Phosphate Glass Fibre Composites for Bone Repair

We investigate high-modulus degradable materials intended to replace metals in biomedical applications.These are typicallycomposites comprising a polylactide(PLA)matrix reinforced with phosphate glass fibres,which provide reinforcementsimilar to E-glass but are entirely degradable in water to produce,principally,calcium phosphate.We have made compositesusing a variety of fibre architectures,from non-woven random mats to unidirectional fibre tapes.Flexural properties in theregion of 30 GPa modulus and 350 MPa strength have been achieved-directly comparable to quoted values for human corticalbone.In collaboration with other groups we have begun to consider the development of foamed systems with structures mimickingcancellous bone and this has shown significant promise.The fibres in these foamed structures provide improved creepresistance and reinforcement of the pore walls.To date the materials have exhibited excellent cellular responses in vitro andfurther studies are due to include consideration of the surface character of the materials and the influence of this on cell interaction,both with the composites and the glass fibres themselves,which show promise as a standalone porous scaffold.     

Histological and histochemical study on mammary gland of Damascus goats through stages of lactation

This research was carried out on seven Damascus goats, to study the relationship between milk production, during advancing lactation, and the changes in secretory mammary cells frequency and cellular activity. Biopsies were obtained from the mammary gland at the three stages of lactation, early, mid and late, for histological and histochemical studies. The histological structures of the mammary gland showed clear differences between lactation stages—being more developed in the early and the mid stages, compared to the late stage of lactation. The number of the alveolar secretory cells increased from the early to the mid stage of lactation by 12.9% and then was reduced at the late stage by 35.9% from that at the mid stage. The milk yield increased by 51.3% from the early to the mid stage, and then was reduced at the late stage by 71.4% from that of the mid stage. The total sectional areas of plate equal to 1.22 mm²/plate of the alveoli were the smallest during late lactation (0.36 mm²/plate) compared to that during the early and the mid stage of lactation (0.50 and 1.17 mm²/plate, respectively). Numerous loci of alkaline phosphatase enzyme (AP) were apparent on the outer surface of the alveolar secretory cells at the early and the mid stages of lactation—suggesting that this enzyme plays an important physiological role in the apical membrane of the alveolar epithelial cells during lactation. Dense protein staining of these cells as well as increased frequency of DNA expression denote great development and increased numbers of these cells at early and mid stages of lactation. This was accompanied by a high level of milk secretion reaching 939.3 ± 130 and 1421.4 ± 123.4 ml/head/day, respectively. In contrast, at the late stage of lactation, the size of alveoli was reduced and few alveoli showed weak AP activity, weak protein manifestation and the lowest frequency of DNA loci. This coincided with the reduction in milk yield (407 ml/head/day). It could be concluded that the stages of the lactation influence the cell number and activity of the mammary parenchyma.     

Expression and characterization of <Emphasis Type="Italic">Trichoderma virens</Emphasis> UKM-1 endochitinase in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg²⁺ and Ca²⁺ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)₃, (GlcNAc)₂ and GlcNAc, in which (GlcNAc)₂ was the main product.     

Potent biphenyl- and 3-phenyl pyridine-based inhibitors of acetyl-CoA carboxylase

We report the synthesis and enzymatic evaluation of potent inhibitors of acetyl-CoA carboxylases (ACCs) containing biphenyl or 3-phenyl pyridine cores. These compounds inhibit both ACC1 and ACC2, or are moderately selective for either enzyme, depending on side chain substitution. Typical activities of the most potent compounds in this class are in the low double-digit to single-digit nanomolar range in in vitro assays using human ACC1 and ACC2 enzymes.     

Differential effect of metals/metalloids on the growth and element uptake of mesquite plants obtained from plants grown at a copper mine tailing and commercial seeds

The selection of appropriate seeds is essential for the success of phytoremediation/restoration projects. In this research, the growth and elements uptake by the offspring of mesquite plants (Prosopis sp.) grown in a copper mine tailing (site seeds, SS) and plants derived from vendor seeds (VS) was investigated. Plants were grown in a modified Hoagland solution containing a mixture of Cu, Mo, Zn, As(III) and Cr(VI) at 0, 1, 5 and 10 mg L⁻¹ each. After one week, plants were harvested and the concentration of elements was determined by using ICP-OES. At 1 mg L⁻¹, plants originated from SS grew faster and longer than control plants (0 mg L⁻¹); whereas plants grown from VS had opposite response. At 5 mg L⁻¹, 50% of the plants grown from VS did not survive, while plants grown from SS had no toxicity effects on growth. Finally, plants grown from VS did not survive at 10 mg L⁻¹ treatment, whilst 50% of the plants grown from SS survived. The ICP-OES data demonstrated that at 1 mg L⁻¹ the concentration of all elements in SS plants was significantly higher compared to control plants and VS plants. While at 5 mg L⁻¹, the shoots of SS plants had significantly more Cu, Mo, As, and Cr. The results suggest that SS could be a better source of plants intended to be used for phytoremediation of soil impacted with Cu, Mo, Zn, As and Cr.     

Diversity of Novel Glutenin Subunits in Bread Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Glutenin is a major determinant of baking performance and viscoelasticity, which are responsible for high-quality bread with a light porous crumb structure of a well-leavened loaf. We analyzed the diversity of glutenin genes from six wheat cultivars (Korean cvs. Keumgang and Jinpum, Chinese cvs. China-108 and Yeonnon-78, and Japanese cvs. Norin-61 and Kantou-107). Glutenins contain two types of isoforms such as high molecular weight glutenin subunit (HMW-GS) and low molecular weight glutenin subunit (LMW-GS). Glutenin fractions were extracted from wheat endosperm using Osborne solubility method. A total of 217 protein spots were separated on two-dimensional gel electrophoresis with isoelectric focusing (wide range of pH 3–10). The proteins spots were subjected to tryptic digestion and identified by matrix assisted laser desorption/ionization–time of flight mass spectrometry. HMW-GS (43 isoforms) and LMW-GS (seven isoforms) are directly responsible for producing high-quality bread and noodles. Likewise, all the seed storage proteins are digested to provide nutrients for the embryo during seed germination and seedling growth. We identified the diverse glutenin subunits in wheat cultivars and compared the gluten isoforms among different wheat cultivars according to quality. This work gives an insight on the quality improvement in wheat crop.     

Molecular Mimicry by an F-Box Effector of Legionella pneumophila Hijacks a Conserved Polyubiquitination Machinery within Macrophages and Protozoa

The ability of Legionella pneumophila to proliferate within various protozoa in the aquatic environment and in macrophages indicates a remarkable evolution and microbial exploitation of evolutionarily conserved eukaryotic processes. Ankyrin B (AnkB) of L. pneumophila is a non-canonical F-box-containing protein, and is the only known Dot/Icm-translocated effector of L. pneumophila essential for intra-vacuolar proliferation within both macrophages and protozoan hosts. We show that the F-box domain of AnkB and the ⁹L¹⁰P conserved residues are essential for intracellular bacterial proliferation and for rapid acquisition of polyubiquitinated proteins by the Legionella-containing vacuole (LCV) within macrophages, Dictyostelium discoideum, and Acanthamoeba. Interestingly, translocation of AnkB and recruitment of polyubiquitinated proteins in macrophages and Acanthamoeba is rapidly triggered by extracellular bacteria within 5 min of bacterial attachment. Ectopically expressed AnkB within mammalian cells is localized to the periphery of the cell where it co-localizes with host SKP1 and recruits polyubiquitinated proteins, which results in restoration of intracellular growth to the ankB mutant similar to the parental strain. While an ectopically expressed AnkB-⁹L¹⁰P/AA variant is localized to the cell periphery, it does not recruit polyubiquitinated proteins and fails to trans-rescue the ankB mutant intracellular growth defect. Direct in vivo interaction of AnkB but not the AnkB-⁹L¹⁰P/AA variant with the host SKP1 is demonstrated. Importantly, RNAi-mediated silencing of expression of SKP1 renders the cells non-permissive for intracellular proliferation of L. pneumophila. The role of AnkB in exploitation of the polyubiquitination machinery is essential for intrapulmonary bacterial proliferation in the mouse model of Legionnaires'' disease. Therefore, AnkB exhibits a novel molecular and functional mimicry of eukaryotic F-box proteins that exploits conserved polyubiquitination machinery for intracellular proliferation within evolutionarily distant hosts.     

Multiplex PCR assay for rapid identification of three endangered snake species of India

Species identification has been the core issue in all approaches of conservation of endangered wild life. In this regard molecular techniques for species authentication have proved indispensable. A novel multiplex PCR assay for the identification of three Indian snake species Python morulus, Ptyas mucosus, and Naja naja is successfully demonstrated using 16S rRNA gene. Three reverse primers and a common forward primer were designed to generate three different size species-specific PCR fragments. Absence of any PCR amplification in non-target species proves the specificity of the primers. These four primers were combined in a multiplex assay to enable identification of three snake species in a single reaction. The assay described here shows its utility in identifying unknown snake specimen and in case of samples yielding low quality DNA. This multiplex PCR technique using novel primers is an unprecedented approach offered for forensic identification of exhibits originating from three Indian snake species. It is expected that this endeavor will help strengthening conservation efforts for these species.     

Molecular identification of crocodile species using novel primers for forensic analysis

All crocodilians are under varying degrees of threat due to over exploitation and these species have been listed in Appendix I or II of CITES. The lack of molecular techniques for the identification of confiscated samples makes it difficult to enforce the law. Conclusive forensic identification of species requires a complete gene sequence which is difficult in case of degraded samples. We have developed two novel sets of primers to amplify two partial cytochrome b gene sequences of six crocodile species i.e. Crocodylus palustris, Crocodylus porosus, Crocodylus siamensis, Crocodylus niloticus, Gavialis gangeticus and Caiman crocodilus. These partial sequences were edited to give a complete cyt b gene sequence, which can be used as an effective tool for forensic authentication of crocodile species. A phylogeny of crocodile species was reconstructed using these sequences. The described primers hold great promise in forensic identification of crocodile species, which can aid in the effective enforcement of law and conservation of these ancient species.     

Patch area, substrate depth, and richness affect giving-up densities: a test with mourning doves and cottontail rabbits

We compared the foraging behavior of mourning doves Zenaida macroura and cottontail rabbits Sylvilagus floridanus in patches that varied in initial food abundance, surface area and substrate depth. We measured giving‐up densities (GUD), food harvest and proportion of food harvested to investigate their ability to respond to characteristics of resource patches. GUDs have been analyzed in three ways: grams of per patch, grams per unit surface area (GUD_AREA), and grams per unit volume of sand (GUD_VOL). Mourning doves and cottontails exhibited similar responses to resource density and sand depth. Both foragers detected and responded to variation in initial food abundance. The proportion of food harvested from a patch increased from 40.7, 43.8 to 48.3% (for the doves) and 34.9, 35.8 to 38.4% (for the rabbits) in patches of low, medium and high initial food abundance, respectively. Deeper substrates reduced the foragers’ encounter probability with food, decreased patch quality and resulted in higher GUDs (60% higher in the deepest relative to shallowest substrate) and lower harvests. A significant interaction between initial food abundance and substrate depth showed that both species were willing to dig deeper in patches with higher resource density. Patch size (surface area) had no effect on food harvest or the proportion of food harvested. Consequently, GUD_AREA and GUD_VOL increased in patches with a smaller surface area. Smaller patches appeared to hamper the dove's and cottontail's movement across the surface. Our results revealed that mourning doves and cottontails forage under imperfect information. Both species were able to respond to patch properties by biasing their feeding efforts toward rich and easy opportunities, however, mourning doves were more efficient at food harvesting. The interaction of patch area, volume and food abundance directly influenced food harvest. Such resource characters occur under natural situations where food varies in abundance, area of distribution, and accessibility.