SARS-CoV-2 papain-like protease inhibits ISGylation of the viral nucleocapsid protein to evade host anti-viral immunity.

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes mild-to-severe respiratory symptoms, including acute respiratory distress. Despite remarkable efforts to investigate the virological and pathological impacts of SARS-CoV-2, many of the characteristics of SARS-CoV-2 infection still remain unknown. The interferon-inducible ubiquitin-like protein ISG15 is covalently conjugated to several viral proteins to suppress their functions. It was reported that SARS-CoV-2 utilizes its papain-like protease (PLpro) to impede ISG15 conjugation, ISGylation. However, the role of ISGylation in SARS-CoV-2 infection remains unclear. We aimed to elucidate the role of ISGylation in SARS-CoV-2 replication. We observed that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation in cultured cells. Site-directed mutagenesis reveals that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation, alongside conserved lysine residue in MERS-CoV (K372) and SARS-CoV (K375). We also observed that the nucleocapsid-ISGylation results in the disruption of nucleocapsid oligomerization, thereby inhibiting viral replication. Knockdown of ISG15 mRNA enhanced SARS-CoV-2 replication in the SARS-CoV-2 reporter replicon cells, while exogenous expression of ISGylation components partially hampered SARS-CoV-2 replication. Taken together, these results suggest that SARS-CoV-2 PLpro inhibits ISGylation of the nucleocapsid protein to promote viral replication by evading ISGylation-mediated disruption of the nucleocapsid oligomerization.IMPORTANCEISG15 is an interferon-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation in many viruses. However, the role of ISGylation in SARS-CoV-2 infection remains largely unclear. Here, we demonstrated that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation. We also found that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation. We obtained evidence suggesting that nucleocapsid-ISGylation results in the disruption of nucleocapsid-oligomerization, thereby suppressing SARS-CoV-2 replication. We discovered that SARS-CoV-2 papain-like protease inhibits ISG15 conjugation of nucleocapsid protein via its de-conjugating enzyme activity. The present study may contribute to gaining new insight into the roles of ISGylation-mediated anti-viral function in SARS-CoV-2 infection and may lead to the development of more potent and selective inhibitors targeted to SARS-CoV-2 nucleocapsid protein.

Phase II clinical trial of docetaxel and trastuzumab for HER2-positive advanced extramammary Paget's disease (EMPD-HER2DOC).

BACKGROUND: No consensus has been reached regarding the optimal chemotherapy for metastatic extramammary Paget's disease (EMPD), a rare cutaneous adenocarcinoma, because of the lack of solid evidence from prospective trials. However, the immunohistochemical profile of EMPD reportedly resembles that of breast cancer, particularly in terms of human epidermal growth factor receptor 2 (HER2) expression, suggesting that HER2 is a promising therapeutic target for advanced HER2-positive EMPD. METHODS: In this phase II single-arm trial, 13 Japanese patients received intravenous trastuzumab (loading dose of 8 mg/kg and maintenance dose of 6 mg/kg) and docetaxel (75 mg/m2) every 3 weeks for up to 2 years. The docetaxel dose was reduced or discontinued according to its toxicity. The primary trial endpoints were objective response rate (ORR) after 3 cycles of treatment and safety throughout the study period. RESULTS: All 13 patients completed 3 cycles of combination therapy. The median follow-up was 27.9 months. The ORR was 76.9% (n = 10/13; 90% CI, 50.5-93.4). Frequently observed adverse events were neutropenia (100%), hypoalbuminemia (84.6%), and mucocutaneous infection (84.6%), all of which were well tolerated. CONCLUSION: The combination of docetaxel and trastuzumab demonstrated a favorable clinical effect and acceptable tolerability, which makes it a good treatment option for HER2-positive metastatic EMPD (ClinicalTrials.gov Identifier: UMIN000021311, jRCTs031180073).

[Redo Mitral Valve Replacement for Prosthetic Valve Endocarditis in a Patient with End Stage Colon Cancer:Report of a Case].

Prosthetic valve endocarditis (PVE) is rare but devastating. A 69-year old man admitted for active endocarditis caused by Streptococcus pasteurianus. Antibiotic therapy was started, but the patient developed bowel obstruction owing to cancer with multiple liver metastases, and underwent transverse colectomy. Following colectomy, antibiotic agent was given continued for 4 weeks after and mitral valve replacement( MVR) using a bioprosthesis was performed. Oral antibiotic therapy was continued for six months after MVR to avoid infection recurrence. One year after MVR, the size of multiple liver metastases increased despite oral anticancer drugs administration. A totally implantable central venous access port( CV port) was placed and intravenous chemotherapy was started for progressive metastatic colorectal cancer. But the CV port was removed due to device infection caused by multiple drug resistant Staphyrococcus lugdunensis one month later, but the patient developed prosthetic valve endocarditits( PVE) due to the same bacterium, that caused valve stenosis. Redo MVR was indicated because of progressive dyspnea and uncontrollable fever. The patient was discharged one month after redo MVR, but suffered carcinomatous peritonitis, and eventually died eight months post-discharge. Chemotherapy needs caution because of potential risk of PVE in patients with prosthetic valves, especially for those with a history of infectious endocarditis.

Time to symptom onset and manual reduction outcomes as predictors of bowel viability in incarcerated obturator hernias.

The current study aimed to identify the indications for manual reduction in incarcerated obturator hernias (OH). Further, whether time to symptom onset and manual reduction outcomes can be predictors of bowel viability and the need for bowel resection in incarcerated OH were examined. This retrospective study included 26 patients with incarcerated OH who underwent surgery. All patients underwent manual reduction, and computed tomography scan after manual reduction confirmed hernia release. Multivariate analyses were performed to determine the predictors of bowel resection. The bowel resection group had a significantly longer average time to symptom onset than the nonbowel resection group (88 vs 36 h). Further, the bowel resection group was more likely to have failed manual reduction than the nonbowel resection group. A time to symptom onset of ≥ 72 h and failed manual reduction were significant predictors of bowel viability. Age, sex, hernia localization, American Society of Anesthesiologists physical status score, and laboratory findings did not differ significantly between the bowel resection and nonbowel resection groups. Time to symptom onset and manual reduction outcomes are significant predictors of bowel viability in incarcerated OH. Patients with a time to symptom onset of ≥ 72 h and failed manual reduction require surgical evaluation due to a high risk of bowel nonviability. Therefore, a cautious approach is required in the management of OH, and further research on optimized treatment protocols should be conducted.

Development of a Machine Learning-Based Model for Predicting the Incidence of Peripheral Intravenous Catheter-Associated Phlebitis.

Introduction: Early and accurate identification of high-risk patients with peripheral intravascular catheter (PIVC)-related phlebitis is vital to prevent medical device-related complications. Aim of the study: This study aimed to develop and validate a machine learning-based model for predicting the incidence of PIVC-related phlebitis in critically ill patients. Materials and methods: Four machine learning models were created using data from patients ≥ 18 years with a newly inserted PIVC during intensive care unit admission. Models were developed and validated using a 7:3 split. Random survival forest (RSF) was used to create predictive models for time-to-event outcomes. Logistic regression with least absolute reduction and selection operator (LASSO), random forest (RF), and gradient boosting decision tree were used to develop predictive models that treat outcome as a binary variable. Cox proportional hazards (COX) and logistic regression (LR) were used as comparators for time-to-event and binary outcomes, respectively. Results: The final cohort had 3429 PIVCs, which were divided into the development cohort (2400 PIVCs) and validation cohort (1029 PIVCs). The c-statistic (95% confidence interval) of the models in the validation cohort for discrimination were as follows: RSF, 0.689 (0.627-0.750); LASSO, 0.664 (0.610-0.717); RF, 0.699 (0.645-0.753); gradient boosting tree, 0.699 (0.647-0.750); COX, 0.516 (0.454-0.578); and LR, 0.633 (0.575-0.691). No significant difference was observed among the c-statistic of the four models for binary outcome. However, RSF had a higher c-statistic than COX. The important predictive factors in RSF included inserted site, catheter material, age, and nicardipine, whereas those in RF included catheter dwell duration, nicardipine, and age. Conclusions: The RSF model for the survival time analysis of phlebitis occurrence showed relatively high prediction performance compared with the COX model. No significant differences in prediction performance were observed among the models with phlebitis occurrence as the binary outcome.

Cellular Release of Infectious Hepatitis C Virus Particles via Endosomal Pathways.

Hepatitis C virus (HCV) is a positive-sense, single-stranded RNA virus that causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The release of infectious HCV particles from infected hepatocytes is a crucial step in viral dissemination and disease progression. While the exact mechanisms of HCV particle release remain poorly understood, emerging evidence suggests that HCV utilizes intracellular membrane trafficking and secretory pathways. These pathways include the Golgi secretory pathway and the endosomal trafficking pathways, such as the recycling endosome pathway and the endosomal sorting complex required for transport (ESCRT)-dependent multivesicular bodies (MVBs) pathway. This review provides an overview of recent advances in understanding the release of infectious HCV particles, with a particular focus on the involvement of the host cell factors that participate in HCV particle release. By summarizing the current knowledge in this area, this review aims to contribute to a better understanding of endosomal pathways involved in the extracellular release of HCV particles and the development of novel antiviral strategies.