Computational Design of Inhibitory Agents of BMP-Noggin Interaction to Promote Osteogenesis

Bone growth factors, such as Bone Morphogenic Protein-2 (BMP-2) have been approved by the FDA to replace grafting for some surgical interventions, but the high dose requirement limits its use in patients. Noggin, an extracellular protein, blocks the effect of BMP-2 by binding to BMP. Preventing the BMP-2/noggin interaction will help increase the free concentration of BMP-2 and therefore should enhance its efficacy to induce bone formation. The work presented here involves computational design of novel small molecule inhibitory agents of BMP-2/noggin interaction, based on our current understanding of BMP-2, and its known putative ligands (receptors and antagonists). A successful acquisition of such an inhibitory agent of BMP-2/noggin interaction would allow clinicians to reduce the dose required of BMP-2 protein in clinical applications to promote osteogenesis. The available crystal structures of the BMPs, its receptors, and the binding partner noggin were analyzed to identify the critical residues involved in their interaction. In presenting this study, LUDI de novo design method was utilized to perform virtual screening of a large number of compounds from a commercially available library against the binding sites of noggin to identify the lead chemical compounds that could potentially block BMP-noggin interaction with a high specificity.

Differential Sensitivity of Nitrogen-Fixing, Filamentous Cyanobacterial Species to an Organochlorine Insecticide - 6, 7, 8, 9, 10, 10- Hexachloro-1, 5, 5a, 6, 9, 9a-Hexahydro-6, 9- Methano-2, 4, 3-Benzodioxathiepine-3-Oxide

Application of pesticides in the paddy fields has deleterious effects on non-target organisms including cyanobacteria which are photosynthesizing and nitrogen fixing micro-organisms contributing significantly towards soil fertility and crop yield. Pesticide contamination in the paddy fields has manifested into a serious global environmental concern. To study the effect of one such pesticide, three cyanobacterial strains; Anabaena fertilissima, Aulosira fertilissima and Westiellopsis prolifica were selected for their stress responses to an Organochlorine insecticide - 6, 7, 8, 9, 10, 10-hexachloro-1, 5, 5a, 6, 9, 9a-hexahydro-6, 9-methano-2, 4, 3- benzodioxathiepine-3-oxide, with reference to their photosynthesic pigments-chlorophyll-a and carotenoids as well as accessory pigments-phycobiliproteins (phycocyanin, allophycocyanin and phycoerythrin), stress induced biochemical metabolites like carbohydrates, proteins, amino acids, phenols and enzymes-nitrate reductase, glutamine synthetase and succinate dehydrogenase. All the three cyanobacterial strains were adversely affected by the insecticide doses and inhibition was dose dependent. Reduction in photosynthetic and accessory pigments, metabolites, nitrogen fixing and respiratory enzymes of the test organisms were accompanied with an initial increase in their total protein at lower Organochlorine doses. On the other hand, increased amount of phenols in all the insecticide treated concentrations was indicative of stressed activities of the organisms.

Bone Generation through Mechanical Loading

Bones are dynamic and responsive organs, they regulate their strength and mass according to the loads which they are subjected. Because, the Wnt/β-catenin pathway has profound effects on the regulation of bone mass, we hypothesized that mechanical loading of bone cells stimulates Wnt/β-catenin signaling, which results in the generation of new bone mass. Mechanical loading triggers the secretion of the Wnt molecule, which after binding to transmembrane proteins, causes GSK-3β (Glycogen synthase kinase 3 beta) to cease the phosphorylation of β-catenin. β-catenin accumulation in the cytoplasm, followed by its transport into the nucleus, binding to transcription factors (TCF/LEF) that initiate transcription of genes related to bone formation. To test this hypothesis, we used TOPGAL (Tcf Optimal Promoter β-galactosidase) mice in an experiment in which cyclic loads were applied to the forearm. TOPGAL mice are reporters for cells effected by the Wnt/β-catenin signaling pathway. TOPGAL mice are genetically engineered mice in which transcriptional activation of β- catenin, results in the production of an enzyme, β-galactosidase. The presence of this enzyme allows us to localize transcriptional activation of β-catenin to individual cells, thereby, allowing us to quantify the effects that mechanical loading has on the Wnt/β-catenin pathway and new bone formation. The ulnae of loaded TOPGAL mice were excised and transverse slices along different parts of the ulnar shaft were assayed for the presence of β-galactosidase. Our results indicate that loading increases β-catenin transcriptional activity in regions where this pathway is already primed (i.e. where basal activity is already higher) in a load magnitude dependent manner. Further experiments are needed to determine the temporal and spatial activation of this signaling in relation to bone formation.

Real-Time Detecting Concentration of Mycobacterium Tuberculosis by CNTFET Biosensor

Aptamers are useful tools in microorganism researches, diagnoses, and treatment. Aptamers are specific target molecules formed by oligonucleic acid molecules, and are not decomposed by alcohol. Aptamers used to detect Mycobacterium tuberculosis (MTB) have been proved to have specific affinity to the outer membrane proteins of MTB. This article presents a biosensor chip set with aptamers for early detection of MTB with high specificity and sensitivity, even in very low concentration. Meanwhile, we have already made a modified hydrophobic facial mask module with internal rendering hydrophobic for effectively collecting M. tuberculosis.

A Study of the Cyclic Variations of the Enzyme and the Electrolyte Activity in Uterine and Oviducal Secretions during an Estrous Cycle of the Ewe

Uterine and oviducal fluids are necessary for capacitation of the spermatozoa and early embryonic development. The aim of the present study was to determine the effects of estrous cycle phases (follicular and luteal) on some biological parameters (enzymes, electrolytes and total proteins) in uterine and oviducal secretions of ewes. Oviducal and uterine fluids were collected, diluted and centrifuged. According to our results, concentrations of GPT, G6PDH, total proteins, K and Na were significantly (P

A Novel Nucleus-Based Classifier for Discrimination of Osteoclasts and Mesenchymal Precursor Cells in Mouse Bone Marrow Cultures

Bone remodeling occurs by the balanced action of bone resorbing osteoclasts (OC) and bone-building osteoblasts. Increased bone resorption by excessive OC activity contributes to malignant and non-malignant diseases including osteoporosis. To study OC differentiation and function, OC formed in in vitro cultures are currently counted manually, a tedious procedure which is prone to inter-observer differences. Aiming for an automated OC-quantification system, classification of OC and precursor cells was done on fluorescence microscope images based on the distinct appearance of fluorescent nuclei. Following ellipse fitting to nuclei, a combination of eight features enabled clustering of OC and precursor cell nuclei. After evaluating different machine-learning techniques, LOGREG achieved 74% correctly classified OC and precursor cell nuclei, outperforming human experts (best expert: 55%). In combination with the automated detection of total cell areas, this system allows to measure various cell parameters and most importantly to quantify proteins involved in osteoclastogenesis.

Effect of Calcium Chloride on Rheological Properties and Structure of Inulin - Whey Protein Gels

The rheological properties, structure and potential synergistic interactions of whey proteins (1-6%) and inulin (20%) in mixed gels in the presence of CaCl2 was the aim of this study. Whey proteins have a strong influence on inulin gel formation. At low concentrations (2%) whey proteins did not impair in inulin gel formation. At higher concentration (4%) whey proteins impaired inulin gelation and inulin impaired the formation of a Ca2+-induced whey protein network. The presence of whey proteins at a level allowing for protein gel network formation (6%) significantly increased the rheological parameters values of the gels. SEM micrographs showed that whey protein structure was coated by inulin moieties which could make the mixed gels firmer. The protein surface hydrophobicity measurements did not exclude synergistic interactions between inulin and whey proteins, however. The use of an electrophoretic technique did not show any stable inulin-whey protein complexes.

Quantitative Genetics Researches on Milk Protein Systems of Romanian Grey Steppe Breed

The paper makes part from a complex research project on Romanian Grey Steppe, a unique breed in terms of biological and cultural-historical importance, on the verge of extinction and which has been included in a preservation programme of genetic resources from Romania. The study of genetic polymorphism of protean fractions, especially kappa-casein, and the genotype relations of these lactoproteins with some quantitative and qualitative features of milk yield represents a current theme and a novelty for this breed. In the estimation of the genetic parameters we used R.E.M.L. (Restricted Maximum Likelihood) method. The main lactoprotein from milk, kappa - casein (K-cz), characterized in the specialized literature as a feature having a high degree of hereditary transmission, behaves as such in the nucleus under study, a value also confirmed by the heritability coefficient (h2 = 0.57 %). We must mention the medium values for milk and fat quantity (h2=0.26, 0.29 %) and the fat and protein percentage from milk having a high hereditary influence h2 = 0.71 - 0.63 %. Correlations between kappa-casein and the milk quantity are negative and strong. Between kappa-casein and other qualitative features of milk (fat content 0.58-0.67 % and protein content 0.77- 0.87%), there are positive and very strong correlations. At the same time, between kappa-casein and β casein (β-cz), β lactoglobulin (β- lg) respectively, correlations are positive having high values (0.37 – 0.45 %), indicating the same causes and determining factors for the two groups of features.

The Impact of Germination and In Vitro Digestion on the Formation of Angiotensin Converting Enzyme (ACE) Inhibitory Peptides from Lentil Proteins Compared to Whey Proteins

Biologically active peptides are of particular interest in food science and human nutrition because they have been shown to play several physiological roles. In vitro gastrointestinal digestion of lentil and whey proteins in this study produced high angiotensin-I converting enzyme inhibitory activity with 75.5±1.9 and 91.4±2.3% inhibition, respectively. High ACE inhibitory activity was observed in lentil after 5 days of germination (84.3±1.2%). Fractionation by reverse phase chromatography gave inhibitory activities as high as 86.3±2.0 for lentil, 94.8±1.8% for whey and 93.7±1.7% at 5th day of germination. Further purification by HPLC resulted in several inhibitory peptides with IC50 values ranging from 0.064 to 0.164 mg/ml. These results demonstrate that lentil proteins are a good source of peptides with ACE inhibitory activity that can be released by germination or gastrointestinal digestion. Despite the lower bioactivity in comparison with whey proteins, incorporation of lentil proteins in functional food formulations and natural drugs look promising.