Spheroids formation in large drops suspended in superhydrophobic paper cones.

The utilization of 3D cell culture for spheroid formation holds significant implications in cancer research, contributing to a fundamental understanding of the disease and aiding drug development. Conventional methods such as the hanging drop technique and other alternatives encounter limitations due to smaller drop volumes, leading to nutrient starvation and restricted culture duration. In this study, we present a straightforward approach to creating superhydrophobic paper cones capable of accommodating large volumes of culture media drops. These paper cones have sterility, autoclavability, and bacterial repellent properties. Leveraging these attributes, we successfully generate large spheroids of ovarian cancer cells and, as a proof of concept, conduct drug screening to assess the impact of carboplatin. Thus, our method enables the preparation of flexible superhydrophobic surfaces for laboratory applications in an expeditious manner, exemplified here through spheroid formation and drug screening demonstrations.

Proteomic Analysis of the Mycobacterium tuberculosis Outer Membrane for Potential Implications in Uptake of Small Molecules.

Increased resistance to current antimycobacterial agents and a potential bias toward relatively hydrophobic chemical entities highlight an urgent need to understand how current anti-TB drugs enter the tubercle bacilli. While inner membrane proteins are well-studied, how small molecules cross the impenetrable outer membrane remains unknown. Here, we employed mass spectrometry-based proteomics to show that octyl-ß-d-glucopyranoside selectively extracts the outer membrane proteins of Mycobacterium tuberculosis. Differentially expressed proteins between nutrient-replete and nutrient-depleted conditions were enriched to identify proteins involved in nutrient uptake. We demonstrate cell surface localization of seven new proteins using immunofluorescence and show that overexpression of the proteins LpqY and ProX leads to hypersensitivity toward streptomycin, while overexpression of SubI, SpmT, and Rv2041 exhibited higher membrane permeability, assessed through an EtBr accumulation assay. Further, proton NMR metabolomics suggests the role of six outer membrane proteins in glycerol uptake. This study identifies several outer membrane proteins that are involved in the permeation of small hydrophilic molecules and are potential targets for enhancing the uptake and efficacy of anti-TB drugs.

Redefining genetic essentiality in Mycobacterium tuberculosis.

Sequencing transposon mutant libraries have been pivotal in annotating essential and non-essential genes in bacteria. This is particularly very helpful in the case of Mycobacterium tuberculosis with a large part of its genome without known function. It is not known whether there are any variations in the essentiality states as a function of optimal growth in the absence of any selection pressure. We here grow a high-density mutant library of M. tuberculosis through serial cultures and monitor the temporal fluctuations in insertion frequencies across all TA dinucleotides in the genome. Genes that cause morphological and physiological heterogeneity or enable metabolic bypass were found to gradually lose insertions, while genes comprising the toxin-antitoxin systems were found to get enriched with insertions during growth in nutrient replete conditions. High levels of fluctuations were observed in genes involved in cell wall and cell processes, intermediary metabolism, and genes involved in virulence, suggesting new modes of adaptation undertaken by the mutants. We also report the essentiality status of several newly annotated genetic features.

Peptidoglycan in Mycobacteria: chemistry, biology and intervention.

Peptidoglycan, a major glycoconjugate in the mycobacterial cell envelope provides strength to resist osmotic stress and plays a pivotal role in maintaining the cellular morphology. Several unique growth stage specific structural alterations occur in its constituent monosaccharides and peptides that allow Mycobacterium to survive nutrient starvation and environmental stress. Here, we discuss the enzymes involved in its intricate biosynthesis that are novel targets for therapeutic intervention and provide an opportunity for potential antibiotic adjuvants. We also revisit the enzymatic steps which are critical for maintaining the equilibrium between peptidoglycan synthesis and hydrolysis during cellular growth and division specifically focused on the importance of cell wall remodelling during "exit from dormancy" in Mycobacterium, a phenomenon with tremendous physiological and therapeutic importance for intervention in mycobacterial infections.

Gloriosa superba Mediated Synthesis of Platinum and Palladium Nanoparticles for Induction of Apoptosis in Breast Cancer.

Green chemistry approaches for designing therapeutically significant nanomedicine have gained considerable attention in the past decade. Herein, we report for the first time on anticancer potential of phytogenic platinum nanoparticles (PtNPs) and palladium nanoparticles (PdNPs) using a medicinal plant Gloriosa superba tuber extract (GSTE). The synthesis of the nanoparticles was completed within 5 hours at 100°C which was confirmed by development of dark brown and black colour for PtNPs and PdNPs, respectively, along with enhancement of the peak intensity in the UV-visible spectra. High-resolution transmission electron microscopy (HRTEM) showed that the monodispersed spherical nanoparticles were within a size range below 10 nm. Energy dispersive spectra (EDS) confirmed the elemental composition, while dynamic light scattering (DLS) helped to evaluate the hydrodynamic size of the particles. Anticancer activity against MCF-7 (human breast adenocarcinoma) cell lines was evaluated using MTT assay, flow cytometry, and confocal microscopy. PtNPs and PdNPs showed 49.65 ± 1.99% and 36.26 ± 0.91% of anticancer activity. Induction of apoptosis was most predominant in the underlying mechanism which was rationalized by externalization of phosphatidyl serine and membrane blebbing. These findings support the efficiency of phytogenic fabrication of nanoscale platinum and palladium drugs for management and therapy against breast cancer.