Development of nested multiplex polymerase chain reaction (PCR) assay for the detection of Toxocara canis, Toxocara cati and Ascaris suum contamination in meat and organ meats

Ascarid Larva Migrans Syndrome (ascarid LMS) is a clinical syndrome in humans, caused by the migration of animal roundworm larvae such as Toxocara canis, Toxocara cati and Ascaris suum. Humans may acquire infection by ingesting embryonated eggs, or infective larvae of these parasites in contaminated meat and organ meats. To detect these pathogenic contaminations, a novel nested multiplex PCR system was developed. Our novel nested multiplex PCR assay showed specific amplification of T. canis, T. cati and Ascaris spp. Detection limit of the nested multiplex PCR was tested with serial dilution of T. canis, T. cati or A. suum genomic DNA (gDNA) from 100?pg to 100 ag and found to be 10?fg, 1?fg and 100?fg, respectively. When larvae were spiked into chicken liver tissue, DNA of T. canis and A. suum was detected from the liver spiked with a single larva, while the assay required at least 2 larvae of T. cati. Moreover, the ascarid DNA was detected from the liver of mice infected with 100 and 300 eggs of T. canis, T. cati or A. suum. This nested multiplex PCR assay could be useful for the detection of contamination with ascarid larvae in meat and organ meats.     

不同能源柳无性系对土壤镉污染的抗性研究

运为了比较不同能源柳无性系对土壤镉抗性的差异,采用盆栽方法,设置土壤镉含量5个梯度(0、10、20、40、80 mg·kg^-1),以乡土旱柳为对照,对能柳1、能柳2、能柳E、能柳C的扦插幼苗对土壤镉污染的生理生态反应进行了系统测定,分析了不同无性系的抗性。结果显示,低浓度处理（≤20 mg·kg^-1）促进苗木生长,其中能柳2在10 mg·kg^-1处理下根和单株生物量增幅最大,可达到对照的132.0%和120.0%;随着镉浓度的增加,生长量下降,在最高浓度处理下,能柳C生长量降低最少,根、枝、叶和单株生物量分别为对照的86.1%、85.3%、82.9%、84.9%,旱柳的枝、叶生物量降低最多,为对照（无镉）的43.8%、45.0%,而能柳1的根、单株生物量降低最多,仅为对照的36.1%、44.4%;随着镉浓度的增加,不同能源柳无性系叶片的SOD、POD、CAT活性和根系活力基本呈现先升后降的趋势,其中,CAT活性变化较为平缓;关于SOD活性和根系活力,能柳1、能柳2在20 mg·kg^-1时已显著下降,旱柳在40 mg·kg^-1时显著下降,而能柳E、能柳C在40 mg·kg^-1时才显著下降。采用隶属函数对5个无性系在镉胁迫条件下的生长和酶指标进行综合分析,不同无性系对土壤镉污染的抗性顺序为能柳C>能柳2>能柳1>旱柳>能柳E。不同无性系对镉均有抗性,都可在镉污染区推广种植,栽培中,应根据土壤污染和绿化目标,做出适当选择。     

The application of strand invasion phenomenon,directed by peptide nucleic acid (PNA) and single‐stranded DNA binding protein (SSB) for the recognition of specific sequences of human endogenous retroviral HERV‐W family

The HERV‐W family of human endogenous retroviruses represents a group of numerous sequences that show close similarity in genetic composition. It has been documented that some members of HERV‐W–derived expression products are supposed to play significant role in humans' pathology, such as multiple sclerosis or schizophrenia. Other members of the family are necessary to orchestrate physiological processes (eg, ERVWE1 coding syncytin‐1 that is engaged in syncytiotrophoblast formation). Therefore, an assay that would allow the recognition of particular form of HERV‐W members is highly desirable. A peptide nucleic acid (PNA)–mediated technique for the discrimination between multiple sclerosis‐associated retrovirus and ERVWE1 sequence has been developed. The assay uses a PNA probe that, being fully complementary to the ERVWE1 but not to multiple sclerosis‐associated retrovirus (MSRV) template, shows high selective potential. Single‐stranded DNA binding protein facilitates the PNA‐mediated, sequence‐specific formation of strand invasion complex and, consequently, local DNA unwinding. The target DNA may be then excluded from further analysis in any downstream process such as single‐stranded DNA‐specific exonuclease action. Finally, the reaction conditions have been optimized, and several PNA probes that are targeted toward distinct loci along whole HERV‐W env sequences have been evaluated. We believe that PNA/single‐stranded DNA binding protein–based application has the potential to selectively discriminate particular HERV‐W molecules as they are at least suspected to play pathogenic role in a broad range of medical conditions, from psycho‐neurologic disorders (multiple sclerosis and schizophrenia) and cancers (breast cancer) to that of an auto‐immunologic background (psoriasis and lupus erythematosus).     

Pitfalls in the sample preparation and analysis of N-acylethanolamines

N-acylethanolamines (NAEs) are a group of lipid mediators synthesized in response to a number of physiological and pathological stimuli. Because of the low tissue concentrations of NAEs, analyses often include liquid extraction followed by solid-phase extraction and subsequent quantitation by LC/MS or GC/MS. Reported levels of NAEs vary considerably, however, and often no explanation is given for these discrepancies. Brought on by difficulties encountered during method development, the effects of using four different brands of silica-containing solid phase extraction (SPE) columns and five different brands of chloroform for sample preparation were investigated. Considerable variation in the retention and recoveries of seven NAEs and 2-arachidonoylglycerol existed between the SPE columns. Furthermore, it was found that some chloroforms contained quantifiable amounts of N-palmitoylethanolamine and N-stearoylethanolamine. Finally, it was found that use of one of the chloroforms resulted in a loss of N-oleoylethanolamine from solution due to addition of chlorine to the ω-9 bond. The identity of this reaction product was confirmed by LC-MS/MS and NMR. It is recommended that these aspects of sample preparation and analysis should be thoroughly validated during method development and the relevant information on specific brands used be reported in future communications in order to better estimate the validity of reported quantitative data.     

Tracking down human contamination in ancient human teeth

DNA contamination arising from the manipulation of ancient calcified tissue samples is a poorly understood, yet fundamental, problem that affects the reliability of ancient DNA (aDNA) studies. We have typed the mitochondrial DNA hypervariable region I of the only 6 people involved in the excavation, washing, and subsequent anthropological and genetic study of 23 Neolithic remains excavated from Granollers (Barcelona, Spain) and searched for their presence among the 572 clones generated during the aDNA analyses of teeth from these samples. Of the cloned sequences, 17.13% could be unambiguously identified as contaminants, with those derived from the people involved in the retrieval and washing of the remains present in higher frequencies than those of the anthropologist and genetic researchers. This finding confirms, for the first time, previous hypotheses that teeth samples are most susceptible to contamination at their initial excavation. More worrying, the cloned contaminant sequences exhibit substitutions that can be attributed to DNA damage after the contamination event, and we demonstrate that the level of such damage increases with time: contaminants that are >10 years old have approximately 5 times more damage than those that are recent. Furthermore, we demonstrate that in this data set, the damage rate of the old contaminant sequences is indistinguishable from that of the endogenous DNA sequences. As such, the commonly used argument that miscoding lesions observed among cloned aDNA sequences can be used to support data authenticity is misleading in scenarios where the presence of old contaminant sequences is possible. We argue therefore that the typing of those involved in the manipulation of the ancient human specimens is critical in order to ensure that generated results are accurate.     

Benzodiazepines in the brain

Great progress has been made in the last 5 yr in demonstrating the presence of benzodiazepines (BDZs) in mammalian tissues, in beginning studies on the origin of these natural compounds, and in elucidating their possible biological roles. Many unanswered questions remain regarding the sources and biosynthetic pathways responsible for the presence of BDZs in brain and their different physiological and/or biochemical actions. This essay will focus on recent findings supporting that: (1) BDZs are of natural origin; (2) mammalian brain contains BDZs in concentrations ranging between 5.10⁻¹⁰–10⁻⁸ M; (3) dietary source of BDZs might be a plausible explanation for their occurrence in animal tissues, including man; (4) the formation of BDZ-like molecules in brain is a possibility, experimentally supported; (5) BDZ-like molecules including diazepam andN-desmethyldiazepam are elevated in hepatic encephalopathy; and (6) natural BDZs in the brain are involved in the modulation of memory processes. Future studies using the full range of biochemical, physiological, behavioral, and molecular biological techniques available to the neuroscientist will hopefully continue to yield exciting and new information concerning the biological roles that BDZs might play in the normal and pathological functioning of the brain.     

贵州玉米籽粒镰刀菌污染情况调查

【背景】镰刀菌引起的作物病害在贵州省内时有发生,由于该地区低温高湿的气候特征并不适宜镰刀菌生长,之前未系统开展镰刀菌侵染和分布情况调查。【目的】调查贵州省玉米籽粒中镰刀菌病害的污染情况。【方法】收集贵州省58个县/县级市的玉米籽粒样品78份,利用原核表达的可用于检测镰刀菌的Fv SG7-AP融合蛋白对样品进行快速免疫学检测,并对部分样品进行生物学培养鉴定。【结果】共有67份样品检测结果为阳性,污染率高达85.90%,生物学培养后显微镜观察到镰刀菌菌丝和孢子。检测出镰刀菌污染的玉米样品分布在贵州省51个县/县级市,其中19个地区检测到轻度污染样品,20个地区检测到中度污染样品,12个地区检测到重度污染样品。【结论】贵州省绝大部分地区存在不同程度的镰刀菌污染,有必要在作物耕种、收获及贮藏期间采取有效的防控措施,以保障食品安全与人畜健康。     

Pesticide Residues in Agricultural Produce in Hubei Province,PR China

Pesticide residues in agricultural produce were examined in two villages of Qianjiang municipality, Hubei province, PR China. Six food groups were sampled from the fields prior to harvesting from the farmers' market and from farmers' homes. Organochlorine residues were detected in almost all examined food groups, with residue levels significantly lower than the national tolerances (MRL). Total BHC residues were on average 31.7?µg/kg and DDT 102.5 (µg/kg. Organophosphorus residues were detected in vegetable samples. Mean residue levels of phoxim and methamidophos in vegetables were 89.9?µg/kg and 36.5?µg/kg, respectively, both exceeding the MRL. Estimated daily intakes (EDIs) of pesticide residues per person, based on the six food groups sampled, were 4.88?mg for total DDT, 2.04 (µg for total BHC, and 19.33 (µg for methamidophos. The overall results show that the increased use and misuse of pesticides for crop protection in Qianjiang municipality, notably in vegetable production, have led to worrisome levels of pesticide contamination of agricultural produce that may carry certain risks for human health.     

苹果坐果的激素调控

以红富士苹果为试材,花朵数为5的花序,其坐果率明显高于花朵数为4的花序。开花前,花朵数为5的花序,其花朵子房内GAs,、Z浓度明显高于后者;开花后,后者GAs仍然低于前者,但Z浓度、Z／GAs比值则迅速增加并明显高于前者。蕾期喷施GA4 7DCPTA、Glu、SA可以明显提高花朵数为4的花序的坐果率。