrichment ratio is up to 1500 when the ion concentration is 0.01 mM, which is much higher than precedent researches typically ranging from tens to hundreds. Besides, we found that the forward voltage bias will produce ions enrichment and the reverse voltage bias will produce ions depletion. The ion enrichment ratio is higher at the larger voltage bias, absolute surface charge density and smaller nanochannel height. In addition, the ion enrichment performance is more sensitive to the change of charged wall length and not sensitive to the change of uncharged wall length. The research report offers important information and instructions for the design and optimum on ion enrichment performance.

Hydroxyphenylpyruvate Dioxygenase Is a Metabolic Immune Checkpoint for UTX-deficient Colorectal Cancer.

BACKGROUND & AIMS: Aberrant epigenetic events mediated by histone methyltransferases and demethylases contribute to malignant progression of colorectal cancer (CRC). However, the role of the histone demethylase ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) in CRC remains poorly understood. METHODS: UTX conditional knockout mice and UTX-silenced MC38 cells were used to investigate UTX function in tumorigenesis and development of CRC. We performed time of flight mass cytometry to clarify the functional role of UTX in remodeling immune microenvironment of CRC. To investigate metabolic interaction between myeloid-derived suppressor cells (MDSCs) and CRC, we analyzed metabolomics data to identify metabolites secreted by UTX-deficient cancer cells and taken up by MDSCs. RESULTS: We unraveled a tyrosine-mediated metabolic symbiosis between MDSC and UTX-deficient CRC. Loss of UTX in CRC resulted in methylation of phenylalanine hydroxylase, preventing its degradation and subsequently increasing tyrosine synthesis and secretion. Tyrosine taken up by MDSCs was metabolized to homogentisic acid by hydroxyphenylpyruvate dioxygenase. Homogentisic acid modified protein inhibitor of activated STAT3 via carbonylation of Cys 176, and relieved the inhibitory effect of protein inhibitor of activated STAT3 on signal transducer and activator of transcription 5 transcriptional activity. This in turn, promoted MDSC survival and accumulation, enabling CRC cells to acquire invasive and metastatic traits. CONCLUSIONS: Collectively, these findings highlight hydroxyphenylpyruvate dioxygenase as a metabolic checkpoint to restrict immunosuppressive MDSCs and to counteract malignant progression of UTX-deficient CRC.

ST6GAL1 inhibits metastasis of hepatocellular carcinoma via modulating sialylation of MCAM on cell surface.

The poor prognosis of hepatocellular carcinoma (HCC) is mainly because of its high rate of metastasis. Thus, elucidation of the molecular mechanisms underlying HCC metastasis is of great significance. Glycosylation is an important post-translational modification that is closely associated with tumor progression. Altered glycosylation including the altered sialylation resulting from aberrant expression of ß-galactoside α2,6 sialyltransferase 1 (ST6GAL1) has long been considered as an important feature of cancer cells. However, there is limited information on the roles of ST6GAL1 and α2,6 sialylation in HCC metastasis. Here, we found that ST6GAL1 and α2,6 sialylation were negatively correlated with the metastatic potentials of HCC cells. Moreover, ST6GAL1 overexpression inhibited migration and invasion of HCC cells in vitro and suppressed HCC metastasis in vivo. Using a metabolic labeling-based glycoproteomic strategy, we identified a list of sialylated proteins that may be regulated by ST6GAL1. In particular, an increase in α2,6 sialylation of melanoma cell adhesion molecule (MCAM) inhibited its interaction with galectin-3 and decreased its expression on cell surface. In vitro and in vivo analysis showed that ST6GAL1 exerted its function in HCC metastasis by regulating MCAM expression. Finally, we found the relative intensity of sialylated MCAM was negatively correlated with tumor malignancy in HCC patients. Taken together, these results demonstrate that ST6GAL1 may be an HCC metastasis suppressor by affecting sialylation of MCAM on cell surface, which provides a novel insight into the roles of ST6GAL1 in HCC progression and supports the functional complexity of ST6GAL1 in a cancer type- and tissue type-specific manner.

A novel matrine derivative, WM130, inhibits activation and movement of human hepatic stellate LX-2 cells by targeting cofilin 1.

Matrine, one of the active ingredients of Sophora flavescens Ait., has a protective effect in animal models on acute liver injury and liver fibrosis. However, since the protective effects are short-lived, a structural modification of matrine is needed to improve its anti-fibrotic effects. In the previous study we obtained a stable, highly active new matrine derivative, WM130, and explored its anti-fibrotic effects on the human hepatic stellate cell line, LX-2. CCK-8, wound healing, and transwell assays were used to investigate cell proliferation and migration, while 3D mimic study was used to determine the target of WM130. Western blots investigated the levels of α-SMA, cofilin 1, p-cofilin 1, F-actin, PI3K, p-Akt, Akt, and PTEN in LX-2 cells treated with MW130. The results revealed that WM130 can significantly inhibit the proliferation of LX-2 cells at an IC50 of 60 µg/ml. At 30 µg/ml, matrine or WM130 significantly inhibited the migration of LX-2 cells. Moreover, WM130 significantly reduced the expression of α-SMA, cofilin 1, F-actin, PI3K, and p-Akt, and increased PTEN levels. In conclusion, WM130 inhibits the proliferation, activation, and migration of human hepatic stellate LX-2 cells by targeting cofilin 1. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00548-w.

NSUN5 promotes progression and predicts poor prognosis in hepatocellular carcinoma.

The 5-methylcytosine (m5C) RNA methyltransferase NOP2/Sun RNA methyltransferase 5 (NSUN5) has been reported to serve important roles in numerous diseases. However, the functions and clinical significance of NSUN5 in hepatocellular carcinoma (HCC) remain unknown. Clinical information and NSUN5 mRNA sequencing data for 374 patients with HCC were downloaded from The Cancer Genome Atlas (TCGA) database, and NSUN5 mRNA and protein expression levels in 120 patients with HCC (present study cohorts) were assessed using reverse transcription-quantitative PCR, western blotting or immunohistochemistry. The association between NSUN5 mRNA and protein expression levels and the clinical characteristics (or prognosis) of patients with HCC was analyzed using the χ2 or log-rank test. The functions of NSUN5 in HCC were evaluated using in vitro and in vivo experiments, and the mechanism by which NSUN5 affected the progression of HCC was assessed using bioinformatics analysis using LinkedOmics. NSUN5 was significantly upregulated and predicted poor prognosis in HCC according to data from both TCGA database and present study cohorts. NSUN5 significantly promoted HCC proliferation and migration in vitro and significantly induced HCC tumor growth in vivo. Bioinformatics analysis demonstrated that NSUN5 was positively correlated with genes associated with translation in HCC. It was hypothesized that overexpression of NSUN5 strengthened ribosome functions and global protein translation, which may promote the proliferation and migration of HCC. In conclusion, NSUN5 may promote the progression of HCC by enhancing translation, thus making it a potential target for HCC treatment.

Deep Sequencing of HPV16 E6 Region Reveals Unique Mutation Pattern of HPV16 and Predicts Cervical Cancer.

The genetic diversity of human papillomavirus (HPV) 16 within cervical cells and tissue is usually associated with persistent virus infection and precancerous lesions. To explore the HPV16 mutation patterns contributing to the cervical cancer (CC) progression, a total of 199 DNA samples from HPV16-positive cervical specimens were collected and divided into high-grade squamous intraepithelial lesion (HSIL) and the non-HSIL(NHSIL) groups. The HPV16 E6 region (nt 7125-7566) was sequenced using next-generation sequencing. Based on HPV16 E6 amino acid mutation features selected by Lasso algorithm, four machine learning approaches were used to establish HSIL prediction models. The receiver operating characteristic was used to evaluate the model performance in both training and validation cohorts. Western blot was used to detect the degradation of p53 by the E6 variants. Based on the 13 significant mutation features, the logistic regression (LR) model demonstrated the best predictive performance in the training cohort (AUC = 0.944, 95% CI: 0.913-0.976), and also achieved a high discriminative ability in the independent validation cohort (AUC = 0.802, 95% CI: 0.601-1.000). Among these features, the E6 D32E and H85Y variants have higher ability to degrade p53 compared to the E6 wildtype (P < 0.05). In conclusion, our study provides evidence for the first time that HPV16 E6 sequences contain vital mutation features in predicting HSIL. Moreover, the D32E and H85Y variants of E6 exhibited a significantly higher ability to degrade p53, which may play a vital role in the development of CC. IMPORTANCE The study provides evidence for the first time that HPV16 E6 sequences contain vital mutation features in predicting the high-grade squamous intraepithelial lesion and can reduce even more unneeded colposcopies without a loss of sensitivity to detect cervical cancer. Moreover, the D32E and H85Y variants of E6 exhibited a significantly higher ability to degrade p53, which may play a vital role in the development of cervical cancer.

Sparse logistic regression revealed the associations between HBV PreS quasispecies and hepatocellular carcinoma.

BACKGROUND: Chronic infection with hepatitis B virus (HBV) has been proved highly associated with the development of hepatocellular carcinoma (HCC). AIMS: The purpose of the study is to investigate the association between HBV preS region quasispecies and HCC development, as well as to develop HCC diagnosis model using HBV preS region quasispecies. METHODS: A total of 104 chronic hepatitis B (CHB) patients and 117 HBV-related HCC patients were enrolled. HBV preS region was sequenced using next generation sequencing (NGS) and the nucleotide entropy was calculated for quasispecies evaluation. Sparse logistic regression (SLR) was used to predict HCC development and prediction performances were evaluated using receiver operating characteristic curves. RESULTS: Entropy of HBV preS1, preS2 regions and several nucleotide points showed significant divergence between CHB and HCC patients. Using SLR, the classification of HCC/CHB groups achieved a mean area under the receiver operating characteristic curve (AUC) of 0.883 in the training data and 0.795 in the test data. The prediction model was also validated by a completely independent dataset from Hong Kong. The 10 selected nucleotide positions showed significantly different entropy between CHB and HCC patients. The HBV quasispecies also classified three clinical parameters, including HBeAg, HBVDNA, and Alkaline phosphatase (ALP) with the AUC value greater than 0.6 in the test data. CONCLUSIONS: Using NGS and SLR, the association between HBV preS region nucleotide entropy and HCC development was validated in our study and this could promote the understanding of HCC progression mechanism.

Genetic studies discover novel coding and non-coding mutations in patients with Wilson's disease in China.

OBJECTIVES: Wilson disease (WD) is a rare autosomal recessive genetic disorder associated with various mutations in the ATP7B gene and leads to significant disability or death if untreated. Early diagnosis and proper therapy usually predict a good prognosis, especially in pre-symptomatic WD. Genetic testing provides an accurate and effective diagnostic method for the early diagnosis of WD. METHODS: We recruited 18 clinically diagnosed WD patients from 16 unrelated families and two independent individuals. The next-generation sequencing of the ATP7B gene was performed. The 293T cell lines were divided into wild-type (WT) ATP7B and mutated ATP7B groups. Cell proliferation was determined by Cell Counting Kit-8 (CCK-8) assay and apoptosis was detected by Annexin V/propidium iodide (PI) assays. RESULTS: Pedigree analysis showed that compound heterozygous variants (17/18, 94.44%) were present in the majority of WD patients. A total of 33 ATP7B gene variants were identified, including three variants with uncertain significance (VUS) [two splice mutations (c.51+2T>G, c.1543+40G>A) and one frameshift mutation (c.3532_3535del)]. The CCK-8 and apoptosis assays demonstrated that the VUS of ATP7B could significantly affect the transportation of copper. CONCLUSIONS: The study revealed genetic defects of 16 Chinese families and two independent individuals with WD, which enriched the mutation spectrum of the ATP7B gene worldwide and provided valuable information for studying the mutation types of ATP7B in the Chinese populations. Genetic testing in WD patients is necessary to shorten the time to initiate therapy, reduce damage to the liver and improve the prognosis.

Common DNA methylation changes in biliary tract cancers identify subtypes with different immune characteristics and clinical outcomes.

BACKGROUND: DNA methylation-associated studies on biliary tract cancer (BTC), including cholangiocarcinoma (CCA) and gallbladder cancer (GBC), may improve the BTC classification scheme. We proposed to identify the shared methylation changes of BTCs and investigate their associations with genomic aberrations, immune characteristics, and survival outcomes. METHODS: Multi-dimensional data concerning mutation, DNA methylation, immune-related features, and clinical data of 57 CCAs and 48 GBCs from Eastern Hepatobiliary Surgery Hospital (EHSH) and 36 CCAs in the TCGA-CHOL cohort were analyzed. RESULTS: In our cohort including 24 intrahepatic CCAs (iCCAs), 20 perihilar CCAs (pCCAs), 13 distal CCAs (dCCAs), and 48 GBCs, 3369 common differentially methylated regions (DMRs) were identified by comparing tumor and non-tumor samples. A lower level of methylation changes of these common DMRs was associated with fewer copy number variations, fewer mutational burden, and remarkably longer overall survival (OS, hazard ratio [HR] = 0.07, 95% confidence interval [CI] 0.01-0.65, P = 0.017). Additionally, a 12-marker model was developed and validated for prognostication after curative surgery (HR = 0.21, 95% CI 0.10-0.43, P < 0.001), which exhibited undifferentiated prognostic effects in subgroups defined by anatomic location (iCCAs, d/pCCAs, GBCs), TNM stage, and tumor purity. Its prognostic utility remained significant in multivariable analysis (HR = 0.26, 95% CI 0.11-0.59, P = 0.001). Moreover, the BTCs with minimal methylation changes exhibited higher immune-related signatures, infiltration of CD8+ lymphocytes, and programmed death-ligand 1 (PD-L1) expression, indicating an inflamed tumor immune microenvironment (TIME) with PD-L1 expression elicited by immune attack, potentially suggesting better immunotherapy efficacy. CONCLUSIONS: In BTCs, DNA methylation is a powerful tool for molecular classification, serving as a robust indicator of genomic aberrations, survival outcomes, and tumor immune microenvironment. Our integrative analysis provides insights into the prognostication after curative surgery and patient selection for immunotherapy.