Glycosylation as a tool for rational vaccine design

The discovery of broadly neutralizing antibodies that can neutralize multiple strains or subtypes of a pathogen has renewed interest in the development of broadly protective vaccines. To that end, there has been an interest in designing immunofocusing strategies to direct the immune response to specific, conserved regions on antigenic proteins. Modulation of glycosylation is one such immunofocusing strategy; extensive glycosylation is often exploited by pathogens for immune evasion. Masking epitopes on protein immunogens with “self” glycans can also shield the underlying protein surface from humoral immune surveillance. We review recent advances in applying glycosylation as an immunofocusing tool. We also highlight recent interesting work in the HIV-1 field involving the identification and elicitation of broadly neutralizing antibodies that incorporate glycans into their binding epitopes.     

Anatomical and biochemical aspects of interaction between roots of chickpea and Fusarium oxysporum f. sp. ciceris race 2

Roots of the susceptible “JG-62” and resistant “WR-315” chickpeas (Cicer arietinum L.) were inoculated with a conidial suspension of Fusarium oxysporum f. sp. ciceris. Anatomical and biochemical studies were carried out in a time-course manner to elucidate the infection process and plant defence reactions. Scanning electron microscope images revealed fungal colonisation in the root hair region. Early occurrence of fungal biofilms associated with the infected “JG-62” root epidermis was also visualised. After 96 h of inoculation, a gradual accumulation of polysaccharide positive deposits was observed in the xylem vessels of the infected “JG-62” roots. Fungal mycelium was observed in the vessel lumen of infected “JG-62” after 22 days of inoculation. Due to fungal invasion during this period, some of the vessels also appeared collapsed in “JG-62”, whereas vessels in “WR-315” remained intact. The host plant defence responses specifically linked to the susceptible interactions were the induction of ascorbate peroxidase, guaiacol peroxidase and superoxide dismutase in roots and shoots.     

FT-IR,FT-Raman spectra and DFT vibrational analysis of 2-aminobiphenyl

In this work, the Fourier transform infrared (FT-IR) and Fourier transform Raman (FT-Raman) spectra of 2-aminobiphenyl (2ABP) were recorded in the solid phase. The optimised geometry, frequency and intensity of the vibrational bands of 2ABP were obtained by the density functional theory (BLYP and B3LYP) methods with complete relaxation in the potential energy surface using 6-31G(d) basis set. The harmonic vibrational frequencies were calculated and the scaled values have been compared with experimental FT-IR and FT-Raman spectra. The observed and the calculated frequencies are found to be in good agreement. The experimental spectra also coincide satisfactorily with those of theoretically constructed spectrograms.     

A Study of the Interaction of Cr(III) Complexes and Their Selective Binding with B-DNA: A Molecular Modeling Approach

Abstract

Molecular modeling and energy minimisation calculations have been used to investigate the interaction of chromium(III) complexes in different ligand environments with various sequences of B-DNA. The complexes are [Cr(salen)(H₂O)₂]⁺; salen denotes 1, 2 bis-salicylideneaminoethane, [Cr(salprn)(H₂O)₂]⁺; salprn denotes 1, 3 bis- salicylideneamino-propane, [Cr(phen)₃]³⁺; phen denotes 1, 10 phenanthroline and [Cr(en)₃]³⁺; en denotes eth- ylenediamine. All the chromium(III) complexes are interacted with the minor groove and major groove of d(AT)₁₂, d(CGCGAATTCGCG)₂ and d(GC)₁₂ sequences of DNA. The binding energy and hydrogen bond parameters of DNA-Cr complex adduct in both the groove have been determined using molecular mechanics approach. The binding energy and formation of hydrogen bonds between chromium(III) complex and DNA has shown that all complexes of chromium(III) prefer minor groove interaction as the favourable binding mode.     

Vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars

For the first time we have developed a reliable and efficient vacuum infiltration-assisted Agrobacterium-mediated genetic transformation (VIAAT) protocol for Indian soybean cultivars and recovered fertile transgenic soybean plants through somatic embryogenesis. Immature cotyledons were used as an explant and three Agrobacterium tumefaciens strains (EHA 101, EHA 105, and KYRT 1) harbouring the binary vector pCAMBIA1301 were experimented in the co-cultivation. The immature cotyledons were pre-cultured in liquid somatic embryo induction medium prior to vacuum infiltration with the Agrobacterium suspension and co-cultivated for 3 days on co-cultivation medium containing 50 mg l^?1 citric acid, 100 µM acetosyringone, and 100 mg l^?1 l-cysteine. The transformed somatic embryos were selected in liquid somatic embryo induction medium containing 10 mg l^?1 hygromycin and the embryos were germinated in basal medium containing 20 mg l^?1 hygromycin. The presence and integration of the hpt II and gus genes into the soybean genome were confirmed by GUS histochemical assay, polymerase chain reaction, and Southern hybridization. Among the different combinations tested, high transformation efficiency (9.45 %) was achieved when immature cotyledons of cv. Pusa 16 were pre-cultured for 18 h and vacuum infiltrated with Agrobacterium tumefaciens KYRT 1 for 2 min at 750 mm of Hg. Among six Indian soybean cultivars tested, Pusa 16 showed highest transformation efficiency of 9.45 %. The transformation efficiency of this method (VIAAT) was higher than previously reported sonication-assisted Agrobacterium-mediated transformation. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into soybean has been developed.     

Microbial Communities in Chesapeake & Ohio Canal National Historical Park and Their Function as Indicators of Water Quality

Abstract

The objective of this study was to establish meaningful relationships between prokaryotic community profiles and water quality parameters in different water bodies (spring, stream, cave, and mine) in the middle reach of the Chesapeake & Ohio Canal National Historic Park (C&O), Maryland. The microbial profiles in the water samples were determined using metagenomic analysis. The relationships between microbial phylogenetic profiles and water quality parameters were investigated using principal component analysis (PCA) and redundancy analysis (RDA). The most abundant phyla identified in most samples were Proteobacteria (55.4%), Bacteroidetes (12.3%), Actinobacteria (10.6%), Firmicutes (2.4%), Planktomycetes (1.8%), Verrucomicrobia (1.5%), Chloroflexi (1.5%), and Acidobacteria (1.3%), which are major bacterial and archaeal groups typically observed in natural freshwater environments. PCA showed that water chemistry was determined primarily by the geology of the site and the type of water source (i.e., spring, stream, cave, or mine). Most samples located in carbonate formations correlated with high alkalinity, inorganic carbon, and calcium, representing the typical karstic geochemistry. RDA shows that pH, electrical conductivity, temperature and nutrients including nitrate, phosphate, and sulfate, were significant determinants of the microbial ecology.     

A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data

Microsomal cytochrome b₅ (cytb₅) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb₅ (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb₅ NMR resonances and site-directed mutagenesis data were used to characterize the cytb₅ interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb₅ using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb₅ and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb₅. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb₅. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb₅-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes.     

The Peptidyl-prolyl Isomerase Pin1 Up-regulation and Proapoptotic Function in Dopaminergic Neurons: RELEVANCE TO THE PATHOGENESIS OF PARKINSON DISEASE*

Parkinson disease (PD) is a chronic neurodegenerative disease characterized by a slow and progressive degeneration of dopaminergic neurons in substantia nigra. The pathophysiological mechanisms underlying PD remain unclear. Pin1, a major peptidyl-prolyl isomerase, has recently been associated with certain diseases. Notably, Ryo et al. (Ryo, A., Togo, T., Nakai, T., Hirai, A., Nishi, M., Yamaguchi, A., Suzuki, K., Hirayasu, Y., Kobayashi, H., Perrem, K., Liou, Y. C., and Aoki, I. (2006) J. Biol. Chem. 281, 4117–4125) implicated Pin1 in PD pathology. Therefore, we sought to systematically characterize the role of Pin1 in PD using cell culture and animal models. To our surprise we observed a dramatic up-regulation of Pin1 mRNA and protein levels in dopaminergic MN9D neuronal cells treated with the parkinsonian toxicant 1-methyl-4-phenylpyridinium (MPP⁺) as well as in the substantia nigra of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. Notably, a marked expression of Pin1 was also observed in the substantia nigra of human PD brains along with a high co-localization of Pin1 within dopaminergic neurons. In functional studies, siRNA-mediated knockdown of Pin1 almost completely prevented MPP⁺-induced caspase-3 activation and DNA fragmentation, indicating that Pin1 plays a proapoptotic role. Interestingly, multiple pharmacological Pin1 inhibitors, including juglone, attenuated MPP⁺-induced Pin1 up-regulation, α-synuclein aggregation, caspase-3 activation, and cell death. Furthermore, juglone treatment in the MPTP mouse model of PD suppressed Pin1 levels and improved locomotor deficits, dopamine depletion, and nigral dopaminergic neuronal loss. Collectively, our findings demonstrate for the first time that Pin1 is up-regulated in PD and has a pathophysiological role in the nigrostriatal dopaminergic system and suggest that modulation of Pin1 levels may be a useful translational therapeutic strategy in PD.