Identification of Drug and Vaccine Target in Mycobacterium leprae : A Reverse Vaccinology Approach

Mycobacterium leprae, an infectious agent of chronic infection so-called Leprosy. It is a prime healthconcern in various countries including India. India i     

Surface Enhanced Raman Scattering from Single-Walled Carbon Nanotube Decorated on Ag Nanowires

Plasmonics - Surface-enhanced Raman spectroscopy (SERS) is a surface sensitive technique which gives enhanced Raman signal intensity of the molecules by roughening metal surfaces. It can be used to...     

Meeting report: SMART timing—principles of single molecule techniques course at the University of Michigan 2014

Four days after the announcement of the 2014 Nobel Prize in Chemistry for “the development of super‐resolved fluorescence microscopy” based on single molecule detection, the Single Molecule Analysis in Real‐Time (SMART) Center at the University of Michigan hosted a “Principles of Single Molecule Techniques 2014” course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 296–302, 2015.     

Molecular Consequences of the SERPINH1/HSP47 Mutation in the Dachshund Natural Model of Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a heritable connective tissue disease characterized by bone fragility and increased risk of fractures. Up to now, mutations in at least 18 genes have been associated with dominant and recessive forms of OI that affect the production or post-translational processing of procollagen or alter bone homeostasis. Among those, SERPINH1 encoding heat shock protein 47 (HSP47), a chaperone exclusive for collagen folding in the ER, was identified to cause a severe form of OI in dachshunds (L326P) as well as in humans (one single case with a L78P mutation). To elucidate the disease mechanism underlying OI in the dog model, we applied a range of biochemical assays to mutant and control skin fibroblasts as well as on bone samples. These experiments revealed that type I collagen synthesized by mutant cells had decreased electrophoretic mobility. Procollagen was retained intracellularly with concomitant dilation of ER cisternae and activation of the ER stress response markers GRP78 and phospho-eIF2α, thus suggesting a defect in procollagen processing. In line with the migration shift detected on SDS-PAGE of cell culture collagen, extracts of bone collagen from the OI dog showed a similar mobility shift, and on tandem mass spectrometry, the chains were post-translationally overmodified. The bone collagen had a higher content of pyridinoline than control dog bone. We conclude that the SERPINH1 mutation in this naturally occurring model of OI impairs how HSP47 acts as a chaperone in the ER. This results in abnormal post-translational modification and cross-linking of the bone collagen.     

Mycobacterium tuberculosis class II apurinic/apyrimidinic‐endonuclease/3′‐5′ exonuclease III exhibits DNA regulated modes of interaction with the sliding DNA β‐clamp

The class‐II AP‐endonuclease (XthA) acts on abasic sites of damaged DNA in bacterial base excision repair. We identified that the sliding DNA β‐clamp forms in vivo and in vitro complexes with XthA in Mycobacterium tuberculosis. A novel ₂₃₉QLRFPKK₂₄₅ motif in the DNA‐binding domain of XthA was found to be important for the interactions. Likewise, the peptide binding‐groove (PBG) and the C‐terminal of β‐clamp located on different domains interact with XthA. The β‐clamp‐XthA complex can be disrupted by clamp binding peptides and also by a specific bacterial clamp inhibitor that binds at the PBG. We also identified that β‐clamp stimulates the activities of XthA primarily by increasing its affinity for the substrate and its processivity. Additionally, loading of the β‐clamp onto DNA is required for activity stimulation. A reduction in XthA activity stimulation was observed in the presence of β‐clamp binding peptides supporting that direct interactions between the proteins are necessary to cause stimulation. Finally, we found that in the absence of DNA, the PBG located on the second domain of the β‐clamp is important for interactions with XthA, while the C‐terminal domain predominantly mediates functional interactions in the substrate's presence.     

Revisiting the membrane interaction mechanism of a membrane‐damaging β‐barrel pore‐forming toxin Vibrio cholerae cytolysin

Vibrio cholerae cytolysin (VCC) permeabilizes target cell membranes by forming transmembrane oligomeric β‐barrel pores. VCC has been shown to associate with the target membranes via amphipathicity‐driven spontaneous partitioning into the membrane environment. More specific interaction(s) of VCC with the membrane components have also been documented. In particular, specific binding of VCC with the membrane lipid components is believed to play a crucial role in determining the efficacy of the pore‐formation process. However, the structural basis and the functional implications of the VCC interaction with the membrane lipids remain unclear. Here we show that the distinct loop sequences within the membrane‐proximal region of VCC play critical roles to determine the functional interactions of the toxin with the membrane lipids. Alterations of the loop sequences via structure‐guided mutagenesis allow amphipathicity‐driven partitioning of VCC to the membrane lipid bilayer. Alterations of the loop sequences, however, block specific interactions of VCC with the membrane lipids and abort the oligomerization, membrane insertion, pore‐formation and cytotoxic activity of the toxin. Present study identifies the structural signatures in VCC implicated for its functional interactions with the membrane lipid components, a process that presumably acts to drive the subsequent steps of the oligomeric β‐barrel pore‐formation and cytotoxic responses.     

Molecular characterization of lapinized classical Swine Fever vaccine strain by full-length genome sequencing and analysis

The complete genome of a lapinized classical swine fever virus (CSFV) vaccine strain was amplified into nine overlapping fragments by RT-PCR, and nucleotide sequences were determined. Complete genome sequence alignment and phylogenetic analysis indicated 92.6-98.6% identities at the nucleotide level with other reported CSFV strains and could be grouped into subgroup 1.1 along with other attenuated strains of CSFV. The 5'-UTR demonstrated >97.0% nucleotide similarity with most of vaccine CSFV strains from China. Further, its 3'-UTR sequence indicated a length similar to all the CSFV strains from China with >98.0% nucleotide similarity, although high length heterogeneity of 3'-UTR was reported among different CSFV strains. There was 12 nt (TTTTCTTTTTTT) insertion in 3'-UTR similar to other reported attenuated vaccine strains. However, secondary structure of 3'-UTR indicated that Indian CSFV strain requires further passage to obtain a 3'-UTR structure similar to most of the attenuated strains.