In an in vitro model of human tuberculosis, monocyte-microglial networks regulate matrix metalloproteinase-1 and -3 gene expression and secretion via a p38 mitogen activated protein kinase-dependent pathway.

BACKGROUND: Tuberculosis (TB) of the central nervous system (CNS) is characterized by extensive tissue inflammation, driven by molecules that cleave extracellular matrix such as matrix metalloproteinase (MMP)-1 and MMP-3. However, relatively little is known about the regulation of these MMPs in the CNS. METHODS: Using a cellular model of CNS TB, we stimulated a human microglial cell line (CHME3) with conditioned medium from Mycobacterium tuberculosis-infected primary human monocytes (CoMTb). MMP-1 and MMP-3 secretion was detected using ELISAs confirmed with casein zymography or western blotting. Key results of a phospho-array profile that detects a wide range of kinase activity were confirmed with phospho-Western blotting. Chemical inhibition (SB203580) of microglial cells allowed investigation of expression and secretion of MMP-1 and MMP-3. Finally we used promoter reporter assays employing full length and MMP-3 promoter deletion constructs. Student's t-test was used for comparison of continuous variables and multiple intervention experiments were compared by one-way ANOVA with Tukey's correction for multiple pairwise comparisons. RESULTS: CoMTb up-regulated microglial MMP-1 and MMP-3 secretion in a dose- and time-dependent manner. The phospho-array profiling showed that the major increase in kinase activity due to CoMTb stimulation was in p38 mitogen activated protein kinase (MAPK), principally the α and γ subunits. p38 phosphorylation was detected at 15 minutes, with a second peak of activity at 120 minutes. High basal extracellular signal-regulated kinase activity was further increased by CoMTb. Secretion and expression of MMP-1 and MMP-3 were both p38 dependent. CoMTb stimulation of full length and MMP-3 promoter deletion constructs demonstrated up-regulation of activity in the wild type but a suppression site between -2183 and -1612 bp. CONCLUSIONS: Monocyte-microglial network-dependent MMP-1 and MMP-3 gene expression and secretion are dependent upon p38 MAPK in tuberculosis. p38 is therefore a potential target for adjuvant therapy in CNS TB

Do female association preferences predict the likelihood of reproduction?

Sexual selection acting on male traits through female mate choice is commonly inferred from female association preferences in dichotomous mate choice experiments. However, there are surprisingly few empirical demonstrations that such association preferences predict the likelihood of females reproducing with a particular male. This information is essential to confirm association preferences as good predictors of mate choice. We used green swordtails (<i>Xiphophorus helleri</i>) to test whether association preferences predict the likelihood of a female reproducing with a male. Females were tested for a preference for long- or short-sworded males in a standard dichotomous choice experiment and then allowed free access to either their preferred or non-preferred male. If females subsequently failed to produce fry, they were provided a second unfamiliar male with similar sword length to the first male. Females were more likely to reproduce with preferred than non-preferred males, but for those that reproduced, neither the status (preferred/non-preferred) nor the sword length (long/short) of the male had an effect on brood size or relative investment in growth by the female. There was no overall preference based on sword length in this study, but male sword length did affect likelihood of reproduction, with females more likely to reproduce with long- than short-sworded males (independent of preference for such males in earlier choice tests). These results suggest that female association preferences are good indicators of female mate choice but that ornament characteristics of the male are also important

Homozygosity for a missense mutation in the 67 kDa isoform of glutamate decarboxylase in a family with autosomal recessive spastic cerebral palsy: parallels with Stiff-Person Syndrome and other movement disorders

Background Cerebral palsy (CP) is an heterogeneous group of neurological disorders of movement and/or posture, with an estimated incidence of 1 in 1000 live births. Non-progressive forms of symmetrical, spastic CP have been identified, which show a Mendelian autosomal recessive pattern of inheritance. We recently described the mapping of a recessive spastic CP locus to a 5 cM chromosomal region located at 2q24-31.1, in rare consanguineous families. Methods Here we present data that refine this locus to a 0.5 cM region, flanked by the microsatellite markers D2S2345 and D2S326. The minimal region contains the candidate gene GAD1, which encodes a glutamate decarboxylase isoform (GAD67), involved in conversion of the amino acid and excitatory neurotransmitter glutamate to the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Results A novel amino acid mis-sense mutation in GAD67 was detected, which segregated with CP in affected individuals. Conclusions This result is interesting because auto-antibodies to GAD67 and the more widely studied GAD65 homologue encoded by the GAD2 gene, are described in patients with Stiff-Person Syndrome (SPS), epilepsy, cerebellar ataxia and Batten disease. Further investigation seems merited of the possibility that variation in the GAD1 sequence, potentially affecting glutamate/GABA ratios, may underlie this form of spastic CP, given the presence of anti-GAD antibodies in SPS and the recognised excitotoxicity of glutamate in various contexts

Matrix metalloproteinase-9 activity and a downregulated Hedgehog pathway impair blood-brain barrier function in an <i>in vitro</i> model of CNS tuberculosis

Central nervous system tuberculosis (CNS TB) has a high mortality and morbidity associated with severe inflammation. The blood-brain barrier (BBB) protects the brain from inflammation but the mechanisms causing BBB damage in CNS TB are uncharacterized. We demonstrate that Mycobacterium tuberculosis (Mtb) causes breakdown of type IV collagen and decreases tight junction protein (TJP) expression in a co-culture model of the BBB. This increases permeability, surface expression of endothelial adhesion molecules and leukocyte transmigration. TJP breakdown was driven by Mtb-dependent secretion of matrix metalloproteinase (MMP)-9. TJP expression is regulated by Sonic hedgehog (Shh) through transcription factor Gli-1. In our model, the hedgehog pathway was downregulated by Mtb-stimulation, but Shh levels in astrocytes were unchanged. However, Scube2, a glycoprotein regulating astrocyte Shh release was decreased, inhibiting Shh delivery to brain endothelial cells. Activation of the hedgehog pathway by addition of a Smoothened agonist or by addition of exogenous Shh, or neutralizing MMP-9 activity, decreased permeability and increased TJP expression in the Mtb-stimulated BBB co-cultures. In summary, the BBB is disrupted by downregulation of the Shh pathway and breakdown of TJPs, secondary to increased MMP-9 activity which suggests that these pathways are potential novel targets for host directed therapy in CNS TB

A pilot study of rivastigmine in the treatment of delirium after stroke: A safe alternative

<p>Abstract</p> <p>Background</p> <p>Delirium is a common disorder in the early phase of stroke. Given the presumed cholinergic deficiency in delirium, we tested treatment with the acetylcholinesterase inhibitor rivastigmine.</p> <p>Methods</p> <p>This pilot study was performed within an epidemiological study. In 527 consecutive stroke patients presence of delirium was assessed during the first week with the confusion assessment method. Severity was scored with the delirium rating scale (DRS). Sixty-two patients developed a delirium in the acute phase of stroke. Only patients with a severe and persistent delirium (defined as a DRS of 12 or more for more than 24 hours) were enrolled in the present study. In total 26 fulfilled these criteria of whom 17 were treated with orally administered rivastigmine with a total dose between 3 and 12 mg a day. Eight patients could not be treated because of dysphagia and one because of early discharge.</p> <p>Results</p> <p>No major side effects were recorded. In 16 patients there was a considerable decrease in severity of delirium. The mean DRS declined from 14.8 on day one to 8.5 after therapy and 5.6 after tapering. The mean duration of delirium was 6.7 days (range; 2–17).</p> <p>Conclusion</p> <p>Rivastigmine is safe in stroke patients with delirium even after rapid titration. In the majority of patients the delirium improved after treatment. A randomized controlled trial is needed to establish the usefulness of rivastigmine in delirium after stroke.</p> <p>Trial registration</p> <p>Nederlands Trial Register NTR1395</p

A new framework for designing programmes of assessment

Research on assessment in medical education has strongly focused on individual measurement instruments and their psychometric quality. Without detracting from the value of this research, such an approach is not sufficient to high quality assessment of competence as a whole. A programmatic approach is advocated which presupposes criteria for designing comprehensive assessment programmes and for assuring their quality. The paucity of research with relevance to programmatic assessment, and especially its development, prompted us to embark on a research project to develop design principles for programmes of assessment. We conducted focus group interviews to explore the experiences and views of nine assessment experts concerning good practices and new ideas about theoretical and practical issues in programmes of assessment. The discussion was analysed, mapping all aspects relevant for design onto a framework, which was iteratively adjusted to fit the data until saturation was reached. The overarching framework for designing programmes of assessment consists of six assessment programme dimensions: Goals, Programme in Action, Support, Documenting, Improving and Accounting. The model described in this paper can help to frame programmes of assessment; it not only provides a common language, but also a comprehensive picture of the dimensions to be covered when formulating design principles. It helps identifying areas concerning assessment in which ample research and development has been done. But, more importantly, it also helps to detect underserved areas. A guiding principle in design of assessment programmes is fitness for purpose. High quality assessment can only be defined in terms of its goals

Avoiding Dangerous Missense: Thermophiles Display Especially Low Mutation Rates

Rates of spontaneous mutation have been estimated under optimal growth conditions for a variety of DNA-based microbes, including viruses, bacteria, and eukaryotes. When expressed as genomic mutation rates, most of the values were in the vicinity of 0.003–0.004 with a range of less than two-fold. Because the genome sizes varied by roughly 104-fold, the mutation rates per average base pair varied inversely by a similar factor. Even though the commonality of the observed genomic rates remains unexplained, it implies that mutation rates in unstressed microbes reach values that can be finely tuned by evolution. An insight originating in the 1920s and maturing in the 1960s proposed that the genomic mutation rate would reflect a balance between the deleterious effect of the average mutation and the cost of further reducing the mutation rate. If this view is correct, then increasing the deleterious impact of the average mutation should be countered by reducing the genomic mutation rate. It is a common observation that many neutral or nearly neutral mutations become strongly deleterious at higher temperatures, in which case they are called temperature-sensitive mutations. Recently, the kinds and rates of spontaneous mutations were described for two microbial thermophiles, a bacterium and an archaeon. Using an updated method to extrapolate from mutation-reporter genes to whole genomes reveals that the rate of base substitutions is substantially lower in these two thermophiles than in mesophiles. This result provides the first experimental support for the concept of an evolved balance between the total genomic impact of mutations and the cost of further reducing the basal mutation rate

Multifaceted SlyD from Helicobacter pylori: implication in [NiFe] hydrogenase maturation

SlyD belongs to the FK506-binding protein (FKBP) family with both peptidylprolyl isomerase (PPIase) and chaperone activities, and is considered to be a ubiquitous cytosolic protein-folding facilitator in bacteria. It possesses a histidine- and cysteine-rich C-terminus binding to selected divalent metal ions (e.g., Ni2+, Zn2+), which is important for its involvement in the maturation processes of metalloenzymes. We have determined the solution structure of C-terminus-truncated SlyD from Helicobacter pylori (HpSlyDΔC). HpSlyDΔC folds into two well-separated, orientation-independent domains: the PPIase-active FKBP domain and the chaperone-active insert-in-flap (IF) domain. The FKBP domain consists of a four-stranded antiparallel β-sheet with an α-helix on one side, whereas the IF domain folds into a four-stranded antiparallel β-sheet accompanied by a short α-helix. Intact H. pylori SlyD binds both Ni2+ and Zn2+, with dissociation constants of 2.74 and 3.79 μM respectively. Intriguingly, binding of Ni2+ instead of Zn2+ induces protein conformational changes around the active sites of the FKBP domain, implicating a regulatory role of nickel. The twin-arginine translocation (Tat) signal peptide from the small subunit of [NiFe] hydrogenase (HydA) binds the protein at the IF domain. Nickel binding and the recognition of the Tat signal peptide by the protein suggest that SlyD participates in [NiFe] hydrogenase maturation processes