Evolution of the coronavirus spike protein in the full-length genome and defective viral genome under diverse selection pressures.

How coronaviruses evolve by altering the structures of their full-length genome and defective viral genome (DVG) under dynamic selection pressures has not been studied. In this study, we aimed to experimentally identify the dynamic evolutionary patterns of the S protein sequence in the full-length genome and DVG under diverse selection pressures, including persistence, innate immunity and antiviral drugs. The evolutionary features of the S protein sequence in the full-length genome and in the DVG under diverse selection pressures are as follows: (i) the number of nucleotide (nt) mutations does not necessarily increase with the number of selection pressures; (ii) certain types of selection pressure(s) can lead to specific nt mutations; (iii) the mutated nt sequence can be reverted to the wild-type nt sequence under the certain type of selection pressure(s); (iv) the DVG can also undergo mutations and evolve independently of the full-length genome; and (v) DVG species are regulated during evolution under diverse selection pressures. The various evolutionary patterns of the S protein sequence in the full-length genome and DVG identified in this study may contribute to coronaviral fitness under diverse selection pressures.

Associations of Chinese Herbal Medicine Use with the Risks of Acute Exacerbation and Mortality in Patients with Chronic Obstructive Pulmonary Disease: A Nationwide Retrospective Cohort Study.

Objectives: This study aimed to assess the correlation of exacerbation and the mortality rate in patients with chronic obstructive pulmonary disease (COPD) between biomedical treatments with or without Chinese herbal medicine (CHM) as an adjunct. Design: A total of 81,261 COPD patients were identified from the National Health Insurance Research Database in Taiwan between 2001 and 2012. After screening and matching, 3176 COPD patients were included in the study. Statistical analyses were performed to assess the differences in the baseline characteristics. The authors used the Cox proportional hazard regression analysis to calculate the risks of mortality and hospitalization due to acute exacerbation of COPD within 1 year between a CHM user cohort and non-CHM user cohort. The cumulative incidence of mortality in COPD patients with or without CHM treatment was calculated by the Kaplan-Meier method. Results: COPD patients in the CHM user cohort demonstrated a significantly lower risk of mortality (p < 0.001) and acute exacerbation (p < 0.05), compared with the non-CHM user cohort. In addition, the CHM users exhibited a reduced cumulative incidence of mortality compared with the non-CHM user cohort (p < 0.001). Xiao Qing Long Tang and Fritillariae thunbergii were the most common Chinese herbal formula and single Chinese herb prescribed for COPD patients. Conclusion: Combining CHM with biomedical treatment might reduce the risk of acute exacerbation and incidence of mortality in patients with COPD.

Benefits of Metformin Combined with Pemetrexed-Based Platinum Doublets as a First-Line Therapy for Advanced Lung Adenocarcinoma Patients with Diabetes.

Lung cancer remains a challenge in daily practice. Chemotherapy is first considered for advanced lung adenocarcinoma bearing no active driver mutations. Maintaining drug efficacy and overcoming drug resistance are essential. This study aimed to explore the real-world use of anti-diabetic agent metformin in combination with pemetrexed-based platinum doublets in a first-line setting. We retrospectively collected data during 2004~2013 from TaiwaN's National Health Insurance Research Database to access the survival benefit of metformin combined with pemetrexed-based platinum doublets as a first-line therapy for diabetic patients with advanced lung adenocarcinoma. Demographic data and information regarding platinum reagents, diabetes medications, and metformin doses were gathered, and overall survival status regarding metformin use was analyzed. Overall survival status based on the daily dose and the calculated cumulative defined daily dose (DDD) of metformin prescribed during the first 3 months after lung cancer was diagnosed was also assessed. A total of 495 patients were enrolled with a mean age of 67 years old, and the majority of the patients were male. After adjusting for age, sex, diabetes medication, and platinum reagents used, the adjusted hazard ratio (HR) for the metformin-user group was 0.61 (95% confidence interval (CI); 0.46~0.79; p < 0.001). The metformin-user group had a survival benefit (log-rank p < 0.001). We analyzed metformin dosing during the first 3 months after lung cancer diagnosis, and for a daily dose ≥ 1500 mg, the adjusted hazard ratio (aHR) was 0.42 (95% CI; 0.27~0.65; p < 0.001). Regarding the cumulative DDD of metformin, a DDD equal to or exceeding 21 resulted in aHR of 0.48 (95% CI; 0.34~0.69; p < 0.001). In this study, we found that the combination of metformin and pemetrexed-based platinum doublets provides a robust survival benefit as a first-line therapy for diabetic patients with advanced lung adenocarcinoma. It is worth conducting a large and randomized clinical trial to further investigate the antitumor effects of metformin on advanced lung adenocarcinoma when used as a first-ling therapy, including in non-diabetic patients.

Additive Antiproliferative and Antiangiogenic Effects of Metformin and Pemetrexed in a Non-Small-Cell Lung Cancer Xenograft Model.

Lung cancer is heterogeneous and challenging to cope with once it has progressed. Chemotherapy is the first step once no active driver mutation has been discovered. Non-antitumor drugs have been found to be beneficial when used as adjuvants to chemotherapy. In this study, the additive effect and mechanism of metformin combined with pemetrexed in non-small-cell lung cancer (NSCLC) cells were elucidated. Three NSCLC cell lines, A549, H1975, and HCC827, were used to analyze tumor cell proliferation, colony formation and the cell cycle in vitro when exposed to metformin alone, pemetrexed alone or their combination. We found that combination treatment in three cell lines exerted antiproliferative effects through cell cycle arrest in the S phase. An ex vivo chicken chorioallantoic membrane (CAM) assay was used to examine the antiangiogenic effect of metformin combined with pemetrexed on vascular structure formation. We further created an A549 orthotopic xenograft model with an in vivo imaging system (IVIS) and explored the associated indicators involved in the tumorigenic process. The in vitro results showed that the combination of metformin and pemetrexed exhibited an antiproliferative effect in reducing cell viability and colony formation, the downregulation of cyclin D1 and A2 and the upregulation of CDKN1B, which are involved in the G1/S phase. For antiangiogenic effects, the combination therapy inhibited the vascular structure, as proven by the CAM assay. We elucidated that combination therapy could target VEGFA and Endoglin by RT-qPCR, ELISA and histopathological findings in an A549 orthotopic NSCLC xenograft model. Our research demonstrated the additive antiproliferative and antiangiogenic effects of the combination of metformin with pemetrexed in NSCLC and could be applied to clinical lung cancer therapy.

Explainable Machine Learning to Predict Successful Weaning Among Patients Requiring Prolonged Mechanical Ventilation: A Retrospective Cohort Study in Central Taiwan.

Objective: The number of patients requiring prolonged mechanical ventilation (PMV) is increasing worldwide, but the weaning outcome prediction model in these patients is still lacking. We hence aimed to develop an explainable machine learning (ML) model to predict successful weaning in patients requiring PMV using a real-world dataset. Methods: This retrospective study used the electronic medical records of patients admitted to a 12-bed respiratory care center in central Taiwan between 2013 and 2018. We used three ML models, namely, extreme gradient boosting (XGBoost), random forest (RF), and logistic regression (LR), to establish the prediction model. We further illustrated the feature importance categorized by clinical domains and provided visualized interpretation by using SHapley Additive exPlanations (SHAP) as well as local interpretable model-agnostic explanations (LIME). Results: The dataset contained data of 963 patients requiring PMV, and 56.0% (539/963) of them were successfully weaned from mechanical ventilation. The XGBoost model (area under the curve [AUC]: 0.908; 95% confidence interval [CI] 0.864-0.943) and RF model (AUC: 0.888; 95% CI 0.844-0.934) outperformed the LR model (AUC: 0.762; 95% CI 0.687-0.830) in predicting successful weaning in patients requiring PMV. To give the physician an intuitive understanding of the model, we stratified the feature importance by clinical domains. The cumulative feature importance in the ventilation domain, fluid domain, physiology domain, and laboratory data domain was 0.310, 0.201, 0.265, and 0.182, respectively. We further used the SHAP plot and partial dependence plot to illustrate associations between features and the weaning outcome at the feature level. Moreover, we used LIME plots to illustrate the prediction model at the individual level. Additionally, we addressed the weekly performance of the three ML models and found that the accuracy of XGBoost/RF was ~0.7 between weeks 4 and week 7 and slightly declined to 0.6 on weeks 8 and 9. Conclusion: We used an ML approach, mainly XGBoost, SHAP plot, and LIME plot to establish an explainable weaning prediction ML model in patients requiring PMV. We believe these approaches should largely mitigate the concern of the black-box issue of artificial intelligence, and future studies are warranted for the landing of the proposed model.

The Long-Term Effectiveness of Omalizumab in Adult Patients with Severe Allergic Asthma: Continuous Treatment Versus Boosting Treatment.

The implications of boosting Omalizumab treatment (OT) in patients with severe allergic asthma (SAA) remain unclear. The study aimed to explore and compare the 12-month effectiveness between continuous, at least 10-month OT (continuation group) and four-month boost of Omalizumab (boost group) in adult patients with SAA. In this retrospective cohort study, clinical data were collected for further analysis. Of all participants (n = 124), a significant reduction in annual exacerbations (baseline = 0.8 ± 1.5, follow-up = 0.5 ± 1.0, p = 0.047 *) and improvement in small airway ventilation as evaluated by forced expiratory flow at 25-75% (baseline = 55.1 ± 11.1%, follow-up = 59.4 ± 8.4%, p < 0.001 *) were found in the continuation group (n = 110). By contrast, the boost group (n = 14) had significantly increased annual exacerbations (baseline = 0.7 ± 1.4, follow-up = 2.9 ± 3.6, p = 0.031 *) and impaired small airway function (baseline = 55.3 ± 12.9, follow-up = 52.1 ± 12.5, p = 0.026 *). Furthermore, the continuation group rather than the boost group had significant decreases in the frequency of oral corticosteroid (OCS) use as controllers (baseline = 32.7%, follow-up = 20.0%, p = 0.047 *; baseline = 50.0%, follow-up = 21.4%, p = 0.237, respectively) and OCS maintenance dose (mg/month) (baseline = 85.9 ± 180.8, follow-up = 45.8 ± 106.6, p = 0.020 *; baseline = 171.4 ± 221.5, follow-up = 50.0 ± 104.3, p = 0.064, respectively), and increases in asthma control test scores (baseline = 16.0 ± 3.0, follow-up = 19.8 ± 4.4, p < 0.001 *; baseline = 14.6 ± 3.8, follow-up = 19.7 ± 4.7, p = 0.050, respectively). Continuous OT would be beneficial for adult patients with SAA, while boost of Omalizumab would worsen their long-term outcomes.

Predictors of a Minimal Clinically Important Difference Following Omalizumab Treatment in Adult Patients With Severe Allergic Asthma.

Several factors have been found to be predictors of a good response following omalizumab treatment in patients with severe allergic asthma (SAA). However, it remains unclear whether clinical characteristics can predict a minimal clinically important difference (MCID) following omalizumab treatment in this population. Therefore, the aim of this study was to investigate the features associated with an MCID following omalizumab treatment in adult patients with SAA. Of the 124 participants enrolled in this retrospective, cross-sectional study, 94, 103, 20 and 53 achieved the MCID following treatment with omalizumab and were considered to be responders of exacerbation reduction (no exacerbation during the 1-year follow-up period or ≧50% reduction in exacerbations from baseline), oral corticosteroid (OCS) sparing (no use of OCS to control asthma during the study period or a reduction of the monthly OCS maintenance dose to <50% of baseline), lung function (an increase of ≧230 ml in the forced expiratory volume in 1 s from baseline) and asthma control (an increase of ≧3 points in the asthma control test score from baseline), respectively. Normal weight [<25 vs. ≧30 kg/m2, odds ratio (OR) = 3.86, p = 0.024] was predictive of a responder of reduction in exacerbations following omalizumab treatment while subjects with a blood eosinophil level of <300 cells/µL (<300 vs. ≧300 cells/µL, OR = 5.81, p = 0.001) were more likely to exhibit an MCID in OCS sparing. No factor was found to be a predictor of lung function or asthma control. When choosing treatment for adult patients with SAA, our findings may help to select those who may benefit the most from omalizumab treatment.

Shared prenatal impacts among childhood asthma, allergic rhinitis and atopic dermatitis: a population-based study.

BACKGROUND: Increasing prevalence of childhood allergic diseases including asthma is a global health concern, and we aimed to investigate prenatal risk factors for childhood asthma and to address the potential shared prenatal impacts among childhood asthma, allergic rhinitis (AR) and atopic dermatitis (AD). METHODS: We used two claim databases, including Taiwan Birth Cohort Study (TBCS) and National Health Insurance Research Database (NHIRD), to identify independent paired mother-child data (mother-child dyads) between 2006 and 2009. The association between prenatal factors and asthma was determined by calculating adjusted odds ratio (aOR) with 95% confidence interval (CI) using conditional logistic regression analysis. RESULTS: A total of 628,878 mother-child dyads were included, and 43,915 (6.98%) of children developed asthma prior to age 6. We found that male gender (aOR 1.50, 95% CI 1.47-1.53), maternal asthma (aOR 1.80, 95% CI 1.71-1.89), maternal AR (aOR 1.33, 95% CI 1.30-1.37), preterm birth (aOR 1.32, 95% CI 1.27-1.37), low birth weight (aOR 1.14, 95% CI 1.10-1.19) and cesarean section (aOR 1.10, 95% CI 1.08-1.13) were independent predictors for childhood asthma. A high urbanization level and a low number of older siblings were associated with asthma in a dose-response manner. Notably, we identified that the association between maternal asthma and childhood asthma (aOR 1.80, 95% CI 1.71-1.89) was stronger compared with those between maternal asthma and childhood AR (aOR 1.67, 95% CI 1.50-1.87) as well as childhood AD (aOR 1.31, 95% CI 1.22-1.40). Similarly, the association between maternal AR and childhood AR (aOR 1.62, 95% CI 1.53-1.72) was higher than those between maternal AR and childhood asthma (aOR 1.33, 95% CI 1.30-1.37) as well as childhood AD (aOR 1.35, 95% CI 1.31-1.40). Furthermore, the number of maternal allergic diseases was associated with the three childhood allergic diseases in a dose-response manner. CONCLUSIONS: In conclusion, this population-based study provided evidence of prenatal impacts on childhood asthma and demonstrated the shared maternal impacts among childhood asthma, AR, and AD. These findings highlight the shared prenatal impacts among allergic diseases, and studies are warranted to address the pivotal pathway in allergic diseases.

Kefir peptides alleviate particulate matter <4 µm (PM4.0)-induced pulmonary inflammation by inhibiting the NF-κB pathway using luciferase transgenic mice.

Kefir peptides, generated by kefir grain fermentation of milk proteins, showed positive antioxidant effects, lowered blood pressure and modulated the immune response. In this study, kefir peptide was evaluated regarding their anti-inflammatory effects on particulate matter <4 µm (PM4.0)-induced lung inflammation in NF-κB-luciferase+/+ transgenic mice. The lungs of mice under 20 mg/kg or 10 mg/kg PM4.0 treatments, both increased significantly the generation of reactive oxygen species (ROS) and inflammatory cytokines; increased the protein expression levels of p-NF-κB, NLRP3, caspase-1, IL-1ß, TNF-α, IL-6, IL-4 and α-SMA. Thus, we choose the 10 mg/kg of PM4.0 for animal trials; the mice were assigned to four treatment groups, including control group (saline treatment), PM4.0 + Mock group (only PM4.0 administration), PM4.0 + KL group (PM4.0 + 150 mg/kg low-dose kefir peptide) and PM4.0 + KH group (PM4.0 + 500 mg/kg high-dose kefir peptide). Data showed that treatment with both doses of kefir peptides decreased the PM4.0-induced inflammatory cell infiltration and the expression of the inflammatory mediators IL-lß, IL-4 and TNF-α in lung tissue by inactivating NF-κB signaling. The oral administrations of kefir peptides decrease the PM4.0-induced lung inflammation process through the inhibition of NF-κB pathway in transgenic luciferase mice, proposing a new clinical application to particulate matter air pollution-induced pulmonary inflammation.

Using Dual Fluorescence Reporting Genes to Establish an In Vivo Imaging Model of Orthotopic Lung Adenocarcinoma in Mice.

PURPOSE: Lung adenocarcinoma is characterized by a poor prognosis and high mortality worldwide. In this study, we purposed to use the live imaging techniques and a reporter gene that generates highly penetrative near-infrared (NIR) fluorescence to establish a preclinical animal model that allows in vivo monitoring of lung cancer development and provides a non-invasive tool for the research on lung cancer pathogenesis and therapeutic efficacy. PROCEDURES: A human lung adenocarcinoma cell line (A549), which stably expressed the dual fluorescence reporting gene (pCAG-iRFP-2A-Venus), was used to generate subcutaneous or orthotopic lung cancer in nude mice. Cancer development was evaluated by live imaging via the NIR fluorescent signals from iRFP, and the signals were verified ex vivo by the green fluorescence of Venus from the gross lung. The tumor-bearing mice received miR-16 nucleic acid therapy by intranasal administration to demonstrate therapeutic efficacy in this live imaging system. RESULTS: For the subcutaneous xenografts, the detection of iRFP fluorescent signals revealed delicate changes occurring during tumor growth that are not distinguishable by conventional methods of tumor measurement. For the orthotopic xenografts, the positive correlation between the in vivo iRFP signal from mice chests and the ex vivo green fluorescent signal from gross lung tumors and the results of the suppressed tumorigenesis by miR-16 treatment indicated that lung tumor size can be accurately quantified by the emission of NIR fluorescence. In addition, orthotopic lung tumor localization can be accurately visualized using iRFP fluorescence tomography in vivo, thus revealing the trafficking of lung tumor cells. CONCLUSIONS: We introduced a novel dual fluorescence lung cancer model that provides a non-invasive option for preclinical research via the use of NIR fluorescence in live imaging of lung.

Lung tumorigenesis induced by human vascular endothelial growth factor (hVEGF)-A165 overexpression in transgenic mice and amelioration of tumor formation by miR-16.

Many studies have shown that vascular endothelial growth factor (VEGF), especially the human VEGF-A165 (hVEGF-A165) isoform, is a key proangiogenic factor that is overexpressed in lung cancer. We generated transgenic mice that overexpresses hVEGF-A165 in lung-specific Clara cells to investigate the development of pulmonary adenocarcinoma. In this study, three transgenic mouse strains were produced by pronuclear microinjection, and Southern blot analysis indicated similar patterns of the foreign gene within the genomes of the transgenic founder mice and their offspring. Accordingly, hVegf-A165 mRNA was expressed specifically in the lung tissue of the transgenic mice. Histopathological examination of the lung tissues of the transgenic mice showed that hVEGF-A165 overexpression induced bronchial inflammation, fibrosis, cysts, and adenoma. Pathological section and magnetic resonance imaging (MRI) analyses demonstrated a positive correlation between the development of pulmonary cancer and hVEGF expression levels, which were determined by immunohistochemistry, qRT-PCR, and western blot analyses. Gene expression profiling by cDNA microarray revealed a set of up-regulated genes (hvegf-A165, cyclin b1, cdc2, egfr, mmp9, nrp-1, and kdr) in VEGF tumors compared with wild-type lung tissues. In addition, overexpressing hVEGF-A165 in Clara cells increases CD105, fibrogenic genes (collagen α1, α-SMA, TGF-ß1, and TIMP1), and inflammatory cytokines (IL-1, IL-6, and TNF-α) in the lungs of hVEGF-A165-overexpressing transgenic mice as compared to wild-type mice. We further demonstrated that the intranasal administration of microRNA-16 (miR-16) inhibited lung tumor growth by suppressing VEGF expression via the intrinsic and extrinsic apoptotic pathways. In conclusion, hVEGF-A165 transgenic mice exhibited complex alterations in gene expression and tumorigenesis and may be a relevant model for studying VEGF-targeted therapies in lung adenocarcinoma.

Key process indicators of mortality in the implementation of protocol-driven therapy for severe sepsis.

BACKGROUND/PURPOSE: Severe sepsis and septic shock are life-threatening disorders. Integrating treatments into a bundle strategy has been proposed to facilitate timely resuscitation, but is difficult to implement. We implemented protocol-driven therapy for severe sepsis, and analyzed retrospectively the key process indicators of mortality in managing sepsis. METHODS: Continuous quality improvement was begun to implement a tailored protocol-driven therapy for sepsis in a 24-bed respiratory intensive care unit (RICU) of Taichung Veterans General Hospital from January 2007 to February 2008. Patients, who were admitted to the RICU directly, or within 24 hours, were enrolled if they met the criteria for severe sepsis and septic shock. Disease severity [Acute Physiology and Chronic Health Evaluation (APACHE) II and lactate level], causes of sepsis, comorbidity and site of sepsis onset were recorded. Process-of-care indicators included resuscitation time (Tr-s), RICU bed availability (Ti-s) and the ratio of completing the elements of the protocol at 1, 2, 4 and 6 hours. The structure and process-of-care indicators reflated to mortality at 7 days after RICU admission and at RICU discharge were identified retrospectively. RESULTS: Eighty-six patients (mean age, 71 +/- 14 years, 72 men, 14 women, APACHE II, 25.0 +/- 7.0) were enrolled. APACHE II scores and lactate levels were higher for mortality than survival at 7 days after RICU admission (p < 0.01). For the process-of-care indicators, Ti-s (562.2 +/- 483.3 vs.1017.3 +/- 557.8 minutes, p = 0.03) and Tr-s (60.7 +/- 207.8 vs. 248.5 +/- 453.1 minutes, p = 0.07) were shorter for survival than mortality at 7 days after RICU admission. The logistic regression study showed that Tr-s was an important indicator. The ratio of completing the elements of protocols at 1, 2, 4 and 6 hours ranged from 70% to 90% and was not related to mortality. CONCLUSION: Protocol-driven therapy for sepsis was put into clinical practice. Early resuscitation and ICU bed availability were key process indicators in managing sepsis, to reduce mortality.