Efficacy and safety of GST-HG171 in adult patients with mild to moderate COVID-19: a randomised, double-blind, placebo-controlled phase 2/3 trial.

Background: GST-HG171 is a potent, broad-spectrum, orally bioavailable small-molecule 3C like protease inhibitor that has demonstrated greater potency and efficacy compared to Nirmatrelvir in pre-clinical studies. We aimed to evaluate the efficacy and safety of orally administered GST-HG171 plus Ritonavir in patients with coronavirus disease 2019 (COVID-19) infected with emerging XBB and non-XBB variants. Methods: This randomised, double-blind, placebo-controlled phase 2/3 trial was conducted in 47 sites in China among adult patients with mild-to-moderate COVID-19 with symptoms onset ≤72 h. Eligible patients were randomised 1:1 to receive GST-HG171 (150 mg) plus Ritonavir (100 mg) or corresponding placebo tablets twice daily for 5 days, with stratification factors including the risk level of disease progression and vaccination status. The primary efficacy endpoint was time to sustained recovery of clinical symptoms within 28 days, defined as a score of 0 for 11 COVID-19-related target symptoms for 2 consecutive days, assessed in the modified intention-to-treat (mITT) population. This trial was registered at ClinicalTrials.gov (NCT05656443) and Chinese Clinical Trial Registry (ChiCTR2200067088). Findings: Between Dec 19, 2022, and May 4, 2023, 1525 patients were screened. Among 1246 patients who underwent randomisation, most completed basic (21.2%) or booster (74.9%) COVID-19 immunization, and most had a low risk of disease progression at baseline. 610 of 617 who received GST-HG171 plus Ritonavir and 603 of 610 who received placebo were included in the mITT population. Patients who received GST-HG171 plus Ritonavir showed shortened median time to sustained recovery of clinical symptoms compared to the placebo group (13.0 days [95.45% confidence interval 12.0-15.0] vs. 15.0 days [14.0-15.0], P = 0.031). Consistent results were observed in both SARS-CoV-2 XBB (45.7%, 481/1053 of mITT population) and non-XBB variants (54.3%, 572/1053 of mITT population) subgroups. Incidence of adverse events was similar in the GST-HG171 plus Ritonavir (320/617, 51.9%) and placebo group (298/610, 48.9%). The most common adverse events in both placebo and treatment groups were hypertriglyceridaemia (10.0% vs. 14.7%). No deaths occurred. Interpretation: Treatment with GST-HG171 plus Ritonavir has demonstrated benefits in symptom recovery and viral clearance among low-risk vaccinated adult patients with COVID-19, without apparent safety concerns. As most patients were treated within 2 days after symptom onset in our study, confirming the potential benefits of symptom recovery for patients with a longer duration between symptom onset and treatment initiation will require real-world studies. Funding: Fujian Akeylink Biotechnology Co., Ltd.

Transpancreatic Sphincterotomy After Double Guidewire Technique Was Noninferior to Primary Transpancreatic Sphincterotomy in Difficult Biliary Cannulation.

BACKGROUND: When unintentional pancreatic duct access occurs during difficult biliary cannulation, the double guidewire (DGW) or transpancreatic sphincterotomy (TPS) may be utilized. DGW can be easily switched to TPS due to the existing guidewire in the pancreatic duct. However, the efficacy of TPS after DGW, named sequential DGW-TPS technique, versus primary TPS has not been assessed. AIMS: Our aim was to compare the benefits and adverse events of sequential DGW-TPS technique and primary TPS. METHODS: We performed a comparative retrospective cohort study that enrolled a total of 117 patients with native papillae. The patients were divided into one of 2 groups according to the primary bile duct access technique (sequential DGW-TPS or primary TPS), both with pancreatic stenting. RESULTS: Between November 2017 and May 2023, a total of 84 patients were grouped into sequential DGW-TPS and 33 into primary TPS. The overall post-ERCP pancreatitis (PEP) rate was 4.3% in the entire cohort, with no statistical differences were observed between the groups in terms of PEP rates (P = 0.927), PEP severity (P = 1.000), first biliary cannulation success (P = 0.621), overall cannulation success (P = 1.000), hyperamylasemia incidence (P = 0.241), elevated amylase levels (P = 0.881), and postoperative hospital stay (P = 0.185). Furthermore, these results remained consistent in multivariable regression analysis. CONCLUSIONS: The sequential DGW-TPS technique showed a comparable safety and biliary cannulation success rate to primary TPS in difficult biliary cannulation. Given the potential long-term complications associated with TPS, DGW should be first if inadvertent pancreatic access occurs, with TPS serving as second only if DGW fails.

Preclinical evaluation of the SARS-CoV-2 Mpro inhibitor RAY1216 shows improved pharmacokinetics compared with nirmatrelvir.

Although vaccines are available for SARS-CoV-2, antiviral drugs such as nirmatrelvir are still needed, particularly for individuals in whom vaccines are less effective, such as the immunocompromised, to prevent severe COVID-19. Here we report an α-ketoamide-based peptidomimetic inhibitor of the SARS-CoV-2 main protease (Mpro), designated RAY1216. Enzyme inhibition kinetic analysis shows that RAY1216 has an inhibition constant of 8.4 nM and suggests that it dissociates about 12 times slower from Mpro compared with nirmatrelvir. The crystal structure of the SARS-CoV-2 Mpro:RAY1216 complex shows that RAY1216 covalently binds to the catalytic Cys145 through the α-ketoamide group. In vitro and using human ACE2 transgenic mouse models, RAY1216 shows antiviral activities against SARS-CoV-2 variants comparable to those of nirmatrelvir. It also shows improved pharmacokinetics in mice and rats, suggesting that RAY1216 could be used without ritonavir, which is co-administered with nirmatrelvir. RAY1216 has been approved as a single-component drug named 'leritrelvir' for COVID-19 treatment in China.

Recent advances in CRISPR-Cas9-based genome insertion technologies.

Programmable genome insertion (or knock-in) is vital for both fundamental and translational research. The continuously expanding number of CRISPR-based genome insertion strategies demonstrates the ongoing development in this field. Common methods for site-specific genome insertion rely on cellular double-strand breaks repair pathways, such as homology-directed repair, non-homologous end-joining, and microhomology-mediated end joining. Recent advancements have further expanded the toolbox of programmable genome insertion techniques, including prime editing, integrase coupled with programmable nuclease, and CRISPR-associated transposon. These tools possess their own capabilities and limitations, promoting tremendous efforts to enhance editing efficiency, broaden targeting scope and improve editing specificity. In this review, we first summarize recent advances in programmable genome insertion techniques. We then elaborate on the cons and pros of each technique to assist researchers in making informed choices when using these tools. Finally, we identify opportunities for future improvements and applications in basic research and therapeutics.

Clinical prediction models for cervical lymph node metastasis of papillary thyroid carcinoma.

PURPOSE: Accurate preoperative diagnosis of lymph node metastasis (LNM) in papillary thyroid carcinoma (PTC) remains an unsolved problem. This study aimed to construct a nomogram and scoring system for predicting LNM based on the clinical characteristics of patients with PTC. METHODS: 1400 patients with PTC who underwent thyroidectomy and lymph node dissection at the First Affiliated Hospital of Sun Yat-sen University were retrospectively enrolled and randomly divided into training and internal testing sets. Furthermore, 692 patients with PTC from three other medical centers were collected as external testing sets. Least absolute shrinkage and selection operator (LASSO) was used to screen the predictors, and a nomogram was constructed. In addition, a scoring system was constructed using 10-fold cross-validation. The performances of the two models were verified among datasets and compared with preoperative ultrasound (US). RESULTS: Six independent predictors were included in the multivariate logistic model: age, sex, US diagnosis of LNM, tumor diameter, location, and thyroid peroxidase antibody level. The areas under the receiver operating characteristic curve (AUROC) (95% confidence interval) of this nomogram in the training, internal testing, and three external testing sets were 0.816 (0.791-0.840), 0.782 (0.727-0.837), 0.759 (0.699-0.819), 0.749 (0.667-0.831), and 0.777 (0.726-0.828), respectively. The AUROC of the scoring system were 0.810 (0.785-0.835), 0.772 (0.718-0.826), 0.736 (0.675-0.798), 0.717 (0.635-0.799) and 0.756 (0.704-0.808), respectively. The prediction performances were both significantly superior to those of preoperative US (P < 0.001). CONCLUSION: The nomogram and scoring system performed well in different datasets and significantly improved the preoperative prediction of LNM than US alone.

[An evaluation of carrier detection for Spinal muscular atrophy using digital PCR assay].

OBJECTIVE: To assess the effectiveness and feasibility of carrier detection for Spinal muscular atrophy (SMA) by using digital PCR assay. METHODS: Peripheral blood samples were collected from 214 pregnant women who were routinely screened for SMA carriers, of which 204 were randomly selected samples and 10 were samples with known copy numbers of SMN1 exons 7 and 8. Samples with known copy numbers of SMN1 exons 7 and 8 were randomly mixed into the experiment to validate the performance of the digital PCR assay. RESULTS: The copy numbers of SMN1 exons 7 and 8 and SMN2 exons 7 and 8 in peripheral blood samples were detected by digital PCR assay. The results of SMN1 exons 7 and 8 were compared with those of the quantitative PCR method to assess the reliability and clinical performance of the digital PCR assay. Among the 204 random samples, digital PCR has detected five samples with simultaneous heterozygous deletion of SMN1 exons 7 and 8, three samples with heterozygous deletion of SMN1 exon 8 only, and 196 samples with no deletion of SMN1 exons 7 and 8. Ten samples with known SMN1 exons 7 and 8 copy numbers were detected with the expected values. The digital PCR test results were fully consistent with that of the quantitative PCR. CONCLUSION: The results of digital PCR for the detection of copy number variation of SMN1 exons 7 and 8 were consistent with qPCR. Digital PCR assay was able to clearly distinguish the copy number of the target genes, therefore can be used for SMA carrier screening. Moreover, it can also detect copy number of SMN2 exons 7 and 8, which can provide more information for genetic counseling.

Structure-based discovery of dual pathway inhibitors for SARS-CoV-2 entry.

Since 2019, SARS-CoV-2 has evolved rapidly and gained resistance to multiple therapeutics targeting the virus. Development of host-directed antivirals offers broad-spectrum intervention against different variants of concern. Host proteases, TMPRSS2 and CTSL/CTSB cleave the SARS-CoV-2 spike to play a crucial role in the two alternative pathways of viral entry and are characterized as promising pharmacological targets. Here, we identify compounds that show potent inhibition of these proteases and determine their complex structures with their respective targets. Furthermore, we show that applying inhibitors simultaneously that block both entry pathways has a synergistic antiviral effect. Notably, we devise a bispecific compound, 212-148, exhibiting the dual-inhibition ability of both TMPRSS2 and CTSL/CTSB, and demonstrate antiviral activity against various SARS-CoV-2 variants with different viral entry profiles. Our findings offer an alternative approach for the discovery of SARS-CoV-2 antivirals, as well as application for broad-spectrum treatment of viral pathogenic infections with similar entry pathways.

Energy-economy-environment assessments of refrigerants R152a and R134a in a vapor compression refrigeration system using a variable displacement oil-free linear compressor.

In light of the adverse environmental impact of the R134a refrigerant, replacing it with a more environmentally friendly refrigerant has become imperative than ever. This study presents an experimental investigation into the utilization of R152a and R134a refrigerants in a vapor compression refrigeration system employing a variable displacement oil-free linear compressor. The potential for the replacement of R134a with R152a was examined based on energy, environmental, and economic performance analyses. The outcomes indicated that R152a exhibited a higher coefficient of performance (COP) in comparison to R134a under identical operating conditions. Specifically, when the pressure ratio was 2.0 and the piston stroke was 11 mm, R152a's COP was 13.0% higher than R134a. It was also discovered that reducing the operating stroke and increasing the pressure ratio could effectively lower CO2 emissions and total costs. Under the 2.0 pressure ratio and 9-mm piston stroke, R134a produced 1082.4 kg more CO2 emissions than R152a, representing a 209% increase. In addition, the R152a and R134a total cost was reduced by 8.3% with the 2.5 pressure ratio and 11-mm piston stroke. Notably, the results of the current study demonstrated that R152a outperformed R134a in energy consumption, environmental friendliness, and economy in oil-free linear compressor refrigeration systems. R152a used less electric power, generated fewer CO2 emissions, and naturally reduced predicted running costs in order to maintain the same COP.

RNA-binding motif protein 24 inhibits HBV replication in vivo.

Despite the extensive use of effective vaccines and antiviral drugs, chronic hepatitis B virus (HBV) infection continues to pose a serious threat to global public health. Therapies with novel mechanisms of action against HBV are being explored for achieving a functional cure. In this study, five murine models of HBV replication were used to investigate the inhibitory effect of RNA binding motif protein 24 (RBM24) on HBV replication. The findings revealed that RBM24 serves as a host restriction factor and suppresses HBV replication in vivo. The transient overexpression of RBM24 in hydrodynamics-based mouse models of HBV replication driven by the CMV or HBV promoters suppressed HBV replication. Additionally, the ectopic expression of RBM24 decreased viral accumulation and the levels of HBV covalently closed circular DNA (cccDNA) in an rcccDNA mouse model. The liver-directed transduction of adeno-associated viruses (AAV)-RBM24 mediated the stable hepatic expression of RBM24 in pAAV-HBV1.2 and HBV/tg mouse models, and markedly reduced the levels of HBV cccDNA and other viral indicators. Altogether, these findings revealed that RBM24 inhibits the replication of HBV in vivo, and RBM24 may be a potential therapeutic target for combating HBV infections.

A new vaccination regimen using adenovirus-vectored vaccine confers effective protection against African swine fever virus in swine.

African swine fever (ASF) is an acute and highly contagious lethal infectious disease in swine that severely threatens the global pig industry. At present, a safe and efficacious vaccine is urgently required to prevent and control the disease. In this study, we evaluated the safety and immunogenicity of replication-incompetent type-2 adenoviruses carrying African swine fever virus (ASFV) antigens, namely CP204L (p30), E183L (p54), EP402R (CD2v), B646L (p72), and B602L (p72 chaperone). A vaccine cocktail delivered by simultaneous intramuscular (IM) and intranasal (IN) administration robustly elicited both systemic and mucosal immune responses against AFSV in mice and swine and provided highly effective protection against the circulating ASFV strain in farmed pigs. This multi-antigen cocktail vaccine was well tolerated in the vaccinated animals. No significant interference among antigens was observed. The combined IM and IN vaccination using this adenovirus-vectored antigen cocktail vaccine warrants further evaluation for providing safe and effective protection against ASFV infection and transmission.

Discovery and characterization of potent pan-variant SARS-CoV-2 neutralizing antibodies from individuals with Omicron breakthrough infection.

The SARS-CoV-2 Omicron variant evades most currently approved neutralizing antibodies (nAbs) and caused drastic decrease of plasma neutralizing activity elicited by vaccination or prior infection, urging the need for the development of pan-variant antivirals. Breakthrough infection induces a hybrid immunological response with potentially broad, potent and durable protection against variants, therefore, convalescent plasma from breakthrough infection may provide a broadened repertoire for identifying elite nAbs. We performed single-cell RNA sequencing (scRNA-seq) and BCR sequencing (scBCR-seq) of B cells from BA.1 breakthrough-infected patients who received 2 or 3 previous doses of inactivated vaccine. Elite nAbs, mainly derived from the IGHV2-5 and IGHV3-66/53 germlines, showed potent neutralizing activity across Wuhan-Hu-1, Delta, Omicron sublineages BA.1 and BA.2 at picomolar NT50 values. Cryo-EM analysis revealed diverse modes of spike recognition and guides the design of cocktail therapy. A single injection of paired antibodies cocktail provided potent protection in the K18-hACE2 transgenic female mouse model of SARS-CoV-2 infection.

Development of a novel virus-like particle-based vaccine for preventing tick-borne encephalitis virus infection.

Tick-borne encephalitis virus (TBEV) is an important tick-borne pathogen that poses as a serious public health concern. The coverage and immunogenicity of the currently available vaccines against TBEV are relatively low; therefore, it is crucial to develop novel and effective vaccines against TBEV. The present study describes a novel strategy for the assembly of virus-like particles (VLPs) by co-expressing the structural (core/prM/E) and non-structural (NS2B/NS3Pro) proteins of TBEV. The efficacy of the VLPs was subsequently evaluated in C57BL/6 mice, and the resultant IgG serum could neutralize both Far-Eastern and European subtypes of TBEV. These findings indicated that the VLP-based vaccine elicited the production of cross-subtype reactive antibodies. The VLPs provided protection to mice lacking the type I interferon receptor (IFNAR-/-) against lethal TBEV challenge, with undetectable viral load in brain and intestinal tissues. Furthermore, the group that received the VLP vaccine did not exhibit significant pathological changes and the inflammatory factors were significantly suppressed compared to the control group. Immunization with the VLP vaccine induced the production of multiple-cytokine-producing antiviral CD4+ T cells in vivo, including TNF-α+, IL-2+, and IFN-γ+ T cells. Altogether, the findings suggest that noninfectious VLPs can serve as a potentially safe and effective vaccine candidate against diverse subtypes of TBEV.

Mitochondrial Glycerol-3-Phosphate Dehydrogenase Restricts HBV Replication via the TRIM28-Mediated Degradation of HBx.

Hepatitis B virus (HBV) infection affects hepatic metabolism. Serum metabolomics studies have suggested that HBV possibly hijacks the glycerol-3-phosphate (G3P) shuttle. In this study, the two glycerol-3-phosphate dehydrogenases (GPD1 and GPD2) in the G3P shuttle were analyzed for determining their role in HBV replication and the findings revealed that GPD2 and not GPD1 inhibited HBV replication. The knockdown of GPD2 expression upregulated HBV replication, while GPD2 overexpression reduced HBV replication. Moreover, the overexpression of GPD2 significantly reduced HBV replication in hydrodynamic injection-based mouse models. Mechanistically, this inhibitory effect is related to the GPD2-mediated degradation of HBx protein by recruiting the E3 ubiquitin ligase TRIM28 and not to the alterations in G3P metabolism. In conclusion, this study revealed GPD2, a key enzyme in the G3P shuttle, as a host restriction factor in HBV replication. IMPORTANCE The glycerol-3-phosphate (G3P) shuttle is important for the delivery of cytosolic reducing equivalents into mitochondria for oxidative phosphorylation. The study analyzed two key components of the G3P shuttle and identified GPD2 as a restriction factor in HBV replication. The findings revealed a novel mechanism of GPD2-mediated inhibition of HBV replication via the recruitment of TRIM28 for degrading HBx, and the HBx-GPD2 interaction could be another potential therapeutic target for anti-HBV drug development.

Somatically hypermutated antibodies isolated from SARS-CoV-2 Delta infected patients cross-neutralize heterologous variants.

SARS-CoV-2 Omicron variants feature highly mutated spike proteins with extraordinary abilities in evading antibodies isolated earlier in the pandemic. Investigation of memory B cells from patients primarily with breakthrough infections with the Delta variant enables isolation of a number of neutralizing antibodies cross-reactive to heterologous variants of concern (VOCs) including Omicron variants (BA.1-BA.4). Structural studies identify altered complementarity determining region (CDR) amino acids and highly unusual heavy chain CDR2 insertions respectively in two representative cross-neutralizing antibodies-YB9-258 and YB13-292. These features are putatively introduced by somatic hypermutation and they are heavily involved in epitope recognition to broaden neutralization breadth. Previously, insertions/deletions were rarely reported for antiviral antibodies except for those induced by HIV-1 chronic infections. These data provide molecular mechanisms for cross-neutralization of heterologous SARS-CoV-2 variants by antibodies isolated from Delta variant infected patients with implications for future vaccination strategy.

Sterile 20-like kinase 3 promotes tick-borne encephalitis virus assembly by interacting with NS2A and prM and enhancing the NS2A-NS4A association.

Tick-borne encephalitis virus (TBEV) is the causative agent of a potentially fatal neurological infection in humans. Investigating virus-host interaction is important for understanding the pathogenesis of TBEV and developing effective antiviral drugs against this virus. Here, we report that mammalian ste20-like kinase 3 (MST3) is involved in the regulation of TBEV infection. The knockdown or knockout of MST3, but not other mammalian ste20-like kinase family members, inhibited TBEV replication. The knockdown of MST3 also significantly reduced TBEV replication in mouse primary astrocytes. Life cycle analysis indicated that MST3 remarkably impaired virion assembly efficiency and specific infectivity by respectively 59% and 95% in MST3-knockout cells. We further found that MST3 interacts with the viral proteins NS2A and prM; and MST3 enhances the interaction of NS2A-NS4A. Thus, MST3-NS2A complex plays a major role in recruiting prM-E heterodimers and NS4A and mediates the virion assembly. Additionally, we found that MST3 was biotinylated and combined with other proteins (e.g., ATG5, Sec24A, and SNX4) that are associated with the cellular membrane required for TBEV infection. Overall, our study revealed a novel function for MST3 in TBEV infection and identified as a novel host factor supporting TBEV assembly.

Parallelism error analysis and its effect on modulation depth based on a rotating parallel mirror Fourier spectrometer.

The stability of the moving mirror of a Michelson Fourier transform spectrometer (M-FTS) has a non-negligible influence on its spectral quality, which limits its application. We proposed a spectrometer scheme with a pair of rotating parallel mirrors (RPM-FTS), which has advantages of fast response and high stability. The influence of the parallelism error of parallel mirrors on interference was analyzed by establishing a rotation vector model between the parallelism error, rotation angle, and optical path. The modulation depth of the RPM-FTS is more insensitive with the same installation error of the M-FTS; thus, more spectral details can be displayed easily.

Potential threat of human pathogenic orthopoxviruses to public health and control strategies.

Orthopoxviruses (OPXVs) belong to a group of nucleo-cytoplasmic large DNA viruses. Human pathogenic OPXVs (hpOPXVs) include at least five viruses, among which smallpox virus and monkeypox virus are the most dangerous viral pathogens. Both viruses are classified as category-one human infectious pathogens in China. Although smallpox was globally eradicated in the 1980 s, it is still a top biosecurity threat owing to the possibility of either being leaked to the outside world from a laboratory or being weaponized by terrorists. Beginning in early May 2022, a sudden outbreak of monkeypox was concurrently reported in more than 100 disparate geographical areas, representing a public health emergency of international concern, as declared by the World Health Organization (WHO). In this review, we present the reasons for hpOPXVs such as monkeypox virus presenting a potential threat to public health. We then systematically review the historical and recent development of vaccines and drugs against smallpox and monkeypox. In the final section, we highlight the importance of viromics studies as an integral part of a forward defense strategy to eliminate the potential threat to public health from emerging or re-emerging hpOPXVs and their variants.

Limited disassembly of cytoplasmic hepatitis B virus nucleocapsids restricts viral infection in murine hepatic cells.

BACKGROUND AND AIMS: Murine hepatic cells cannot support hepatitis B virus (HBV) infection even with supplemental expression of viral receptor, human sodium taurocholate cotransporting polypeptide (hNTCP). However, the specific restricted step remains elusive. In this study, we aimed to dissect HBV infection process in murine hepatic cells. APPROACH AND RESULTS: Cells expressing hNTCP were inoculated with HBV or hepatitis delta virus (HDV). HBV pregenomic RNA (pgRNA), covalently closed circular DNA (cccDNA), and different relaxed circular DNA (rcDNA) intermediates were produced in vitro . The repair process from rcDNA to cccDNA was assayed by in vitro repair experiments and in mouse with hydrodynamic injection. Southern blotting and in situ hybridization were used to detect HBV DNA. HBV, but not its satellite virus HDV, was restricted from productive infection in murine hepatic cells expressing hNTCP. Transfection of HBV pgRNA could establish HBV replication in human, but not in murine, hepatic cells. HBV replication-competent plasmid, cccDNA, and recombinant cccDNA could support HBV transcription in murine hepatic cells. Different rcDNA intermediates could be repaired to form cccDNA both in vitro and in vivo . In addition, rcDNA could be detected in the nucleus of murine hepatic cells, but cccDNA could not be formed. Interestingly, nuclease sensitivity assay showed that the protein-linked rcDNA isolated from cytoplasm was completely nuclease resistant in murine, but not in human, hepatic cells. CONCLUSIONS: Our results imply that the disassembly of cytoplasmic HBV nucleocapsids is restricted in murine hepatic cells. Overcoming this limitation may help to establish an HBV infection mouse model.