Overexpression of wild-type HRAS drives non-alcoholic steatohepatitis to hepatocellular carcinoma in mice.

Hepatocellular carcinoma (HCC), a prevalent solid carcinoma of significant concern, is an aggressive and often fatal disease with increasing global incidence rates and poor therapeutic outcomes. The etiology and pathological progression of non-alcoholic steatohepatitis (NASH)-related HCC is multifactorial and multistage. However, no single animal model can accurately mimic the full NASH-related HCC pathological progression, posing considerable challenges to transition and mechanistic studies. Herein, a novel conditional inducible wild-type human HRAS overexpressed mouse model (HRAS-HCC) was established, demonstrating 100% morbidity and mortality within approximately one month under normal dietary and lifestyle conditions. Advanced symptoms of HCC such as ascites, thrombus, internal hemorrhage, jaundice, and lung metastasis were successfully replicated in mice. In-depth pathological features of NASH- related HCC were demonstrated by pathological staining, biochemical analyses, and typical marker gene detections. Combined murine anti-PD-1 and sorafenib treatment effectively prolonged mouse survival, further confirming the accuracy and reliability of the model. Based on protein-protein interaction (PPI) network and RNA sequencing analyses, we speculated that overexpression of HRAS may initiate the THBS1-COL4A3 axis to induce NASH with severe fibrosis, with subsequent progression to HCC. Collectively, our study successfully duplicated natural sequential progression in a single murine model over a very short period, providing an accurate and reliable preclinical tool for therapeutic evaluations targeting the NASH to HCC continuum.

Characterization of a pangolin SARS-CoV-2-related virus isolate that uses the human ACE2 receptor.

Various SARS-CoV-2-related coronaviruses have been increasingly identified in pangolins, showing a potential threat to humans. Here we report the infectivity and pathogenicity of the SARS-CoV-2-related virus, PCoV-GX/P2V, which was isolated from a Malayan pangolin (Manis javanica). PCoV-GX/P2V could grow in human hepatoma, colorectal adenocarcinoma cells, and human primary nasal epithelial cells. It replicated more efficiently in cells expressing human angiotensin-converting enzyme 2 (hACE2) as SARS-CoV-2 did. After intranasal inoculation to the hACE2-transgenic mice, PCoV-GX/P2V not only replicated in nasal turbinate and lungs, but also caused interstitial pneumonia, characterized by infiltration of mixed inflammatory cells and multifocal alveolar hemorrhage. Existing population immunity established by SARS-CoV-2 infection and vaccination may not protect people from PCoV-GX/P2V infection. These findings further verify the hACE2 utility of PCoV-GX/P2V by in vivo experiments using authentic viruses and highlight the importance for intensive surveillance to prevent possible cross-species transmission.

Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development.

To discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.

Induction of balanced immunity in BALB/c mice by vaccination with a recombinant fusion protein containing a respiratory syncytial virus G protein fragment and a CTL epitope.

Respiratory syncytial virus (RSV), an important pathogen of the lower respiratory tract, is responsible for severe illness both in new born and young children and in elderly people. However, development of a RSV vaccine has been hampered by the outcome of the infant trials in the 1960s with a formalin-inactivated RSV (FI-RSV) preparation. Previous studies in mice indicated that G protein immunization resulted in antibody and Th2-type response and failed to induce MHC I-restricted CD8(+) T-cell response. Vaccines designed to induce CD8(+) T-cell along with antibody response might be ideal. In the present report, a fusion protein G1F/M2 containing a RSV-G protein fragment (G: 125-225 amino acid) and a CD8(+) T-cell epitope from RSV-M2 protein was investigated. G1F/M2 was cloned, expressed in E. coli, purified and renaturated. In BALB/c mice, G1F/M2 induced not only humoral immunity but also cellular immunity. In addition, interestedly, G1F/M2 elicited balanced IgG1/IgG2a response. These results suggest that the fusion protein G1F/M2 is potential as a RSV subunit vaccine.

[Sequence variation of the chloroplast gene ndh D region in cytoplasmic male sterile sorghum].

A fragment SAAU-02(700) was amplified specifically from total DNA of seven sorghun varieties with male-fertile cytoplasm (N-cytoplasm). PCR assays indicated that it was amplified from chloroplast (cp) DNA. Sequence analysis revealed this newly cloned fragment contained a portion of chloroplast gene psa C (88 bp) and part of ndh D gene (192 bp). Total DNA, mitochondrial (mt) DNA, and cpDNA were digested with EcoR I + Hind III and probed with fragment SAAU-02(700). The Southern hybridization patterns displayed a 0.74 kb band both in total DNA and cpDNA, but an additional faint band 0.45 kb in size was found only in the latter. No polymorphic hybridization signal between the N-cytoplasm and male-sterile cytoplasm (S-cytoplasm) was observed. Southern hybridization of total DNA of CMS line A1 Tx623 and fertile line Tx623 digested with Hae III gave a band 4.9 kb in size in the former and a 4.45 kb band in the latter. This revealed that the sequence of ndh D from CMS line was likely altered. Further studies designed to determine whether or not the variation has some effect on the metabolism of mitochondria and chloroplast, even on the occurrence of male sterility in sorghum are underway.