A novel recombinant multiepitope protein as a hepatitis C diagnostic intermediate of high sensitivity and specificity

A novel recombinant multiepitope protein has been designed that consists of six linear, immunodominant, and phylogenetically conserved epitopes from hepatitis C virus. Five of these antigens (core, NS3, NS4I, NS4II, and NS5) are being used in many of the third-generation kits while sixth epitope (core3g) is an additional sequence from a newly identified Indian isolate. The genes for these epitopes have been joined together to code for a single multiepitope protein that has been evaluated for its diagnostic potential for the detection of anti-HCV antibodies in human plasma. Two separate synthetic genes have been designed, both encoding the same six epitopes in a single open reading frame along with spacers having additional amino acids to function as flexible (r-HCV-F-MEP) or rigid (r-HCV-R-MEP) linkers. High-level expression of hepatitis C multiepitope protein in Escherichia coli has been achieved. The protein has been purified using a single affinity step yielding >25 mg pure protein/liter culture and used as the coating antigen in anti-HCV EIA. The use of this multiepitope protein eliminates the requirement for multiple diagnostic intermediates for the development of anti-HCV diagnostic kit. The sensitivity and specificity of the HCV multiepitope protein was evaluated by Boston Biomedica Worldwide Performance Panels, HCV Seroconversion Panels and Viral Co-infection Panels, and was found to be comparable with commercially available anti-HCV EIA kits. This analysis indicated its unequivocal performance as capture antigen in anti-HCV EIA. The high epitope density, careful choice of epitopes and use of E. coli system for expression, coupled with simple purification protocol provides the potential for the development of an inexpensive diagnostic test with high degree of sensitivity and specificity.     

Influence of supplemental UV-B radiation on photosynthetic characteristics of rice plants

In a field experiment with rice (Oryza sativa L. cv. Saket 4) grown under ambient and supplemental ultraviolet-B (UV-B) radiation at 20 % ozone depletion, differences in gas exchange, concentrations of photosynthetic pigments, anthocyanins and flavonoids, biomass accumulation, catalase and peroxidase activities, and contents of ascorbic acid and phenol were determined. Decline in photosynthesis was associated with reductions in stomatal conductance and concentrations of photosynthetic pigments. Enhanced UV-B radiation (eUV-B) increased the contents of flavonoid and phenolic compounds in leaves. Peroxidase activity increased and catalase activity was always lower at eUV-B. The total plant biomass decreased at eUV-B.     

Probing WW Domains to Uncover and Refine Determinants of Specificity in Ligand Recognition

Understanding the specificity of protein-protein interaction mediated by domains and their ligands will have strong impact on basic and applied research. Visual inspection of WW domain sequences prompted a general classification of the domains into two large subfamilies. One subfamily contains two consecutive aromatic residues in the beta 2 strand of the domain whereas the other contains three or four consecutive aromatic residues in the same position. In the recent past, we proposed a rule of ‘two vs. three aromatics’ in the beta 2 strand of WW domains as a molecular discriminator between Class I and Class II WW domains, which recognize PPxY or PPLP motifs, respectively. Using phage display libraries expressing WW domains with random sequences replacing a part of the beta 2 strand, we provided additional evidence supporting our rule. We conclude that three consecutive aromatic amino acids within the beta 2 strand of WW domain are required but not always sufficient for the WW domain to belong to Class II.     

Calculating Climate Effects on Birds and Mammals: Impacts on Biodiversity, Conservation, Population Parameters, and Global Community Structure

This paper describes how climate variation in time and spacecan constrain community structure on a global scale. We explorebody size scaling and the energetic consequences in terms ofabsorbed mass and energy and expended mass and energy. We explainhow morphology, specific physiological properties, and temperaturedependent behaviors are key variables that link individual energeticsto population dynamics and community structure. This paper describes an integrated basic principles model formammal energetics and extends the model to bird energetics.The model additions include molar balance models for the lungsand gut. The gut model couples food ingested to respiratorygas exchanges and evaporative water loss from the respiratorysystem. We incorporate a novel thermoregulatory model that yieldsmetabolic calculations as a function of temperature. The calculationsmimic empirical data without regression. We explore the differencesin the quality of insulation between hair and feathers withour porous media model for insulation. For mammals ranging in size from mice to elephants we show thatcalculated metabolic costs are in agreement with experimentaldata. We also demonstrate how we can do the same for birds rangingin size from hummingbirds to ostriches. We show the impact ofchanging posture and changing air temperatures on energeticcosts for birds and mammals. We demonstrate how optimal bodysize that maximizes the potential for growth and reproductionchanges with changing climatic conditions and with diet quality.Climate and diet may play important roles in constraining communitystructure (collection of functional types of different bodysizes) at local and global scales. Thus, multiple functionaltypes may coexist in a locality in part because of the temporaland spatial variation in climate and seasonal food variation.We illustrate how the models can be applied in a conservationand biodiversity context to a rare and endangered species ofparrot, the Orange-bellied Parrot of Australia and Tasmania.     

Energy functions for protein design I: efficient and accurate continuum electrostatics and solvation

Electrostatics and solvation energies are important for defining protein stability, structural specificity, and molecular recognition. Because these energies are difficult to compute quickly and accurately, they are often ignored or modeled very crudely in computational protein design. To address this problem, we have developed a simple, fast, and accurate approximation for calculating Born radii in the context of protein design calculations. When these approximate Born radii are used with the generalized Born continuum dielectric model, energies calculated by the 10(6)-fold slower finite difference Poisson-Boltzmann model are faithfully reproduced. A similar approach can be used for estimating solvent-accessible surface areas (SASAs). As an independent test, we show that these approximations can be used to accurately predict the experimentally determined pK(a)s of >200 ionizable groups from 15 proteins.     

Leukocyte Function-associated Antigen-1/Intercellular Adhesion Molecule-1 Interaction Induces a Novel Genetic Signature Resulting in T-cells Refractory to Transforming Growth Factor-β Signaling

The immunesuppressive cytokine TGF-β plays crucial regulatory roles in the induction and maintenance of immunologic tolerance and prevention of immunopathologies. However, it remains unclear how circulating T-cells can escape from the quiescent state maintained by TGF-β. Here, we report that the T-cell integrin leukocyte function-associated antigen-1 (LFA-1) interaction with its ligand intercellular adhesion molecule-1 (ICAM-1) induces a genetic signature associated with reduced TGF-β responsiveness via up-regulation of SKI, E3 ubiquitin-protein ligase SMURF2, and SMAD7 (mothers against decapentaplegic homolog 7) genes and proteins. We confirmed that the expression of these TGF-β inhibitory molecules was dependent on STAT3 and/or JNK activation. Increased expression of SMAD7 and SMURF2 in LFA-1/ICAM-1 cross-linked T-cells resulted in impaired TGF-β-mediated phosphorylation of SMAD2 and suppression of IL-2 secretion. Expression of SKI caused resistance to TGF-β-mediated suppression of IL-2, but SMAD2 phosphorylation was unaffected. Blocking LFA-1 by neutralizing antibody or specific knockdown of TGF-β inhibitory molecules by siRNA substantially restored LFA-1/ICAM-1-mediated alteration in TGF-β signaling. LFA-1/ICAM-1-stimulated human and mouse T-cells were refractory to TGF-β-mediated induction of FOXP3(+) (forkhead box P3) and RORγt(+) (retinoic acid-related orphan nuclear receptor γt) Th17 differentiation. These mechanistic data suggest an important role for LFA-1/ICAM-1 interactions in immunoregulation concurrent with lymphocyte migration that may have implications at the level of local inflammatory response and for anti-LFA-1-based therapies.     

Contour abnormalities of the abdomen after breast reconstruction with abdominal flaps: the role of muscle preservation.

The purpose of the present study was to determine whether contour abnormalities of the abdomen after breast reconstruction with abdominal flaps are related to the harvest of the rectus abdominis muscle. Abdominal contour was analyzed in 155 women who had breast reconstruction with abdominal flaps; 108 women had free transverse rectus abdominis muscle (TRAM) flaps, 37 had pedicled TRAM flaps, and 10 had deep inferior epigastric perforator (DIEP) flaps. The reconstruction was unilateral in 110 women and bilateral in 45 women. Three methods of muscle-sparing were used; they are classified as preservation of the lateral muscle, preservation of the medial and lateral muscle, or preservation of the entire muscle. One of these three methods of muscle-sparing was used in 91 women (59 percent) and no muscle-sparing was used in 64 women (41 percent). Postoperative contour abnormalities occurred in 15 woman and included epigastric fullness in five, upper bulge in three, and lower bulge in 10. One woman experienced two abnormalities, one woman experienced three, and no woman developed a hernia. Of these abnormalities, 11 occurred after the free TRAM flap, seven after the pedicled TRAM flap, and none after the DIEP flap. Bilateral reconstruction resulted in 11 abnormalities in nine women, and unilateral reconstruction resulted in seven abnormalities in six women. chi2 analysis of the free and pedicled TRAM flaps demonstrates that muscle-sparing explains the observed differences in upper bulge and upper fullness (p = 0.02), with a trend toward significance for lower bulge (p = 0.06). chi2 analysis of the free TRAM and DIEP flaps does not explain the observed difference in abnormal abdominal contour. Analysis of muscle-sparing and non-muscle-sparing methods demonstrates that the observed difference between the techniques is only explained for a lower bulge after the bilateral free TRAM flap (p = 0.04).     

Synthesis of s-triazine-based thiazolidinones as antimicrobial agents

A novel series of thiazolidinone derivatives, namely 4-{4-dimethylamino-6-[4-oxo-2-phenyl-5-(4-pyridin-2-yl-piperazin-1-ylmethyl)-thiazolidin-3-yl]-[1,3,5]-triazin-2-yloxy}-1-methyl-1H-quinolin-2-ones, have been synthesized from the key intermediate 4-(4-amino-6-dimethylamino-[1,3,5]-triazin-2-yloxy)-1-methyl-1H-quinolin-2-one (5). Compound 5 was condensed with various aldehydes to give Schiff base derivatives, which after cyclization gave thiazolidinones that were linked with 1-pyridin-2-yl-piperazine to obtain the target compounds. The newly synthesized compounds were evaluated for their antimicrobial activity against eight bacteria (Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Proteus vulgaris, Shigella flexneri) and four fungi (Aspergillus niger, Candida albicans, Aspergillus fumigatus, Aspergillus clavatus).     

H2O2 pretreated rice seedlings specifically reduces arsenate not arsenite: difference in nutrient uptake and antioxidant defense response in a contrasting pair of rice cultivars

The study investigated the reduction in metalloid uptake at equimolar concentrations (~53.3 μM) of As(III) and As(V) in contrasting pair of rice seedlings by pretreating with H₂O₂ (1.0 μM) and SA (1.0 mM). Results obtained from the contrasting pair (arsenic tolerant vs. sensitive) of rice seedlings (cv. Pant Dhan 11 and MTU 7029, respectively) shows that pretreatment of H₂O₂ and H₂O₂ + SA reduces As(V) uptake significantly in both the cultivars, while no reduction in the As(III) uptake. The higher growth inhibition, higher H₂O₂ and TBARS content in sensitive cultivar against As(III) and As(V) treatments along with higher As accumulation (~1.2 mg g⁻¹ dw) than in cv. P11, unravels the fundamental difference in the response between the sensitive and tolerant cultivar. In the H₂O₂ pretreated plants, the translocation of As increased in tolerant cultivar against AsIII, whereas, it decreased in sensitive cultivar both against AsIII and AsV. In both the cultivars translocation of Mn increased in the H₂O₂ pretreated plants against As(III), whereas, the translocation of Cu increased against As(V). In tolerant cultivar the translocation of Fe increased against As(V) with H₂O₂ pretreatment whereas, it decreased in the sensitive cultivar. In both the cultivars, Zn translocation increased against As(III) and decreased against As(V). The higher level of H₂O₂ and SOD (EC 1.15.1.1) activity in sensitive cultivar whereas, higher, APX (EC 1.11.1.11), GR (EC 1.6.4.2) and GST (EC 1.6.4.2) activity in tolerant cultivar, also demonstrated the differential anti-oxidative defence responses between the contrasting rice cultivars.

Electronic supplementary material

The online version of this article (doi:10.1007/s12298-014-0255-1) contains supplementary material, which is available to authorized users.