Green synthesis and physiochemical characterization of nickel oxide nanoparticles: Interaction studies with Calf thymus DNA

One of the most promising applications of nanomaterials is that of nanobiosensors, using biomolecules such as nucleic acids as receptors. This study aimed to synthesize nickel oxide nanoparticles (NiO NPs) by an environmentally friendly green synthesis, using the extract of the herb Coriandrum sativum (coriander). The synthesized NPs were characterized using UV–Visible spectroscopy, Fourier transform infrared spectroscopy, X‐ray diffraction, X‐ray photon spectroscopy, field emission scanning electron microscopy coupled with energy dispersive spectroscopy, dynamic light scattering, zeta potential and transmission electron microscopy. All results confirmed the synthesis of pure, spherical, positively charged NiO NPs of around 95 nm in diameter with prominent hydroxyl groups attached to the surface. Furthermore, interaction studies of synthesized NiO NPs with calf thymus DNA (CT DNA) were performed using UV–Visible spectroscopy, UV–thermal melting, circular dichroism, and fluorescence spectroscopy. CT DNA served as a substitute for nucleic acid biosensors. All experimental studies indicated that the NiO NPs bound electrostatically with CT DNA. These studies may facilitate exploring the potential of NiO NP–nucleic acid conjugated materials to be used as nanobiosensors for various applications, especially in pharmacological, epidemiological, and environmental diagnostic applications, and in detection.     

Characterization and structure-guided engineering of the novel versatile terpene monooxygenase CYP109Q5 from Chondromyces apiculatus DSM436

One of the major challenges in chemical synthesis is the selective oxyfunctionalization of non-activated C-H bonds, which can be enabled by biocatalysis using cytochrome P450 monooxygenases. In this study, we report on the characterization of the versatile CYP109Q5 from Chondromyces apiculatus DSM436, which is able to functionalize a wide range of substrates (terpenes, steroids and drugs), including the ring of β-ionone in non-allylic positions. The crystal structure of CYP109Q5 revealed flexibility within the active site pocket that permitted the accommodation of bulky substrates, and enabled a structure-guided approach to engineering the enzyme. Some variants of CYP109Q5 displayed a switch in selectivity towards the non-allylic positions of β-ionone, allowing the simultaneous production of 2- and 3-hydroxy-β-ionone, which are chemically challenging to synthesize and are important precursors for carotenoid synthesis. An efficient whole-cell system finally enabled the production of up to 0.5 g l⁻¹ hydroxylated products of β-ionone; this system can be applied to product identification in further biotransformations. Overall, CYP109Q5 proved to be highly evolvable and active. The studies in this work demonstrate that, using rational mutagenesis, the highly versatile CYP109Q5 generalist can be progressively evolved to be an industrially valuable specialist for the synthesis of specific products.     

Effect of Organic Selenium Supplementation on Growth, Se Uptake, and Nutrient Utilization in Guinea Pigs

Forty weaned male guinea pigs (Cavia porcellus) of 152.6?±?7.96 g mean body weight were divided into four equal groups and fed a common basal diet comprised of 25% ground cowpea (Vigna unguiculata) hay, 30% ground maize (Zea mays) grain, 22% ground gram (Cicer arietinum) grain, 9.5% deoiled rice (Oryza sativa) bran, 6% soybean (Glycine max) meal, 6% fish meal, 1.5% mineral mixture (without Se), and ascorbic acid at 200 mg/kg to meet their nutrient requirements along with 0, 0.1, 0.2, and 0.3 ppm of organic selenium (Se) in groups I, II, III, and IV, respectively. Experimental feeding lasted for a period of 10 weeks, during which, daily feed intake and weekly body weights were recorded. Intake and digestibility of dry matter, organic matter, ether extract, crude fiber, and nitrogen-free extract as well as uptake of calcium and phosphorus were similar (P?>?0.05) among the four groups. Feed:gain ratio was also similar (P?>?0.05) in the four groups. However, digestibility of crude protein was significantly (P?<?0.001) higher in group II supplemented with 0.1 ppm organic Se as compared to other three group. Intake and absorption of Se was significantly (P?<?0.001) higher in all the Se supplemented groups as compared to control group. Average daily gain (ADG) was significantly (P?<?0.05) higher in group II (3.16 g/day) and III (3.38 g/day) as compared to group I (2.88 g/day). However, ADG in group IV (supplemented 0.3 ppm organic Se) was significantly (P?<?0.05) lower (2.83 g/day) than group II and III, but comparable (P?>?0.05) to group I. Findings of the present experiment suggests that Se requirements of guinea pigs are ≥0.2 ppm, as supplementation of 0.1 ppm organic Se in the diet (having 0.1 ppm Se) not only enhanced their growth rate but also improved the protein utilization.     

A study of 7-deaza-2'-deoxyguanosine 2'-deoxycytidine base pairing in DNA

The incorporation of 7-deazaguanine modifications into DNA is frequently used to probe protein recognition of H-bonding information in the major groove of DNA. While it is generally assumed that 7-deazaguanine forms a normal Watson–Crick base pair with cytosine, detailed thermodynamic and structural analyses of this modification have not been reported. The replacement of the 7-N atom on guanine with a C–H, alters the electronic properties of the heterocycle and eliminates a major groove cation-binding site that could affect the organization of salts and water in the major groove. We report herein the characterization of synthetic DNA oligomers containing 7-deazaguanine using a variety of complementary approaches: UV thermal melting, differential scanning calorimetry (DSC), circular dichroism (CD), chemical probing and NMR. The results indicate that the incorporation of a 7-deazaguanine modification has a significant effect on the dynamic structure of the DNA at the flanking residue. This appears to be mediated by changes in hydration and cation organization.     

Subproteomic analysis of metal-interacting proteins in human B cells

Metal-protein interactions are vitally important in all living organisms. Metalloproteins, including structural proteins and metabolic enzymes, participate in energy transfer and redox reactions or act as metallochaperones in metal trafficking. Among metal-associated diseases, T cell mediated allergy to nickel (Ni) represents the most common form of human contact hypersensitivity. With the aim to elucidate disease-underlying mechanisms such as Ni-specific T cell activation, we initiated a proteomic approach to identify Ni-interacting proteins in human B cells. As antigen presenting cells, B cells are capable of presenting MHC-associated Ni-epitopes to T cells, a prerequisite for hapten-specific T cell activation. Using metal-affinity enrichment, 2-DE and MS, 22 Ni-interacting proteins were identified. In addition to known Ni-binding molecules such as tubulin, actin or cullin-2, we unexpectedly discovered that at least nine of these 22 proteins belong to stress-inducible heat shock proteins or chaperonins. Enrichment was particularly effective for the hetero-oligomeric TRiC/CCT complex, which is involved in MHC class I processing. Blue Native/SDS electrophoresis analysis revealed that Ni-NTA-beads specifically retained the complete protein machinery, including the associated chaperonin substrate tubulin. The apparent Ni-affinity of heat shock proteins suggests a new function of these molecules in human Ni allergy, by linking innate and adaptive immune responses.     

T-cell antigen-receptor stoichiometry: pre-clustering for sensitivity

The T-cell antigen receptor (TCR x CD3) is a multi-subunit complex that is responsible for triggering an adaptive immune response. It shows high specificity and sensitivity, while having a low affinity for the ligand. Furthermore, T cells respond to antigen over a wide concentration range. The stoichiometry and architecture of TCR x CD3 in the membrane have been under intense scrutiny because they might be the key to explaining its paradoxical properties. This review highlights new evidence that TCR x CD3 is found on intact unstimulated T cells in a monovalent form (one ligand-binding site per receptor) as well as in several distinct multivalent forms. This is in contrast to the TCR x CD3 stoichiometries determined by several biochemical means; however, these data can be explained by the effects of different detergents on the integrity of the receptor. Here, we discuss a model in which the multivalent receptors are important for the detection of low concentrations of ligand and therefore confer sensitivity, whereas the co-expressed monovalent TCR x CD3s allow a wide dynamic range.     

Identification of Novel HIV 1- Protease Inhibitors: Application of Ligand and Structure Based Pharmacophore Mapping and Virtual Screening

A combined ligand and structure-based drug design approach provides a synergistic advantage over either methods performed individually. Present work bestows a good assembly of ligand and structure-based pharmacophore generation concept. Ligand-oriented study was accomplished by employing the HypoGen module of Catalyst in which we have translated the experimental findings into 3-D pharmacophore models by identifying key features (four point pharmacophore) necessary for interaction of the inhibitors with the active site of HIV-1 protease enzyme using a training set of 33 compounds belonging to the cyclic cyanoguanidines and cyclic urea derivatives. The most predictive pharmacophore model (hypothesis 1), consisting of four features, namely, two hydrogen bond acceptors and two hydrophobic, showed a correlation (r) of 0.90 and a root mean square of 0.71 and cost difference of 56.59 bits between null cost and fixed cost. The model was validated using CatScramble technique, internal and external test set prediction. In the second phase of our study, a structure-based five feature pharmacophore hypothesis was generated which signifies the importance of hydrogen bond donor, hydrogen bond acceptors and hydrophobic interaction between the HIV-1 protease enzyme and its inhibitors. This work has taken a significant step towards the full integration of ligand and structure-based drug design methodologies as pharmacophoric features retrieved from structure-based strategy complemented the features from ligand-based study hence proving the accuracy of the developed models. The ligand-based pharmacophore model was used in virtual screening of Maybridge and NCI compound database resulting in the identification of four structurally diverse druggable compounds with nM activities.     

Identification of pathways associated with invasive behavior by ovarian cancer cells using multidimensional protein identification technology (MudPIT)

Proteomic profiling has emerged as a useful tool for identifying tissue alterations in disease states including malignant transformation. The aim of this study was to reveal expression profiles associated with the highly motile/invasive ovarian cancer cell phenotype. Six ovarian cancer cell lines were subjected to proteomic characterization using multidimensional protein identification technology (MudPIT), and evaluated for their motile/invasive behavior, so that these parameters could be compared. Within whole cell extracts of the ovarian cancer cells, MudPIT identified proteins that mapped to 2245 unique genes. Western blot analysis for selected proteins confirmed the expression profiles revealed by MudPIT, demonstrating the fidelity of this high-throughput analysis. Unsupervised cluster analysis partitioned the cell lines in a manner that reflected their motile/invasive capacity. A comparison of protein expression profiles between cell lines of high (group 1) versus low (group 2) motile/invasive capacity revealed 300 proteins that were differentially expressed, of which 196 proteins were significantly upregulated in group 1. Protein network and KEGG pathway analysis indicated a functional interplay between proteins up-regulated in group 1 cells, with increased expression of several key members of the actin cytoskeleton, extracellular matrix (ECM) and focal adhesion pathways. These proteomic expression profiles can be utilized to distinguish highly motile, aggressive ovarian cancer cells from lesser invasive ones, and could prove to be essential in the development of more effective strategies that target pivotal cell signaling pathways used by cancer cells during local invasion and distant metastasis.