Discovery of a Potent Chloroacetamide GPX4 Inhibitor with Bioavailability to Enable Target Engagement in Mice, a Potential Tool Compound for Inducing Ferroptosis In Vivo.

Compounds that inhibit glutathione peroxidase 4 (GPX4) hold promise as cancer therapeutics in their ability to induce a form of nonapoptotic cell death called ferroptosis. Our research identified 24, a structural analog of the potent GPX4 inhibitor RSL3, that has much better plasma stability (t1/2 > 5 h in mouse plasma). The bioavailability of 24 provided efficacious plasma drug concentrations with IP dosing, thus enabling in vivo studies to assess tolerability and efficacy. An efficacy study in mouse using a GPX4-sensitive tumor model found that doses of 24 up to 50 mg/kg were tolerated for 20 days but had no effect on tumor growth, although partial target engagement was observed in tumor homogenate.

Considerations from the Innovation and Quality Induction Working Group in Response to Drug-Drug Interaction Guidance from Regulatory Agencies: Guidelines on Model Fitting and Recommendations on Time Course for In Vitro Cytochrome P450 Induction Studies Including Impact on Drug Interaction Risk Assessment.

Translational and ADME Sciences Leadership Group Induction Working Group (IWG) presents an analysis on the time course for cytochrome P450 induction in primary human hepatocytes. Induction of CYP1A2, CYP2B6, and CYP3A4 was evaluated by seven IWG laboratories after incubation with prototypical inducers (omeprazole, phenobarbital, rifampicin, or efavirenz) for 6-72 hours. The effect of incubation duration and model-fitting approaches on induction parameters (Emax and EC50) and drug-drug interaction (DDI) risk assessment was determined. Despite variability in induction response across hepatocyte donors, the following recommendations are proposed: 1) 48 hours should be the primary time point for in vitro assessment of induction based on mRNA level or activity, with no further benefit from 72 hours; 2) when using mRNA, 24-hour incubations provide reliable assessment of induction and DDI risk; 3) if validated using prototypical inducers (>10-fold induction), 12-hour incubations may provide an estimate of induction potential, including characterization as negative if <2-fold induction of mRNA and no concentration dependence; 4) atypical dose-response ("bell-shaped") curves can be addressed by removing points outside an established confidence interval and %CV; 5) when maximum fold induction is well defined, the choice of nonlinear regression model has limited impact on estimated induction parameters; 6) when the maximum fold induction is not well defined, conservative DDI risk assessment can be obtained using sigmoidal three-parameter fit or constraining logistic three- or four-parameter fits to the maximum observed fold induction; 7) preliminary data suggest initial slope of the fold induction curve can be used to estimate Emax/EC50 and for induction risk assessment. SIGNIFICANCE STATEMENT: Regulatory agencies provide inconsistent guidance on the optimum length of time to evaluate cytochrome P450 induction in human hepatocytes, with EMA recommending 72 hours and FDA suggesting 48-72 hours. The Induction Working Group analyzed a large data set generated by seven member companies and determined that induction response and drug-drug risk assessment determined after 48-hour incubations were representative of 72-hour incubations. Additional recommendations are provided on model-fitting techniques for induction parameter estimation and addressing atypical concentration-response curves.

Metabolic disposition of H3B-8800, an orally available small-molecule splicing modulator, in rats, monkeys, and humans.

H3B-8800, a novel orally available modulator of the SF3b complex, which potently and preferentially kills spliceosome-mutant tumor cells, is in clinical development for the treatment of advanced myeloid malignancies. We characterized the pharmacokinetics, metabolism and disposition of H3B-8800 in rats, monkeys and humans.In vitro, H3B-8800 is a substrate of CYP3A4/5, flavin-containing monooxygenases (FMOs) and P-glycoprotein (P-gp), and showed a favorable drug-drug interaction profile as a perpetrator.Following oral dosing of 14C-H3B-8800 in bile-duct cannulated SD rats, 54.7% of the dosed radioactivity was excreted in the bile, with less found in feces (36.8%). The low amount in urine (3.7%), suggests that renal elimination is a minor pathway of clearance for H3B-8800.In Long-Evans rats, radioactivity derived from 14C-H3B-8800 was rapidly absorbed, with the highest distribution in the ocular, metabolic/excretory, and gastrointestinal tract tissues. No radioactivity was detected in the central nervous system.Seven metabolites were observed in human plasma following 4 daily doses of 40 mg H3B-8800. H3B-68736 (N-desmethyl), H3B-77176 (N-oxide), and unchanged H3B-8800 were the prominent components in human plasma, at 27.3%, 18.1%, and 33.2%, respectively, of the total drug-related material in a pooled AUC0-24h sample. The same 7 metabolites were observed in monkey plasma.

Nomination of controls of various layouts for heavy machinery.

BACKGROUND: Control selection in heavy machinery may be a problem due to the poor compatibility of the commonly-used single line of vertical levers for controls. OBJECTIVE: This study examined the effects of control arrangements on directional compatibility and participant responses in terms of choice/function of controls. METHODS: We investigated experimentally, for a number of new designs of control arrangements, the selection of controls for requested functions of four different machines: fork lift trucks, excavators, tower cranes, and telescopic cranes. Control arrangements were designed with different levels of directional compatibility and participants were requested to nominate which control was related to each of the machine motions and to make ratings of certainty of response. A second part required participants to rank the various control arrangements in terms of their compatibility. RESULTS: The in-line control arrangement was worst for each machine and increased directional compatibility improved the stereotype strength, certainty of response and ranking of control arrangement. CONCLUSIONS: Directional compatibility is the main factor in the design of control/machine output arrangements due to its effect on stereotype strength and correct selection of control of a given function.

Prehospital electrocardiogram shortens ischaemic time in patients with ST-segment elevation myocardial infarction.

INTRODUCTION: Total ischaemic time should be shortened as much as possible in patients with ST-segment elevation myocardial infarction (STEMI). This study evaluated whether prehospital 12-lead electrocardiogram (ECG) could shorten system delay in STEMI management. METHODS: From November 2015 to November 2017, 15 ambulances equipped with X Series Monitor/ Defibrillator (Zoll Medical Corporation) were used in the catchment area of Queen Mary Hospital, Hong Kong. Prehospital ECG was performed for patients with chest pain; the data were tele-transmitted to attending emergency physicians at the Accident and Emergency Department (AED) for rapid assessment. Data from patients with STEMI who were transported by these 15 ambulances were compared with data from patients with STEMI who were transported by ambulances without prehospital ECG or who used self-arranged transport. RESULTS: Data were analysed from 197 patients with STEMI. The median patient delay for activation of the emergency response system was 90 minutes; 12% of patients experienced a delay of >12 hours. There was a significant difference in delay between patients transported by ambulance and those who used self-arranged transport (P<0.001). For system delay, the use of prehospital ECG shortened the median time from ambulance on scene to first ECG (P<0.001). When performed upon ambulance on scene, prehospital ECG was available 5 minutes earlier than if performed in ambulance compartment before departure. Use of prehospital ECG significantly shortened AED door-to-triage time, AED door-to-first AED ECG time, AED door-to-physician consultation time, and length of stay in the AED (P<0.001 for all comparisons). CONCLUSION: Prehospital ECG shortened ischaemic time prior to hospital admission.

Authentic Modeling of Human Respiratory Virus Infection in Human Pluripotent Stem Cell-Derived Lung Organoids.

Infectious viruses so precisely fit their hosts that the study of natural viral infection depends on host-specific mechanisms that affect viral infection. For human parainfluenza virus 3, a prevalent cause of lower respiratory tract disease in infants, circulating human viruses are genetically different from viruses grown in standard laboratory conditions; the surface glycoproteins that mediate host cell entry on circulating viruses are suited to the environment of the human lung and differ from those of viruses grown in cultured cells. Polarized human airway epithelium cultures have been used to represent the large, proximal airways of mature adult airways. Here we modeled respiratory virus infections that occur in children or infect the distal lung using lung organoids that represent the entire developing infant lung. These 3D lung organoids derived from human pluripotent stem cells contain mesoderm and pulmonary endoderm and develop into branching airway and alveolar structures. Whole-genome sequencing analysis of parainfluenza viruses replicating in the organoids showed maintenance of nucleotide identity, suggesting that no selective pressure is exerted on the virus in this tissue. Infection with parainfluenza virus led to viral shedding without morphological changes, while respiratory syncytial virus infection induced detachment and shedding of infected cells into the lung organoid lumens, reminiscent of parainfluenza and respiratory syncytial virus in human infant lungs. Measles virus infection, in contrast, induced syncytium formation. These human stem cell-derived lung organoids may serve as an authentic model for respiratory viral pathogenesis in the developing or infant lung, recapitulating respiratory viral infection in the host.IMPORTANCE Respiratory viruses are among the first pathogens encountered by young children, and the significant impact of these viral infections on the developing lung is poorly understood. Circulating viruses are suited to the environment of the human lung and are different from those of viruses grown in cultured cells. We modeled respiratory virus infections that occur in children or infect the distal lung using lung organoids that represent the entire developing infant lung. These 3D lung organoids, derived from human pluripotent stem cells, develop into branching airway and alveolar structures and provide a tissue environment that maintains the authentic viral genome. The lung organoids can be genetically engineered prior to differentiation, thereby generating tissues bearing or lacking specific features that may be relevant to viral infection, a feature that may have utility for the study of host-pathogen interaction for a range of lung pathogens.

Prehospital 12-lead electrocardiogram for patients with chest pain: a pilot study.

INTRODUCTION: After ST-segment elevation myocardial infarction (STEMI), it is vital to shorten reperfusion time. This study examined data from a pilot project to shorten the door-to-balloon (D2B) time by using prehospital 12-lead electrocardiogram (ECG). METHODS: Fifteen ambulances equipped with X Series® Monitor/Defibrillator (Zoll Medical Corporation) were deployed to the catchment area of Queen Mary Hospital, Hong Kong, from November 2015 to December 2016. For patients with chest pain, prehospital 12-lead ECG was performed and tele-transmitted to attending physicians at the accident and emergency department for immediate interpretation. The on-call cardiologist was called before patient arrival if STEMI was suspected. Data from this group of patients with STEMI were compared with data from patients with STEMI who were transported by ambulances without prehospital ECG or by self-arranged transport. RESULTS: From 841 patients with chest pain, 731 gave verbal consent and prehospital ECG was performed and transmitted. Of these, 25 patients with clinically diagnosed STEMI required emergency coronary angiogram with or without primary percutaneous coronary intervention. The mean D2B time for these 25 patients (93 minutes) was significantly shorter (P=0.003) than that for 58 patients with STEMI transported by ambulances without prehospital ECG (112 minutes) and that for 41 patients with STEMI with self-arranged transport (138 minutes). However, shorter reperfusion time was only recorded during daytime hours (08:00-17:59). No statistically significant difference in 30-day mortality was found. CONCLUSION: Prehospital ECG is technologically feasible in Hong Kong and shortens the D2B time. However, shorter reperfusion time was only recorded during daytime hours.

Celecoxib is a substrate of CYP2D6: Impact on celecoxib metabolism in individuals with CYP2C9*3 variants.

Celecoxib was characterized as a substrate of human cytochrome P450 (CYP) 2D6 in vitro. In recombinant CYP2D6, celecoxib hydroxylation showed atypical substrate inhibition kinetics with apparent Km, Ki, and Vmax of 67.2 µM, 12.6 µM, and 1.33 µM/min, respectively. In human liver microsomes (HLMs), a concentration-dependent inhibition of celecoxib hydroxylation by quinidine was observed after CYP2C9 and CYP3A4 were inhibited. In individual HLMs with variable CYP2D6 activities, a significant correlation was observed between celecoxib hydroxylation and CYP2D6-selective dextromethorphan O-demethylation when CYP2C9 and CYP3A4 activities were suppressed (r = 0.97, P < 0.0001). Molecular modeling showed two predominant docking modes of celecoxib with CYP2D6, resulting in either a substrate or an inhibitor. A second allosteric binding antechamber, which stabilized the inhibition mode, was revealed. Modeling results were consistent with the observed substrate inhibition kinetics. Using HLMs from individual donors, the relative contribution of CYP2D6 to celecoxib metabolism was found to be highly variable and dependent on CYP2C9 genotypes, ranging from no contribution in extensive metabolizers with CYP2C9*1*1 genotype to approximately 30% in slow metabolizers with allelic variants CYP2C9*1*3 and CYP2C9*3*3. These results demonstrate that celecoxib may become a potential victim of CYP2D6-associated drug-drug interactions, particularly in individuals with reduced CYP2C9 activity.

Considerations from the Innovation and Quality Induction Working Group in Response to Drug-Drug Interaction Guidances from Regulatory Agencies: Focus on CYP3A4 mRNA In Vitro Response Thresholds, Variability, and Clinical Relevance.

The Innovation and Quality Induction Working Group presents an assessment of best practice for data interpretation of in vitro induction, specifically, response thresholds, variability, application of controls, and translation to clinical risk assessment with focus on CYP3A4 mRNA. Single concentration control data and Emax/EC50 data for prototypical CYP3A4 inducers were compiled from many human hepatocyte donors in different laboratories. Clinical CYP3A induction and in vitro data were gathered for 51 compounds, 16 of which were proprietary. A large degree of variability was observed in both the clinical and in vitro induction responses; however, analysis confirmed in vitro data are able to predict clinical induction risk. Following extensive examination of this large data set, the following recommendations are proposed. a) Cytochrome P450 induction should continue to be evaluated in three separate human donors in vitro. b) In light of empirically divergent responses in rifampicin control and most test inducers, normalization of data to percent positive control appears to be of limited benefit. c) With concentration dependence, 2-fold induction is an acceptable threshold for positive identification of in vitro CYP3A4 mRNA induction. d) To reduce the risk of false positives, in the absence of a concentration-dependent response, induction ≥ 2-fold should be observed in more than one donor to classify a compound as an in vitro inducer. e) If qualifying a compound as negative for CYP3A4 mRNA induction, the magnitude of maximal rifampicin response in that donor should be ≥ 10-fold. f) Inclusion of a negative control adds no value beyond that of the vehicle control.

Pharmacokinetic/pharmacodynamic drug-drug interactions of avatrombopag when coadministered with dual or selective CYP2C9 and CYP3A interacting drugs.

AIMS: Avatrombopag, a thrombopoietin receptor agonist, is a substrate of cytochrome P450 (CYP) 2C9 and CYP3A. We assessed three drug-drug interactions of avatrombopag as a victim with dual or selective CYP2C9/3A inhibitors and inducers. METHODS: This was a three-part, open-label study. Forty-eight healthy subjects received single 20 mg doses of avatrombopag alone or with one of 3 CYP2C9/3A inhibitors or inducers: fluconazole 400 mg once daily for 16 days, itraconazole 200 mg twice daily on Day 1 and 200 mg once daily on Days 2-16, or rifampicin 600 mg once daily for 16 days. Pharmacokinetics, pharmacodynamics (platelet count) and safety of avatrombopag were evaluated. RESULTS: Coadministration of a single 20-mg dose of avatrombopag with fluconazole at steady-state resulted in 2.16-fold increase of AUC of avatrombopag, prolonged terminal elimination phase half-life (from 19.7 h to 39.9 h) and led to a clinically significant increase in maximum platelet count (1.66-fold). Itraconazole had a mild increase on both avatrombopag pharmacokinetics and pharmacodynamics compared to fluconazole. Coadministration of rifampicin caused a 0.5-fold decrease in AUC and shortened terminal elimination phase half-life (from 20.3 h to 9.84 h), but has no impact on maximum platelet count. Coadministration with interacting drugs was found to be generally safe and well-tolerated. CONCLUSIONS: The results from coadministration of fluconazole or itraconazole suggest that CYP2C9 plays a more predominant role in metabolic clearance of avatrombopag than CYP3A. To achieve comparable platelet count increases when avatrombopag is coadministered with CYP3A and CYP2C9 inhibitors, an adjustment in the dose or duration of treatment is recommended, while coadministration with strong inducers is not currently recommended.

Equivalent magnetic vector potential model for low-frequency magnetic exposure assessment.

In this paper, a novel source model based on a magnetic vector potential for the assessment of induced electric field strength in a human body exposed to the low-frequency (LF) magnetic field of an electrical appliance is presented. The construction of the vector potential model requires only a single-component magnetic field to be measured close to the appliance under test, hence relieving considerable practical measurement effort-the radial basis functions (RBFs) are adopted for the interpolation of discrete measurements; the magnetic vector potential model can then be directly constructed by summing a set of simple algebraic functions of RBF parameters. The vector potentials are then incorporated into numerical calculations as the equivalent source for evaluations of the induced electric field in the human body model. The accuracy and effectiveness of the proposed model are demonstrated by comparing the induced electric field in a human model to that of the full-wave simulation. This study presents a simple and effective approach for modelling the LF magnetic source. The result of this study could simplify the compliance test procedure for assessing an electrical appliance regarding LF magnetic exposure.

Considerations from the IQ Induction Working Group in Response to Drug-Drug Interaction Guidance from Regulatory Agencies: Focus on Downregulation, CYP2C Induction, and CYP2B6 Positive Control.

The European Medicines Agency (EMA), the Pharmaceutical and Medical Devices Agency (PMDA), and the Food and Drug Administration (FDA) have issued guidelines for the conduct of drug-drug interaction studies. To examine the applicability of these regulatory recommendations specifically for induction, a group of scientists, under the auspices of the Drug Metabolism Leadership Group of the Innovation and Quality (IQ) Consortium, formed the Induction Working Group (IWG). A team of 19 scientists, from 16 of the 39 pharmaceutical companies that are members of the IQ Consortium and two Contract Research Organizations reviewed the recommendations, focusing initially on the current EMA guidelines. Questions were collated from IQ member companies as to which aspects of the guidelines require further evaluation. The EMA was then approached to provide insights into their recommendations on the following: 1) evaluation of downregulation, 2) in vitro assessment of CYP2C induction, 3) the use of CITCO as the positive control for CYP2B6 induction by CAR, 4) data interpretation (a 2-fold increase in mRNA as evidence of induction), and 5) the duration of incubation of hepatocytes with test article. The IWG conducted an anonymous survey among IQ member companies to query current practices, focusing specifically on the aforementioned key points. Responses were received from 19 companies. All data and information were blinded before being shared with the IWG. The results of the survey are presented, together with consensus recommendations on downregulation, CYP2C induction, and CYP2B6 positive control. Results and recommendations related to data interpretation and induction time course will be reported in subsequent articles.

Out-of-hospital cardiac arrest in Hong Kong: a territory-wide study.

INTRODUCTION: Out-of-hospital cardiac arrest is a global health care problem. Like other cities in the world, Hong Kong faces the impact of such events. This study is the first territory-wide investigation of the epidemiology and outcomes of out-of-hospital cardiac arrest in Hong Kong. It is hoped that the findings can improve survival of patients with cardiac arrest. METHODS: This study was a retrospective analysis of the prospectively collected data on out-of-hospital cardiac arrest managed by the emergency medical service from 1 August 2012 to 31 July 2013. The characteristics of patients and cardiac arrests, timeliness of emergency medical service attendance, and survival rates were reported with descriptive statistics. Predictors of 30-day survival were evaluated with logistic regression. RESULTS: A total of 5154 cases of out-of-hospital cardiac arrest were analysed. The median age of patients was 80 years. Most arrests occurred at the patient's home. Ventricular fibrillation or ventricular tachycardia was identified in 8.7% of patients. The median time taken for the emergency services to reach the patient was 9 minutes. The median time to first defibrillation was 12 minutes. Of note, 2.3% of patients were alive at 30 days or survived to hospital discharge; 1.5% had a good neurological outcome. Location of arrest, initial electrocardiogram rhythm, and time to first defibrillation were independent predictors of survival at 30 days. CONCLUSION: The survival rate of out-of-hospital cardiac arrest patients in Hong Kong is low. Territory-wide public access defibrillation programme and cardiopulmonary resuscitation training may help improve survival.

Impact of Probe Substrate Selection on Cytochrome P450 Reaction Phenotyping Using the Relative Activity Factor.

Accurately assessing the contribution of cytochrome P450 (P450) isoforms to overall metabolic clearance is important for prediction of clinical drug-drug interactions (DDIs). The relative activity factor (RAF) approach in P450 reaction phenotyping assumes that the interaction between P450-selective probes and testing systems is the same as the interaction of drug candidate with those systems. To test this assumption, an intersystem clearance ratio (ICR) was created to evaluate the difference in values between RAF-scaled intrinsic clearance (CLint) and measured CLint in human liver microsomes (HLMs). The RAF value for CYP3A4 or CYP2C9 derived from a particular P450-selective probe reaction was applied to calculate RAF-scaled CLint for other probe reactions of the same P450 isoform in a crossover manner and compared with the measured HLM CLint When RAF derived from midazolam or nifedipine was used for CYP3A4, the ICR for testosterone 6ß-hydroxylation was 31 and 25, respectively, suggesting significantly diverse interactions of CYP3A4 probes with the testing systems. Such ICR differences were less profound among probes for CYP2C9. In addition, these RAF values were applied to losartan and meloxicam, whose metabolism is mostly CYP2C9 mediated. Only using the RAF derived from testosterone for CYP3A4 produced the expected CYP2C9 contribution of 72%-87% and 47%-69% for metabolism of losartan and meloxicam, respectively. RAF derived from other CYP3A4 probes would have attributed predominantly to CYP3A4 and led to incorrect prediction of DDIs. Our study demonstrates a significant impact of probe substrate selection on P450 phenotyping using the RAF approach, and the ICR may provide a potential solution.

Smoking and mortality in a prospective cohort study of elderly Chinese in Hong Kong.

BACKGROUND AND AIMS: Large cohort studies on smoking and mortality in elderly people are scarce, and few studies examined smokers aged 85+ years separately. We estimated the risks of all-cause and cause-specific mortality due to smoking in an elderly Chinese cohort in Hong Kong. DESIGN: A population-based prospective cohort of 65,510 Chinese enrolled from 1998 to 2001 and followed until May 2012. SETTING: All 18 Elderly Health Service centres in Hong Kong, China. PARTICIPANTS: Elderly people aged 65+ years. MEASUREMENTS: Self-reported smoking status was assessed at baseline interview and categorized as never, former and current smokers. FINDINGS: Compared with never smokers, after adjustment for sex, age, education, social security assistance, housing type, monthly expenditure, alcohol use, depressive symptoms and health status, the hazard ratio (HR) for current smokers was 1.89 [95% confidence interval (CI) = 1.81-1.98] for all participants aged 65+ years at baseline, corresponding to an attributable fraction (AF) of about 50%, which is based on AF = (HR-1)/HR. As the effect of smoking varied with age (P for age interaction <0.001), subgroup analysis by age group showed that the adjusted HR for current smokers aged 65-84 years was 1.93 (95% CI = 1.84-2.03), and for 85+ years was 1.29 (95% CI = 1.05-1.58). All the risk estimates did not vary by sex (P for sex interaction ranged 0.74-0.89). CONCLUSIONS: In Hong Kong, the risk of death from smoking appears to be the same for Chinese women as it is for men. Half of all deaths in Chinese smokers aged 65 years and older and a quarter of all deaths in Chinese smokers aged 85 years and older are caused by smoking-attributable diseases.

Intraconal orbital dermoid cyst in an adult patient: a rare location.

Orbital dermoid cyst is a rare cause of orbital tumor in the adult population. Most of the orbital dermoids are extraconal, arising adjacent to bone suture and occurring in the pediatric patients. Deep orbital dermoid cyst located entirely intraconally is extremely rare. We report a case of intraconal orbital dermoid in an adult patient with CT and MRI evaluation.

Socioenvironmental factors associated with constipation in Hong Kong children and Rome III criteria.

OBJECTIVE: Data regarding the prevalence of constipation in the general population of Asian children using internationally standardized definitions are scarce. Environmental factors surrounding a child's day to day living may trigger or perpetuate constipation and encourage postponement of defecation. METHODS: A territory-wide cross-sectional questionnaire survey was conducted in 2318 Hong Kong Chinese elementary school students. Constipation was defined by pediatric Rome III criteria. RESULTS: The mean age of the children was 9 ± 1.9 years; 51% were boys. Two hundred eighty-two children (12.2%, 95% confidence interval [CI] 10.9%-13.5%) were found to have constipation. Children ages 6 to 7 years had the highest prevalence (16.8%, 95% CI 13.8%-19.8%). There was no difference in prevalence between boys and girls (11.6% vs 12.3%; P > 0.05) and between obese and nonobese children (11.5% vs 11.1%; P > 0.05). In univariate analysis, constipation was found to be significantly more prevalent among those children who lived with neither parent, had inadequate company of parents at home, refused to pass bowel movements in school, spent long hours doing homework, had inadequate sleep, and had decreased fiber intake and frequent consumption of fast food (P < 0.05). Multivariate analysis identified refusal to pass bowel movements in school toilets (odds ratio [OR] 1.97, 95% CI 1.42%-2.74%), having dinner with one/both parents <50% of time (OR 1.52, 95% CI 1.01%-2.31%), nighttime sleep <7 hours (OR 1.87, 95% CI 1.04%-3.33%), and frequent consumption of fast food (OR 1.14, 95% CI 1.03%-1.26%) to be independent factors associated with constipation. CONCLUSIONS: Socioenvironmental factors are associated with childhood constipation, and bringing them to the awareness of the public may help prevent or stop the progression of childhood constipation at its early stages.

The median effective dose of bupivacaine, levobupivacaine, and ropivacaine after intrathecal injection in lower limb surgery.

BACKGROUND: Intrathecal anesthesia is commonly used for lower limb surgery. Bupivacaine, levobupivacaine, and ropivacaine have all been used as intrathecal drugs, but their relative potency in this context has not been fully determined. In this study, we determined the median effective dose (ED(50)) of these three local anesthetics for intrathecal anesthesia in lower limb surgery and hence their relative potencies. METHODS: Seventy-five patients scheduled for lower limb surgery under combined spinal-epidural anesthesia were randomly allocated to one of three groups receiving intrathecal bupivacaine, levobupivacaine, or ropivacaine. The dose of local anesthetic was varied using up-down sequential allocation technique. The dose for the first patient in each group was 8 mg, and the dosing increment was set at 1 mg. Subsequent doses in each group were determined by the outcome in the previous patient using success or failure of the spinal anesthesia as the primary end point. A success was recorded if a bilateral T12 sensory block to cold was attained within 20 min after intrathecal injection, and the surgery proceeded successfully until at least 50 min after the intrathecal injection without supplementary epidural injection. The ED(50) was calculated using the method of Dixon and Massey. RESULTS: The ED(50)s were 5.50 mg for bupivacaine (95% confidence interval [CI]: 4.90-6.10 mg), 5.68 mg for levobupivacaine (95% CI: 4.92-6.44 mg), and 8.41 mg for ropivacaine (95% CI: 7.15-9.67 mg) in intrathecal anesthesia. The relative anesthetic potency ratios are 0.97 (95% CI: 0.81-1.17) for levobupivacaine/bupivacaine, 0.65 (95% CI: 0.54-0.80) for ropivacaine/bupivacaine, and 0.68 (95% CI: 0.55-0.84) for ropivacaine/levobupivacaine. CONCLUSION: This study suggests that in intrathecal anesthesia for lower limb surgery, ropivacaine is less potent than levobupivacaine and bupivacaine, whereas the potency is similar between levobupivacaine and bupivacaine.

The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles.

The production of virus-like particles (VLPs) constitutes a relevant and safe model to study molecular determinants of virion egress. The minimal requirement for the assembly of VLPs for the coronavirus responsible for severe acute respiratory syndrome in humans (SARS-CoV) is still controversial. Recent studies have shown that SARS-CoV VLP formation depends on either M and E proteins or M and N proteins. Here we show that both E and N proteins must be coexpressed with M protein for the efficient production and release of VLPs by transfected Vero E6 cells. This suggests that the mechanism of SARS-CoV assembly differs from that of other studied coronaviruses, which only require M and E proteins for VLP formation. When coexpressed, the native envelope trimeric S glycoprotein is incorporated onto VLPs. Interestingly, when a fluorescent protein tag is added to the C-terminal end of N or S protein, but not M protein, the chimeric viral proteins can be assembled within VLPs and allow visualization of VLP production and trafficking in living cells by state-of-the-art imaging technologies. Fluorescent VLPs will be used further to investigate the role of cellular machineries during SARS-CoV egress.