An Integrated approach for Proteomic and Immunological analysis of Cell wall and Cell membrane proteins of Mycobacterium tuberculosis.

Mycobacterium cell wall and cell membrane proteins serve as key modulators in the host immune response. Some of these proteins, like MPT63, are known as immunodominant antigens, and others, such as LpqS and LpqH, are being explored as subunit vaccine candidates. However, due to their hydrophobic nature, conventional separation methods are not able to solubilize and isolate these proteins individually, leading to limited research on their systematic separation and immunological characterization. In this study, we successfully employed a combination of Preparative Isoelectric focusing and Preparatory SDS-PAGE to separate mycobacterial cell wall and membrane proteins into 234 fractions. These fractions were pooled based on the proteins molecular weight, resulting in 24 pooled fractions labelled as Ag1 to Ag24. Immunological analyses on these pooled fractions were carried out in 3 different study groups (subjects with latent tuberculosis infection (LTBI, N=20), tuberculosis-diseased patients (PTB, N=20), and healthy subjects (HC, N=20). In these study groups, Interferon-γ and TNF-α responses against these antigens were analysed using the cytokine ELISA method. Notably, one pooled fraction (Ag24 - Cell wall high molecular weight proteins pool) induced significantly higher IFN gamma response in LTBI compared to PTB, while also triggering significantly higher TNF-alpha response in PTB compared to LTBI. Additionally, three other pooled fractions (Ag6 - Cell Membrane Medium molecular weight proteins Pool, Ag11 - Cell Membrane high molecular weight proteins Pool, Ag23 - Cell wall high molecular weight proteins pool) induced significantly higher IFN gamma response in LTBI compared to PTB. Proteomic analysis of the Ag24 fraction identified four proteins (ACN, KatG, Rv2623, and Rv0404). Among them, KatG and Rv0404 were previously reported as immunodominant T-cell antigens. On the other hand, ACN lacks immunological information, and limited information is available on protein Rv2623. In conclusion, extensive immunological studies on these four proteins could enhance their development as tuberculosis prognostic biomarkers, while our systematic 2-dimensional separation method shows promising potential for characterizing cell wall and membrane proteins in other pathogen

Optical, photoconducting, thermal and anisotropic mechanical behaviours of Benzimidazolium salicylate single crystals

Protonated organic single crystals of Benzimidazolium salicylate (BSL) were successfully grown by the slow evaporation solution technique at room temperature. The crystal structure and lattice parameters of the as-grown single crystal have been identified and calculated by the powder X-ray diffraction (PXRD) method. Microanalysis has confirmed the elemental compositions and the Fourier transform infrared (FTIR) spectroscopy technique has revealed vibrations of the functional groups in these crystals. The optical band gap was calculated indicating that the as-grown crystal exhibits 75% of optical transparency in the 350–800 nm range. Fluorescence study has suggested the blue emission nature of the crystal. The thermal stability of the as-grown crystal has been elaborated from the thermogrativity differential thermo-analysis (TG-DTA) technique. Vickers hardness measurement has revealed the mechanically soft nature of the crystal, which is driven by the reverse indentation size effect (RISE) and shows an anisotropic behaviour. A linear increase of the photocurrent on illumination suggests the positive photoconductivity of the crystal. The third-order non-linear optical property characteristics, such as the absorption coefficient (β), the refractive index (n2) and the susceptibility (χ(3)) have been examined using the Z-scan technique