Precision measurements of the RSA method using a phantom model of hip prosthesis

Radiostereometric analysis (RSA) has become one of the recommended techniques for pre-market evaluation of new joint implant designs. In this study we evaluated the effect of repositioning of X-ray tubes and phantom model on the precision of the RSA method. In precision measurements, we utilized mean error of rigid body fitting (ME) values as an internal control for examinations. ME value characterizes relative motion among the markers within each rigid body and is conventionally used to detect loosening of a bone marker. Three experiments, each consisting of 10 double examinations, were performed. In the first experiment, the X-ray tubes and the phantom model were not repositioned between one double examination. In experiments two and three, the X-ray tubes were repositioned between one double examination. In addition, the position of the phantom model was changed in experiment three. Results showed that significant differences could be found in 2 of 12 comparisons when evaluating the translation and rotation of the prosthetic components. Repositioning procedures increased ME values mimicking deformation of rigid body segments. Thus, ME value seemed to be a more sensitive parameter than migration values in this study design. These results confirmed the importance of standardized radiographic technique and accurate patient positioning for RSA measurements. Standardization and calibration procedures should be performed with phantom models in order to avoid unnecessary radiation dose of the patients. The present model gives the means to establish and to follow the intra-laboratory precision of the RSA method. The model is easily applicable in any research unit and allows the comparison of the precision values in different laboratories of multi-center trials.     

"Plasmo2D": an ancillary proteomic tool to aid identification of proteins from Plasmodium falciparum

Bioinformatics tools to aid gene and protein sequence analysis have become an integral part of biology in the post-genomic era. Release of the Plasmodium falciparum genome sequence has allowed biologists to define the gene and the predicted protein content as well as their sequences in the parasite. Using pI and molecular weight as characteristics unique to each protein, we have developed a bioinformatics tool to aid identification of proteins from Plasmodium falciparum. The tool makes use of a Virtual 2-DE generated by plotting all of the proteins from the Plasmodium database on a pI versus molecular weight scale. Proteins are identified by comparing the position of migration of desired protein spots from an experimental 2-DE and that on a virtual 2-DE. The procedure has been automated in the form of user-friendly software called "Plasmo2D". The tool can be downloaded from http://144.16.89.25/Plasmo2D.zip.     

ProteoMod: A new tool to quantitate protein post-translational modifications

Post-translational modifications (PTMs) are known to regulate biological processes by controlling protein function. The effect of a PTM on protein function depends critically on the position and the number of modifications. While there are convenient methods available to qualitatively examine modifications like phosphorylation, glycosylation, acetylation and methylation, methods available for their quantitative assessment are cumbersome. We have developed a new tool that allows quantitation of the number of phosphorylation events in proteins with ease. The "ProteoMod" tool depends on shifts in the isoelectric points of proteins upon post-translational change. The extent of shift exhibited upon phosphorylation is algorithmically converted into the number of phosphorylations conferred. The validity of ProteoMod was confirmed by examining proteins with previously known number of phosphorylations. The list of proteins examined included HSP27, HSP70 and tumor suppressor p53. The approach can also be applied to estimate modifications like acetylation, methylation and sialylation in proteins. We analyzed shifts in isoelectric points due to sialylation events in N-glycoproteins. Using influenza hemagglutinin we show that shifts in isoelectric points correlate with intracellular distribution of this model membrane protein. In addition to extending the application of two dimensional gel electrophoresis to quantitate modifications, our study also highlights its potential use in cell biology.     

Telocytes within human skeletal muscle stem cell niche

Human skeletal muscle tissue displays specific cellular architecture easily damaged during individual existence, requiring multiple resources for regeneration. Congruent with local prerequisites, heterogeneous muscle stem cells (MuSCs) are present in the muscle interstitium. In this study, we aimed to characterize the properties of human muscle interstitial cells that had the characteristic morphology of telocytes (TCs). Immunocytochemistry and immunofluorescence showed that cells with TC morphology stained positive for c-kit/CD117 and VEGF. C-kit positive TCs were separated with magnetic-activated cell sorting, cultured in vitro and expanded for study. These cells exhibited high proliferation capacity (60% expressed endoglin/CD105 and 80% expressed nuclear Ki67). They also exhibited pluripotent capacity limited to Oct4 nuclear staining. In addition, 90% of c-kit positive TCs expressed VEGF. C-kit negative cells in the MuSCs population exhibited fibroblast-like morphology, low trilineage differential potential and negative VEGF staining. These results suggested that c-kit/CD117 positive TCs represented a unique cell type within the MuSC niche.     

Peroxisomal multifunctional enzyme type 2 from the fruitfly: dehydrogenase and hydratase act as separate entities, as revealed by structure and kinetics

All of the peroxisomal β-oxidation pathways characterized thus far house at least one MFE (multifunctional enzyme) catalysing two out of four reactions of the spiral. MFE type?2 proteins from various species display great variation in domain composition and predicted substrate preference. The gene CG3415 encodes for Drosophila melanogaster MFE-2 (DmMFE-2), complements the Saccharomyces cerevisiae MFE-2 deletion strain, and the recombinant protein displays both MFE-2 enzymatic activities in vitro. The resolved crystal structure is the first one for a full-length MFE-2 revealing the assembly of domains, and the data can also be transferred to structure-function studies for other MFE-2 proteins. The structure explains the necessity of dimerization. The lack of substrate channelling is proposed based on both the structural features, as well as by the fact that hydration and dehydrogenation activities of MFE-2, if produced as separate enzymes, are equally efficient in catalysis as the full-length MFE-2.     

New methods for the analysis of binarized BIOLOG GN data of Vibrio species: minimization of stochastic complexity and cumulative classification

We apply minimization of stochastic complexity and the closely related method of cumulative classification to analyse the extensively studied BIOLOG GN data of Vibrio spp. Minimization of stochastic complexity provides an objective tool of bacterial taxonomy as it produces classifications that are optimal from the point of view of information theory. We compare the outcome of our results with previously published classifications of the same data set. Our results both confirm earlier detected relationships between species and discover new ones.     

The Potential of <Emphasis Type="Italic">Lactobacillus casei</Emphasis> and <Emphasis Type="Italic">Entercoccus faecium</Emphasis> Combination as a Preventive Probiotic Against <Emphasis Type="Italic">Entamoeba</Emphasis>

Travellers’ diarrhoea caused by enteric protozoa like Entamoeba histolytica is among the most common protozoan diseases in developing countries. In developing countries, amoebiasis is the second most prevalent protozoan disease. This protozoan parasite is often known to coexist as a part of the normal gut microbiota. It is estimated that around 50–60 % of population in developing countries might be harbouring Entamoeba in an asymptomatic manner. Due to physiological perturbation or upon immuno-compromise, it can become virulent and then cause diarrhoea, bloody stools and may invade other organs if left untreated. Nitroimidazole drugs, namely metronidazole and tinidazole, are widely used to treat protozoan infections. These drugs often show dose-dependent side effects. With emerging antibiotic resistance, novel therapeutics to prevent parasitic infections is required. This study aims to study effect of probiotics on prevention of Amoebiasis. In this study, we have investigated the effect of selected probiotics on the growth of Entamoeba. From the list of probiotics being currently used, five bacterial strains were selected for testing. These probiotic strains were co-cultured with Entamoeba, and their effect on Entamoeba proliferation was checked. Of the five probiotics chosen, individual treatments of Lactobacillus casei and Enterococcus faecium showed a significant reduction of up to 71 % in parasite survival only at higher CFUs. When the two probiotics were used in combination, the percentage of survival reduced gradually further to 80 % at a total CFU of 10⁹ cells/ml of bacteria. The study lays the foundation for providing cost-effective prophylactic treatment for amoebiasis without the overuse of antibiotics.     

Oxidative folding and assembly with transthyretin are sequential events in the biogenesis of retinol binding protein in the endoplasmic reticulum

Retinol-binding protein (RBP) is secreted out of the cell in its ligand-bound holo-form. The apo-form of RBP is selectively retained within the endoplasmic reticulum (ER) by a mechanism that remains unknown. Using isolated microsomal system, we have recapitulated the biogenesis of RBP involving its oxidative folding and assembly with transthyretin in the ER. In addition to dissecting its pathway of disulfide oxidation, we have analyzed association of its early folding intermediates with ER-chaperones. Our results show that of the three intramolecular disulfides present in RBP (4–160, 70–174, and 120–129) the smallest loop (120–129) was most critical for RBP to fold. Its absence caused RBP to aggregate into an intermolecular disulfide-linked structure. After acquisition of the small loop, formation of one of the two big disulfides (4–160 or 70–174) was sufficient for RBP to acquire a folded state. Using cross-linking in intact microsomes and sedimentation on sucrose gradients, we show that newly synthesized RBP is associated with a complex of chaperones consisting of Grp94, BiP, PDI, and calnexin. The complex was constitutively present in the ER, independent of the presence of folding substrates. RBP dissociated from this complex coincident with the formation of one of the two big disulfide loops, whereas RBP mutant lacking both the large disulfides showed persistent association. While highlighting the matrix-like characteristics of ER in isolated microsomal system our results provide insight into RBP folding and assembly mechanisms that will aid our understanding of its complex secretion properties.