Setting out the frame conditions for feasible use of FFPE derived RNA

Introduction

The usage of formalin-fixed paraffin embedded (FFPE) tissue is characterized by its long shelf-life and simple handling. Therefore it is the most commonly available tissue specimen in routine diagnostics and histological studies. Formaldehyde fixation may result in RNA degradation and cross linking with proteins, while storage conditions also affect RNA integrity. The present study was designed to investigate the influence of these factors on RNA analysis.

Detection of cytomegaloviral DNA in human milk cells and cell free milk whey by nested PCR

Human cytomegalovirus (HCMV) DNA can be detected in different compartments of human milk. A protocol for the preparation of milk whey free of fat and cells for the detection of human cytomegalovirus (HCMV) by nested PCR is presented. This is based upon the experience of the separation of more than 200 milk specimens of healthy seropositive breast feeding mothers. HCMV DNA could be detected in freshly centrifuged and filtrated milk whey specimens without contamination by cellular DNA. In limiting dilution experiments using HCMV plasmid DNA, the effect of different DNA extraction procedures from native milk and milk whey on the detection limit of cytomegaloviral DNA was demonstrated. About 200 viral genome equivalents/ml in milk whey or native milk were detectable by classical organic phenol/chloroform extraction or a spin column method, respectively. The detection of viral DNA in milk cells depended on a minimum number of milk cells (10⁵–2×10⁵) available for DNA extraction. In contrast to the findings of cytomegaloviral DNA in native sera or plasma of immunosuppressed patients we failed to amplify low level viral DNA from native breast milk by nested PCR due to an inhibition of Taq polymerase by lipid components. Finally, the course of cell associated and cell free DNAlactia was monitored. Analyzing sequential milk specimens, in some cases the presence of HCMV DNA in colostrum could be demonstrated. DNAlactia of milk cells and whey was partially discordant. Onset (week 1–4 after delivery) and duration (2 weeks up to more than 3 months) of DNAlactia showed distinct individual patterns. The methods described, allow further analysis of the mechanisms involved in the postnatal HCMV transmission by breast feeding seropositive mothers.     

Comparison of agar-based media for primary isolation of glycopeptide-resistant enterococci

Objective: To compare four vancomycin-containing agar media for the isolation of glycopeptide-resistant enterococci (GRE) from clinical fecal specimens: kanamycin-aesculin-azide (KAA) agar; bile-aesculin-polymixin (BAP) agar; aztreonam-amphotericin blood (CBAA) agar; and neomycin blood (CBN) agar.
Methods: Fecal specimens from 125 patients were inoculated onto each medium. Media were examined for enterococci after incubation for up to 48 h. Enterococci were identified to species level, and glycopeptide phenotypes were determined by measuring minimum inhibitory concentrations of vancomycin and teicoplanin.
Results: GRE were isolated from 44/125 samples. Enterococcus faecalis and Enterococcus faecium isolates, expressing glycopeptide resistance of the VanA or VanB phenotypes, were recovered from 27/33 (82%) specimens on BAP medium, 26/33 (79%) on KAA medium, and 21/33 (64%) on CBN and CBAA media. Enterococcus gallinarum and Enterococcus casseliflavus isolates expressing low-level glycopeptide resistance (VanC phenotype) were recovered from 14/15 (93%) specimens on CBAA medium, 7/15 (47%) on KAA and CBN media, and 6/15 (40%) on BAP medium.
Conclusions: The media tested in this study, with the exception of CBN medium, detected at least 75% of patients colonized by GRE. Further development of BAP, CBAA and KAA media is warranted to improve growth and selectivity.     

Rare-male mating advantage: An artifact caused by differential storage conditions?

Virgin males (or females) of some species ofDrosophila, when stored singly, are known to be superior in mating to males (or females) stored in groups. This may create a spurious rare-male effect on some occasions. When no account is taken of this storage effect in an experimental setup designed to show rare-male mating advantage, bias in favor of a raremale effect may result. It is shown that merely by storing the rare males singly and the common males in groups, with males not differing in any other respect, a very strong spurious rare-male effect can be produced. Similarly, it is shown that a spurious rare-female effect is possible too. It is proposed that the very strong rare-male effect for pepperment scent as found by Dal Molin [(1979).Am. Nat. 113:951–954] is merely a result of such a bias. The relevance for natural populations of the mating advantage associated with the single housing condition is discussed. In the experiments designed to show a spurious rare-male effect, mating chambers of two different sizes were used. It is shown that sexual selection is more severe in the small chambers, for both males and females.     

Rewarding effects of the optical isomers of 3,4-methylenedioxy-methylamphetamine ('Ecstasy') and 3,4-methylenedioxy-ethylamphetamine ('Eve') measured by conditioned place preference in rats

3,4-methylenedioxy-methylamphetamine (MDMA) (‘Ecstasy’) and its analogue 3,4-methylenedioxy-methylamphetamine (MDE) (‘Eve’) are well known illicit street drugs mainly abused by young people. In spite of the actual research going on, the classification of their abuse potential remains unclear. Since secondary reinforcers are the main factors responsible for craving and relapse, the aim of our study was to assess the potency of MDMA and MDE in a second order reinforcement paradigm, i.e. conditioned place preference (CPP). For the general assessment of our study conditions, we compared MDMA with amphetamine. Unexpectedly, no significant CPP for MDMA was found in contrast to amphetamine. Detailed analysis of current literature led us to the working hypothesis that social environment is crucial for the development of CPP. In a subsequent experiment we tested the influence of housing conditions on CPP using MDMA and demonstrated that isolated animals show significant CPP compared to group-housed ones. In order to better understand the rewarding mechanisms of Ecstasy-derivatives, we tested both the racemic drugs and the pure isomers in the CPP paradigm. Both MDMA's optical isomers and racemic MDMA showed significant CPP without notable differences, while MDE and its isomers completely failed to show any significant CPP. In conclusion, the mechanism by which MDMA induces addiction is much more complicated than assumed so far and more pronounced in isolated animals. The fact that both optical isomers of MDMA led to CPP implies that at least two pathways by which MDMA induces craving behaviour exist.     

汉区少数民族学生文化疏离感研究

目的:提出文化疏离感的操作性定义和初步的理论构想,为我国少数民族的文化认同和文化适应,以及少数民族青少年的教育培养提供比较重要的参考资料,为疏离感理论的丰富起到一定积极作用.方法:自编<汉区少数民族学生文化疏离感量表>,进行信效度考察以后对678名汉区少数民族学生进行调查.结果:1.文化疏离感及其四个子维度在地区主效应上均表现出显著差异,在年龄主效应上差异不显著;文化分离感和不和谐感在性别主效应上差异显著.2.文化疏离感及其两个子维度(文化孤立感和文化分离感)在年龄和地区,以及在年龄、地区和性别的交互作用上均表现出显著差异.3.影响文化疏离感及其文化孤立感、文化分离感、被控感三个维度的主要变量是学生小学前后居住地区的改变,而影响不和谐感的主要变量是性别.结论:小学前后居住地的改变会影响少数民族学生文化疏离感.     

Cell cultures for diagnosis of arbovirus infections in livestock and wildlife

Summary Arboviruses can be isolated in serially propagated cells derived from various vertebrates and invertebrates. Cell cultures can be used for direct detection of antigen by fluorescent antibody and enzyme-linked immunosorbent assays, for nucleic acid hybridization, and for visualization of viruses with electron microscopy. Reagents for enzyme-linked immunosorbent assays for IgM and IgG antibodies, hemagglutination-inhibition, complement fixation, and serum dilution-plaque reduction neutralization tests can be prepared in cell cultures infected with these viruses. Thus, cell cultures can be used as laboratory hosts for essentially all isolation, identification, and serodiagnostic procedures for arboviruses. This paper outlines current methods for diagnosis of arbovirus infections in livestock and wildlife, describes certain of these techniques, and provides references for others.     

The effects of storage of blood and isolated DNA on the integrity of DNA

Long-term storage of DNA is required for a number of genetic studies; prior to extraction, blood samples may be subject to elevated temperatures for variable intervals. We have studied the effect of temperatures ranging from ?70°C to +65°C on human blood and on DNA extracted from it. DNA in solution stored at ambient temperatures up to 37°C for 6 months was digestible by three different restriction endonucleases, whereas storage at 45°C is deleterious after 6-7 weeks. DNA can be extracted from blood samples stored at ?70°C for at least 2 months or at 23°C for a week or more, but blood stored at these temperatures may yield less high-molecular-weight DNA. Cell pellets from which plasma has been removed also can serve as a source of DNA. Isolated DNA stored dry for years (up to 30) is difficult to dissolve and may appear degraded, but a sample stored dry for 13 years and then in solution at ?20°C for 7 years appeared to be intact.