Osr2 functions as a biomechanical checkpoint to aggravate CD8+ T cell exhaustion in tumor.

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.

Design of a dual Ir-Eu tag for fluorescent visualization and ICP-MS quantification of SIRPα and its host cells.

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.

Quantification of active selenols in cells: a selenol-specific recognition europium-switched signal-amplification ICP-MS approach.

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.

Prospective, randomized, controlled, noninferiority clinical trial to evaluate the safety and efficacy of absorbable macroporous polysaccharide composites as adjunct to hemostasis during open surgery.

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.

Competing endogenous RNA network and prognostic nomograms for hepatocellular carcinoma patients who underwent R0 resection.

The prognosis of hepatocellular carcinoma (HCC) after R0 resection is unsatisfactory due to the high rate of recurrence. In this study, we investigated the recurrence-related RNAs and the underlying mechanism. The long noncoding RNA (lncRNA), microRNA (miRNA), and messenger RNA (mRNA) expression data and clinical information of 247 patients who underwent R0 resection patients with HCC were obtained from The Cancer Genome Atlas. Comparing the 1-year recurrence group (n = 56) with the nonrecurrence group (n = 60), we detected 34 differentially expressed lncRNAs (DElncRNAs), five DEmiRNAs, and 216 DEmRNAs. Of these, three DElncRNAs, hsa-mir-150-5p, and 11 DEmRNAs were selected for constructing the competing endogenous RNA (ceRNA) network. Next, two nomogram models were constructed based separately on the lncRNAs and mRNAs that were further selected by Cox and least absolute shrinkage and selection operator regression analysis. The two nomogram models that showed a high prediction accuracy for disease-free survival with the concordance indexes at 0.725 and 0.639. Further functional enrichment analysis of DEmRNAs showed that the mRNAs in the ceRNA network and nomogram models were associated with immune pathways. Hence, we constructed a hsa-mir-150-5p-centric ceRNA network and two effective nomogram prognostic models, and the related RNAs may be useful as potential biomarkers for predicting recurrence in patients with HCC.

GABPA predicts prognosis and inhibits metastasis of hepatocellular carcinoma.

BACKGROUND: Increasing evidence indicates that abnormal expression of GABPA is associated with tumor development and progression. However, the function and clinicopathological significance of GABPA in hepatocellular carcinoma (HCC) remain obscure. METHODS: The mRNA and protein expression of GABPA in HCC clinical specimens and cell lines was examined by real-time PCR and western blotting, respectively. Follow-up data were used to uncover the relationship between GABPA expression and the prognosis of HCC patients. HCC cell lines stably overexpressing or silencing GABPA were established to explore the function of GABPA in HCC cell migration and invasion by Transwell and wound healing assays in vitro and in a xenograft model in vivo. Restoration of function analysis was used to examine the underlying molecular mechanisms. RESULTS: GABPA was downregulated at the protein and mRNA levels in HCC tissues compared with adjacent normal tissues. Decreased GABPA expression was correlated with alpha-fetoprotein levels (P = 0.001), tumor grade (P = 0.017), and distant metastasis (P = 0.021). Kaplan-Meier survival analysis showed that patients with lower GABPA expression had significantly shorter survival times than those with higher GABPA (P = 0.031). In vivo and in vitro assays demonstrated that GABPA negatively regulated HCC cell migration and invasion, and the effect of GABPA on HCC cell migration was mediated at least partly by the regulation of E-cadherin. CONCLUSIONS: Collectively, our data indicate that GABPA inhibits HCC cell migration by modulating E-cadherin and could serve as a novel biomarker for HCC prognosis. GABPA may act as a tumor suppressor during HCC progression and metastasis, and is a potential therapeutic target in HCC.

Analysis of risk factors related to gastrointestinal fistula in patients with severe acute pancreatitis: a retrospective study of 344 cases in a single Chinese center.

BACKGROUND: Gastrointestinal fistula (GIF) in severe acute pancreatitis (SAP) is considered as a sparse episode and studied sporadically in the literature. There is paucity of data on the prediction of the effect on risk of GIF in patient with SAP. This study was aimed to investigate risk factors related to GIF in the development of SAP. METHODS: The clinical data of 344 patients with SAP from 2011 to 2016 were reviewed retrospectively. All patients were divided into the GIF group and the non-GIF group, and their data analyzed with respect to 15 parameters were applied to explore potential risk factors for GIF in patients with SAP. RESULTS: Of the 344 eligible patients, 52 (15.12%) progressed to GIF. Only occurrence of infected pancreatic and extra-pancreatic necrosis (IPN) (P = 0.004, OR = 3.012) and modified CT severity index (MCTSI) (P = 0.033, OR = 1.183) were proved to be independent risk factors for GIF in patients with SAP, and blood type B (P = 0.048, OR = 2.096, 95% CI: 0.748-3.562) indicated weaker association of risk factor for GIF. The early (48-72 h after admission) enteral nutrition (EEN) (P = 0.016, OR = 0.267) acted as a protective factor. CONCLUSIONS: Occurrence of IPN and high MCTSI are independent risk factors for the development of GIF in patients with SAP, blood type B reveals a potential correlation with GIF in patients with SAP. EEN is helpful to prevent the progression of GIF secondary to SAP.

[Ursodeoxycholic acid induced apoptosis of human hepatoma cells HepG2 and SMMC-7721 bymitochondrial-mediated pathway].

OBJECTIVE: To explore the effects and underlying mechanisms of ursodeoxycholic acid on human hepatoma cells. METHODS: HepG2 and SMMC-7721 HCC cell lines were respectively treated with ursodeoxycholic acid. And cell proliferation, apoptosis and the expression of Bax/Bcl-2 gene were detected by methyl thiazolyl tetrazolium (MTT), inverted microscopy, fluorescent microscopy, flow cytometry and Western blot. RESULTS: Ursodeoxycholic acid significantly inhibited the proliferation of human hepatoma cells in a concentration- and time-dependent manner. The half maximal inhibitory concentrations (IC50) of HepG2 and SMMC-7721 were 397.3 and 387.7 µg/ml respectively after a 48-hour treatment of 400 µg /ml ursodeoxycholic acid. And it also induced the apoptosis of HepG2 and SMMC-7721 cells, up-regulated Bax gene and down-regulated Bcl-2 gene. CONCLUSION: Ursodeoxycholic acid inhibits the proliferation of hepatoma cells and induce apoptosis by mitochondrial-mediated pathway.

Single cell glycan-linkages profiling for hepatocellular carcinoma early diagnosis using lanthanide encoded bacteriophage MS2 based ICP-MS.

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 (α-2,3-Sia, α-Gal, α-1,2-Fuc, α-1,6-Fuc, α-2,6-Sia, α-GalNAc, and Gal-ß-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.

TGF-ß1/SMAD3-driven GLI2 isoform expression contributes to aggressive phenotypes of hepatocellular carcinoma.

Hedgehog signaling is activated in response to liver injury, and modulates organogenesis. However, the role of non-canonical hedgehog activation via TGF-ß1/SMAD3 in hepatic carcinogenesis is poorly understood. TGF-ß1/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-ß/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.

Dynamic immune recovery process after liver transplantation revealed by single-cell multi-omics analysis.

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.

Pharmaceutical targeting of OTUB2 sensitizes tumors to cytotoxic T cells via degradation of PD-L1.

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.

Safety and cost-effectiveness analysis of laparoscopic splenectomy by secondary pedicle division using monopolar electrocautery.

BACKGROUND/AIMS: The expense of laparoscopic splenectomy (LS) has limited its use in developing countries, while medical costs are increasing worldwide. In this study, we performed LS by secondary pedicle division using monopolar electrocautery to achieve cost savings. METHODOLOGY: Over seven years, we performed 45 consecutive LSs by secondary pedicle division using monopolar electrocautery (n=17) or ultrasonic shears (n=28) at a single center. These were reviewed to assess outcome and cost. RESULTS: Mean operating time was 179.7min, 7 conversions to open operation (15.6%) were necessary. There were four postoperative complications (8.9%) and no deaths. Twenty-three of 28 (82.1%) patients with idiopathic thrombocytopenic purpura developed a long-term positive response; and mean operative cost was RMB6,577 (US$1,034), which was much lower than that of Endo-GIATM in published reports. Between the monopolar electrocautery and ultrasonic shears groups, there were no significant differences in demographic characteristics or intraoperative and postoperative details, but operative cost was significantly lower in the former (RMB4,416, US$696 vs. RMB7,889, US$1,243; p<0.01). CONCLUSIONS: LS by secondary pedicle division using monopolar electrocautery is safe, efficacious and economical.

Value of Non-tumoral Liver Volume in the Prognosis of Large Hepatocellular Carcinoma Patients After R0 Resection.

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.

Hepatocellular carcinoma detection via targeted enzymatic methyl sequencing of plasma cell-free DNA.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

SPTAN1/NUMB axis senses cell density to restrain cell growth and oncogenesis through Hippo signaling.

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.

Influence of mutations in hepatitis B virus surface protein on viral antigenicity and phenotype in occult HBV strains from blood donors.

BACKGROUND & AIMS: This study aimed at investigating mutations in the hepatitis B surface protein (HBsAg) in occult hepatitis B virus (HBV) infection (OBI) and their influence on viral antigenicity and phenotype. METHODS: The characteristics of 61 carriers with OBI (OBI group), 153 HBsAg(+) carriers with serum HBsAg ≤ 100 IU/ml (HBsAg-L group) and 54 carriers with serum HBsAg >100 IU/ml (HBsAg-H group) from 38,499 blood donors were investigated. Mutations in the major hydrophilic region (MHR) of the viral sequences were determined. Thirteen representative MHR mutations observed in OBI sequences were antigenically characterized with a panel of monoclonal antibodies (MAbs) and commercial HBsAg immunoassays and functionally characterized in HuH7 cells and hydrodynamically injected mice. RESULTS: Of 61 OBI sequences, 34 (55.7%) harbored MHR mutations, which was significantly higher than the frequency in either the HBsAg-L (34.0%, p=0.003) or the HBsAg-H group (17.1%, p<0.001). Alterations in antigenicity induced by the 13 representative MHR mutations identified in the OBI group were assessed by reacting recombinant HBV mutants with 30 different MAbs targeting various epitopes. Four out of the 13 mutations (C124R, C124Y, K141E, and D144A) strongly decreased the analytical sensitivity of seven commercial HBsAg immunoassays, and 10 (G119R, C124Y, I126S, Q129R, S136P, C139R, T140I, K141E, D144A, and G145R) significantly impaired virion and/or S protein secretion in both HuH7 cells and mice. CONCLUSIONS: MHR mutations alter antigenicity and impair virion secretion, both of which may contribute to HBsAg detection failure in individuals with OBI.

Therapeutic angiogenesis using basic fibroblast growth factor in combination with a collagen matrix in chronic hindlimb ischemia.

Although therapeutic angiogenesis by angiogenic cytokines is a feasible strategy to improve regional blood flow in ischemic regions, the optimal delivery mode needs to be established. Here we designed a complex of collagen matrix (CM) and basic fibroblast growth factor (bFGF) and evaluated its proangiogenic effect in ischemic hindlimbs. The bFGF-CM was prepared using lyophilization. The morphology, porosity and toxicity of CM were examined. The bFGF releasing profile and bioactivity of released bFGF were assessed. bFGF-CM was intramuscularly implanted into the rabbit ischemic hindlimb model. Oxygen saturation parameters (OSP) of ischemic hindlimbs was measured to evaluate the extremity perfusion at intervals. Histological examination was performed to evaluate the level of angiogenesis. The CM and bFGF-CM were of identical multiporous structure lacking cytotoxicity. The releasing profile lasted 10 days and the released bFGF remained bioactive. OSP in bFGF-CM group was significantly higher than that in CM, bFGF and ischemic groups at 2 and 4 weeks. The number of capillaries and mature vessels in bFGF-CM group were significantly greater than that in untreated control, CM and bFGF groups. Therefore, bFGF-CM enables the safe and effective long-term release of bFGF with improved angiogenesis in ischemic hindlimbs compared with CM devoid of bFGF.

Genome-wide long noncoding RNA and mRNA expression profiles demonstrate associations between exposure to inorganic elements and the risk of developing hepatocellular carcinoma.

BACKGROUND: Long noncoding RNAs (lncRNAs) are closely associated with the development of hepatocellular carcinoma (HCC). The present study conducted a genome-wide microarray analysis and qPCR validation to obtain comprehensive insights into this issue. METHODS: Thirty male HCC patients with chronic HBV infection were included in the present study. Primary HCC tissue and normal tissue were collected. Double-stranded complementary DNA synthesized from 10 pairs of samples was labeled and hybridized to a microarray chip. Further analyses, such as hierarchical clustering, gene ontology (GO) and pathway analyses, were performed. In addition, qPCR validation was performed on tissue samples and additional serum samples. RESULTS: The microarray analysis identified 946 upregulated and 571 downregulated lncRNAs and 1720 upregulated and 1106 downregulated mRNAs. Among these RNAs, ENST00000583827.1 (fold change: 21) and uc010isf.1 (fold change: 18) were the most over- and underexpressed lncRNAs in the HCC tissues, respectively. For the mRNAs, KIF20A (fold change: 26) and HEPACAM (fold change: 50) were the most over- and underexpressed in the HCC tissues, respectively. The GO analysis demonstrated that the most differentially expressed mRNAs were related to the response of metal ions. The pathway analysis also suggested that the most enriched pathway was mineral absorption. CONCLUSIONS: The subsequent qPCR validation exhibited high consistency with the microarray analysis, except for three lncRNAs. The qPCR analysis also demonstrated that TCONS_00008984 had a 767-fold overexpression level in HCC tissues when compared with normal tissues, and this finding was confirmed in the serum samples; therefore, TCONS_00008984 has the potential to serve as a diagnostic marker or prognostic indicator. The GO and pathway analyses indicated that exposure to inorganic elements may be involved in HCC risk.

Clinical significance and expression of cyclin kinase subunits 1 and 2 in hepatocellular carcinoma.

BACKGROUND: The mammalian cyclin kinase subunit (Cks) family has two members, Cks1 and Cks2, which were identified based on the protein sequence homology to yeast Cks. Overexpression of Cks1 and Cks2 has been reported to be associated with high aggressiveness and a poor prognosis in various malignancies, including gastric, breast and prostate carcinomas. Yet, whether Cks1 and Cks2 are overexpressed in hepatocellular carcinoma (HCC) remains uncharacterized. AIMS: To investigate whether overexpression of the Cks family is clinically relevant to HCC, and whether expression patterns of Cks1 and Cks2 in HCC have diagnostic and prognostic value. METHODS: Real-time quantitative reverse transcriptase polymerase chain reaction, immunostaining and Western blot analyses were used to detect the expression of Cks1 and Cks2 at the mRNA and protein levels respectively. The associations between Cks1 and Cks2 expressions and clinical features, as well as the association between Cks1 or Cks2 and p27(kip1) expressions in HCC, were analysed. RESULTS: Expressions of Cks1 and Cks2 at both mRNA and protein levels were significantly higher in HCC than those in the adjacent noncancerous tissues (including chronic hepatitis and cirrhosis) and normal liver tissues. Overexpressions of Cks1 and Cks2 in HCC were closely associated with poor differentiation features. The expressions of both Cks1 and Cks2 were negatively associated with p27(kip1) at the protein level. CONCLUSIONS: Overexpression of Cks1 and Cks2 is associated with the aggressive tumour behaviours of HCC, and thus has diagnostic and prognostic value. Further efforts are needed to develop novel biomarkers for HCC based on CKs1 and Cks2 expressions.