Objective To investigate the effect of ubiquitin specific peptidase 22 (USP22) on the occurrence and development of esophageal squamous cell carcinoma (ESCC) under hypoxic conditions, and its regulatory relationship with hypoxia-inducible factor-1α (HIF-1α). Methods Western blotting and quantitative polymerase chain reaction were used to detect the differences in USP22 protein and mRNA expression between normal esophageal epithelial cells HEEC and ESCC cell lines KYSE30, KYSE150, EC9706, and TE-1 under normoxic (5% CO2, 20% O2, 75% N2) and hypoxic (5% CO2, 1% O2, 94% N2) conditions. By transfecting USP22 plasmid or siUSP22, ESCC cells were divided into a normoxia control group, a normoxia+USP22 group, a normoxia+siUSP22 group, a hypoxia control group, a hypoxia+USP22 group, and a hypoxia+siUSP22 group. The proliferation and migration abilities of cells in each group were detected. The expression of USP22 and HIF-1α under hypoxic conditions after up-regulating or down-regulating USP22 was detected, and their regulatory relationship was verified. The interaction between USP22 and HIF-1α was verified by co-immunoprecipitation (Co-IP) technique. Results Compared with HEEC cells, the expression of USP22 in ESCC cells was significantly increased (P<0.05). Up-regulation of USP22 expression promoted the proliferation and migration of ESCC cells, while silencing USP22 inhibited the proliferation and migration of ESCC cells (P<0.05). Under hypoxic conditions, the expression of USP22 and HIF-1α increased, and with the up-regulation of USP22 expression, the expression of HIF-1α also significantly increased (P<0.05). Co-IP experiment confirmed the binding between USP22 and HIF-1α. Conclusion Up-regulation of USP22 expression promotes the proliferation and migration of ESCC cells. Hypoxia microenvironment can induce the increase of USP22 expression in ESCC. USP22 may participate in the regulation of the occurrence and development of ESCC by directly binding to HIF-1α.

OBJECTIVE To establish the method for determining the contents of five active components in raw and charred herb pairs of Gardenia jasminoides-Scutellaria baicalensis ， construct their fingerprints， and investigate the effects of G. jasminoides processing on chemical constituents in the h erb pairs. METHODS The HPLC method was used to determine the contents of genipin 1- β -D-gentiobioside， geniposide， crocin Ⅰ， crocin Ⅱ and baicalin in ten batches of raw G. jasminoides-S. baicalensis herb pairs and ten batches of charred G. jasminoides-S. baicalensis herb pairs. Using the same HPLC method， chromatographic fingerprints for the ten batches of raw herb pairs and ten batches of processed herb pairs were established with the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine （2012 edition）. Principal component analysis and orthogonal partial least squares-discriminant analysis were performed. RESULTS Compared with the raw G. jasminoides-S. baicalensis herb pair， the average content of genipin 1- β -D-gentiobioside in the charred G. jasminoides-S. baicalensis herb pair increased by 12.65%； while the average contents of geniposide， crocin Ⅰ， crocin Ⅱ and baicalin decreased by 7.86%， 60.62%， 62.07% and 0.15%， respectively. Chromatographic fingerprints of ten batches of raw herb pairs and ten batches of processed herb pairs shared 18 common peaks with similarities ranging from 0.997 to 1.000 and 0.988 to 1.000， respectively. Five common peaks were identified： genipin 1- β -D-gentiobioside （peak 2）， geniposide （peak 3）， crocin Ⅰ （peak 6）， crocin Ⅱ （peak 9） and baicalin （peak 10）. Principal component analysis and orthogonal partial least squares-discriminant analysis results showed that the raw G. jasminoides-S. baicalensis herb pair and the charred G. jasminoides-S. baicalensis herb pair could be clearly distinguished. Variable importance in the projection （VIP） values for crocin Ⅰ and crocin Ⅱ were both greater than one. CONCLUSIONS A method has been successfully developed for the determination of five active c omponents， including genipin 1- β -D-gentiobioside， in the G. jasminoides-S. baicalensis herb pair， and its fingerprint has been drawn. Processing is found to increase the content of genipin 1- β -D-gentiobioside， while decreasing the levels of geniposide， crocin Ⅰ， crocin Ⅱ and baicalin in the herb pair. Furthermore， crocin Ⅰ and crocin Ⅱ could serve as potential quality markers for the quality control of this herb pair.

Background: s/Aims: Non-invasive models stratifying clinically significant portal hypertension (CSPH) are limited. Herein, we developed a new non-invasive model for predicting CSPH in patients with compensated cirrhosis and investigated whether carvedilol can prevent hepatic decompensation in patients with high-risk CSPH stratified using the new model. Methods: Non-invasive risk factors of CSPH were identified via systematic review and meta-analysis of studies involving patients with hepatic venous pressure gradient (HVPG). A new non-invasive model was validated for various performance aspects in three cohorts, i.e., a multicenter HVPG cohort, a follow-up cohort, and a carvediloltreating cohort. Results: In the meta-analysis with six studies (n=819), liver stiffness measurement and platelet count were identified as independent risk factors for CSPH and were used to develop the new “CSPH risk” model. In the HVPG cohort (n=151), the new model accurately predicted CSPH with cutoff values of 0 and –0.68 for ruling in and out CSPH, respectively. In the follow-up cohort (n=1,102), the cumulative incidences of decompensation events significantly differed using the cutoff values of <–0.68 (low-risk), –0.68 to 0 (medium-risk), and >0 (high-risk). In the carvediloltreated cohort, patients with high-risk CSPH treated with carvedilol (n=81) had lower rates of decompensation events than non-selective beta-blockers untreated patients with high-risk CSPH (n=613 before propensity score matching [PSM], n=162 after PSM). Conclusions Treatment with carvedilol significantly reduces the risk of hepatic decompensation in patients with high-risk CSPH stratified by the new model.

OBJECTIVE To investigate the effects and mechanism of total alkaloids of Corydalis Rhizoma （TAC） on the regulation of cuproptosis in rats with diabetic cardiomyopathy （DCM） based on silence information regulator 1（Sirt1）/tumor protein 53（P53）signaling pathway. METHODS DCM rat model was induced by high-fat and high-sugar diet and intraperitoneal injection of streptozotocin. Thirty-two model rats were randomly divided into model group， TAC low-dose， medium-dose and high-dose groups （7， 10.5， 14 mg/kg）， with 8 rats in each group. An additional 8 rats were assigned to normal control group. Related drugs or normal saline were administered intragastrically in each group， once a day， for 4 weeks. After the last medication， the fasting blood glucose （FBG） levels of the rats were measured. The levels of myocardial creatine kinase （CK）， creatine kinase isoenzyme （CK-MB）， and lactate dehydrogenase （LDH） in serum and myocardial tissue of rats were all detected. The pathological morphology， fibrosis degree， and Cu2+ deposition of myocardial tissue in rats were observed. The levels of Cu2+ and glutathione （GSH） in myocardial tissue， the expressions of Sirt1/P53 signaling pathway-related proteins [Sirt1， P53， solute carrier family 7 membrane 11 （SLC7A11）]， and iron-sulfur cluster-related proteins [ferredoxin 1 （FDX1）， lipoic acid synthetase （LIAS）， aconitase 2 （ACO2）， NADH-ubiquinone oxidoreductase core subunit S8 （NDUFS8）， dihydrolipoamide acetyltransferase （DLAT）， dihydrolipoamide succinyltransferase （DLST）]， and heat shock protein 70 （HSP70） were all determined. RESULTS Compared with normal control group， the model group exhibited significantly elevated levels of FBG， CK， CK-MB and LDH in both serum and myocardial tissue， as well as increased 2+ levels of Cu in myocardial tissue and the expression of P53 and HSP70 proteins （P＜0.05）； the level of GSH and the expression levels of Sirt1， SLC7A11， FDX1， LIAS， ACO2， NDUFS8， DLAT， and DLST proteins in myocardial tissue were all significantly decreased （P＜0.05）； the myocardial tissue exhibited severe pathological damage， with numerous inflammatory cell infiltrations and significant fibrosis， as well as increased deposition of Cu2+. Compared with model group， most of the above quantitative indicators in rats were significantly reversed in TAC groups （P＜0.05）； the pathological damage to the myocardial tissue was alleviated， with reduced fibrosis and Cu2+ deposition. CONCLUSIONS TAC can ameliorate DCM in rats， and its mechanism of action may be related to activating the activity of the Sirt1/P53 signaling pathway， promoting the chelation of GSH with Cu2+， and inhibiting cuproptosis of cardiomyocyte.

Membrane proteins are integral components of cellular membranes, accounting for approximately 30% of the mammalian proteome and serving as targets for 60% of FDA-approved drugs. They are critical to both physiological functions and disease mechanisms. Their functional protein-protein interactions form the basis for many physiological processes, such as signal transduction, material transport, and cell communication. Membrane protein interactions are characterized by membrane environment dependence, spatial asymmetry, weak interaction strength, high dynamics, and a variety of interaction sites. Therefore, in situ analysis is essential for revealing the structural basis and kinetics of these proteins. This paper introduces currently available in situ analytical techniques for studying membrane protein interactions and evaluates the characteristics of each. These techniques are divided into two categories: label-based techniques (e.g., co-immunoprecipitation, proximity ligation assay, bimolecular fluorescence complementation, resonance energy transfer, and proximity labeling) and label-free techniques (e.g., cryo-electron tomography, in situ cross-linking mass spectrometry, Raman spectroscopy, electron paramagnetic resonance, nuclear magnetic resonance, and structure prediction tools). Each technique is critically assessed in terms of its historical development, strengths, and limitations. Based on the authors’ relevant research, the paper further discusses the key issues and trends in the application of these techniques, providing valuable references for the field of membrane protein research. Label-based techniques rely on molecular tags or antibodies to detect proximity or interactions, offering high specificity and adaptability for dynamic studies. For instance, proximity ligation assay combines the specificity of antibodies with the sensitivity of PCR amplification, while proximity labeling enables spatial mapping of interactomes. Conversely, label-free techniques, such as cryo-electron tomography, provide near-native structural insights, and Raman spectroscopy directly probes molecular interactions without perturbing the membrane environment. Despite advancements, these methods face several universal challenges: (1) indirect detection, relying on proximity or tagged proxies rather than direct interaction measurement; (2) limited capacity for continuous dynamic monitoring in live cells; and (3) potential artificial influences introduced by labeling or sample preparation, which may alter native conformations. Emerging trends emphasize the multimodal integration of complementary techniques to overcome individual limitations. For example, combining in situ cross-linking mass spectrometry with proximity labeling enhances both spatial resolution and interaction coverage, enabling high-throughput subcellular interactome mapping. Similarly, coupling fluorescence resonance energy transfer with nuclear magnetic resonance and artificial intelligence (AI) simulations integrates dynamic structural data, atomic-level details, and predictive modeling for holistic insights. Advances in AI, exemplified by AlphaFold’s ability to predict interaction interfaces, further augment experimental data, accelerating structure-function analyses. Future developments in cryo-electron microscopy, super-resolution imaging, and machine learning are poised to refine spatiotemporal resolution and scalability. In conclusion, in situ analysis of membrane protein interactions remains indispensable for deciphering their roles in health and disease. While current technologies have significantly advanced our understanding, persistent gaps highlight the need for innovative, integrative approaches. By synergizing experimental and computational tools, researchers can achieve multiscale, real-time, and perturbation-free analyses, ultimately unraveling the dynamic complexity of membrane protein networks and driving therapeutic discovery.

Objective: To screen the frequency of CD36 antigen expression in platelet donor database in Shaanxi province and analyze the molecular genetic characteristics of samples with CD36 antigen deficiency and low expression. Methods: A total of 525 platelet donors samples were randomly collected during May 2023. CD36-FITC monoclonal antibody was used for immunofluorescence labeling, and flow cytometry was applied to detect the expression of CD36 antigen on platelets. For samples with CD36 antigen deficiency on platelets, the expression of CD36 on monocytes was further detected. Samples with CD36 antigen deficiency and low expression were sequenced and analyzed. Results: Among the 525 blood samples, 99.24% (521/525) showed positive expression of CD36 antigen. There were differences in the expression intensity of CD36 antigen, with low expression accounting for 3.43% (18/525) and CD36 antigen deficiency accounting for 0.76% (4/525), all of which were type Ⅱ deficiency. The exon mutation frequency of CD36 type Ⅱ deficiency and low expression samples was 31.82% (7/22), and the exon mutation types were 121-1_126delGCAAGTT, 329-330delAC, 1142T>G, 1204-1246dupl 43bp, 1221G>A, and 1228-1239delATTGTGCCTATT. All four cases of CD36 type Ⅱ deficiency had a 121-6 T>C mutation in intron 3. All CD36 low expression samples had a mutation of 282-10A>G, and 121-6T>C mutation rate was 61.1%(11/18). Conclusion: There were differences in the expression of CD36 antigen in the platelet donor database in Shaanxi province, which may be caused by multiple molecular genetic variations. The frequency of CD36 antigen deficiency in Shaanxi was lower than that of Han, Zhuang and Yao populations in southern China. This study provides references for solving the CD36 antibody mediated transfusion reaction and auxiliary treatment of diseases caused by CD36 antigen deficiency in the future. It also provides a basis for investigating the molecular mechanisms of CD36 deficiency and low expression.

Background: s/Aims: Sarcomatoid hepatocellular carcinoma (HCC) is a rare histological subtype of HCC characterized by extremely poor prognosis; however, its molecular characterization has not been elucidated. Methods: In this study, we conducted an integrated multiomics study of whole-exome sequencing, RNA-seq, spatial transcriptome, and immunohistochemical analyses of 28 paired sarcomatoid tumor components and conventional HCC components from 10 patients with sarcomatoid HCC, in order to identify frequently altered genes, infer the tumor subclonal architectures, track the genomic evolution, and delineate the transcriptional characteristics of sarcomatoid HCCs. Results: Our results showed that the sarcomatoid HCCs had poor prognosis. The sarcomatoid tumor components and the conventional HCC components were derived from common ancestors, mostly accessing similar mutational processes. Clonal phylogenies demonstrated branched tumor evolution during sarcomatoid HCC development and progression. TP53 mutation commonly occurred at tumor initiation, whereas ARID2 mutation often occurred later. Transcriptome analyses revealed the epithelial–mesenchymal transition (EMT) and hypoxic phenotype in sarcomatoid tumor components, which were confirmed by immunohistochemical staining. Moreover, we identified ARID2 mutations in 70% (7/10) of patients with sarcomatoid HCC but only 1–5% of patients with non-sarcomatoid HCC. Biofunctional investigations revealed that inactivating mutation of ARID2 contributes to HCC growth and metastasis and induces EMT in a hypoxic microenvironment. Conclusions We offer a comprehensive description of the molecular basis for sarcomatoid HCC, and identify genomic alteration (ARID2 mutation) together with the tumor microenvironment (hypoxic microenvironment), that may contribute to the formation of the sarcomatoid tumor component through EMT, leading to sarcomatoid HCC development and progression.

Background: s/Aims: Non-invasive models stratifying clinically significant portal hypertension (CSPH) are limited. Herein, we developed a new non-invasive model for predicting CSPH in patients with compensated cirrhosis and investigated whether carvedilol can prevent hepatic decompensation in patients with high-risk CSPH stratified using the new model. Methods: Non-invasive risk factors of CSPH were identified via systematic review and meta-analysis of studies involving patients with hepatic venous pressure gradient (HVPG). A new non-invasive model was validated for various performance aspects in three cohorts, i.e., a multicenter HVPG cohort, a follow-up cohort, and a carvediloltreating cohort. Results: In the meta-analysis with six studies (n=819), liver stiffness measurement and platelet count were identified as independent risk factors for CSPH and were used to develop the new “CSPH risk” model. In the HVPG cohort (n=151), the new model accurately predicted CSPH with cutoff values of 0 and –0.68 for ruling in and out CSPH, respectively. In the follow-up cohort (n=1,102), the cumulative incidences of decompensation events significantly differed using the cutoff values of <–0.68 (low-risk), –0.68 to 0 (medium-risk), and >0 (high-risk). In the carvediloltreated cohort, patients with high-risk CSPH treated with carvedilol (n=81) had lower rates of decompensation events than non-selective beta-blockers untreated patients with high-risk CSPH (n=613 before propensity score matching [PSM], n=162 after PSM). Conclusions Treatment with carvedilol significantly reduces the risk of hepatic decompensation in patients with high-risk CSPH stratified by the new model.

Background: s/Aims: Sarcomatoid hepatocellular carcinoma (HCC) is a rare histological subtype of HCC characterized by extremely poor prognosis; however, its molecular characterization has not been elucidated. Methods: In this study, we conducted an integrated multiomics study of whole-exome sequencing, RNA-seq, spatial transcriptome, and immunohistochemical analyses of 28 paired sarcomatoid tumor components and conventional HCC components from 10 patients with sarcomatoid HCC, in order to identify frequently altered genes, infer the tumor subclonal architectures, track the genomic evolution, and delineate the transcriptional characteristics of sarcomatoid HCCs. Results: Our results showed that the sarcomatoid HCCs had poor prognosis. The sarcomatoid tumor components and the conventional HCC components were derived from common ancestors, mostly accessing similar mutational processes. Clonal phylogenies demonstrated branched tumor evolution during sarcomatoid HCC development and progression. TP53 mutation commonly occurred at tumor initiation, whereas ARID2 mutation often occurred later. Transcriptome analyses revealed the epithelial–mesenchymal transition (EMT) and hypoxic phenotype in sarcomatoid tumor components, which were confirmed by immunohistochemical staining. Moreover, we identified ARID2 mutations in 70% (7/10) of patients with sarcomatoid HCC but only 1–5% of patients with non-sarcomatoid HCC. Biofunctional investigations revealed that inactivating mutation of ARID2 contributes to HCC growth and metastasis and induces EMT in a hypoxic microenvironment. Conclusions We offer a comprehensive description of the molecular basis for sarcomatoid HCC, and identify genomic alteration (ARID2 mutation) together with the tumor microenvironment (hypoxic microenvironment), that may contribute to the formation of the sarcomatoid tumor component through EMT, leading to sarcomatoid HCC development and progression.

Background: s/Aims: Non-invasive models stratifying clinically significant portal hypertension (CSPH) are limited. Herein, we developed a new non-invasive model for predicting CSPH in patients with compensated cirrhosis and investigated whether carvedilol can prevent hepatic decompensation in patients with high-risk CSPH stratified using the new model. Methods: Non-invasive risk factors of CSPH were identified via systematic review and meta-analysis of studies involving patients with hepatic venous pressure gradient (HVPG). A new non-invasive model was validated for various performance aspects in three cohorts, i.e., a multicenter HVPG cohort, a follow-up cohort, and a carvediloltreating cohort. Results: In the meta-analysis with six studies (n=819), liver stiffness measurement and platelet count were identified as independent risk factors for CSPH and were used to develop the new “CSPH risk” model. In the HVPG cohort (n=151), the new model accurately predicted CSPH with cutoff values of 0 and –0.68 for ruling in and out CSPH, respectively. In the follow-up cohort (n=1,102), the cumulative incidences of decompensation events significantly differed using the cutoff values of <–0.68 (low-risk), –0.68 to 0 (medium-risk), and >0 (high-risk). In the carvediloltreated cohort, patients with high-risk CSPH treated with carvedilol (n=81) had lower rates of decompensation events than non-selective beta-blockers untreated patients with high-risk CSPH (n=613 before propensity score matching [PSM], n=162 after PSM). Conclusions Treatment with carvedilol significantly reduces the risk of hepatic decompensation in patients with high-risk CSPH stratified by the new model.