Genomic Landscape and Phenotypic Assessment of Cronobacter sakazakii Isolated From Raw Material, Environment, and Production Facilities in Powdered Infant Formula Factories in China.

Cronobacter is a foodborne pathogen associated with severe infections and high mortality in neonates. The bacterium may also cause gastroenteritis, septicemia, and urinary tract and wound infectious in adults. A total of 15 Cronobacter isolates collected from 617 raw materials and environment samples from Powdered Infant Formula manufacturing factories during 2016 in Shaanxi, China, were analyzed for antimicrobial susceptibilities, species identification, biofilm formation, and whole-genome sequencing. The results showed that all 15 isolates were Cronobacter sakazakii, while the antimicrobial susceptibility test showed that all 15 C. sakazakii were pan susceptible. Most isolates were able to produce a weak biofilm, and two isolates from soil samples produced a strong biofilm formation. All isolates were classified into seven STs including ST4, ST40, ST64, ST93, ST148, ST256, and ST494, with ST64 (4/15, 26.7%) being dominant, and most were clinically related. The isolates harbored at least 11 virulence genes and two plasmids, with one isolate being positive for all virulence genes. Phylogenetic and ANI analysis showed strong clustering by sequence types and isolates from different sources or regions with a similar genomic background. The fact that isolates were obtained from raw materials and environment samples of PIF facilities shared a close phylogeny with one another suggests that cross-contamination events may have occurred between the processing room and external environments, which may give rise to a recurring risk of a continuous contamination during production.

Utility of Normalized TdP Score System in Drug Proarrhythmic Potential Assessment: A Blinded in vitro Study of CiPA Drugs.

Drugs that prolong QT may cause torsade de pointes (TdP). However, translation of nonclinical assessment of QT prolongation or hERG channel, targeted by QT-prolonging drugs, into clinical TdP risk has been insufficient to date. In this blinded study, we confirmed the utility of a Normalized TdP Score System in predicting drug-induced TdP risks among 34 drugs, including 28 with low, intermediate, and high TdP risks under the Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative plus six compounds with names blinded to the investigators, using the rabbit ventricular wedge assay. Concentration-dependent TdP scores were determined by drug-induced changes in QT, Tp-e , and proarrhythmias. Disclosure of the names and testing concentrations was made after completion of the experiments and report to the sponsors. Drugs' normalized TdP scores were calculated thereafter based on their respective free clinical maximum concentration (Cmax ). Drugs' normalized TdP scores were calculated and ranked for 33 drugs, excluding 1 investigational drug, and the TdP risks of the 28 CiPA drugs were correctly distinguished according to their respective categories of low, intermediate, and high TdP risks under the CiPA initiative. Accordingly, we are able to propose the cutoff values of the normalized TdP scores at 1 × Cmax : ≤ 0, > 0 to < 0.65 and ≥ 0.65, respectively, for low, intermediate, and high risk. This blinded study supports utility of our Normalized TdP Score System in predicting drug-induced TdP risks in 33 drugs, including 28 used for characterization of other assays under the CiPA initiative. However, these results need to be replicated in other laboratories.

How Much Is Too Much? The Influence of Work Hours on Social Development: An Empirical Analysis for OECD Countries.

Work is a cornerstone of social development. Quantifying the impact on development of fluctuations in work hours is important because longer work hours increasingly seem to be the norm. Based on an integrative perspective that combines individual, organizational, and social factors, we constructed a model using data from 31 member countries of the Organisation for Economic Co-operation and Development (OECD). The proposed model was used to test the effect of work hours on different levels and to propose feasible suggestions accordingly. The results show that people in developing countries work more hours per week than those in developed countries, and that males work longer hours than females. Furthermore, regression analysis shows that current work hours are having a negative impact on development in OECD countries, especially in developing countries where people are working longer hours. Longer hours, in other words, do not promote development effectively. Specifically, work hours at the individual level are negatively related to health. At the level of organization, work hours are a reverse indicator of organizational performance, and at the level of society, there is a negative relationship between work hours and economic development. This study provides support for the proposition by the International Labour Organization to reduce work hours, and it facilitates our understanding of the relationship between work hours and social development.

Assessment of drug-induced proarrhythmia: The importance of study design in the rabbit left ventricular wedge model.

In the present study, we investigated an impact of the stimulation rate on the detection of the proarrhythmic potential of 10 reference compounds with effects on different cardiac ion channels in the isolated arterially-perfused rabbit left ventricular wedge preparation. The compounds were tested in the wedge model using two distinct protocols; including baseline stimulation at 1-Hz followed by a brief period at 0.5-Hz, either without an additional brief period of 2-Hz stimulation (i.e. Protocol 1) or with 2-Hz stimulation (i.e. Protocol 2). As expected, QT-prolonging drugs (ibutilide and quinidine) prolonged the QT interval, similarly increased the Torsades de Pointes (TdP) score, and elicited early afterdepolarizations (EADs) in both protocols. HMR1556 and JNJ-303 (IKs blockers) also prolonged the QT interval up to 1µM similarly in both protocols. Nifedipine (Ca(2+) antagonist) shortened the QT interval, and reduced force of contraction similarly in both protocols. However, Na(+) channel blockers (Ia, Ib, Ic) widened the QRS duration more in Protocol 2 than in Protocol 1. Furthermore, it was only possible to detect non-TdP-like ventricular tachycardia/fibrillation (VT/VF) induced by Na(+) blockers and by QT-shortening drugs (levcromakalim and mallotoxin) using the 2-Hz stimulation (Protocol 2). Our data suggest that the inclusion of a brief period of fast stimulation at 2Hz is critical for detecting drug-induced slowing of conduction (QRS widening), QT shortening and associated (non-TdP-like) VT/VF, which are distinct from the QT prolongation/TdP proarrhythmia in isolated, arterially-perfused rabbit left ventricular wedges.

[Study on factors influencing DNA sequencing by automatic genetic analyzer].

OBJECTIVE: To acquire accurate and successful DNA sequencing in a cost-effective way by ABI3500xl automatic genetic analyzer. METHODS: BigDye was diluted to 8, 16 and 32 times in PCR product sequencing. Three different methods including CENTRI-SEP kit, BigDye cleaning beads and ethanol-NaAc-EDTA were used to purify the sequencing PCR products. RESULTS: The results of DNA sequencing were correct when BigDye was diluted up to 16 times. The misreading of nucleic acid bases was found as BigDye was diluted to 32 times. All three purification methods provided acceptable DNA sequencing results. In terms of method for purification of PCR products, the CENTRI-SEP Kit was the most expensive but time-saving (0.5 h), while ethanol-NaAc-EDTA method was the most economical but time-consuming (2 h). The BigDye cleaning beads method was of a suitable purification time (1 h) but not fit for high-throughput DNA sequencing. CONCLUSION: BigDye should be diluted up to 16 times in DNA sequencing by ABI3500xl DNA analyzer. Although all three purification methods may promise DNA sequencing results with good quality, it is necessary to choose an appropriate one to keep the balance between time and cost on the basis of the lab condition.

Preclinical assessment of drug-induced proarrhythmias: role of the arterially perfused rabbit left ventricular wedge preparation.

Drug-induced torsade de pointes (TdP) is a rare but lethal side effect of many cardiovascular and non-cardiovascular drugs. It has led to black box warnings or even withdrawal of many useful compounds from the market and is one of the major stumbling blocks for new drug development. The critical need for a better test that can predict the TdP liability of a candidate drug has led to the development of multiple preclinical models. Each of these models has it own merits and limitations in preclinical testing for TdP liability; however, most of these models have not been adequately validated, so their precise sensitivity and specificity remain largely unknown. Recent blinded validation studies have demonstrated that the rabbit left ventricular wedge preparation can predict drug-induced TdP with an extremely high sensitivity and specificity. As a matter of fact, the wedge technique was initially developed primarily for studying the electrical heterogeneity of myocardium and the cellular basis of QT prolongation and TdP. Naturally then, the electrophysiological data obtained from the wedge takes into account every critical factor associated with the development of TdP. The TdP scores generated using the wedge technique have been shown to assess the torsadogenic potential of the drugs in a predictable fashion. This review elaborates on the current and prospective role of the rabbit left ventricular wedge preparation in preclinical assessment of drug-induced proarrhythmias including but not limited to TdP.

Blinded validation of the isolated arterially perfused rabbit ventricular wedge in preclinical assessment of drug-induced proarrhythmias.

BACKGROUND: The development of preclinical models with high predictive value for the identification of drugs with a proclivity to induce Torsade de Pointes (TdP) in the clinic has long been a pressing goal of academia, industry and regulatory agencies alike. The present study provides a blinded appraisal of drugs, in an isolated arterially-perfused rabbit ventricular wedge preparation, with and without the potential to produce TdP. METHODS AND RESULTS: Thirteen compounds were tested for their potential for TdP using the rabbit left ventricular wedges. All investigators were blinded to the names, concentrations and molecular weights of the drugs. The compounds were prepared by the study sponsor and sent to the investigator as 4 sets of 13 stock solutions with the order within each set being assigned by a random number generator. Each compound was scored semi-quantitatively for its relative potential for TdP based on its effect on ventricular repolarization measured as QT interval, dispersion of repolarization measured as T(p-e)/QT ratio and early afterdepolarizations. Disclosure of the names and concentrations after completion of the study revealed that all compounds known to be free of TdP risk received a score of less or equal to 0.25, whereas those with known TdP risk received a score ranging from 1.00 to 7.25 at concentrations less than 100X their free therapeutic plasma C(max). CONCLUSIONS: Our study provides a blinded evaluation of the isolated arterially-perfused rabbit wedge preparation demonstrating both a high sensitivity and specificity in the assessment of 13 agents with varying propensity for causing TdP.

Assessment of the proarrhythmic potential of the novel antiarrhythmic agent AZD7009 and dofetilide in experimental models of torsades de pointes.

BACKGROUND: This study examined the proarrhythmic potential of the novel antiarrhythmic agent AZD7009 and dofetilide. METHODS AND RESULTS: The electrophysiological and proarrhythmic effects of AZD7009 and dofetilide were assessed in the arterially perfused canine and rabbit left ventricular wedge preparation. The proarrhythmic potential of AZD7009, dofetilide, and azimilide was further assessed in the methoxamine-sensitized rabbit model of torsades de pointes (TdP) in vivo. AZD7009 lengthened the action potential duration (APD) and the QT interval in a bell-shaped manner (15.9 +/- 1.3% in canine wedge and 46.1 +/- 2.9% in rabbit wedge) occurring at 3 and 1 microM. In contrast, dofetilide did not show the bell-shaped concentration response and the QT interval was lengthened more extensively (27.7 +/- 1.6% and 100.8 +/- 10.0%). Furthermore, whereas dofetilide prolonged the midmyocardial and endocardial APD predominantly, resulting in an increased transmural dispersion of repolarization (TDR), AZD7009 prolonged the APD more homogenously in all cell layers. At 1 microM, AZD7009 produced phase 2 early afterdepolarizations (EADs) in 1/4 rabbit preparations but without ventricular R-on-T extrasystoles or TdP. In contrast, starting at 0.03 microM, dofetilide-induced EADs, R-on-T extrasystoles and TdP in 6/6, 5/6, and 4/6 preparations. Following intravenous infusion of AZD7009 (210 nmol/kg/minute), dofetilide (2 nmol/kg/minute) or azimilide (3.33 micromol/kg/minute), TdP was induced in 0/8, 5/8, and 5/8 rabbits (P = 0.026 vs AZD7009), respectively. In 5/5 rabbits, AZD7009 promptly suppressed TdP induced by dofetilide. CONCLUSIONS: In animal models of TdP, AZD7009 delays ventricular repolarization in a self-limited way associated with a low risk of repolarization-related proarrhythmia.