Effects of transient focal cerebral ischemia in mice deficient in puma

Bcl-2 homology domain 3 (BH3)-only pro-apoptotic proteins may play an important role in upstream cell death signaling pathways underlying ischemic brain injury. Puma is a potent BH3-only protein that can be induced via p53, FoxO3a and endoplasmic reticulum stress pathways and is upregulated by global cerebral ischemia. To more completely define the contribution of Puma to ischemic brain injury we measured the expressional response of Puma to transient focal cerebral ischemia in mice and also compared infarct volumes in puma-deficient versus puma-expressing mice. Real-time quantitative PCR determined puma mRNA levels were significantly increased 8 h after 90 min middle cerebral artery (MCA) occlusion in the ipsilateral cortex, while expression remained unchanged contralaterally. Puma protein levels were also increased in the ischemic cortex over the same period. However, cortical and striatal infarct volumes were not significantly different between puma-deficient and puma-expressing mice at 24 h, and no differences between genotypes were found for post-ischemic neurological deficit scores. These data demonstrate that focal cerebral ischemia is associated with puma induction but suggest that Puma does not contribute significantly to lesion development in the present model.     

Postnatal changes in expression of vesicular glutamate transporters in the main olfactory bulb of the rat

Olfactory information is initially processed through intricate synaptic interactions between glutamatergic projection neurons and GABAergic interneurons in the olfactory bulb. Although bulbar neurons and networks have been reported to develop even postnatally, much is yet unknown about the glutamatergic neuron development. To address this issue, we studied the postnatal ontogeny of vesicular glutamate transporters (VGLUT1 and VGLUT2) in the main olfactory bulb of rats, using in situ hybridization, immunohistochemistry, and their combination. In situ hybridization data showed that VGLUT1 mRNA is intensely expressed in differentiating mitral cells and smaller cells of the mitral cell layer (MCL) on postnatal day 1 (P1), and also at lower levels in small- and medium-sized cells, presumably tufted cell populations, of the external plexiform layer (EPL) from P5 onward. VGLUT2 mRNA was expressed in many MCL cell populations on P1, also in small- and medium-sized cells of the EPL at almost the same level as MCL cells between P5 and P7, and became apparently less intense in the MCL than in the EPL from P10 onward. The expression, unlike VGLUT1 mRNA, was also found in small-sized cells of the interglomerular region. In partial agreement with these data, immunohistochemical analyses demonstrated that subsets of mitral and EPL cells are stained for VGLUT1 or VGLUT2, with the former cells coexpressing both subtypes until P5. Moreover, a combined fluorescence in situ hybridization–immunohistochemical dual labeling of the P10 bulb revealed that neither VGLUT1 nor VGLUT2 mRNA is expressed in GABAergic or dopaminergic periglomerular cells, implying their expression in other periglomerular cell subclasses, external tufted cells and/or short-axon cells. Thus, the present study suggests that early in the postnatal development distinct glutamatergic bulbar neurons of rats express spatiotemporally either or both of the two VGLUT subtypes as a specific vesicular transport system, specifically contributing to glutamate-mediated neurobiological events.     

Evaluation of New Preanalysis Sample Treatment Tools and DNA Isolation Protocols To Improve Bacterial Pathogen Detection in Whole Blood

Two novel preanalysis sample treatment tools were evaluated in combination with four DNA extraction kits for the selective isolation of bacterial DNA from whole blood. The combination of performing a preanalysis sample treatment and using a larger sample volume increased the detection limit to 50 CFU per ml.New approaches using molecular technologies are continuously being developed to improve the diagnosis of bloodstream infections. A critical issue in the success of the application of molecular methods is the sample treatment and/or nucleic acid isolation (2). The low concentration of pathogens and the presence of PCR-inhibitory compounds in blood are important challenges that should be dealt with during sample treatment (1, 6, 7, 9, 10). This could be done with so-called preanalysis sample treatment tools, which combine the selective enrichment of bacterial DNA from blood with the integrated, highly efficient removal of PCR inhibitors. The aim of this study was to determine whether the addition of a preanalysis sample treatment to a selective DNA extraction protocol could improve the amplification and detection of bacterial DNA in whole-blood samples.MolYsis Basic (MolZym GmbH & Co. KG, Bremen, Germany) and Looxster (SIRS-lab GmbH, Jena, Germany) were evaluated as novel preanalysis sample treatment tools in combination with the QIAamp DNA blood mini kit (Qiagen GmbH, Hilden, Germany), MagNA Pure LC (MPLC) microbiology kit M^Grade (Roche Diagnostics GmbH, Mannheim, Germany), MolYsis Complete kit (MolZym GmbH & Co. KG, Bremen, Germany), and MagSi-DNA isolation kit for blood (MagnaMedics Diagnostics B.V., Maastricht, The Netherlands). A schematic overview of the performed analytical processes for whole blood spiked with methicillin (meticillin)-resistant Staphylococcus aureus is given in Fig. ​Fig.1.1. Bacterial DNA extraction was followed by amplification in a multiplex real-time PCR assay targeting three methicillin-resistant Staphylococcus aureus genes (mecA, femA, and sa442) (3). Real-time PCR was set up in a final volume of 50 μl with 2× Absolute QPCR ROX (500 nM) mix (Abgene, Epsom, United Kingdom). Primers and probes were purchased from Sigma-Genosys (Haverhill, United Kingdom) and Applied Biosystems (Nieuwerkerk a/d Ijssel, The Netherlands), respectively. Final reactions contained 0.6 μM of mecA primer and 0.3 μM of femA and sa442 primer; 100 nM, 125 nM, and 150 nM of femA, mecA, and sa442 probe, respectively; and 18.85 μl of template DNA. Optimal thermal-cycling conditions were as follows: initial denaturation at 95°C for 15 min, 42 cycles of denaturation for 15 s at 95°C, and annealing at 60°C for 1 min.Open in a separate windowFIG. 1.Schematic overview of the analytical process for whole-blood samples. (A) DNA extraction with two conventional kits, without preanalysis sample treatment. (B) DNA extraction with two conventional kits and one novel kit, following preanalysis sample treatment with MolYsis Basic. (C) DNA extraction with two conventional kits, followed by a preanalysis sample treatment with Looxster. (D) DNA extraction with a novel kit, which combines all the steps of the preanalysis sample treatment (MolYsis Basic) with a complementary matching DNA extraction protocol.The detection limit of all DNA extraction procedures was determined and is presented in Table ​Table1.1. Two conventional DNA extraction kits (QIAamp and MPLC) were tested with and without two different preanalysis sample treatment protocols. Preanalysis sample treatment, with either MolYsis Basic or Looxster, combined with QIAamp extraction, based on a spin-column format, did not result in an increase in detection limit. Both procedures performed equally and were able to detect three target genes at a minimal concentration of 10³ CFU per ml, which was analogous to 16 CFU per PCR. Recently, a study by Horz et al. investigated if MolYsis Basic could indeed eliminate human DNA in oral samples to improve the detection of bacterial DNA (4). They found that the use of MolYsis Basic prior to DNA isolation reduced the level of human DNA. However, this effect was accompanied with a partial loss of bacterial DNA. The MPLC kit performed the same as the QIAamp kit, i.e., a detection limit of 10³ CFU per ml. The MPLC kit in combination with MolYsis Basic could detect 500 CFU per ml. However, when combined with Looxster, the minimal detectable amount of bacteria was 10⁴ CFU per ml (data not shown). This was most likely due to the incompatibility of the Looxster kit and the MPLC elution buffer, which is essential to the performance of the MPLC kit.

TABLE 1.

Lower detection limits of the methicillin-resistant Staphylococcus aureus real-time PCR assay in relation to preanalysis sample treatment with MolYsis Basic/Looxster and the DNA extraction protocol used

Incorporating and evaluating an integrated gender-specific medicine curriculum: a survey study in Dutch GP training

Background  

We recently set standards for gender-specific medicine training as an integrated part of the GP training curriculum. This paper describes the programme and evaluation of this training.     

Policies to improve end-of-life decisions in Flemish hospitals: communication, training of health care providers and use of quality assessments

Background  

The prevalence and implementation of institutional end-of-life policies has been comprehensively studied in Flanders, Belgium, a country where euthanasia was legalised in 2002. Developing end-of-life policies in hospitals is a first step towards improving the quality of medical decision-making at the end-of-life. Implementation of policies through quality assessments, communication and the training and education of health care providers is equally important in improving actual end-of-life practice. The aim of the present study is to report on the existence and nature of end-of-life policy implementation activities in Flemish acute hospitals.     

GABAC receptors are expressed in GABAergic and non-GABAergic neurons of the rat superior colliculus and visual cortex

GABA_C receptors are enriched in the upper grey layers of the mammalian superior colliculus and contribute to synaptic processing. Electrophysiological data suggested that the GABA_C receptor ρ subunits are expressed by GABAergic interneurons which represent about half of the neurons in the stratum griseum superficiale (SGS). Combining in situ hybridization for ρ2 receptor mRNA and the glutamic acid decarboxylase GAD-65 mRNA confirmed this assumption. A majority of ρ-labeled neurons in SGS and pretectum are GABAergic. Combining in situ hybridization with immunohistochemistry for the two projection neuron markers calbindin and parvalbumin revealed that a few ρ2 mRNA expressing cells coexpressed calbindin, but not parvalbumin. In visual cortex, ρ2 mRNA was present in pyramidal neurons and parvalbumin-containing interneurons. The results show that in the SGS primarily GABAergic neurons express GABA_C receptors whereas the majority of tectothalamic calbindin neurons and intrinsically projecting parvalbumin neurons do not.     

Psychological distress, depression and generalised anxiety in Turkish and Moroccan immigrants in Belgium

Background

This study assesses the prevalence of and risk and protective factors for common mental health complaints in a general population sample of Turkish and Moroccan immigrants living in Belgium. Focus is on between- and within-group variation.

Methods

The study is based on pooled data from the Belgian Health Interview Surveys 2001 and 2004 and focuses on the Turkish and Moroccan immigrant population aged 18–65 (N = 147 Turks, N = 359 Moroccans). Mental health status is assessed with the General Health Questionnaire—12 and the Symptom Checklist 90-R subscales for depression and generalised anxiety. Risk and protective factors considered are gender, age, household type, labor market position, educational level, household income, homeownership, being foreign- or native born and social support.

Results

Between-group variance was not significant. Within-group analysis showed significant effects of gender and social support. Although not significant, the results suggested positive associations between social adversity and mood status. In addition, there was a tendency for higher risks for psychological distress, depression and generalised anxiety in foreign-born as compared to Belgian-born Turkish and Moroccan immigrants.     

Patterns of intellectual, adaptive and behavioral functioning in individuals with mild mental retardation

Many researchers have studied the population of individuals with mild mental retardation (MIMR) as if it is a clear entity. Few researchers have investigated potential subtypes within the MIMR population. The purpose of the present study was to investigate which subtypes can be identified on the basis of intellectual, adaptive and behavioral functioning. Seventy-three individuals with MIMR were assessed on measures of intellectual, adaptive and behavioral functioning. An agglomerative hierarchical cluster-analytic technique was used to define potential subgroups with characteristic behavioral patterns. Four subtypes were identified. The behavioral patterns are described and implications for assessment are discussed.