Virological characterization of the 2022 outbreak-causing monkeypox virus using human keratinocytes and colon organoids.

The outbreak-causing monkeypox virus of 2022 (2022 MPXV) is classified as a clade IIb strain and phylogenetically distinct from prior endemic MPXV strains (clades I or IIa), suggesting that its virological properties may also differ. Here, we used human keratinocytes and induced pluripotent stem cell-derived colon organoids to examine the efficiency of viral growth in these cells and the MPXV infection-mediated host responses. MPXV replication was much more productive in keratinocytes than in colon organoids. We observed that MPXV infections, regardless of strain, caused cellular dysfunction and mitochondrial damage in keratinocytes. Notably, a significant increase in the expression of hypoxia-related genes was observed specifically in 2022 MPXV-infected keratinocytes. Our comparison of virological features between 2022 MPXV and prior endemic MPXV strains revealed signaling pathways potentially involved with the cellular damages caused by MPXV infections and highlights host vulnerabilities that could be utilized as protective therapeutic strategies against human mpox in the future.

Evaluation of Broad Anti-Coronavirus Activity of Autophagy-Related Compounds Using Human Airway Organoids.

To deal with the broad spectrum of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that threaten human health, it is essential to not only drugs develop that target viral proteins but also consider drugs that target host proteins/cellular processes to protect them from being hijacked for viral infection and replication. To this end, it has been reported that autophagy is deeply involved in coronavirus infection. In this study, we used airway organoids to screen a chemical library of autophagic modulators to identify compounds that could potentially be used to fight against infections by a broad range of coronaviruses. Among the 80 autophagy-related compounds tested, cycloheximide and thapsigargin reduced SARS-CoV-2 infection efficiency in a dose-dependent manner. Cycloheximide treatment reduced the infection efficiency of not only six SARS-CoV-2 variants but also human coronavirus (HCoV)-229E and HCoV-OC43. Cycloheximide treatment also reversed viral infection-induced innate immune responses. However, even low-dose (1 µM) cycloheximide treatment altered the expression profile of ribosomal RNAs; thus, side effects such as inhibition of protein synthesis in host cells must be considered. These results suggest that cycloheximide has broad-spectrum anti-coronavirus activity in vitro and warrants further investigation.

Association between advanced care planning and emergency department visits: A systematic review.

BACKGROUND: Advance care planning can help provide optimal medical care according to a patient's wishes as a part of patient-centered discussions on end-of-life care. This can prevent undesired transfers to emergency departments. However, the effects of advance care planning on emergency department visits and ambulance calls in various settings or specific conditions remain unclear. AIM: To evaluate whether advanced care planning affected the frequency of emergency department visits and ambulance calls. DESIGN: Systematic review. This study was registered in PROSPERO (CRD42022340109). We assessed risk of bias using RoB 2.0, ROBINS-I, and ROBINS-E. DATA SOURCES: We searched the PubMed, Cochrane CENTRAL, and EMBASE databases from their inception until September 22, 2022 for studies comparing patients with and without advanced care planning and reported the frequency of emergency department visits and ambulance calls as outcomes. RESULTS: Eight studies were included. Regarding settings, two studies on patients in nursing homes showed that advanced care planning significantly reduced the frequency of emergency department visits and ambulance calls. However, two studies involving several medical care facilities reported inconclusive results. Regarding patient disease, a study on patients with depression or dementia showed that advanced care planning significantly reduced emergency department visits; in contrast, two studies on patients with severe respiratory diseases and serious illnesses showed no significant reduction. Seven studies showed a high risk of bias. CONCLUSIONS: Advanced care planning may lead to reduced emergency department visits and ambulance calls among nursing home residents and patients with depression or dementia. Further research is warranted to identify the effectiveness of advanced care planning in specific settings and diseases.

Association between physicians' characteristics and their knowledge, attitudes, and practices regarding advance care planning: a cross-sectional study.

BACKGROUND: Despite physicians' vital role in advance care planning, a limited number of physicians practice it. This study assessed factors associated with physicians' knowledge, attitudes, and practices regarding advance care planning. METHODS: This cross-sectional study used data from an anonymous survey conducted by the Japanese Ministry of Health, Labour and Welfare. Questionnaires were mailed to 4500 physicians in November and December 2022. Data from 1260 respondents were analyzed. RESULTS: Of the respondents, 46.4%, 77.0%, and 82.0% reported good knowledge of advance care planning, agreed with promoting it, and with its provision by medical/care staff, respectively. Male physicians were significantly less likely to support advance care planning (odds ratio: 0.54, 95% confidence interval: 0.35-0.84) or agree to its provision by medical/care staff (odds ratio: 0.47, 95% confidence interval: 0.29-0.78) but significantly more likely to practice it (odds ratio: 1.58, 95% confidence interval: 1.05-2.36). Physicians specialized in surgery or internal/general/palliative medicine were more knowledgeable about advance care planning and more likely to practice it. Physicians working in clinics were significantly less knowledgeable (odds ratio: 0.33, 95% confidence interval: 0.25-0.44) about advance care planning and less likely to support it (odds ratio: 0.37, 95% confidence interval: 0.27-0.50), agree with its provision by medical/care staff (odds ratio: 0.54, 95% confidence interval: 0.39-0.75), or to practice it (odds ratio: 0.16, 95% confidence interval: 0.12-0.22). CONCLUSIONS: Physicians working in clinics had less knowledge of advance care planning, less supportive attitudes, and less likely to practice it. Knowledge, attitudes and practice also varied by gender and specialty. Interventions should target physicians working in clinics.

Root canal sealers affect artifacts on cone-beam computed tomography images.

The purpose of this study was to evaluate the appearance of artifacts by four types of root canal filling sealers on cone-beam computed tomography (CBCT) images. Thirty standardized tooth models were given the radiopacity equivalent to human teeth, and root canal preparation was performed using WaveOne Gold. Root canal filling by a single-point method was performed using WaveOne Gold gutta-percha points and four types of root canal sealers: AH Plus (AH), CANALS (CA), BioRoot RCS (BR), and MTA Fillapex (MTA). Samples were taken by periapical radiography at 60 kV and scanned by CBCT at three tube voltages (70, 85, and 100 kV). The gray-scale values (GVs) of the periapical radiographs were measured and the aluminum equivalents were calculated. On the CBCT axial images, the artifact and dentin area GVs were measured and the rate of change in the GV (RCGV) was calculated as follows: RCGV (%) = (dentin area GV - artifact GV)/dentin area GV × 100. High-density areas with artifacts on the CBCT images were also measured. On the periapical radiographs, the aluminum equivalent was largest for AH and smallest for MTA. On the CBCT images, AH showed the largest values for both RCGV and the high-density areas, while BR and MTA showed comparable values. Correlations were found between the radiopacity on the periapical radiographs and the degree of artifacts on the CBCT images. These findings suggest that the greater the contrast in the 2D image, the higher the artifacts in the 3D image.

Situation, Education, Innovation, and Recommendation: A Large-Scale Systematic Review of Advance Care Planning in the Age of COVID-19.

The COVID-19 pandemic highlighted the need for advance care planning (ACP) as a way to help mitigate the various care concerns that accompanied the healthcare crisis. However, unique obstacles to typical ACP practice necessitated the need for guidance and innovation to help facilitate these vital conversations. The aim of this systematic review was to identify the various ACP barriers and facilitators that arose during the pandemic and determine how ACP practice was affected across different contexts and among different populations. This systematic review (PROSPERO registration number: CRD42022359092), which adheres to the PRISMA guidelines for reporting systematic reviews, examined studies on ACP in the context of the COVID-19 pandemic. The review involved searches of five databases, including MEDLINE and Embase. Of the 843 identified studies, 115 met the inclusion criteria. The extracted ACP barriers and facilitators were codified and quantified. The most frequently occurring ACP barrier codes were: Social distancing measures and visitation restrictions, Uncertainty surrounding the COVID-19 prognosis, and Technological/Telehealth barriers. The most frequently occurring ACP facilitator codes were the following: Telehealth/virtual ACP platforms, Training for clinicians, and Care team collaboration. Identifying the ACP barriers and facilitators is essential for developing effective, resilient ACP promotion strategies and improving its delivery, accessibility, and acceptability.

Barriers to and facilitators of advance care planning implementation for medical staff after the COVID-19 pandemic: an overview of reviews.

OBJECTIVE: The COVID-19 pandemic has impacted the capacity for advance care planning (ACP) among patients, families and healthcare teams. We sought to identify and review the barriers to and facilitators of ACP implementation for medical staff in different settings (eg, hospitals, outpatient palliative care, nursing and care homes) during the pandemic. DESIGN: This study employed an overview of reviews design. We searched the MEDLINE, CENTRAL, Web of Science and Embase databases for studies published between 8 December 2019 and 30 July 2023. We used AMSTAR 2 to assess the risk of bias. RESULTS: We included seven reviews. Common barriers to ACP implementation included visitation restrictions, limited resources and personnel and a lack of coordination among healthcare professionals. In care and nursing homes, barriers included a dearth of palliative care physicians and the psychological burden on facility staff. Using telemedicine for information sharing was a common facilitator across settings. In hospitals, facilitators included short-term training in palliative care and palliative care physicians joining the acute care team. In care and nursing homes, facilitators included ACP education and emotional support for staff. CONCLUSIONS: Visitation restrictions and limited resources during the pandemic posed obstacles; however, the implementation of ACP was further hindered by insufficient staff education on ACP in hospitals and facilities, as well as a scarcity of information sharing at the community level. These pre-existing issues were magnified by the pandemic, drawing attention to their significance. Short-term staff training programmes and immediate information sharing could better enable ACP. PROSPERO REGISTRATION NUMBER: CRD42022351362.

Elucidation of the liver pathophysiology of COVID-19 patients using liver-on-a-chips.

SARS-CoV-2 induces severe organ damage not only in the lung but also in the liver, heart, kidney, and intestine. It is known that COVID-19 severity correlates with liver dysfunction, but few studies have investigated the liver pathophysiology in COVID-19 patients. Here, we elucidated liver pathophysiology in COVID-19 patients using organs-on-a-chip technology and clinical analyses. First, we developed liver-on-a-chip (LoC) which recapitulating hepatic functions around the intrahepatic bile duct and blood vessel. We found that hepatic dysfunctions, but not hepatobiliary diseases, were strongly induced by SARS-CoV-2 infection. Next, we evaluated the therapeutic effects of COVID-19 drugs to inhibit viral replication and recover hepatic dysfunctions, and found that the combination of anti-viral and immunosuppressive drugs (Remdesivir and Baricitinib) is effective to treat hepatic dysfunctions caused by SARS-CoV-2 infection. Finally, we analyzed the sera obtained from COVID-19 patients, and revealed that COVID-19 patients, who were positive for serum viral RNA, are likely to become severe and develop hepatic dysfunctions, as compared with COVID-19 patients who were negative for serum viral RNA. We succeeded in modeling the liver pathophysiology of COVID-19 patients using LoC technology and clinical samples.

Machine learning models predicting undertriage in telephone triage.

BACKGROUND: Undertriaged patients have worse outcomes than appropriately triaged patients. Machine learning provides better triage prediction than conventional triage in emergency departments, but no machine learning-based undertriage prediction models have yet been developed for prehospital telephone triage. We developed and validated machine learning models for telephone triage. MATERIALS AND METHODS: We conducted a retrospective cohort study with the largest after-hour house-call (AHHC) service dataset in Japan. Participants were ≥16 years and used the AHHC service between 1 November 2018 and 31 January 2021. We developed five prediction models based on support vector machine (SVM), lasso regression (LR), random forest (RF), gradient-boosted decision tree (XGB), and deep neural network (DNN). The primary outcome was undertriage, and predictors were telephone triage level and routinely available telephone-based data, including age, sex, 80 chief complaint categories and 10 comorbidities. We measured the area under the receiver operating characteristic curve (AUROC) for all the models. RESULTS: We identified 15,442 eligible patients (age: 38.4 ± 16.6, male: 57.2%), including 298 (1.9%; age: 58.2 ± 23.9, male: 55.0%) undertriaged patients. RF and XGB outperformed the other models, with the AUROC values (95% confidence interval; 95% CI) of the SVM, LR, RF, XGB and DNN for undertriage being 0.62 (0.55-0.69), 0.79 (0.74-0.83), 0.81 (0.76-0.86), 0.80 (0.75-0.84) and 0.77 (0.73-0.82), respectively. CONCLUSIONS: We found that RF and XGB outperformed other models. Our findings suggest that machine learning models can facilitate the early detection of undertriage and early intervention to yield substantially improved patient outcomes.KEY MESSAGESUndertriaged patients experience worse outcomes than appropriately triaged patients; thus, we developed machine learning models for predicting undertriage in the prehospital setting. In addition, we identified the predictors of risk factors associated with undertriage.Random forest and gradient-boosted decision tree models demonstrated better prediction performance, and the models identified the risk factors associated with undertriage.Machine learning models aid in the early detection of undertriage, leading to significantly improved patient outcomes and identifying undertriage-associated risk factors, including chief complaint categories, could help prioritize conventional telephone triage protocol revision.

Comparison of the Characteristics and Outcomes of COVID-19 Patients Treated by a Hospital-at-Home Service in Japan during the Alpha and Delta Waves.

Coronavirus infections occurred in repeated waves caused by different variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with the number of patients increasing during each wave. A private after-hours house-call (AHHC) service provides hospital-at-home (HaH) services to patients in Japan requiring oxygen when hospital beds are in short supply. This retrospective study aimed to compare the characteristics of COVID-19 patients treated by the AHHC service during the COVID-19 waves caused by the Alpha (March−June 2021) and Delta (July−December 2021) SARS-CoV-2 variants. All patients with COVID-19 treated by the AHHC service from March to December 2021 while awaiting hospitalization were included. The data were collected from medical records and follow-up telephone interviews. The AHHC service treated 55 and 273 COVID-19 patients during the Alpha and Delta waves, respectively. The patients treated during the Delta wave were significantly younger than those treated during the Alpha wave (median: 63 years and 47 years, respectively; p < 0.001). Disease severity did not differ significantly between the two waves, but the crude case-fatality rate was significantly higher during the Alpha wave (10/55, 18.2%) than during the Delta wave (4/273, 1.4%; p < 0.001). The patient characteristics and outcomes differed between the Alpha and Delta waves.

Exocyst complex component 2 is a potential host factor for SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused an epidemic and spread rapidly all over the world. Because the analysis of host factors other than receptors and proteases has not been sufficiently performed, we attempted to identify and characterize host factors essential for SARS-CoV-2 infection using iPS cells and airway organoids (AO). Based on previous CRISPR screening and RNA-seq data, we found that exocyst complex component 2 (EXOC2) is one important host factor for SARS-CoV-2 infection. The intracellular SARS-CoV-2 nucleocapsid (N) expression level was decreased to 3.7% and the virus copy number in cell culture medium was decreased to 1.6% by EXOC2 knockdown. Consistently, immunostaining results showed that N protein-positive cells were significantly decreased by EXOC2 knockdown. We also found that EXOC2 knockdown downregulates SARS-CoV-2 infection by regulating IFNW1 expression. In conclusion, controlling the EXOC2 expression level may prevent SARS-CoV-2 infection and deserves further study.

Cell response analysis in SARS-CoV-2 infected bronchial organoids.

The development of an in vitro cell model that can be used to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research is expected. Here we conducted infection experiments in bronchial organoids (BO) and an BO-derived air-liquid interface model (BO-ALI) using 8 SARS-CoV-2 variants. The infection efficiency in BO-ALI was more than 1,000 times higher than that in BO. Among the bronchial epithelial cells, we found that ciliated cells were infected with the virus, but basal cells were not. Ciliated cells died 7 days after the viral infection, but basal cells survived after the viral infection and differentiated into ciliated cells. Fibroblast growth factor 10 signaling was essential for this differentiation. These results indicate that BO and BO-ALI may be used not only to evaluate the cell response to SARS-CoV-2 and coronavirus disease 2019 (COVID-19) therapeutic agents, but also for airway regeneration studies.

Modeling SARS-CoV-2 infection and its individual differences with ACE2-expressing human iPS cells.

Genetic differences are a primary reason for differences in the susceptibility and severity of COVID-19. As induced pluripotent stem (iPS) cells maintain the genetic information of the donor, they can be used to model individual differences in SARS-CoV-2 infection in vitro. We found that human iPS cells expressing the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) (ACE2-iPS cells) can be infected w SARS-CoV-2. In infected ACE2-iPS cells, the expression of SARS-CoV-2 nucleocapsid protein, budding of viral particles, and production of progeny virus, double membrane spherules, and double-membrane vesicles were confirmed. We performed SARS-CoV-2 infection experiments on ACE2-iPS/ embryonic stem (ES) cells from eight individuals. Male iPS/ES cells were more capable of producing the virus compared with female iPS/ES cells. These findings suggest that ACE2-iPS cells can not only reproduce individual differences in SARS-CoV-2 infection in vitro but also are a useful resource to clarify the causes of individual differences in COVID-19 due to genetic differences.

Dual inhibition of TMPRSS2 and Cathepsin Bprevents SARS-CoV-2 infection in iPS cells.

It has been reported that many receptors and proteases are required for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Although angiotensin-converting enzyme 2 (ACE2) is the most important of these receptors, little is known about the contribution of other genes. In this study, we examined the roles of neuropilin-1, basigin, transmembrane serine proteases (TMPRSSs), and cathepsins (CTSs) in SARS-CoV-2 infection using the CRISPR interference system and ACE2-expressing human induced pluripotent stem (iPS) cells. Double knockdown of TMPRSS2 and cathepsin B (CTSB) reduced the viral load to 0.036% ± 0.021%. Consistently, the combination of the CTPB inhibitor CA-074 methyl ester and the TMPRSS2 inhibitor camostat reduced the viral load to 0.0078% ± 0.0057%. This result was confirmed using four SARS-CoV-2 variants (B.1.3, B.1.1.7, B.1.351, and B.1.1.248). The simultaneous use of these two drugs reduced viral load to less than 0.01% in both female and male iPS cells. These findings suggest that compounds targeting TMPRSS2 and CTSB exhibit highly efficient antiviral effects independent of gender and SARS-CoV-2 variant.

Usability of Polydimethylsiloxane-Based Microfluidic Devices in Pharmaceutical Research Using Human Hepatocytes.

A liver-on-a-chip (liver-chip) is a microfluidic device carrying liver cells such as human hepatocytes. It is used to reproduce a part of liver function. Many microfluidic devices are composed of polydimethylsiloxane (PDMS), which is a type of silicone elastomer. PDMS is easy to process and suitable for cell observation, but its high hydrophobicity carries the risk of drug absorption. In this study, we evaluated drug absorption to the PDMS device and investigated the drug responsiveness of human hepatocytes cultured in the PDMS device (hepatocyte-chips). First, the absorption rates of 12 compounds to the PDMS device were measured. The absorption rates of midazolam, bufuralol, cyclosporine A, and verapamil were 92.9, 71.7, 71.4, and 99.6%, respectively, but the other compounds were poorly absorbed. Importantly, the absorption rate of the compounds was correlated with their octanol/water distribution coefficient (log D) values (R2 = 0.76). Next, hepatocyte-chips were used to examine the response to drugs, which are typically used to evaluate hepatic functions. Using the hepatocyte-chips, we could confirm the responsiveness of drugs including cytochrome P450 (CYP) inducers and farnesoid X receptor (FXR) ligands. We believe that our findings will contribute to drug discovery research using PDMS-based liver-chips.

Fatal acute hypernatremia resulting from a massive intake of seasoning soy sauce.

BACKGROUND: Hypernatremia due to salt poisoning is clinically rare and standard care procedures have not been established. We report a case of salt poisoning due to massive intake of seasoning soy sauce. CASE PRESENTATION: A 40-year-old woman presented to the emergency department with seizures and remarkable hypernatremia with a serum sodium concentration of 183 mEq/L. The initial brain computed tomography scan showed brain shrinkage, which could occur during the acute phase of hypernatremia. We reduced her serum sodium level rapidly, rather than at the recommended slow rate. On day 3, the patient's brain computed tomography scan showed widespread low-density areas and edema. The patient died 8 days after admission. CONCLUSION: After reviewing instances of resuscitation following salt intoxication, aggressive rapid correction of serum sodium concentration should only be considered in acute phases of hypernatremia within a few hours from ingestion, and 2-3 h could be one of the criteria.

Acute mitral valve regurgitation causing severe alveolar hemorrhage.

BACKGROUND: Acute mitral regurgitation could occur without common symptoms like hemodynamic instability, but with dyspnea, hemoptysis, and right-sided infiltration on radiography. We report a case of severe alveolar hemorrhage caused by acute mitral regurgitation, which occurred in the absence of shock. CASE PRESENTATION: A 40-year-old man presented with dyspnea with bloody phlegm and hypoxia, despite being hemodynamically stable. Chest radiography revealed right-sided infiltration, and bronchoscopy showed fresh bloody phlegm in his tracheae. No specific findings were detected with any tests. After treatment with several medications and support with extracorporeal membrane oxygenation, his condition improved, although the etiology of the disease remained unknown. Transthoracic and transesophageal echocardiogram revealed severe mitral valve regurgitation with ruptured mitral chordae tendineae. These suggested that the sudden onset of mitral valve regurgitation had caused severe alveolar hemorrhage. CONCLUSION: Severe alveolar hemorrhage, especially with right-sided infiltration on chest radiography, should be considered a symptom of acute mitral regurgitation.

Comparison of commercially available media for hepatic differentiation and hepatocyte maintenance.

Human hepatocytes are essential materials in pharmaceutical researches. Not only primary human hepatocytes (PHH) but also human iPS cell-derived hepatocyte-like cells (human iPS-HLCs) are expected to be applied as materials for pharmaceutical researches. To date, several culture media have been developed for culturing human hepatocytes. However, there have been no reports comparing these media to determine which is most suitable for culturing human hepatocytes. In this study, we compared five commercial media (Hepatocyte Culture Medium (HCM), HepatoZYME-SFM, Cellartis Power Primary HEP Medium, DMEM/F12, and William's E Medium (WEM)) to determine which is most suitable for culturing PHH and human iPS-HLCs. In hepatic differentiation of human iPS cells (day 14-25 of differentiation), albumin (ALB) and urea secretion abilities and CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM or WEM. During maintenance of human iPS-HLCs, ALB and urea producing abilities and CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM. Importantly, we found that human iPS-HLCs cultured in HCM were maintained for 3 weeks or more without impairment of their hepatic functions. These results suggest that it is necessary to select an optimal medium for hepatic differentiation and maintenance of human iPS-HLCs. In the case of PHH culture, there was little difference in hepatic functions among the five media. However, the CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM and WEM. In conclusion, it is important to select the optimal medium for specific application when carrying out pharmaceutical researches using human hepatocytes.

Photoactivatable oncolytic adenovirus for optogenetic cancer therapy.

Virotherapy using oncolytic adenovirus is an effective anticancer strategy. However, the tumor selectivity of oncolytic adenoviruses is not enough high. To develop oncolytic adenovirus with a low risk of off-tumor toxicity, we constructed a photoactivatable oncolytic adenovirus (paOAd). In response to blue light irradiation, the expression of adenoviral E1 genes, which are necessary for adenoviral replication, is induced and replication of this adenovirus occurs. In vitro, efficient lysis of various human cancer cell lines was observed by paOAd infection followed by blue light irradiation. Importantly, there was no off-tumor toxicity unless the cells were irradiated by blue light. In vivo, tumor growth in a subcutaneous tumor model and a mouse model of liver cancer was significantly inhibited by paOAd infection followed by blue light irradiation. In addition, paOAd also showed a therapeutic effect on cancer stem cells. These results suggest that paOAd is useful as a safe and therapeutically effective cancer therapy.