Plasma methylated GNB4 and Riplet as a novel dual-marker panel for the detection of hepatocellular carcinoma.

Early detection of hepatocellular carcinoma (HCC) can greatly improve the survival rate of patients. We aimed to develop a novel marker panel based on cell-free DNA (cfDNA) methylation for the detection of HCC. The differentially methylated CpG sites (DMCs) specific for HCC blood diagnosis were selected from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, then validated by the whole genome bisulphite sequencing (WGBS) of 12 paired HCC and paracancerous tissues. The clinical performance of the panel was evaluated using tissue samples [32 HCC, chronic liver disease (CLD), and healthy individuals] and plasma cohorts (173 HCC, 199 CLD, and 98 healthy individuals). The combination of G protein subunit beta 4 (GNB4) and Riplet had the optimal area under the curve (AUC) in seven candidates through TCGA, GEO, and WGBS analyses. In tissue validation, the GNB4 and Riplet showed an AUC of 100% with a sensitivity and specificity of 100% for detecting any-stage HCC. In plasma, it demonstrated a high sensitivity of 84.39% at 91.92% specificity, with an AUC of 92.51% for detecting any-stage HCC. The dual-marker panel had a higher sensitivity of 78.26% for stage I HCC than alpha-fetoprotein (AFP) of 47.83%, and a high sensitivity of 70.27% for detecting a single tumour (size ≤3 cm). In conclusion, we developed a novel dual-marker panel that demonstrates high accuracy in detecting HCC, surpassing the performance of AFP testing.

Small change, big difference: A promising praziquantel derivative designated P96 with broad-spectrum antischistosomal activity for chemotherapy of schistosomiasis japonica.

BACKGROUND: Praziquantel (PZQ) has been the first line antischistosomal drug for all species of Schistosoma, and the only available drug for schistosomiasis japonica, without any alternative drugs since the 1980s. However, PZQ cannot prevent reinfection, and cannot cure schistosomiasis thoroughly because of its poor activity against juvenile schistosomes. In addition, reliance on a single drug is extremely dangerous, the development and spread of resistance to PZQ is becoming a great concern. Therefore, development of novel drug candidates for treatment and control of schistosomiasis is urgently needed. METHODOLOGYS/PRINCIPAL FINDINGS: One of the PZQ derivative christened P96 with the substitution of cyclohexyl by cyclopentyl was synthesized by School of Pharmaceutical Sciences of Shandong University. We investigated the in vitro and in vivo activities of P96 against different developmental stages of S. japonicum. Parasitological studies and scanning electron microscopy were used to study the primary action characteristics of P96 in vitro. Both mouse and rabbit models were employed to evaluate schistosomicidal efficacy of P96 in vivo. Besides calculation of worm reduction rate and egg reduction rate, quantitative real-time PCR was used to evaluate the in vivo antischistosomal activity of P96 at molecular level. In vitro, after 24h exposure, P96 demonstrated the highest activities against both juvenile and adult worm of S. japonicum in comparison to PZQ. The antischistosomal efficacy was concentration-dependent, with P96 at 50µM demonstrating the most evident schistosomicidal effect. Scanning electron microscopy demonstrated that P96 caused more severe damages to schistosomula and adult worm tegument compared to PZQ. In vivo, our results showed that P96 was effective against S. japonicum at all developmental stages. Notably, its efficacy against young stage worms was significantly improved compared to PZQ. Moreover, P96 retained the high activity comparable to PZQ against the adult worm of S. japonicum. CONCLUSIONS: P96 is a promising drug candidate for chemotherapy of schistosomiasis japonica, which has broad spectrum of action against various developmental stage, potentially addressing the deficiency of PZQ. It might be promoted as a drug candidate for use either alone or in combination with PZQ for the treatment of schistosomiasis.

Hypermethylated CDO1 and ZNF454 in Cytological Specimens as Screening Biomarkers for Endometrial Cancer.

We aimed to estimate the diagnostic value of DNA methylation levels in cytological samples of endometrial cancer (EC) and atypical hyperplasia (AH). Two hypermethylated genes, namely, cysteine dioxygenase type 1 (CDO1) and zinc finger protein 454 (ZNF454), in patients with EC were identified from The Cancer Genome Atlas database. In 103 endometrial histological specimens (the training set), the methylation levels of candidate genes were verified by quantitative methylation-specific polymerase chain reaction (qMSP). The methylation levels of another 120 cytological specimens (the testing set) were evaluated. Sensitivity (Se), specificity (Sp), accuracy, and area under the curve (AUC) were determined, with diagnosis verified by histopathological results. CDO1 and ZNF454 verified hypermethylation in histological specimens of patients with EC and AH compared with those with benign and normal endometrium (P < 0.001). In cytological specimens, hypermethylated CDO1 showed 86.36% Se and 90.79% Sp with the cutoff value of 6.0 to distinguish between malignant and benign groups; ZNF454 showed 79.55% Se and 93.42% Sp with the cutoff value of 7.1. When the two genes were combined, Se increased to 90.91% and Sp was 86.84%. AUC reached 0.931 (95% CI: 0.885-0.976). The diagnostic accuracy with cytology had no significant difference with endometrial tissue (P = 0.847 for CDO1, P = 0.108 for ZNF454, and P = 0.665 for their combination). Hypermethylated CDO1 and ZNF454 in endometrial cytology showed high Se, Sp, and AUC to detect EC and AH. Methylation analysis of endometrial cytology is promising biomarker for the screening of EC and AH.

Tea polyphenol/glycerol-treated double-network hydrogel with enhanced mechanical stability and anti-drying, antioxidant and antibacterial properties for accelerating wound healing.

Frequent dressing changes can result in secondary wound damage. Therefore, it is of great significance to construct a wound dressing that can be used for a long time without changing. Here, a double-network hydrogel was synthesized through hydrogen bonding interactions of tea polyphenol (TP)/glycerol with photo-crosslinked N-acryloyl glycinamide (NAGA), gelatin methacrylate (GelMA), and nanoclay hydrogel. The glycerol/water solvent slowed the diffusion of TP into the NAGA/GelMA/Laponite (NGL)hydrogel, thereby avoiding excessive crosslinking, and forming a uniform network. The hydrogel exhibited excellent water retention (84% within 28 days). Additionally, due to the hygroscopicity of glycerol, the hydrogel's mechanical strength (0.73-1.14 MPa) and tensile strain (207%-353%) increased further after 14 days in an open environment. Additionally, the hydrogel exhibited superior anti-ultraviolet and antioxidant properties, which effectively alleviated the wound site's oxidative stress and accelerated wound healing. Moreover, antibacterial activity was observed against both E. coli and S. aureus in the hydrogel wound dressing. Thus, by promoting wound closure, angiogenesis and collagen deposition, the double-network NGLG20/TG hydrogel dressing can successfully accelerate wound healing. The multifunctional double-network hydrogel, therefore, shows immense potential as an ideal candidate for wound dressings because it is long-lasting and prevents secondary damage caused by frequent dressing changes.

A Single Vaccine Protects against SARS-CoV-2 and Influenza Virus in Mice.

The ongoing SARS-CoV-2 pandemic poses a severe global threat to public health, as do influenza viruses and other coronaviruses. Here, we present chimpanzee adenovirus 68 (AdC68)-based vaccines designed to universally target coronaviruses and influenza. Our design is centered on an immunogen generated by fusing the SARS-CoV-2 receptor-binding domain (RBD) to the conserved stalk of H7N9 hemagglutinin (HA). Remarkably, the constructed vaccine effectively induced both SARS-CoV-2-targeting antibodies and anti-influenza antibodies in mice, consequently affording protection from lethal SARS-CoV-2 and H7N9 challenges as well as effective H3N2 control. We propose our AdC68-vectored coronavirus-influenza vaccine as a universal approach toward curbing respiratory virus-causing pandemics. IMPORTANCE The COVID-19 pandemic exemplifies the severe public health threats of respiratory virus infection and influenza A viruses. The currently envisioned strategy for the prevention of respiratory virus-causing diseases requires the comprehensive administration of vaccines tailored for individual viruses. Here, we present an alternative strategy by designing chimpanzee adenovirus 68-based vaccines which target both the SARS-CoV-2 receptor-binding-domain and the conserved stalk of influenza hemagglutinin. When tested in mice, this strategy attained potent neutralizing antibodies against wild-type SARS-CoV-2 and its emerging variants, enabling an effective protection against lethal SARS-CoV-2 challenge. Notably, it also provided complete protection from lethal H7N9 challenge and efficient control of H3N2-induced morbidity. Our study opens a new avenue to universally curb respiratory virus infection by vaccination.

Facile extrusion 3D printing of gelatine methacrylate/Laponite nanocomposite hydrogel with high concentration nanoclay for bone tissue regeneration.

The extrusion 3D printing of hydrogels has evolved as a promising approach that can be applied for specific tissue repair. However, the printing process of hydrogel scaffolds with high shape fidelity is inseparable from the complex crosslinking strategy, which significantly increases the difficulty and complexity of printing. The aim of this study was to develop a printable hydrogel that can extrude at room temperature and print scaffolds with high shape fidelity without any auxiliary crosslinking during the printing process. To this end, a novel formulation consisting of a Laponite suspension with a high solid concentration and a gelatine methacrylate (GelMA) nanocomposite hydrogel was developed. A homogeneously dispersed high-concentration (up to 20% w/v) Laponite suspension was obtained by stirring at 0 °C. The addition of Laponite with high concentration improved the rheological properties, the degradation stability, and the mechanical strength of the hydrogel. The formulation of 15% (w/v) GelMA and 8% (w/v) Laponite nanocomposite hydrogel exhibited desirable printability and biocompatibility. The GelMA/Laponite hydrogels significantly promoted bone marrow mesenchymal stem cell (BMSC) proliferation and osteogenic differentiation. Both desirable printability under mild conditions and cyto-compatibility enable composite hydrogel a potential candidate as biomaterial inks to be applied for bone tissue regeneration.

Vaccinia virus-based vector against infectious diseases and tumors.

Vaccinia virus was used to prevent smallpox. After the World Health Organization declared smallpox extinct, vaccinia virus has been explored for the development of vaccines against a variety of infectious diseases. It also finds a new place in oncolytic therapy. Here we provide a brief review of the history, current status, and future prospect of vaccinia virus-based vaccine and oncolytic virus. New advancements, including a single vaccine targeting multiple viruses, strategies of arming vaccinia viruses to enhance anti-tumor activity, the promise and challenge of combining vaccinia-based virotherapy with immunotherapy, are discussed as special focus.

Methylation of SDC2/TFPI2 and Its Diagnostic Value in Colorectal Tumorous Lesions.

Background: SDC2 methylation is a feasible biomarker for colorectal cancer detection. Its specificity for colorectal cancer is higher than 90%, but the sensitivity is normally lower than 90%. This study aims to improve the sensitivity of SDC2 detection through finding a high positive target from the false-negative samples of SDC2 detection based on analysis of the bowel subsite difference in methylation. Methods: Hypermethylated TFPI2 was identified in SDC2 hypomethylated colorectal cancer samples retrieved from TCGA database with the methylation level lower than 0.2. The methylation-specific PCR assay was developed and then evaluated using tissue samples (184 cancer and 54 healthy control samples) and stool samples (289 cancer, 190 adenoma, and 217 healthy control samples). Results: TFPI2 was hypermethylated in most SDC2 hypomethylated colorectal cancer samples. When the SDC2/TFPI2-combined PCR assay was performed in stool specimens, the AUC value of cancer vs. control was 0.98, with the specificity of 96.40% and sensitivity of 96.60%, and the AUC value of adenoma vs. control was 0.87, with the specificity of 95.70% and the sensitivity of 80.00%. The improvement in sensitivity was the most momentous in the left colon. As the detection index, the Ct value was better in improving the sensitivity of detection than the methylation level based on the 2-ΔΔCt value. Conclusion: TFPI2 can improve the sensitivity of SDC2 methylation-specific detection of colorectal tumorous lesions while maintaining high specificity, in particular reducing the missed detection of left colon cancer and adenoma.

RASSF4 promotes EV71 replication to accelerate the inhibition of the phosphorylation of AKT.

Enterovirus 71 (EV71) is a neurotropic virus that causes hand, foot and mouth disease (HFMD), occasionally leading to death. As a member of the RAS association domain family (RASSFs), RASSF4 plays important roles in cell death, tumor development and signal transduction. However, little is known about the relationship between RASSF4 and EV71. Our study reveals for the first time that RASSF4 promotes EV71 replication and then accelerates AKT phosphorylation inhibition in EV71-infected 293T cells, suggesting that RASSF4 may be a potential new target for designing therapeutic measures to prevent and control EV71 infection.

An artemisinin derivative of praziquantel as an orally active antischistosomal agent.

BACKGROUND: Schistosomiasis is a major health problem in tropical and sub-tropical areas caused by species of trematode belonging to the genus Schistosoma. The treatment and control of this disease has been relying on the use of a single drug praziquantel. However, the drug resistance concern urged the development of new drugs against schistosoma. Here, we report our systematic biological evaluation of DW-3-15, a new lead compound developed based on our conjugation design rationale as an effective anti-schistosomal agent. METHODOLOGY/PRINCIPAL FINDINGS: The antischistosomal activity of DW-3-15 was systematically evaluated in S. japonicum infected mouse model for its stage-sensitivity and dose response. The results revealed that DW-3-15 exhibited 60-85% worm reduction rate against different development stage of worm. Scanning electron microscopy (SEM) observation indicated that DW-3-15 may damage to the tegument of male schistosomes. CONCLUSIONS/SIGNIFICANCE: Our results demonstrated that DW-3-15 showed potent anti-schistosomal activities in vivo. The results strongly support our conjugation design strategy of artemisinin analogs and further development of DW-3-15 as a new lead compound as anti-schistosomal agent.

Development of chiral praziquantel analogues as potential drug candidates with activity to juvenile Schistosoma japonicum.

A series of chiral praziquantel analogues were synthesized and evaluated against Schistosoma japonicum both in vitro and in vivo. All compounds exhibited low to considerable good activity in vivo. Remarkably, worm reduction rate of R-3 was 60.0% at a single oral dose of 200mg/kg against juvenile stage of Schistosoma japonicum. The target compounds displayed in vivo antischistosomal activity against both Schistosoma japonicum and Schistosoma mansoni. Furthermore, all R-isomers displayed stronger antischistosomal activity than S-isomers in vivo, indicating R-isomers were the active enantiomers, while S-isomers were less active ones. This structure activity relationship (SAR) could have important implications in further drug development for schistosomiasis.

Identification of Pirh2E and Pirh2F, two additional novel isoforms of Pirh2 ubiquitin ligase from human hepatocellular liver carcinoma cell line.

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, especially in China. Recent researches have shown that E3 ubiquitin ligases Pirh2 (p53-induced RING-H2 protein) is highly expressed in HCC cell lines and is correlated with poor survival and prognostic in patients with HCC; however, the function of Pirh2 in the genesis and the development of HCC remains unclear. Pirh2, a member of the RING finger family, can target p53 for degradation and thereby repress a diverse group of biological activities and involved in many signalling pathways related to the genesis and evolution of cancer. Up to now, four Pirh2 variants had been reported and named Pirh2A, Pirh2B, Pirh2C and Pirh2D. Here we report the existence of two additional isoforms from human hepatocellular liver carcinoma cell line (HepG2 cell) and named as Pirh2E and Pirh2F. Compared to full-length Pirh2A, Pirh2E lacks amino acids 235-261, while Pirh2F is missing C-terminal amino acids 227-261 and both isoforms harbor the RING domain; therefore, we speculate that ubiquitin ligase activity maybe reversed by them. Further studies are required to determine whether Pirh2E and Pirh2F functions in a manner similar to Pirh2A.

A novel oncoprotein Pirh2: rising from the shadow of MDM2.

Pirh2 (p53-induced RING-H2) is an E3 ubiquitin ligase that can target p53 for degradation and thereby repress a diverse group of biological activities regulated by p53. Notably, Pirh2, rather than MDM2, is the primary degrader of active p53 under conditions of DNA damage. Moreover, Pirh2 is highly expressed in multiple cancer cell lines regardless of p53 status. Recent research has shown that Pirh2 is involved in many signalling pathways related to the genesis and evolution of cancer. This review aims to summarize a comprehensive picture of the role of Pirh2 in cellular processes and its significance to tumorigenesis. Furthermore, this review focuses on its potential role as a cancer therapeutic target.

[Interference of HIF-1alpha by RNA reduces the expression of matrix metalloproteinase-2 in human cervical carcinoma HeLa cells].

BACKGROUND & OBJECTIVE: Hypoxia inducible factor-1 alpha (HIF-1alpha) is a hypoxia-responsive factor, which commonly exists in the tissues of solid tumors. It plays crucial roles in maintaining cell energy metabolism, tumor angiogenesis and metastasis, cell proliferation and apoptosis. This study was to investigate the effect of silencing HIF-1alpha by RNA interference on the expression of matrix metalloproteinase-2 (MMP-2) in human cervical carcinoma HeLa cells, and to further explore the mechanism. METHODS: Fifteen nude mice were divided into three groups randomly, and were inoculated with HeLa cells, pGenesil-1-HeLa cells and HIF-1alpha-HeLa cells, respectively. Expressions of HIF-1alpha, E-cadherin, beta-catenin, MMP-2 in the xenograft tumors of nude mice were detected by immunohistochemistry. pGenesil-1-HeLa or HIF-1alpha-HeLa cells were cultured under a hypoxic(1% O2) or normoxic environment for 48 h, while HeLa cells were cultured under the same condition as control. Expressions of HIF-1alpha, E-cadherin, beta-catenin and MMP-2 were measured by Western blot. RESULTS: In HeLa, pGenesil-1-HeLa and HIF-1alpha-HeLa cell xenograft groups, the positive rates of HIF-1alpha protein were (69.0+/-12.1)%, (62.8+/-12.3)% and (17.4+/-8.8)%, respectively; expressions of E-cadherin were 1.00+/-0.07, 1.00+/-0.10, 3.21+/-0.25; expressions of beta-catenin were 4.21+/-0.92, 4.31+/-0.87, 1.29+/-0.72; and expressions of MMP-2 were 4.84+/-0.67, 4.50+/-0.71 and 1.00+/-0.83. HIF-1alpha and MMP-2 were positively correlated (correlation coefficient=0.521). Expressions of HIF-1alpha, beta-catenin and MMP-2 protein were low and that of E-cadherin protein was high in HIF-1alpha-HeLa cells under hypoxia or normoxia for 48 h; while opposite results were obtained in HeLa and pGenesil-1-HeLa cells. CONCLUSION: Inhibition of HIF-1alpha could effectively down-regulate the expression of MMP-2 in human cervical carcinoma Hela cells.

Signaling through G(alpha)13 switch region I is essential for protease-activated receptor 1-mediated human platelet shape change, aggregation, and secretion.

This study investigated the involvement of Galpha(13) switch region I (SRI) in protease-activated receptor 1 (PAR1)-mediated platelet function and signaling. To this end, myristoylated peptides representing the Galpha(13) SRI (Myr-G(13)SRI(pep)) and its random counterpart were evaluated for their effects on PAR1 activation. Initial studies demonstrated that Myr-G(13)SRI(pep) and Myr-G(13)SRI(Random-pep) were equally taken up by human platelets and did not interfere with PAR1-ligand interaction. Subsequent experiments revealed that Myr-G(13)SRI(pep) specifically bound to platelet RhoA guanine nucleotide exchange factor (p115RhoGEF) and blocked PAR1-mediated RhoA activation in platelets and human embryonic kidney cells. These results suggest a direct interaction of Galpha(13) SRI with p115RhoGEF and a mechanism for Myr-G(13)SRI(pep) inhibition of RhoA activation. Platelet function studies demonstrated that Myr-G(13)SRI(pep) specifically inhibited PAR1-stimulated shape change, aggregation, and secretion in a dose-dependent manner but did not inhibit platelet activation induced by either ADP or A23187. It was also found that Myr-G(13)SRI(pep) inhibited low dose, but not high dose, thrombin-induced aggregation. Additional experiments showed that PAR1-mediated calcium mobilization was partially blocked by Myr-G(13)SRI(pep) but not by the Rho kinase inhibitor Y-27632. Finally, Myr-G(13)SRI(pep) effectively inhibited PAR1-induced stress fiber formation and cell contraction in endothelial cells. Collectively, these results suggest the following: 1) interaction of Galpha(13) SRI with p115RhoGEF is required for G(13)-mediated RhoA activation in platelets; 2) signaling through the G(13) pathway is critical for PAR1-mediated human platelet functional changes and low dose thrombin-induced aggregation; and 3) G(13) signaling elicits calcium mobilization in human platelets through a Rho kinase-independent mechanism.