Audit of therapeutic interventions in inpatient children using two scores: are they evidence-based in developing countries?

BACKGROUND: The evidence base of clinical interventions in paediatric hospitals of developing countries has not been formally assessed. We performed this study to determine the proportion of evidence-based therapeutic interventions in a paediatric referral hospital of a developing country METHODS: The medical records of 167 patients admitted in one-month period were revised. Primary diagnosis and primary therapeutic interventions were determined for each patient. A systematic search was performed to assess the level of evidence for each intervention. Therapeutic interventions were classified using the Ellis score and the Oxford Centre for Evidence Based Medicine Levels of Evidence RESULTS: Any dehydration due to diarrhoea (59 cases) and pneumonia (42 cases) were the most frequent diagnoses. Based on Ellis score, level I evidence supported the primary therapeutic intervention in 21%, level II in 73% and level III in 6% cases. Using the Oxford classification 16%, 8%, 1% and 75% therapeutic interventions corresponded to grades A, B, C, and D recommendations, respectively. Overall, according to Ellis score, 94% interventions were evidence based. However, out of the total, 75% interventions were based on expert opinion or basic sciences. Most children with mild to moderate dehydration (52 cases) were inappropriately treated with slow intravenous fluids, and most children with non-complicated community acquired pneumonia (42 cases) received intravenous antibiotics CONCLUSIONS: Most interventions were inappropriate, despite the availability of effective therapy for several of them. Diarrhoeal dehydration and community acquired pneumonia were the most common diagnoses and were inappropriately managed. Existing effective interventions for dehydration and pneumonia need to be put into practice at referral hospitals of developing countries. For the remaining problems, there is the need to conduct appropriate clinical studies. Caution must be taken when assigning the level of evidence supporting therapeutic interventions, as commonly used classifications may be misleadin

Phos-Tag-Based Analysis of Myosin Regulatory Light Chain Phosphorylation in Human Uterine Myocytes

The 'phosphate-binding tag' (phos-tag) reagent enables separation of phospho-proteins during SDS-PAGE by impeding migration proportional to their phosphorylation stoichiometry. Western blotting can then be used to detect and quantify the bands corresponding to the phospho-states of a target protein. We present a method for quantification of data regarding phospho-states derived from phos-tag SDS-PAGE. The method incorporates corrections for lane-to-lane loading variability and for the effects of drug vehicles thus enabling the comparison of multiple treatments by using the untreated cellular set-point as a reference. This method is exemplified by quantifying the phosphorylation of myosin regulatory light chain (RLC) in cultured human uterine myocytes.We have evaluated and validated the concept that, when using an antibody (Ab) against the total-protein, the sum of all phosphorylation states in a single lane represents a 'closed system' since all possible phospho-states and phosphoisotypes are detected. Using this approach, we demonstrate that oxytocin (OT) and calpeptin (Calp) induce RLC kinase (MLCK)- and rho-kinase (ROK)-dependent enhancements in phosphorylation of RLC at T18 and S19. Treatment of myocytes with a phorbol ester (PMA) induced phosphorylation of S1-RLC, which caused a mobility shift in the phos-tag matrices distinct from phosphorylation at S19.We have presented a method for analysis of phospho-state data that facilitates quantitative comparison to a reference control without the use of a traditional 'loading' or 'reference' standard. This analysis is useful for assessing effects of putative agonists and antagonists where all phospho-states are represented in control and experimental samples. We also demonstrated that phosphorylation of RLC at S1 is inducible in intact uterine myocytes, though the signal in the resting samples was not sufficiently abundant to allow quantification by the approach used here

PDGF regulates the actin cytoskeleton through hnRNP-K-mediated activation of the ubiquitin E-3-ligase MIR

PDGF is a potent chemotactic mitogen and a strong inductor of fibroblast motility. In Swiss 3T3 fibroblasts, exposure to PDGF but not EGF or IGF-1 causes a rapid loss of actin stress fibers (SFs) and focal adhesions (FAs), which is followed by the development of retractile dendritic protrusions and induction of motility. The PDGF-specific actin reorganization was blocked by inhibition of Src-kinase and the 26S proteasome. PDGF induced Src-dependent association between the multifunctional transcription/translation regulator hnRNP-K and the mRNA-encoding myosin regulatory light-chain (MRLC)-interacting protein (MIR), a E3-ubiquitin ligase that is MRLC specific. This in turn rapidly increased MIR expression, and led to ubiquitination and proteasome-mediated degradation of MRLC. Downregulation of MIR by RNA muting prevented the reorganization of actin structures and severely reduced the migratory and wound-healing potential of PDGF-treated cells. The results show that activation of MIR and the resulting removal of diphosphorylated MRLC are essential for PDGF to instigate and maintain control over the actin-myosin-based contractile system in Swiss 3T3 fibroblasts. The PDGF induced protein destabilization through the regulation of hnRNP-K controlled ubiquitin-ligase translation identifies a novel pathway by which external stimuli can regulate phenotypic development through rapid, organelle-specific changes in the activity and stability of cytoskeletal regulators