Domain swapping in human αA and αB crystallins affects oligomerization and enhances chaperone-like activity

αA and αB crystallins, members of the small heat shock protein family, prevent aggregation of proteins by their chaperone-like activity. These two proteins, although very homologous, particularly in the C-terminal region, which contains the highly conserved "α-crystallin domain," show differences in their protective ability toward aggregation-prone target proteins. In order to investigate the differences between αA and αB crystallins, we engineered two chimeric proteins, αANBC and αBNAC, by swapping the N-terminal domains of αA and αB crystallins. The chimeras were cloned and expressed in Escherichia coli. The purified recombinant wild-type and chimeric proteins were characterized by fluorescence and circular dichroism spectroscopy and gel permeation chromatography to study the changes in secondary, tertiary, and quaternary structure. Circular dichroism studies show structural changes in the chimeric proteins. αBNAC binds more 8-anilinonaphthalene-1-sulfonic acid than the αANBC and the wild-type proteins, indicating increased accessible hydrophobic regions. The oligomeric state of αANBC is comparable to wild-type αB homoaggregate. However, there is a large increase in the oligomer size of the αBNAC chimera. Interestingly, swapping domains results in complete loss of chaperone-like activity of αANBC, whereas αBNAC shows severalfold increase in its protective ability. Our findings show the importance of the N- and C-terminal domains of αA and αB crystallins in subunit oligomerization and chaperone-like activity. Domain swapping results in an engineered protein with significantly enhanced chaperone-like activity

Redox-regulated chaperone function and conformational changes of Escherichia coli Hsp33

We have studied the chaperone activity and conformation of Escherichia coli heat shock protein (Hsp)33, whose activity is known to be switched on by oxidative conditions. While oxidized Hsp33 completely prevents the heat-induced aggregation of ζ-crystallin at 42°C at a ratio of 1:1 (w/w), the reduced form exhibits only a marginal effect on the aggregation. Far UV–circular dichroism (CD) spectra show that reduced Hsp33 contains a significant α-helical component. Oxidation results in significant changes in the far UV–CD spectrum. Near UV–CD spectra show changes in tertiary structural packing upon oxidation. Polarity-sensitive fluorescent probes report enhanced hydrophobic surfaces in the oxidized Hsp33. Our studies show that the oxidative activation of the chaperone function of Hsp33 involves observable conformational changes accompanying increased exposure of hydrophobic pockets

Surgical Management of a Rare Case of Scrotal Lymphangioma in Ongole Bull

An unusual case of scrotal lymphangioma in a ongole bull is described. A 5 years old ongole bull was presented with a eight month history of swelling at the neck of scrotum. On palpation the swelling was hard in consistency. Fine needle aspiration revealed no sign of malignancy. On surgical exploration of scrotal swelling, a hard mass was observed adhering to the outerlayers of spermatic cord. The mass was excised without disturbing the testes and the associated structures. Based on cytological and histopathological examination the mass was diagnosed as lymphangioma. The surgical treatment was successful and the animal had uneventful recovery without any complications

COMPARATIVE STUDIES ON THE IN VITRO ANTIOXIDANT PROPERTIES OF METHANOLIC LEAFY EXTRACTS FROM SIX EDIBLE LEAFY VEGETABLES OF INDIA

Objective: Antioxidants are vital substances which possess the ability to protect the body from damage caused by free radical induced oxidative stress. Epidemiological studies specify that intake of fruits and vegetables have the ability to inhibit the damaging behavior of free radicals in the human body. In this study, we assessed antioxidative properties of the methanolic extracts of Mentha arvensis, Moringa oleifera, Spinacia oleracea, Trigonella foenum-graecum, Tamarindus indica, and Amaranthus viridis. Methods: The methanolic extracts were studied for phytochemical screening and antioxidant properties by different in-vitro experiments including DPPH radical assay, ABTS radical assay, Total antioxidant assay, Reducing activity assay for ascorbic acid equivalents, Total Phenolic content for gallic acid equivalents and Total flavonoid content for quercetin equivalent. Results: The present study revealed that Mentha arvensis extract exhibited the highest DPPH radical scavenging activity (IC50 value of 28 µg/ml), Reducing activity (1.731±0.072), Total antioxidant activity (208 µg/ml expressed as ascorbic acid equivalents), Total phenolic content (75 µg/ml expressed as gallic acid equivalents) and Total flavonoid contents (674 µg/ml expressed as quercetin equivalents) and Tamarindus indica extract showed highest ABTS radical scavenging activity (IC50 value of 35 µg/ml), The results obtained in the present study indicate that the leaves of Mentha arvensis showed potential antioxidant and free radical scavenging activity. Conclusion: The results obtained in the present study indicate that leaves of Mentha arvensis, Moringa oleifera plant materials have potent, Trigonella foenum-graecum, Tamarindus indica have moderate and Amaranthus viridis, Spinacia oleracea have mild antioxidant activity and/or free radical scavenging activity. Key words: Leafy vegetables, Methanolic extracts, In-vitro antioxidant activit

A Comparative Study on the Dimensional Error of 3D CAD Model and SLS RP Model for Reconstruction of Cranial Defect

Rapid Prototyping (RP) is a technology that produces models and prototype parts from 3D CAD model data, CT/MRI scan data, and model data created from 3D object digitizing systems. There are several RP process like Stereolithography (SLA), Solid Ground Curing (SGC), Selective Laser Sintering (SLS), Fused Deposition Modelling (FDM), 3D Printing (3DP) among them SLS and FDM RP processes are used to fabricate pattern of custom cranial implant. RP technology is useful in engineering and biomedical application. This is helpful in engineering for product design, tooling and manufacture etc. RP biomedical applications are design and development of medical devices, instruments, prosthetics and implantation; it is also helpful in planning complex surgical operation. The traditional approach limits the full appreciation of various bony structure movements and therefore the custom implants produced are difficult to measure the anatomy of parts and analyse the changes in facial appearances accurately. Cranioplasty surgery is a surgical correction of a defect in cranial bone by implanting a metal or plastic replacement to restore the missing part. This paper aims to do a comparative study on the dimensional error of CAD and SLS RP Models for reconstruction of cranial defect by comparing the virtual CAD with the physical RP model of a cranial defect

Filamentation without intensity clamping

We present measurements of the supercontinuum emission (SCE) from ultrashort Ti:Saph laser pulse filamentation in air in a tightly focused geometry. The spectral broadening of SCE indicates that peak intensities exceed the clamping value of a few 1013 W/cm2 obtained for filamentation in a loose focusing geometry by at least one order of magnitude. We provide an interpretation for this regime of filamenation without intensity clamping

Preformulation Characterization and the Effect of Ionic Excipients on the Stability of a Novel DB Fusion Protein

Shigella ssp cause bacillary dysentery (shigellosis) which has high global morbidity in young children and the elderly. The virulence of Shigella relies upon a type III secretion system (T3SS) which injects host altering effector proteins into targeted intestinal cells. The Shigella T3SS contains two components, invasion plasmid antigen D (IpaD) and invasion plasmid antigen B (IpaB), that were previously identified as broadly protective antigens. When IpaD and IpaB were co-expressed to give the DB fusion (DBF) protein, vaccine efficacy was further improved. Biophysical characterization under various pH conditions showed that DBF is most stable at pH 7 and 8 and loses its conformational integrity at 48 and 50 °C respectively. Forced degradation studies revealed significant effects on the secondary structure, tertiary structure and conformational stability of DBF. In the presence of phosphate buffers as well as other anionic excipients, DBF demonstrated a concentration dependent conformational stabilization. Molecular docking revealed potential polyanion binding sites in DBF that may interact with phytic acid. These sites can be exploited to stabilize the DBF protein. This work highlights potential destabilizing and stabilizing factors, which not only improves our understanding of the DBF protein but helps in future development of a stable Shigella vaccine

Identification and Characterization of Genetic Determinants of Isoniazid and Rifampicin Resistance in Mycobacterium tuberculosis in Southern India

Abstract: Drug-resistant tuberculosis (TB), one of the leading causes of death worldwide, arises mainly from spontaneous mutations in the genome of Mycobacterium tuberculosis. There is an urgent need to understand the mechanisms by which the mutations confer resistance in order to identify new drug targets and to design new drugs. Previous studies have reported numerous mutations that confer resistance to anti-TB drugs, but there has been little systematic analysis to understand their genetic background and the potential impacts on the drug target stability and/or interactions. Here, we report the analysis of whole-genome sequence data for 98 clinical M. tuberculosis isolates from a city in southern India. The collection was screened for phenotypic resistance and sequenced to mine the genetic mutations conferring resistance to isoniazid and rifampicin. The most frequent mutation among isoniazid and rifampicin isolates was S315T in katG and S450L in rpoB respectively. The impacts of mutations on protein stability, protein-protein interactions and protein-ligand interactions were analysed using both statistical and machine-learning approaches. Drug-resistant mutations were predicted not only to target active sites in an orthosteric manner, but also to act through allosteric mechanisms arising from distant sites, sometimes at the protein-protein interface

Development of a Broadly Protective, Self-Adjuvanting Subunit Vaccine to Prevent Infections by Pseudomonas aeruginosa

Infections caused by the opportunistic pathogen Pseudomonas aeruginosa can be difficult to treat due to innate and acquired antibiotic resistance and this is exacerbated by the emergence of multi-drug resistant strains. Unfortunately, no licensed vaccine yet exists to prevent Pseudomonas infections. Here we describe a novel subunit vaccine that targets the P. aeruginosa type III secretion system (T3SS). This vaccine is based on the novel antigen PaF (Pa Fusion), a fusion of the T3SS needle tip protein, PcrV, and the first of two translocator proteins, PopB. Additionally, PaF is made self-adjuvanting by the N-terminal fusion of the A1 subunit of the mucosal adjuvant double-mutant heat-labile enterotoxin (dmLT). Here we show that this triple fusion, designated L-PaF, can activate dendritic cells in vitro and elicits strong IgG and IgA titers in mice when administered intranasally. This self-adjuvanting vaccine expedites the clearance of P. aeruginosa from the lungs of challenged mice while stimulating host expression of IL-17A, which may be important for generating a protective immune response in humans. L-PaF’s protective capacity was recapitulated in a rat pneumonia model, further supporting the efficacy of this novel fusion vaccine