The Novel Use of a Heterozygous Knockout Mouse for Embryofetal Development Assessment of a Glucokinase Activator

Glucokinase activators (GKAs), such as AZD1656, are designed as antihyperglycemic agents for diabetics and can cause dose‐limiting hypoglycemia in normal animals used in embryofetal development studies. Genetically modified heterozygous GK knockout (gk^del/wt) mice are less susceptible to severe GKA‐induced hypoglycemia than wild‐type mice due to their elevated baseline glucose levels. In this study, the gk^del/wt mouse was used as an alternative rodent strain for embryofetal development studies with AZD1656. Heterozygous global knockout gk^del/wt females were dosed with 20, 50, or 130 mg/kg/day of AZD1656 or vehicle for a minimum of 14 consecutive days before mating with wild‐type males and throughout organogenesis. Maternal effects were confined to slightly reduced food consumption, reduced body weight gain, and the pharmacologic effect of decreased plasma glucose. Fetuses were genotyped. Fetal weights at the high dose were slightly reduced but there was no effect on fetal survival. There were two specific major malformations, omphalocele and right‐sided aortic arch, with increased fetal incidence in mid‐ and high‐dose fetuses (e.g., omphalocele fetal incidence of 0.6, 0.7, 4.6, and 2% across the dose groups) plus increased incidences of minor abnormalities and variants indicative of either delayed or disturbed development. Fetal weight and abnormalities were unaffected by fetal genotype. The fetal effects are considered hypoglycemia related. There was no effect on embryofetal survival in the gk^del/wt mouse at AZD1656 exposures, which were 70× higher than those causing 75% fetal death in rabbits. This illustrates the value of genetically modified animals in unraveling target versus chemistry‐related effects.     

Project-based learning in a collaborative group can enhance student skill and ability in the biochemical laboratory: a case study

ABSTRACT

Experimental courses for undergraduate students majoring in biochemistry or related subjects often do not provide students with systematic and research-based experiences. To help students develop abilities related to laboratory techniques, data analysis, and systematic thought in biology, we performed an exploratory program that employs project-based learning in collaborative groups. The participants (total of 18 students) organized themselves into groups of 2–4 students, and each group researched an enzyme that had not been described previously. The program began with a literature survey of enzyme and bioinformatics analysis. The students cloned the gene encoding the enzyme, purified the enzyme, and, finally, analyzed the enzyme’s catalytic characteristics. The students explained the catalytic mechanism, integrating their experimental data and other knowledge. An instructor provided support and training during the process to support effective teamwork and to cultivate a habit of independence that is believed to be useful for the students’ future careers. The assessment showed that the pilot program yielded an improvement in the participant’ laboratory skills, scientific presentation ability, and experimental design ability. These analyses indicated that the small-scale practice in this study provided benefits to the students and the methods may be popularized to a large extent.     

Estimating time to pregnancy from current durations in a cross-sectional sample

A new design for estimating the distribution of time to pregnancy is proposed and investigated. The design is based on recording current durations in a cross-sectional sample of women, leading to statistical problems similar to estimating renewal time distributions from backward recurrence times. Non-parametric estimation is studied in some detail and a parametric approach is indicated. The results are illustrated on Monte Carlo simulations and on data from a recent European collaborative study. The role and applicability of this approach is discussed.     

The case-only odds ratio as a causal parameter

In the simplest case-only design, cases of a disease are cross-classified into a 2 x 2 table describing a genotype attribute and exposure to some environmental agent. In some instances, the genetic attribute has described inherited genes; in other instances, it has described mutations, for instance, damage to proto-oncogenes or tumor suppressor genes leading to cancer. Here, the population case-only odds ratio is written as a causal parameter in terms of potential outcomes with and without exposure to the agent. It is shown that the case-only odds ratio makes sense as a causal parameter with inherited genes, but its magnitude does not have a causal interpretation with mutations, although deviations from 1 do provide information. The difference is that the environmental agent certainly did not cause an individual to inherit particular genes, but it may have caused the mutation.     

Where to sit in a waiting room: density, age and gender effects on proxemic choices

We studied proxemic behaviour in an hospital waiting room by recording who chose to stand and who chose to sit and which chair, row and position. We distinguished sex and two categories of age, mature and old. We found that fenales sit together more easily than males and more frequently chose to sit close, especially mature ones. Males tend to keep more distances from neighbours by standing up. In general, proxemic behaviour in a waiting room seems to be an indicator confirming the sexual startegies in partner choice described in the evolutionary psychology paradigm.     

Coordination properties towards palladium(II) of a tridentate dianionic ligand acting as a N- or a N,O-donor

The new palladium(II) complex Pd[C₅H₃N-2,6-(CONPh)₂](η¹-NCMe) (1), prepared from N,N′-diphenyl-2,6-pyridinedicarboxamide and Pd(OAc)₂ in acetonitrile, has been characterized via IR, ¹H NMR and single-crystal X-ray diffraction. In this compound the palladium centre is coordinated to three nitrogen donors of the anionic ligand and to the nitrogen atom of acetonitrile.Moreover, the already known Pd[C₅H₃N-2,6-(CONCH₂CH₂Ph)₂](η¹-NCMe) (2) has been studied by ¹H NMR spectrometry and found to readily convert into the macrocyclic tetranuclear species 3, {Pd[C₅H₃N-2,6-(CONCH₂CH₂Ph)₂]}₄ which has been isolated and characterized by IR, ¹H and ¹³C{¹H} NMR, ¹H-¹³C HETCOR and mass spectrometry, as well as by single-crystal X-ray diffraction. In 3, of S₄ symmetry, each palladium atom is coordinated to the three nitrogen atoms of the anionic ligand, while the fourth coordination position is occupied by the amidato oxygen atom of an adjacent unit. This structure is apparently maintained in CDCl₃ solution. The substitution reactions of acetonitrile in 2 with the ligands EEt₂ (E = S, Se) afford Pd[C₅H₃N-2,6-(CONCH₂CH₂Ph)₂](EEt₂) (4, E = S; 5, E = Se); these products can also be obtained by the addition of EEt₂ to 3, as shown by means of ¹H- and, in the case of E = Se, ⁷⁷Se{¹H} NMR spectroscopy in CDCl₃ solution. These results show that the Pd-O bonds of the tetranuclear species are readily broken by weakly coordinating ligands such as acetonitrile and diethylchalcogenides. Nevertheless, we are dealing with equilibrium reactions and, in some solvents, 3 can be obtained from 2, 4 or 5 being favoured by its low solubility.     

Analysis of a two-stage case-control study with cluster sampling of controls: application to nonmelanoma skin cancer

We present a pseudolikelihood approach for analyzing a two-stage population-based case-control study with cluster sampling of controls. These methods were developed to analyze data from a study of nonmelanoma skin cancer (NMSC). This study was designed to evaluate the role of ultraviolet radiation (UVB) on NMSC risk while adjusting for age group, which is known for all subjects, and for other individual-level factors, such as susceptibility to sunburn, which are known only for participants in the case-control study. The methods presented yield estimates of relative and absolute risk, with standard errors, while accounting naturally for the two-stage sampling of the cohort and cluster sampling of controls.     

Pulmonary function of a firemen-diver population: a longitudinal study

Non smoking, male professional firemen-divers (n = 20) underwent two pulmonary function tests (PFT) separated by 8–9 years. Measured data were compared to European Coal Steel Community recommended reference values to permit cross-sectional and then longitudinal studies. Higher vital capacity (VC) and forced expiratory volume in 1 s (FEV₁; bothP<0.001), and lower residual volume (P<0.01) were observed in both PFT. Longitudinal analysis showed a smaller VC reduction than FEV₁ reduction, leading to a FEV₁/VC percentage decrease with time. Maximal mid expiratory flow (MMEF) and MMEF/VC changes during this 9-year period showed an unusually pronounced decrease, suggesting possible chronic effects of diving on small airways. Thus, it is suggested from our observations that a hyperbaric stimulus compensates in part for the effects of aging on VC and that obstructive disease could occur in subjects with long diving experience.     

The value of juvenile animal studies: a Japanese industry perspective

Pharmaceuticals have been used on adults and children; however, they were previously investigated only by adult human clinical studies and adult animal nonclinical studies. The US FDA finalized the guidance of juvenile animal toxicity studies in 2006, and EMEA was finalized in 2008. At that point, juvenile animal toxicity studies were encouraged to investigate the safety of the pediatric population. In Japan, the awareness of the development of pediatric drugs is increasing, and many scientific meetings about juvenile animal studies are being held. A Japanese guideline for juvenile animal toxicity studies has been long awaited by many Japanese pharmaceutical companies because concrete directionality has not been available in Japan thus far. The Ministry of Health, Labour, and Welfare started to prepare the guideline for nonclinical safety studies in juvenile animals since October 2010. After completion of the Japanese guideline, guidelines would exist in the three regions: Japan, US, and Europe. Then, global development of pediatric pharmaceuticals would be accelerated effectively.     

Hydrophobic substituents increase the potency of salacinol,a potent α-glucosidase inhibitor from Ayurvedic traditional medicine ‘Salacia’

Using an in silico method, seven analogs bearing hydrophobic substituents (8a: Me, 8b: Et, 8c: n-Pent, 8d: n-Hept, 8e: n-Tridec, 8f: isoBu and 8g: neoPent) at the 3′-O-position in salacinol (1), a highly potent natural α-glucosidase inhibitor from Ayurvedic traditional medicine ‘Salacia’, were designed and synthesized. In order to verify the computational SAR assessments, their α-glucosidase inhibitory activities were evaluated in vitro. All analogs (8a–8g) exhibited an equal or considerably higher level of inhibitory activity against rat small intestinal α-glucosidases compared with the original sulfonate (1), and were as potent as or higher in potency than the clinically used anti-diabetics, voglibose, acarbose or miglitol. Their activities against human maltase exhibited good relationships to the results obtained with enzymes of rat origin. Among the designed compounds, the one with a 3′-O-neopentyl moiety (8g) was most potent, with an approximately ten fold increase in activity against human maltase compared to 1.     

Pathway analysis of a genome-wide association study in schizophrenia

Objective

The aim of this study was to identify the candidate single nucleotide polymorphisms (SNPs) and candidate mechanisms that contribute to schizophrenia susceptibility and to generate a SNP to gene to pathway hypothesis using an analytical pathway-based approach.

Methods

We used schizophrenia GWAS data of the genotypes of 660,259 SNPs in 1378 controls and 1351 cases of European descent after quality control filtering. ICSNPathway (Identify candidate Causal SNPs and Pathways) analysis was applied to the schizophrenia GWAS dataset. The first stage involved the pre-selection of candidate SNPs by linkage disequilibrium analysis and the functional SNP annotation of the most significant SNPs found. The second stage involved the annotation of biological mechanisms for the pre-selected candidate SNPs using improved-gene set enrichment analysis.

Results

ICSNPathway analysis identified fifteen candidate SNPs, ten candidate pathways, and nine hypothetical biological mechanisms. The most strongly associated potential pathways were as follows. First, rs1644731 and rs1644730 to RDH8 to estrogen biosynthetic process (p < 0.001, FDR < 0.001). The genes involved in this pathway are RDH8 and HSD3B1 (p < 0.05). All-trans-retinol dehydrogenase (RDH8) is a visual cycle enzyme that reduces all-trans-retinal to all-trans-retinol in the presence of NADPH. The chemical reactions and pathways involved result in the formation of estrogens, which are C18 steroid hormones that can stimulate the development of female sexual characteristics. Second, rs1146031 to ACVR1 to mesoderm formation and activin binding (p < 0.001, FDR = 0.032, 0.034). Two of 15 candidate genes are known genes associated with schizophrenia: KCNQ2 and APOL2. One of the 10 candidate pathways, estrogen biosynthetic process, is known to be associated with schizophrenia (p < 0.001, FDR < 0.001). However, 13 of candidate genes (RDH8, ACVR1, PSMD9, KCNAB1, SLC17A3, ARCN1, COG7, STAB2, LRPAP1, STAB1, CXCL16, COL4A4, EXOSC3) and 9 of candidate pathways were novel.

Conclusion

By applying ICSNPathway analysis to schizophrenia GWAS data, we identified candidate SNPs, genes like KCNQ2 and APOL2 and pathways involving the estrogen biosynthetic process may contribute to schizophrenia susceptibility. Further analyses are needed to validate the results of this analysis.     

Cross-sectional Seroprevalence and Genotype of Hepatitis E Virus in Humans and Swine in a High-density Pig-farming Area in Central China

Hepatitis E virus(HEV) infection is a common public health problem in developing countries. However, the current prevalence of HEV and the relationship of HEV genotype between swine and human within high-density pig-farming areas in central China are still inadequately understood. Here, cross-sectional serological and genotypic surveys of HEV among the 1232 general population, 273 workers occupationally exposed to swine, and 276 pigs in a high-density pig-breeding area, were undertaken by ELISA and nested RT-PCR methods. Anti-HEV IgG was detected in 26.22% of general population and 48.35% of occupational workers. The prevalence of swine serum HEV-Ag was 6.52%. The prevalence of anti-HEV IgG was significantly higher among the workers occupationally exposed to swine than among the general population. An increased HEV seropositivity risk among the general population was associated with either being a peasant or male and was very strongly associated with the increase of age. Among the occupationally exposed group, the prevalence of anti-HEV IgG antibodies increased with age and working years. Among the 30 HEV-IgM-positive people,the infection rates of clerks in the public, peasants, pork retailers, and pig farmers were higher than those of others. A phylogenetic analysis revealed that all the isolates belonged to subgenotype 4 d, and four people and four pigs shared97.04%–100% sequence homology. This study revealed a high HEV seroprevalence among the general population and workers occupationally exposed to swine in the Anlu City, and supports the notion that swine are a source of human HEV infection.     

Distribution-free Estimation of a Monotonic pth Quantile Function

When analyzing biological data sets, a frequent problem is to estimate the pth quantile of a distribution, when that quantile is assumed to depend on a covariate; in the present paper the dependence of the quantile on the covariate is assumed to be monotonic. Some properties of an isotonic pth quantile regression, considered as an estimator of an increasing pth quantile function, are presented.     

Psychotic experiences as a predictor of the natural course of suicidal ideation: a Swedish cohort study

Psychotic experiences are far more prevalent in the population than psychotic disorders and are associated with a wide range of depressive, anxiety and behavioral disorders, as well as increased risk for psychotic disorder. Recently, psychotic experiences have been highlighted as a potentially valuable clinical marker of risk for suicidal behavior. There have been few studies to date, however, to assess psychotic experiences as a predictor of suicidality over time. We wished to assess whether young persons with suicidal ideation at baseline assessment who reported psychotic experiences were at higher risk for persistence of suicidal ideation at follow‐up than young persons who also reported suicidal ideation at baseline but who did not report co‐occurring psychotic experiences. A total of 2,263 adolescents were assessed at age 13 to 14 years for psychotic experiences, suicidal ideation and internalizing and externalizing psychopathology. Participants were re‐assessed at ages 16 to 17 years and 19 to 20 years. Among 13‐ to 14‐year olds with suicidal ideation, co‐occurring psychotic experiences did not predict an increased odds of persistence of suicidal ideation to age 16 to 17 years (OR=0.94, 95% CI: 0.19‐4.78). Among 16‐ to 17‐year olds with suicidal ideation, however, co‐occurring psychotic experiences predicted a 6‐fold increased odds of persistence of suicidal ideation to age 19 to 20 years (OR=5.53, 95% CI: 1.33‐23.00). Psychotic experiences are an important but under‐recognized marker of risk for persistence of suicidal ideation, in particular from mid‐adolescence. An increased emphasis on the clinical assessment of psychotic experiences in mental health services should be a priority.     

Genetic susceptibility to heroin addiction: a candidate gene association study

Heroin addiction is a chronic complex disease with a substantial genetic contribution. This study was designed to identify genetic variants that are associated with susceptibility to develop heroin addiction by analyzing 1350 variants in 130 candidate genes. All subjects had Caucasian ancestry. The sample consisted of 412 former severe heroin addicts in methadone treatment, and 184 healthy controls with no history of drug abuse. Nine variants, in six genes, showed the lowest nominal P values in the association tests (P < 0.01). These variants were in noncoding regions of the genes encoding the mu (OPRM1; rs510769 and rs3778151), kappa (OPRK1; rs6473797) and delta (OPRD1; rs2236861, rs2236857 and rs3766951) opioid receptors; the neuropeptide galanin (GAL; rs694066); the serotonin receptor subtype 3B (HTR3B; rs3758987) and the casein kinase 1 isoform epsilon (CSNK1E; rs1534891). Several haplotypes and multilocus genotype patterns showed nominally significant associations (e.g. OPRM1; P = 0.0006 and CSNK1E; P = 0.0007). Analysis of a combined effect of OPRM1 and OPRD1 showed that rs510769 and rs2236861 increase the risk of heroin addiction (P = 0.0005). None of these associations remained significant after adjustment for multiple testing. This study suggests the involvement of several genes and variants in heroin addiction, which is worthy of future study.     

Decreased pulmonary diffusing capacity of divers over a 6-year period

Non-smoking, male, professional firemen divers (n = 15) underwent two pulmonary function tests (PFT) separated by 6 years. Measured data were compared to European Coal Steel Community recommended reference values to permit cross-sectional and then longitudinal study. Higher vital capacity (VC; P < 0.01) and forced expiratory volume in 1 s (FEV₁; P < 0.05), and lower maximal mid-expiratory flow (MMEF) coefficient with VC (MMEF/VC; P < 0.05) were observed in both PFT. Diver's pulmonary diffusing capacity (DL_CO) and the coefficient with alveolar volume (DL_CO/V _A) showed significantly (P < 0.001) different evolution profiles than those expected from predicted values. In divers, DL_CO and DL_CO/V _A decreased from 104.0% to 91.4% and from 106.4% to 91.5% of predicted values respectively. Changes in DL_CO and DL_CO/V _A correlated positively with the initial measurement of DL_CO (r = 0.67, P < 0.01) and DL_CO/V _A (r = 0.74, P < 0.01) respectively, whereas no correlation between changes in pulmonary gas transfer function and age or diving history parameters was found. Thus, it is suggested from our observations that hyperbaric atmosphere exposure increases the effects of aging on pulmonary diffusing capacity and that pulmonary gas transfer function should be regularly tested in professional and recreational divers. Accepted: 22 February 1997     

SNP-based heritability estimation using a Bayesian approach

Heritability is a central element in quantitative genetics. New molecular markers to assess genetic variance and heritability are continually under development. The availability of molecular single nucleotide polymorphism (SNP) markers can be applied for estimation of variance components and heritability on population, where relationship information is unknown. In this study, we evaluated the capabilities of two Bayesian genomic models to estimate heritability in simulated populations. The populations comprised different family structures of either no or a limited number of relatives, a single quantitative trait, and with one of two densities of SNP markers. All individuals were both genotyped and phenotyped. Results illustrated that the two models were capable of estimating heritability, when true heritability was 0.15 or higher and populations had a sample size of 400 or higher. For heritabilities of 0.05, all models had difficulties in estimating the true heritability. The two Bayesian models were compared with a restricted maximum likelihood (REML) approach using a genomic relationship matrix. The comparison showed that the Bayesian approaches performed equally well as the REML approach. Differences in family structure were in general not found to influence the estimation of the heritability. For the sample sizes used in this study, a 10-fold increase of SNP density did not improve precision estimates compared with set-ups with a less dense distribution of SNPs. The methods used in this study showed that it was possible to estimate heritabilities on the basis of SNPs in animals with direct measurements. This conclusion is valuable in cases when quantitative traits are either difficult or expensive to measure.     

发展性阅读障碍的产生机制——从行为到遗传研究

发展性阅读障碍(developmental dyslexia,DD)是一种特殊的学习障碍,探索DD的产生机制有助于寻求DD儿童的鉴别和治疗方法.目前拼音文宁国家对DD的产生机制研究很多,结果也很丰富,但是很多观点还不一致.汉语DD研究起步较晚,各方面还不够深入和完善.简述了DD在认知、脑神经和基因方面的研究进展,对拼音文字DD与汉语DD的研究结果进行了对比,以揭示不同语言文字系统下DD者的认知神经差异.研究认为,应该大力加强对汉语DD的探究,这样不但为中国DD儿童的诊治提供理论基础,也可以为DD的语种特异性问题提供科学依据.