Chronic liver disease (CLD)-related thrombocytopenia can limit the feasibility of invasive procedures. Recombinant human thrombopoietin (rhTPO) has demonstrated a favorable safety profile without hepatotoxicity. We evaluated the efficacy and safety of rhTPO in patients with CLD-related thrombocytopenia who were undergoing elective invasive procedures. In this multicenter, randomized (2:1), double-blind, placebo-controlled phase III trial, 120 adult Chinese patients with CLD-related thrombocytopenia (platelet count < 50 × 109/L) received rhTPO (n = 80) or placebo (n = 40) once daily for up to 5 or 7 days. The primary endpoint was the proportion of patients with sustained platelet counts ≥ 50 × 109/L from 24 h before invasive procedure to 7 days post-procedure, without requiring emergency bleeding management. The primary endpoint was achieved by 85.0% of patients in the rhTPO group versus 12.5% in the placebo group (p < 0.0001). Preoperatively, platelet counts ≥ 50 × 109/L were achieved in 92.5% and 20.0% of patients in the rhTPO and placebo groups, respectively (p < 0.0001). Platelet transfusion was avoided in 92.5% of rhTPO-treated patients versus 25.0% of placebo-treated patients (p < 0.0001). The median duration of platelet counts ≥ 50 × 109/L was significantly longer with rhTPO than with placebo (21.0 vs. 3.0 days, p = 0.0007). Treatment-related treatment-emergent adverse events (TEAEs) occurred in 12.5% of patients in both the rhTPO and placebo groups. No treatment-related serious adverse events were reported. Overall, rhTPO was effective and well tolerated in patients with CLD-related thrombocytopenia and may represent a viable therapeutic option for those undergoing elective invasive procedures. Trial Registration: www.chinadrugtrials.org.cn: number CTR20230919.

Abnormal amino acid metabolism supports cancer cell proliferation, invasion, and immune evasion in hepatocellular carcinoma (HCC). Previous research exploring amino acid metabolism in HCC has primarily focused on the impact of metabolic reprogramming on tumor cells. In this study, we focused on the role of amino acid metabolism dysregulation in the cross-talk between HCC and T cells. HCC cells disrupted lysine uptake in T cells, leading to impaired T-cell immunity. Lysine deprivation decreased STAT3 levels in T cells, inhibiting T-cell proliferation and effector function and ultimately promoting tumor progression. Mechanistically, HCC cells outcompeted T cells for lysine by expressing high levels of the lysine transporter solute carrier family 3 member 2 (SLC3A2). Clinically, elevated SLC3A2 expression correlated with poor survival and was linked to dysregulated T-cell functional gene signatures in patients with HCC. Furthermore, the multikinase inhibitor lenvatinib induced a c-Myc-SLC3A2 regulatory axis that limited the efficacy of lenvatinib treatment. Lysine supplementation enhanced tumor sensitivity to combined treatment with lenvatinib and anti-PD-1 immunotherapy. These findings suggest that lysine supplementation is a potential therapeutic strategy for treating HCC and enhancing the sensitivity of HCC to tyrosine kinase inhibitors and immune checkpoint blockade.Significance: Hepatocellular carcinoma cells impair T-cell activity by consuming lysine, which can be targeted with SLC3A2 inhibition or lysine supplementation as a therapeutic strategy to enhance antitumor immunity and treatment efficacy.

Focal cortical dysplasia type II (FCDII) is a major cause of drug-resistant epilepsy, but genetic factors explain only some cases, suggesting other mechanisms. In this study, we conduct a molecular analysis of brain lesions and adjacent areas in FCDIIb patients. By analyzing over 217,506 single-nucleus transcriptional profiles from 15 individuals, we find significant changes in smooth muscle cells (SMCs) and astrocytes. We identify abnormal vascular malformations and a unique type of SMC that we call "Firework cells", which migrate from blood vessels into the brain parenchyma and associate with VIM+ cells. These abnormalities create localized ischemic-hypoxic (I/H) microenvironments, as confirmed by clinical data, further impairing astrocyte function, activating the HIF-1α/mTOR/S6 pathway, and causing neuronal loss. Using zebrafish models, we demonstrate that vascular abnormalities resulting in I/H environments promote seizures. Our results highlight vascular malformations as a factor in FCDIIb pathogenesis, suggesting potential therapeutic avenues.

Elucidating the temporal process of immune remodeling under immunosuppressive treatment after liver transplantation (LT) is critical for precise clinical management strategies. Here, we performed a single-cell multi-omics analysis of peripheral blood mononuclear cells (PBMCs) collected from LT patients (with and without acute cellular rejection (ACR)) at 13 time points. Validation was performed in two independent cohorts with additional LT patients and healthy controls. Our study revealed a four-phase recovery process after LT and delineated changes in immune cell composition, expression programs, and interactions along this process. The intensity of the immune response differs between the ACR and non-ACR patients. Notably, the newly identified inflamed NK cells, CD14+RNASE2+ monocytes, and FOS-expressing monocytes emerged as predictive indicators of ACR. This study illuminates the longitudinal evolution of the immune cell landscape under tacrolimus-based immunosuppressive treatment during LT recovery, providing a four-phase framework that aids the clinical management of LT patients.

Early diagnosis is paramount for enhancing survival rates and prognosis in the context of malignant diseases. Hepatocellular carcinoma (HCC), the second leading cause of cancer-related deaths worldwide, poses significant challenges for its early detection. In this study, we present an innovative approach which contributed to the early diagnosis of HCC. By lanthanide encoding signal amplification to map glycan-linkages at the single-cell level, the minute quantities of "soft" glycan-linkages on single cell surface were converted into "hard" elemental tags through the use of an MS2 signal amplifier. Harnessing the power of lanthanides encoded within MS2, we achieve nearly three orders of magnitude signal amplification. These encoded tags are subsequently quantified using single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS). Linear discriminant analysis (LDA) identifies seven specific glycan-linkages (alpha-2,3-Sia, alpha-Gal, alpha-1,2-Fuc, alpha-1,6-Fuc, alpha-2,6-Sia, alpha-GalNAc, and Gal beta-1,3-GalNAc) as biomarkers. Our methodology is initially validated at the cellular level with 100% accuracy in discriminating between hepatic carcinoma HepG2 cells and their normal HL7702 cells. We apply this approach to quantify and classify glycan-linkages on the surfaces of 55 clinical surgical HCC specimens. Leveraging these seven glycan-linkages as biomarkers, we achieve precise differentiation between 8 normal hepatic specimens, 40 early HCC specimens, and 7 colorectal metastasis HCC specimens. This pioneering work represents the first instance of employing single-cell glycan-linkages as biomarkers promising for the early diagnosis of HCC with a remarkable 100% predictive accuracy rate, which holds immense potential for enhancing the feasibility and precision of HCC diagnosis in clinical practice.

Hedgehog signaling is activated in response to liver injury, and modulates organogenesis. However, the role of non-canonical hedgehog activation via TGF-β1/SMAD3 in hepatic carcinogenesis is poorly understood. TGF-β1/SMAD3-mediated non-canonical activation was found in approximately half of GLI2-positive hepatocellular carcinoma (HCC), and two new GLI2 isoforms with transactivating activity were identified. Phospho-SMAD3 interacted with active GLI2 isoforms to transactivate downstream genes in modulation of stemness, epithelial-mesenchymal transition, chemo-resistance and metastasis in poorly-differentiated hepatoma cells. Non-canonical activation of hedgehog signaling was confirmed in a transgenic HBV-associated HCC mouse model. Inhibition of TGF-β/SMAD3 signaling reduced lung metastasis in a mouse in situ hepatic xenograft model. In another cohort of 55 HCC patients, subjects with high GLI2 expression had a shorter disease-free survival than those with low expression. Moreover, co-positivity of GLI2 with SMAD3 was observed in 87.5% of relapsed HCC patients with high GLI2 expression, indicating an increased risk of post-resection recurrence of HCC. The findings underscore that suppressing the non-canonical hedgehog signaling pathway may confer a potential strategy in the treatment of HCC.

Alterations in extracellular matrix (ECM) architecture and stiffness represent hallmarks of cancer. Whether the biomechanical property of ECM impacts the functionality of tumor-reactive CD8+ T cells remains largely unknown. Here, we reveal that the transcription factor (TF) Osr2 integrates biomechanical signaling and facilitates the terminal exhaustion of tumor-reactive CD8+ T cells. Osr2 expression is selectively induced in the terminally exhausted tumor-specific CD8+ T cell subset by coupled T cell receptor (TCR) signaling and biomechanical stress mediated by the Piezo1/calcium/CREB axis. Consistently, depletion of Osr2 alleviates the exhaustion of tumor-specific CD8+ T cells or CAR-T cells, whereas forced Osr2 expression aggravates their exhaustion in solid tumor models. Mechanistically, Osr2 recruits HDAC3 to rewire the epigenetic program for suppressing cytotoxic gene expression and promoting CD8+ T cell exhaustion. Thus, our results unravel Osr2 functions as a biomechanical checkpoint to exacerbate CD8+ T cell exhaustion and could be targeted to potentiate cancer immunotherapy.

PD-1 is a co-inhibitory receptor expressed by CD8+ T cells which limits their cytotoxicity. PD-L1 expression on cancer cells contributes to immune evasion by cancers, thus, understanding the mechanisms that regulate PD-L1 protein levels in cancers is important. Here we identify tumor-cell-expressed otubain-2 (OTUB2) as a negative regulator of antitumor immunity, acting through the PD-1/PD-L1 axis in various human cancers. Mechanistically, OTUB2 directly interacts with PD-L1 to disrupt the ubiquitination and degradation of PD-L1 in the endoplasmic reticulum. Genetic deletion of OTUB2 markedly decreases the expression of PD-L1 proteins on the tumor cell surface, resulting in increased tumor cell sensitivity to CD8+ T-cell-mediated cytotoxicity. To underscore relevance in human patients, we observe a significant correlation between OTUB2 expression and PD-L1 abundance in human non-small cell lung cancer. An inhibitor of OTUB2, interfering with its deubiquitinase activity without disrupting the OTUB2-PD-L1 interaction, successfully reduces PD-L1 expression in tumor cells and suppressed tumor growth. Together, these results reveal the roles of OTUB2 in PD-L1 regulation and tumor evasion and lays down the proof of principle for OTUB2 targeting as therapeutic strategy for cancer treatment.

目的 探讨腹腔镜胆囊切除+胆总管切开取石+胆管一期缝合术(LC+LCBDE+PDC)在胆总管结石合并胆囊结石患者中的应用价值.方法 选取2020年6月—2022年6月分别接受腹腔镜胆囊切除+胆总管切开取石+胆管一期缝合术(LC+LCBDE+PDC)治疗的51例胆总管结石合并胆囊结石患者作为LCBDE组,同期选取接受内镜逆行性胰胆管造影(ERCP)+LC治疗的29例胆总管结石合并胆囊结石患者作为ERCP组;对两组患者临床资料进行回顾性分析.结果 LCBDE组手术时间、住院时间、结石清除率均显著优于ERCP组(P<0.05),两组术后肛门排气时间比较差异不显著(P>0.05);治疗前两组焦虑自评量表(SAS)、抑郁自评量表(SDS)、SF-36评分比较差异不显著(P>0.05),治疗后两组SAS、SDS、SF-36评分均得到显著改善(P<0.05),且术后LCBDE组SAS、SDS、SF-36评分均显著优于ERCP组(P<0.05);LCBDE组并发症发生率显著低于ERCP组(P<0.05).结论 与ERCP+LC相比,LC+LCBDE+PDC用于治疗胆总管结石合并胆囊结石有效性和安全性均更高,且能更好地改善患者负面情况和生活质量,值得临床借鉴推广.

Epigenetic variants carried by circulating tumor DNA can be used as biomarkers for early detection of hepatocellular carcinoma (HCC) by noninvasive liquid biopsy. However, traditional methylation analysis method, bisulfite sequencing, with disadvantages of severe DNA damage, is limited in application of low-amount cfDNA analysis. Through mild enzyme-mediated conversion, enzymatic methyl sequencing (EM-seq) is ideal for precise determination of cell-free DNA methylation and provides an opportunity for HCC early detection. EM-seq of methylation control DNA showed that enzymatic conversion of unmethylated C to U was more efficient than bisulfite conversion. Moreover, a relatively large proportion of incomplete converted EM-seq reads contains more than 3 unconverted CH site (CH = CC, CT or CA), which can be removed by filtering to improve accuracy of methylation detection by EM-seq. A cohort of 241 HCC, 76 liver disease, and 279 normal plasma samples were analyzed for methylation value on 1595 CpGs using EM-seq and targeted capture. Model training identified 283 CpGs with significant differences in methylation levels between HCC and non-HCC samples. A HCC screening model based on these markers can efficiently distinguish HCC sample from non-HCC samples, with area under the curve of 0.957 (sensitivity = 90%, specificity = 97%) in the test set, performing well in different stages as well as in serum α-fetoprotein/protein induced by vitamin K absence-II negative samples. Filtering of reads with ≥ 3 CHs derived from incomplete conversion can significantly reduce the noise of EM-seq detection. Based on targeted EM-seq analysis of plasma cell-free DNA, our HCC screening model can efficiently distinguish HCC patients from non-HCC individuals with high sensitivity and specificity.

目的 研究肝细胞肝癌(HCC)组织中 APOBEC1 互补因子(A1CF)和上移码蛋白 1(UPF1)的表达及临床意义.方法 选取 2017 年 1 月至 2020 年 1 月在该院诊治的 118 例HCC患者作为研究对象.应用荧光定量PCR检测组织中A1CF、UPF1 mRNA 表达.应用免疫组织化学检测组织中 A1CF、UPF1 蛋白表达.A1CF、UPF1 mRNA表达的相关性采用 Pearson 相关分析.A1CF、UPF1 蛋白表达的相关性采用 Spearman秩相关分析.分析 HCC 癌组织中 A1CF、UPF1 蛋白表达与临床病理特征的关系.Kaplan-Meier 生存分析A1CF、UPF1 蛋白表达对 HCC患者生存预后的影响.单因素及多因素 COX 回归分析影响 HCC 患者临床生存预后的因素.结果 癌组织中 A1CF mRNA 相对表达量显著高于癌旁组织,差异有统计学意义(P＜0.001).癌组织中UPF1 mRNA相对表达量显著低于癌旁组织,差异有统计学意义P＜0.001).HCC癌组织中A1CF蛋白表达阳性率明显高于癌旁组织(P＜0.001),HCC癌组织中UPF1 蛋白表达阳性率明显低于癌旁组织(P＜0.001).HCC 癌组织中 A1CF 与 UPF1 mRNA 表达呈显著负相关(r=-0.713,P＜0.001).A1CF与UPF1 蛋白表达亦呈显著负相关(rs=-0.782,P＜0.001).不同肿瘤分期、组织学分级 HCC癌组织中A1CF、UPF1 蛋白表达阳性率比较,差异有统计学意义(P＜0.05).A1CF 阳性组患者累积生存明显低于A1CF阴性组,差异有统计学意义(P＜0.05);UPF1 阴性组患者累积生存低于UPF1 阳性组差异有统计学意义(P＜0.05).肿瘤分期Ⅲ期、组织学分级Ⅲ级、A1CF蛋白阳性、UPF1 蛋白阴性是影响患者不良生存预后的独立危险因素.结论 HCC中A1CF表达升高,UPF1 表达降低,二者表达与肿瘤 TNM分期、病理分级有关,是影响 HCC患者不良预后的独立因素.

The loss of contact inhibition is a key step during carcinogenesis. The Hippo-Yes-associated protein (Hippo/YAP) pathway is an important regulator of cell growth in a cell density-dependent manner. However, how Hippo signaling senses cell density in this context remains elusive. Here, we report that high cell density induced the phosphorylation of spectrin α chain, nonerythrocytic 1 (SPTAN1), a plasma membrane-stabilizing protein, to recruit NUMB endocytic adaptor protein isoforms 1 and 2 (NUMB1/2), which further sequestered microtubule affinity-regulating kinases (MARKs) in the plasma membrane and rendered them inaccessible for phosphorylation and inhibition of the Hippo kinases sterile 20-like kinases MST1 and MST2 (MST1/2). WW45 interaction with MST1/2 was thereby enhanced, resulting in the activation of Hippo signaling to block YAP activity for cell contact inhibition. Importantly, low cell density led to SPTAN1 dephosphorylation and NUMB cytoplasmic location, along with MST1/2 inhibition and, consequently, YAP activation. Moreover, double KO of NUMB and WW45 in the liver led to appreciable organ enlargement and rapid tumorigenesis. Interestingly, NUMB isoforms 3 and 4, which have a truncated phosphotyrosine-binding (PTB) domain and are thus unable to interact with phosphorylated SPTAN1 and activate MST1/2, were selectively upregulated in liver cancer, which correlated with YAP activation. We have thus revealed a SPTAN1/NUMB1/2 axis that acts as a cell density sensor to restrain cell growth and oncogenesis by coupling external cell-cell contact signals to intracellular Hippo signaling.

Additional file 2: Table S1. C sites prone to incomplete conversion in EM-seq. Table S2. Quality control metrics for Targeted EM-seq using HCC cell line input from 5 to 20 ng. Table S3. Quality control metrics for Targeted EM-seq from 596 clinical samples. Table S4. Comparison of methylation levels detected from two hybridization batches. Table S5. Summary of patient characteristics. Table S6. Summary of 283 CpG markers. Table S7. Methylation levels of 283 CpGs from clinical samples.

Background and Aims:Hepatectomy is an effective treatment for selected patients with large hepatocellular carcinoma (HCC). This study aimed to develop a nomogram incorporating non-tumoral liver volume (non-TLV) and liver function markers to predict the patients' overall survival (OS) and disease-free survival (DFS).Methods:Data of 198 consecutive large HCC patients who underwent hepatectomy at the Zhongshan Hospital Xiamen University were collected. Another 68 patients from the Mengchao Hepatobiliary Surgery Hospital served as an external validation cohort. The nomograms were developed based on the independent prognostic factors screened by multivariate Cox regression analyses. Concordance index (C-index), calibration curves, and time-dependent receiver operating characteristic (ROC) curves were used to measure the discrimination and predictive accuracy of the models.Results:High HBV DNA level, low non-TLV/ICG, vascular invasion, and a poorly differentiated tumor were confirmed as independent risk factors for both OS and DFS. The model established in this study predicted 5-year post-operative survival and DFS in good agreement with the actual observation confirmed by the calibration curves. The C-indexes of the nomograms in predicting OS and DFS were 0.812 and 0.823 in the training cohort, 0.821 and 0.846 in the internal validation cohort, and 0.724 and 0.755 in the external validation cohort. The areas under the ROC curves (AUCs) of nomograms for predicted OS and DFS at 1, 3, and 5 year were 0.85, 0.86, 0.83 and 0.76, 0.76, 0.63, respectively.Conclusions:Nomograms with non-TLV/ICG predicted the prognosis of single large HCC patients accurately and effectively.

With the expansion of ICP-MS application into the field of bioanalysis, there is an urgent need for novel element tags today. Here, we report the design of a dual-element Ir-Eu tag, opening the door to simultaneous fluorescent imaging and ICP-MS quantification. The ratio of 153Eu/193Ir may serve as a precision control of the labeling process, allowing internal validation of the quantitative results obtained. As for SIRPα and its host cell analysis exemplified here, the Ir-Eu tag demonstrated superior figures of ICP-MS quantification with the LOD (3σ) down to 0.5 (153Eu) and 1.1 (193Ir) pM SIRPα and 220 (153Eu) and 830 (193Ir) RAW264.7 cells more than 130 times more sensitive compared with the LOD (3σ) of 65.2 pM SIRPα at 612 nm using fluorometry. Not limited to these demonstrations, we believe that the design ideas of the dual Ir-Eu tags should be applicable to various cases of bioanalysis when dual optical profiling and ICP-MS quantification are indispensable.

BACKGROUND:To address intraoperative bleeding in cardiac surgery, reducing blood transfusion requirements, is mandatory to achieve effective hemostasis. Hemostatic agents may limit localized persistent bleeding. The introduction of carboxymethyl-chitosan component into the hemostatic agent and the application of the radiation crosslinking technique maintain its capacity for achieving intraoperative hemostasis, thus increasing the clinical utility. METHODS:A prospective, noninferiority and randomized controlled clinical trial to compare the safety and efficacy of absorbable macroporous polysaccharide composites (AMPC, treatment group) with compound microporous polysaccharide hemostatic powder (CMPHP, control group) (2:1 ratio) as adjuncts to hemostasis in open surgery. The main indication was used for hemostasis in various traumatic hemorrhage areas, including cardiothoracic, vascular, and general surgery. The primary endpoint was success rate of hemostasis within 300 s (at a 10% noninferiority margin). The secondary endpoint was hemostasis time. Both endpoints were assessed in the modified intention-to-treat (MITT) population. Safety parameters were assessed. This study is fully compliant with the CONSORT statement. RESULTS:Randomized patients in AMPC and CMPHP groups were 168 and 84, respectively. In MITT population, the success rates of hemostasis within 300 s were 98.8% (163 of 165) in AMPC and 94.0% (78 of 83) in CMPHP (treatment difference 4.8% [95% CI -0.57% to 10.20%]). AMPC was thus noninferior to CMPHP. Hemostasis time (median [interquartile range]) with AMPC (87 [52.5, 180] s) was better than CMPHP (110 [54.5, 181] s). Changes in laboratory parameters over time and shifts to abnormal values were typical of surgeries and similar between two groups. No noticeable adverse effects associated with AMPC or CMPHP were observed. CONCLUSIONS:AMPC is well tolerated as topical hemostatic agent, noninferior to commercial CMPHP, and exhibits excellent safety. This study provides a novel hemostatic agent which appears to offer significant clinical advantage in various hemorrhage areas.

Selenium (Se) is a mysterious thus tempting element playing a dual bio-chemical function, mainly through selenol, during life processes. Quantification of the selenols is thus of great significance for understanding the biological roles of Se, but remains a big challenge. Herein we report a selenol-specific recognition-mediated and europium (Eu) signal-switched amplification inductively coupled plasma mass spectrometry (ICP-MS) approach for quantifying the free active selenols (act-SeH) in cells. A bifunctional molecule, 2,4-dinitrobenzenesulfonyl-piperidin-4-yl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic europium (DNBS-DOTA-Eu), was designed and synthesized for the specific recognition and highly sensitive quantification of act-SeH via switching Se to more sensitive Eu ICP-MS signals. The limit of detection (LOD, 3σ) of 3.41 pg/mL (22.43 pmol/L), corresponding to the absolute mass LOD of 6.82 ag act-SeH per cell, is almost 25 times lower than 83.76 pg/mL (1.06 nmol/L), 167.52 ag, when monitoring 80Se. The results indicate that act-SeH in the selenite-precultured cancerous HepG2 and paracancerous HL7702 cells are 0.090 ± 0.002 pg/cell (n = 7) and 0.021 ± 0.006 pg/cell (n = 7), more than 4.28 times higher in HepG2 than in HL7702. Preliminary application of this approach to the cells from real hepatic tissue samples suggested that act-SeH has a positive relationship with the degree of hepatic disease. act-SeH in cells appears to be a very promising relevant index for understanding the biochemical functions of Se, besides the total Se in cells and blood serum and/or plasma.

The striatum is structurally highly diverse, and its organ functionality critically depends on normal embryonic development. Although several studies have been conducted on the gene functional changes that occur during striatal development, a system-wide analysis of the underlying molecular changes is lacking. Here, we present a comprehensive transcriptome profile that allows us to explore the trajectory of striatal development and identify the correlation between the striatal development and Huntington's disease (HD). Furthermore, we applied an integrative transcriptomic profiling approach based on machine learning to systematically map a global landscape of 277 transcription factor (TF) networks. Most of these TF networks are linked to biological processes, and some unannotated genes provide information about the corresponding mechanisms. For example, we found that the Meis2 and Six3 were crucial for the survival of striatal neurons, which were verified using conditional knockout (CKO) mice. Finally, we used RNA-Seq to speculate their downstream targets.

快速康复外科（enhanced recovery after surgery，ERAS）理念已获得国内大部分外科医生认可，但围绕其实施和应用仍然存在诸多争议和质疑，本文结合1例巨块型肝癌转化切除病例探讨ERAS理念用于肝癌切除术的安全性及有效性。