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Targeted therapy for patients with hepatitis B virus (HBV) infection can lead to objective responses, although response times may be short. At the same time, the response rate to programmed cell death-1 (PD-1) treatment was more durable. It is speculated that HBV targeted therapy can synergistically enhance the antitumor activity with PD-1 blockade. To test this hypothesis, we evaluated the effect of crispr-cas9 on HBV and PD-1 in vitro and in vivo. We found that HBV targeting gRNA/cas9 induced a decrease in the expression of HBsAg, while the PD-1 gene could be knocked out by electroporation targeting gRNA / cas9 by polymerase chain reaction. In HBV transgenic mice, the immunophenotype and cytokine expression of human dendritic cells (DCS) were detected by crispr-cas9 system stimulation, flow cytometry and polymerase chain reaction. These results indicate that gRNA/cas9 treatment upregulates the expression of CD80, CD83 and CD86, and significantly increases the mRNA levels of IL-6, IL-12, IL-23 and tumor necrosis factor alpha. The combination of anti HBV and anti PD-1 therapy can inhibit HBV expression and significantly improve the survival of HBV transgenic mice. In addition, the combination therapy increased the production of interferon by T cells, and then enhanced the expression of Th1 related immunostimulatory genes, thereby reducing the transcription of regulatory / inhibitory immune genes. In general, this response can reshape the tumor microenvironment from immunosuppression to immune stimulation. Finally, anti HBV therapy can induce the expression of interferon dependent programmed cell death ligand-1 in HBV transgenic mice in vivo. To sum up, these results demonstrate that the combination of HBV targeted therapy and PD-1 immune checkpoint block has a strong synergistic effect, thus supporting the transformation potential of this combined therapy strategy in clinical treatment of HBV infection.

The Warburg effect, characterized by energy production through a high rate of aerobic glycolysis, is a metabolic hallmark of cancer cells. We previously found that ginsenoside 20(S)-Rg3 upregulated miR-603 and impaired the malignancy of ovarian cancer cells by inhibiting the Warburg effect. However, the precise functional role of miR-603 in ovarian cancer progression remains poorly defined. Here, we report that the level of miR-603 in ovarian cancer tissues is significantly lower than that in para-tumor tissues. Overexpression of miR-603 in ovarian cancer cells inhibits the Warburg effect as evidenced by a decrease in glucose consumption, lactate production and hexokinase-2 (HK2) expression, reduces cell proliferation in vitro, and weakens their migration and invasion. Further, miR-603 directly targets HK2 as indicated in a luciferase reporter assay. In contrast to agomiR-NC, agomiR-603 treatment significantly inhibits tumor growth in vivo and the Warburg effect, which is illustrated by a decreased uptake of 18F-FDG in subcutaneous xenografts and HK2 downregulation. Finally, miR-603 is negatively regulated by DNMT3A-mediated DNA methylation in the promoter region of its precursor gene, suggesting that 20(S)-Rg3 antagonizes DNMT3A-mediated DNA methylation to impair growth, migration and invasion of ovarian cancer cells. In conclusion, miR-603 is a tumor suppressor targeting HK2 in ovarian cancer and its low level may result from DNMT3A-mediated methylation.

Lateral flow assay (LFA) is user-friendly diagnostic tools but suffering limitations in poorer sensitivities and specificities especially for double-stranded DNA and single-base mutation quantification. Here, to improve the sensitivity and specificity for the LFA based nucleic acid detection, CRISPR-Cas12a mediated Surface enhanced Raman scattering (SERS) LFA was developed. By combination of ultra-sensitive SERS tags and target-specific signal amplification ability of CRISPR-Cas12a, HIV-1 dsDNA can be directly quantified with a LOD of 0.3 fM without any pre-amplification steps, which is almost 4 orders of magnitude lower than that of traditional colorimetric LFA methods. The whole detection process can be finished less than 1 h. Moreover, based on the target specificity of Cas12a, HIV-1 single-based drug resistance mutation (M184V) can be recognized as low as 0.01%. The HIV-1 dsDNA can also be successfully detected in serum samples with good comparable of that in buffer setting. Therefore, the simple and inexpensive paper-based CRISPR-SERS strip has great potential for point-of-care testing (POCT) of nucleic acid targets especially in resource-poor or non-laboratory environments.

Background: Current research focuses on reducing the level of HBV virus. We designed CRISPR/Cas9s that target the HBV genome. Methods: To prevent the emergence of escape mutant viruses, we mixed HBV-CRISPR/Cas9s (HBV-CRISPR/Cas9 complex). These HBV-CRISPR/Cas9s complexes were encapsulated in a nano-liposome complex, which is a hepatocyte-specific drug delivery system. CRISPR/Cas9 was used to assess delivery and knockout efficiencies of nano-liposome-gRNA-HBV complexes in the treatment of mice. The potency of the nano-liposome-gRNA-HBV complexes was evaluated in HepG2.2.15 cells and in HBV-Tg mice. Findings: Effective knockout of targets, efficient delivery of CRISPR/Cas9, and liver-specific delivery were observed with nano-liposome-gRNA-HBV complex treatment. Nano-liposome-gRNA-HBV complexes efficiently reduced HBsAg in vitro and in vivo. However, ETV treatment did not efficiently reduce HBsAg when compared with nano-liposome-gRNA-HBV complex treatment. In addition, the suppressive effects of nano-liposome-gRNA-HBV complexes persisted for 21 days in vitro and in vivo. We indicated that treatment with nano-liposome-gRNA-HBV complexes controlled HBV infection more efficiently than ETV treatment did. Furthermore, the effect of a single dose of nano-liposome-gRNA-HBV complexes persisted for a long time. Interpretation: The results demonstrate that nano-liposome-gRNA-HBV complex may be a promising novel HBV treatment that is more effective than reverse transcriptase inhibitors. Funding Statement: This study was supported by grants from the National Natural Science Foundation of China (Grant nos. 81602295 to Shuai Zhen). This study was supported by grants from Special Talent Training Program in Northwest Women and Children Hospital (2020ZD02) Declaration of Interests: The other authors declare that they have no competing interests. Ethics Approval Statement: All mouse experiments were conducted according to the National Institutes of Health Guidelines for Animal Care. All animal procedures were performed according to the guidelines developed by the China Council on Animal Care and the protocol was approved by the Xi’an jiaotong university.

Objective Carfilzomib is a second-generation tetrapeptide epoxyketone proteasome inhibitor used in current clinical therapy of hematologic malignancies. The mechanism of proteasome inhibition in endometrial cancer is not very clear. Carfilzomib inhibition of type I endometrial carcinoma cell proliferation by inducing cell cycle arrest at the G2/M phase was investigated in our study. Materials and methods HEC-1-A and Ishikawa endometrial carcinoma cell lines and three tumor cell lines were treated by different concentrations of carfilzomib. Methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability. Flow cytometry was used to analyze the cell cycle. Western blot was used to detect proteins involved in cell cycle progression. Results Carfilzomib impaired viability of myelogenous leukemia cell line K562, cervical cancer cell line HeLa, hepatocellular carcinoma cell line SMCC-7721, and endometrial carcinoma cell lines HEC-1-A and Ishikawa. The cell cycle was arrested at the G2/M phase in carfilzomib-treated HEC-1-A endometrial carcinoma cells, while it was arrested at both S and G2/M phases in carfilzomib-treated Ishikawa cells. Carfilzomib treatment significantly induced p21 Waf1/ Cip1 and p27, while substantially reduced cyclin D3 and cyclin-dependent kinase 1. Conclusion This study showed that carfilzomib inhibited endometrial cancer proliferation by upregulating cyclin-dependent kinase inhibitors p21 Waf1/Cip1 and p27 Kip1 , and reducing cyclin-dependent kinase 1 to arrest the cell cycle at the G2/M phase.

Deregulated expression of high-risk human papillomavirus oncogenes (E6 and E7) is a pivotal event for pathogenesis and progression in cervical cancer. Both viral oncogenes are therefore regarded as ideal therapeutic targets. In the hope of developing a gene-specific therapy for HPV-related cancer, we established CRISPR/Cas9 targeting promoter of HPV 16 E6/E7 and targeting E6, E7 transcript, transduced the CRISPR/Cas9 into cervical HPV-16-positive cell line SiHa. The results showed that CRISPR/Cas9 targeting promoter, as well as targeting E6 and E7 resulted in accumulation of p53 and p21 protein, and consequently remarkably reduced the abilities of proliferation of cervical cancer cells in vitro. Then we inoculated subcutaneously cells into nude mice to establish the transplanted tumor animal models, and found dramatically inhibited tumorigenesis and growth of mice incubated by cells with CRISPR/Cas9 targeting (promoter+E6+E7)-transcript. Our results may provide evidence for application of CRISPR/Cas9 targeting HR-HPV key oncogenes, as a new treatment strategy, in cervical and other HPV-associated cancer therapy.

A new type of Fe-C-Si-B alloy was developed. The investigation on microstructure details and the mechanical properties were performed for different austempering combinations. The results indicated that the Fe-C-Si-B alloy comprises ferrite, pearlite and interdendritic eutectic borides in as-cast condition. The distribution of eutectic boride with a chemical formula of M2B (M represents Cr, Fe, Mn or Mo) and is much like that of carbide in high chromium white cast iron. Pure ausferrite structure that is consisted of bainitic ferrite and carbon-riched retained austenite can be obtained by austempering treatment to the Fe-C-Si-B alloy. No carbide would precipitate in the structure and there is not any morphology change of borides. The hardness of the Fe-C-Si-B alloy decreases with increasing of the austempering temperature, and decreases greatly in at the early stages (within 5 to 10 min) of austempering transformation. The transformation is almost finished after about 5 to 10 min when thehardness of the alloy does not change anymore. The Fe-C-Si-B alloy is a new kind of wear resistance material with bright application prospect.