Accretion of bone quantity and quality in the developing mouse skeleton.

In this work, we found that bone mineral formation proceeded very rapidly in mice by 1 day of age, where the degree of mineralization, the tissue mineral density, and the mineral crystallinity reached 36%, 51%, and 87% of the adult values, respectively. However, even though significant mineralization had occurred, the elastic modulus of 1-day-old bone was only 14% of its adult value, indicating that the intrinsic stiffening of the bone lags considerably behind the initial mineral formation. INTRODUCTION: To meet the mechanical challenges during early development, the skeleton requires the rapid accretion of bone quality and bone quantity. Here, we describe early bone development in the mouse skeleton and test the hypothesis that specific compositional properties determine the stiffness of the tissue. MATERIALS AND METHODS: Tibias of female BALB mice were harvested at eight time-points (n = 4 each) distributed between 1 and 40 days of age and subjected to morphometric (muCT), chemical (Fourier transform infrared microspectroscopy), and mechanical (nanoindentation) analyses. Tibias of 450-day-old mice served as fully mineralized control specimens. RESULTS: Bone growth proceeded very rapidly; at 1 day of age, the degree of mineralization (phosphate/protein ratio), the density of mineralized bone (TMD), and mineral crystallinity had reached 36%, 51%, and 87% of the adult (450 days) values, respectively. Spatially, the variability in mineralization across the mid-diaphysis was very high for the early time-points and declined over time. In contrast to the notable changes in mineralization, carbonate substitution into the mineral lattice (carbonate/phosphate ratio) and collagen cross-linking did not show any significant changes over this time period. Even though significant mineralization had occurred, the elastic modulus of 1-day-old bone was only 14% of the adult value and increased to 89% (of its adult value) after 40 days. Between samples of different time-points, significant positive correlations were observed between the elastic modulus and TMD (r(2) = 0.84), phosphate/protein ratio (r(2) = 0.59), and crystallinity (r(2) = 0.23), whereas collagen cross-linking showed a small but significant negative correlation (r(2) = 0.15). CONCLUSIONS: These data indicate that specific chemical and morphometric properties modulate bone's stiffness during early growth. The intrinsic stiffening of the bone, however, lags considerably behind the initial mineral formation, emphasizing the importance of bone mineral quality for optimizing matrix integrity.     

The Repertoire of Na,K-ATPase α and β Subunit Genes Expressed in the Zebrafish, Danio rerio

We have identified a cohort of zebrafish expressed sequence tags encoding eight Na,K-ATPase alpha subunits and five beta subunits. Sequence comparisons and phylogenetic analysis indicate that five of the zebrafish alpha subunit genes comprise an alpha1-like gene subfamily and two are orthologs of the mammalian alpha3 subunit gene. The remaining alpha subunit clone is most similar to the mammalian alpha2 subunit. Among the five beta subunit genes, two are orthologs of the mammalian beta1 isoform, one represents a beta2 ortholog, and two are orthologous to the mammalian beta3 subunit. Using zebrafish radiation hybrid and meiotic mapping panels, we determined linkage assignments for each alpha and beta subunit gene. Na,K-ATPase genes are dispersed in the zebrafish genome with the exception of four of the alpha1-like genes, which are tightly clustered on linkage group 1. Comparative mapping studies indicate that most of the zebrafish Na,K-ATPase genes localize to regions of conserved synteny between zebrafish and humans. The expression patterns of Na,K-ATPase alpha and beta subunit genes in zebrafish are quite distinctive. No two alpha or beta subunit genes exhibit the same expression profile. Together, our data imply a very high degree of Na,K-ATPase isoenzyme heterogeneity in zebrafish, with the potential for 40 structurally distinct alpha/beta subunit combinations. Differences in expression patterns of alpha and beta subunits suggest that many of the isoenzymes are also likely to exhibit differences in functional properties within specific cell and tissue types. Our studies form a framework for analyzing structure function relationships for sodium pump isoforms using reverse genetic approaches.     

Histology-based screen for zebrafish mutants with abnormal cell differentiation.

The power of histology to define states of cell differentiation was used as the basis of a mutagenesis screen in zebrafish. In this screen, 7-day-old parthenogenetic half-tetrad larvae from potential carrier females were screened for mutations affecting cell differentiation in hematoxylin and eosin-stained tissue sections. Seven, noncomplementing, recessive mutations were found. Two mutations affect only the retina: segmented photoreceptors (spr) show a discontinuous photoreceptor cell layer; vestigial outer segments (vos) has fewer photoreceptor cells and degenerated outer segments within this cell layer. Three mutants have gut-specific defects: the epithelial cells of kirby (kby) are replaced by ballooned cells; the intestines of stuffy (sfy) and stuffed (sfd) contain increased luminal mucus. Two mutations affect multiple organs: disordered neural retina (dnr) has disrupted retinal layering and mild nuclear abnormalities in the gut and liver; and in huli hutu (hht), the retinal cell layers are disorganized and multiple organs have mild to severe nuclear abnormalities that are reminiscent of the atypia of human neoplasia. Each mutation appears to be homozygous lethal. This screen is proof of principle for the feasibility of histologic screens to yield novel mutations, including potential models of human disease. The throughput for this type of screen may be enhanced by automation.     

Anterior segment optical coherence tomography documentation of a case of topiramate induced acute angle closure

We present a case report of a 31-year-old female patient who presented to us with a 1 day history of acute bilateral eye pain, blurred vision and headache. She was found to have a myopic shift, raised intraocular pressure (IOP) and shallow anterior chambers in both eyes. She had been commenced on oral topiramate 1 week previously. A number of investigations, including anterior segment optical coherence tomography (AS-OCT) were done and a diagnosis of topiramate induced bilateral acute angle closure (TiAAC) was made. Topiramate was discontinued and she was managed with topical and oral antiglaucoma medications, topical steroids and cyclopegics. Her symptoms subsided dramatically at the next follow-up. The AS-OCT documentation revealed lucidly the improvement in her anterior chamber depth and anterior chamber angle parameters. Her IOP decreased, her myopic shift showed reversal and her AS-OCT findings revealed gross improvement in all the parameters angle opening distance, trabecular iris space area and scleral spur angle. This case report clearly shows with AS OCT documentation the changes which occur in the anterior segment in a case of TiAAC.     

Simple bedside predictors of mechanical ventilation in patients with Guillain-Barre syndrome

Objective

The objective of the study is to develop and validate a predictor score for assessing the requirement of mechanical ventilation (MV) in patients with Guillain-Barre syndrome (GBS).

Study design

The study was conducted in patients admitted with GBS in neurointensive care unit in a tertiary care hospital. The demographic, clinical factors, electrophysiological, and spirometric data of all consecutive patients were prospectively collected. The study was undertaken in 2 stages. In the first stage, data were collected for development of a predictor score. In the second stage, the score developed was validated on a separate set of patient data.

Results

The data collected were compared between the 2 groups (ventilated vs nonventilated). On univariate analysis, time taken to reach maximum deficit, neck weakness, bulbar weakness, facial weakness, single breath count (SBC), forced vital capacity, and phrenic nerve latency predicted the need for MV. On multivariate analysis, only neck weakness, bulbar weakness, SBC, and forced vital capacity were independent predictors of MV. There was a good correlation between SBC and the spirometric tests and phrenic nerve distal motor latency, as reflected in receiver operating characteristics curve. The predictor score developed using the regression coefficient of independent predictors showed that the best cutoff score for prediction of ventilation was 60 (sensitivity, 0.95; 1 – specificity, 0.065). Internal cross validation of the neck weakness, SBC, and bulbar palsy (NSB) score showed good correlation (Pearson R = 0.76; P = .00). There was no statistically significant difference between predicted and observed outcomes (sensitivity, 95%; specificity, 93%).

Conclusion

Several independent risk factors were found to predict the requirement for MV in patients with GBS at admission. However, after scoring and analyzing them, it was found that combining a few of them was more useful to predict the need for MV. A model using NSB score, developed using clinical variables, accurately predicted the requirement of MV. In addition, among the NSB score parameters, simple bedside SBC could adequately assess the adequacy of vital capacity.     

Inhibition of Band 3 tyrosine phosphorylation: a new mechanism for treatment of sickle cell disease

Many hypotheses have been proposed to explain how a glutamate to valine substitution in sickle haemoglobin (HbS) can cause sickle cell disease (SCD). We propose and document a new mechanism in which elevated tyrosine phosphorylation of Band 3 initiates sequelae that cause vaso-occlusion and the symptoms of SCD. In this mechanism, denaturation of HbS and release of heme generate intracellular oxidants which cause inhibition of erythrocyte tyrosine phosphatases, thus permitting constitutive tyrosine phosphorylation of Band 3. This phosphorylation in turn induces dissociation of the spectrin-actin cytoskeleton from the membrane, leading to membrane weakening, discharge of membrane-derived microparticles (which initiate the coagulation cascade) and release of cell-free HbS (which consumes nitric oxide) and activates the endothelium to express adhesion receptors). These processes promote vaso-occlusive events which cause SCD. We further show that inhibitors of Syk tyrosine kinase block Band 3 tyrosine phosphorylation, prevent release of cell-free Hb, inhibit discharge of membrane-derived microparticles, increase sickle cell deformability, reduce sickle cell adhesion to human endothelial cells, and enhance sickle cell flow through microcapillaries. In view of reports that imatinib (a Syk inhibitor) successfully treats symptoms of sickle cell disease, we suggest that Syk tyrosine kinase inhibitors warrant repurposing as potential treatments for SCD.     

Pasireotide (SOM230) shows efficacy and tolerability in the treatment of patients with advanced neuroendocrine tumors refractory or resistant to octreotide LAR: results from a phase II study

Pasireotide (SOM230) is a novel multireceptor-targeted somatostatin (sst) analog with high binding affinity for sst receptor subtype 1, 2, 3 (sst(1,2,3)) and sst(5). Because of this binding profile, pasireotide may offer symptom control in patients with neuroendocrine tumors (NETs) and carcinoid syndrome no longer responsive to octreotide LAR. This was a phase II, open-label, multicenter study of pasireotide in patients with advanced NET whose symptoms of carcinoid syndrome (diarrhea/flushing) were inadequately controlled by octreotide LAR. Patients received s.c. pasireotide 150?μg twice daily (bid), escalated to a maximum dose of 1200?μg bid until a clinical response was achieved. Forty-four patients were evaluated for efficacy and 45 for tolerability. Pasireotide 600-900?μg s.c. bid effectively controlled the symptoms of diarrhea and flushing in 27% of patients. Evaluation of tumor response in 23 patients showed 13 with stable disease and ten with progressive disease at study end. The most common drug-related adverse events were nausea (27%), abdominal pain (20%), weight loss (20%), and hyperglycemia (16%) and most were of mild or moderate severity. Pasireotide 600-900?μg s.c. bid was effective and generally well tolerated in controlling the symptoms of carcinoid syndrome in 27% of patients with advanced NET refractory or resistant to octreotide LAR therapy.