Marginal bone loss around axial and straight implants supported with prefabricated SFI-Bar with mandibular overdentures

To assess the role of prefabricated SFI-Bar in peri-implant bone loss around immediately axially loaded and straight implants. This study comprised of 40 complete denture wearer patients who received two axially parallel implants connected by SFI-Bars in group I and two 15° mesially tilted implants connected by SFI-Bars in group II. Peri- implant bone loss (PiBL) was measured at 1 year, 2 years and 3 years. The mean PiBL at 1 year in group I was 0.21 mm and I group II was 0.22, at 2 years in group I was 0.26 mm and in group II was 0.23 mm and at 3 years, in group I was 0.29 mm and in group II was 0.34 mm. The difference was significant at 3 years (P< 0.05). The mean mesial PIBL at 1 year in group I was 0.18 mm, in group II was 0.20 mm, at 2 years in group I was 0.19 mm and in group II was 0.07 mm and at 3 years, in group I was 0.25 mm and in group II was 0.29 mm. The difference found to be significant in each time duration in both groups (P< 0.05).The mean distal PIBL at 1 year in group I was 0.23 mm, in group II was 0.22 mm, at 2 years in group I was 0.33 mm and in group II was 0.39 mm and at 3 years, in group I was 0.34 mm and in group II was 0.39 mm. The difference found to be significant at 2 and 3 years in both groups (P< 0.05). Authors found that mandibular overdentures retained with Prefabricated SFI-Bar with axial and straight inserted implants may be useful in patients with reduced bone height.     

The Oligonucleotide Binding (OB)-Fold Domain of KREPA4 Is Essential for Stable Incorporation into Editosomes

Most mitochondrial mRNAs in trypanosomatid parasites require uridine insertion/deletion RNA editing, a process mediated by guide RNA (gRNA) and catalyzed by multi-protein complexes called editosomes. The six oligonucleotide/oligosaccharide binding (OB)-fold proteins (KREPA1-A6), are a part of the common core of editosomes. They form a network of interactions among themselves as well as with the insertion and deletion sub-complexes and are essential for the stability of the editosomes. KREPA4 and KREPA6 proteins bind gRNA in vitro and are known to interact directly in yeast two-hybrid analysis. In this study, using several approaches we show a minimal interaction surface of the KREPA4 protein that is required for this interaction. By screening a series of N- and C-terminally truncated KREPA4 fragments, we show that a predicted α-helix of KREPA4 OB-fold is required for its interaction with KREPA6. An antibody against the KREPA4 α-helix or mutations of this region can eliminate association with KREPA6; while a peptide fragment corresponding to the α-helix can independently interact with KREPA6, thereby supporting the identification of KREPA4-KREPA6 interface. We also show that the predicted OB-fold of KREPA4; independent of its interaction with gRNA, is responsible for the stable integration of KREPA4 in the editosomes, and editing complexes co-purified with the tagged OB-fold can catalyze RNA editing. Therefore, we conclude that while KREPA4 interacts with KREPA6 through the α-helix region of its OB-fold, the entire OB-fold is required for its integration in the functional editosome, through additional protein-protein interactions.     

Development of an Aptamer-Based Concentration Method for the Detection of Trypanosoma cruzi in Blood

Trypanosoma cruzi, a blood-borne parasite, is the etiological agent of Chagas disease. T. cruzi trypomastigotes, the infectious life cycle stage, can be detected in blood of infected individuals using PCR-based methods. However, soon after a natural infection, or during the chronic phase of Chagas disease, the number of parasites in blood may be very low and thus difficult to detect by PCR. To facilitate PCR-based detection methods, a parasite concentration approach was explored. A whole cell SELEX strategy was utilized to develop serum stable RNA aptamers that bind to live T. cruzi trypomastigotes. These aptamers bound to the parasite with high affinities (8-25 nM range). The highest affinity aptamer, Apt68, also demonstrated high specificity as it did not interact with the insect stage epimastigotes of T. cruzi nor with other related trypanosomatid parasites, L. donovani and T. brucei, suggesting that the target of Apt68 was expressed only on T. cruzi trypomastigotes. Biotinylated Apt68, immobilized on a solid phase, was able to capture live parasites. These captured parasites were visible microscopically, as large motile aggregates, formed when the aptamer coated paramagnetic beads bound to the surface of the trypomastigotes. Additionally, Apt68 was also able to capture and aggregate trypomastigotes from several isolates of the two major genotypes of the parasite. Using a magnet, these parasite-bead aggregates could be purified from parasite-spiked whole blood samples, even at concentrations as low as 5 parasites in 15 ml of whole blood, as detected by a real-time PCR assay. Our results show that aptamers can be used as pathogen specific ligands to capture and facilitate PCR-based detection of T. cruzi in blood.     

Cardioprotective and antihypertensive effects of Enicostemma littorale Blume extract in fructose-fed rats

We investigated the protective effects of Enicostemma littorale Blume (EL) extract on hypertension and insulin resistance along with its associated cardiovascular complications in high fructose (HF) fed rats. For this, rats were divided among 4 groups: (i) control, fed laboratory chow; (ii) fed with a high level of fructose; (iii) fed with a high level of fructose plus E. littorale extract; and (iv) fed with a high level of fructose plus rosiglitazone (Rg). EL and Rg treatments were given simultaneously with HF diet. The results show that untreated HF-fed rats showed altered oral glucose tolerance, increased fasting insulin, and increased fasting glucose. These rats also exhibited hypertriglyceridemia, moderate hypertension, platelet hyperaggregability, decreased prothrombin time, activated partial thromboplastin time, altered vascular reactivity, and increased serum levels of enzymes (creatine kinase, type muscle-brain (CK-MB), aspartate aminotransferase (SGOT), lactate dehydrogenase (LDH), and alanine aminotransferase (SGPT). This is the first demonstration of platelet hyperaggregation and prothrombotic alteration in HF-fed rats. HF-fed rats treated with EL showed improved insulin resistance, along with reduced hypertriglyceridemia, hypertension, platelet aggregability, blood coagulation, serum enzymes (CK-MB, SGOT, LDH and SGPT), and vascular reactivity. These effects of EL in HF-induced hypertensive rats might be associated with the suppression of hyperinsulinemia and hypertriglyceridemia, along with its antiatherogenic and antithrombogenic potential. These data indicate that the aqueous extract of EL has great therapeutic potential for the prevention and (or) management of insulin resistance and the associated hypertension.     

<Emphasis Type="Italic">In silico</Emphasis> analysis of <Emphasis Type="Italic">Pycnoporus cinnabarinus</Emphasis> laccase active site with toxic industrial dyes

Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in various industrial oxidative processes like production of fine chemicals to bioremediation of contaminated soil and water. In order to understand the mechanisms of substrate binding and interaction between substrates and Pycnoporus cinnabarinus laccase, a homology model was generated. The resulted model was further validated and used for docking studies with toxic industrial dyes- acid blue 74, reactive black 5 and reactive blue 19. Interactions of chemical mediators with the laccase was also examined. The docking analysis showed that the active site always cannot accommodate the dye molecules, due to constricted nature of the active site pocket and steric hindrance of the residues whereas mediators are relatively small and can easily be accommodated into the active site pocket, which, thereafter leads to the productive binding. The binding properties of these compounds along with identification of critical active site residues can be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds.     

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.     

Epoxidation of cottonseed oil by aqueous hydrogen peroxide catalysed by liquid inorganic acids

The kinetics of epoxidation of cottonseed oil by peroxyacetic acid generated in situ from hydrogen peroxide and glacial acetic acid in the presence of liquid inorganic acid catalysts were studied. It was possible to obtain up to 78% relative conversion to oxirane with very less oxirane cleavage by in situ technique. The rate constants for sulphuric acid catalysed epoxidation of cottonseed oil were in the range 0.39-5.4 x 10(-6)L mol(-1)s(-1) and the activation energy was found to be 11.7 kcal mol(-1). Some thermodynamic parameters such as enthalpy, entropy, and free energy of activation were determined to be of 11.0 kcal mol(-1), -51.4 cal mol(-1)K(-1) and 28.1 kcal mol(-1), respectively. The order of effectiveness of catalysts was found to be sulphuric acid>phosphoric acid>nitric acid>hydrochloric acid. Acetic acid was found to be superior to formic acid for the in situ cottonseed oil epoxidation.