Anthraquinone-Based Ligands as MNase Inhibitors: Insights from Inhibition Studies and Generation of a Payload Nanocarrier for Potential Anti-MRSA Therapy.

The present study highlights the prospect of an anthraquinone-based ligand (C1) as an inhibitor of micrococcal nuclease (MNase) enzyme secreted by Staphylococcus aureus. MNase inhibition rendered by 5.0 µM C1 was ∼96 % and the ligand could significantly distort the ß-sheet conformation present in MNase. Mechanistic studies revealed that C1 rendered non-competitive inhibition, reduced the turnover (Kcat ) and catalytic efficiency (Km /Kcat ) of MNase with an IC50 value of 323 nM. C1 could also inhibit nuclease present in the cell-free supernatant (CFS) of a methicillin-resistant Staphylococcus aureus (MRSA) strain. A C1-loaded human serum albumin (HSA)-based nanocarrier (C1-HNC) was developed, which was amicable to protease-triggered release of payload in presence of the CFS of an MRSA strain. Eluates from C1-HNC could effectively reduce the rate of MNase-catalyzed DNA cleavage. The non-toxic nature of C1-HNC in conjunction with the non-competitive mode of MNase inhibition rendered by C1 offers interesting therapeutic prospect in alleviation of MRSA infections.

2-Dodecylmalonic acid-mediated synthesis of mineralized hydroxyapatite amicable for bone cell growth on orthopaedic implant.

The present study illustrates the use of 2-dodecylmalonic acid (MA) as a template in biomineralization-inspired synthesis of hydroxyapatite nanoparticles (HANPs). HANPs synthesized in presence of various concentrations of MA displayed varying particle size and shape. The smallest particle size (22-27 nm) was obtained for MA2-HANP synthesized in presence of 37 µM MA. The critical micelle concentration (CMC) for MA at pH 9.0 relevant for mineralization was ∼35 µM. AFM analysis revealed that at a low concentration of 10 µM and pH 9.0, MA could generate oblong-shaped aggregates. At 40 µM, comparable to the concentration used to generate MA2-HANP, the amphiphile self-assembled to form a spherical soft scaffold, which likely regulated spatial confinement of ions during mineralization and generated small size HANPs. Osteoblast-like MG-63 cells seeded on titanium wire (TW) coated with MA2-HANP-incorporated collagen type I (H-TW) displayed enhanced cell proliferation, high expression of osteogenic differentiation marker genes (Col I, ALP, OCN and Runx2) and copious calcium mineral deposition after 14 days of growth. The nuanced role of the self-assembly process of an amphiphilic template in HANP mineralization unravelled in the present study can guide future scaffold design for biomineralization-inspired synthesis of HANPs tailored for bone tissue engineering applications.

Susceptibility pattern of methicillin resistance Staphylococcus aureus (MRSA) by flow cytometry analysis and characterization of novel lead drug molecule from Streptomyces species.

BACKGROUND: Actinomycetes particularly, Streptomyces species are producing wide variety of natural products with potential bioactivities. The microbial-derived metabolites hold a strong position to combat emerging and re-emerging antimicrobial drug-resistant pathogens. OBJECTIVES: A diverse group of actinomycetes strains were isolated from unexplored regions of mangrove sediment. Further, a polyphasic approach based on 16S rRNA gene sequence analysis and to evaluate their antibacterial potential against a panel of bacterial pathogens and methicillin resistance Staphylococcus aureus (MRSA). METHODS: The mangrove sediment samples were serially diluted with sterile water and plated on inorganic starch agar medium. A total of 20 isolates were pure cultured and 16S rRNA gene sequences were deposited in the public nucleotide databases (GenBank, NCBI). All the isolates were screened for the antibacterial activity by agar overlay method. Further, the susceptibility pattern of MRSA by flow cytometry and fluorescence microscopy was analysed. RESULTS: These twenty different isolates were grouped under nine major clad and they shared 95-99% sequence identity to the 16S rRNA gene sequences of the genus Streptomyces in the public nucleotide databases. Among these strains, the isolates namely JRG-02, JRG-03, JRG-04, JRG-10 and JRG-12 exhibited a broad-spectrum antibacterial activity against Methicillin-resistant Staphylococcus aureus(MRSA) and Gram negative bacteria Klebsiella pneumoniae MTCC109. Furthermore, we have characterized the antibacterial compound production and its properties from the isolate JRG-02, a potential drug candidate. The culture conditions and various nutrient components of strain Streptomyces sp. JRG-02 were optimized for enhanced antibiotics production of the isolate. The FT-IR and LCMS spectrum analysis envisaged the chemical nature of the substance. The effect of antibacterial compound on the viability of MRSA was alone examined by flow cytometry (FACS) and fluorescence microscopy analysis. CONCLUSIONS: The present study clearly shows that the survival of diverse inhabitants of Streptomyces in the mangrove sediments. Hence, the mangrove sediment inhabiting strain Streptomyces sp. JRG-02 has potential pharmaceutical activity and genetic diversity.

Probiotic bacteria cell surface-associated protein mineralized hydroxyapatite incorporated in porous scaffold: In vitro evaluation for bone cell growth and differentiation.

There is a high demand for synthesis of biocompatible hydroxyapatite nanoparticle (HANP), which is a key component in bone tissue engineering scaffolds. The present study describes a facile route of HANP synthesis through mineralization of the cell surface-associated protein (CSP) from the human probiotic lactic acid bacteria (LAB) Lactobacillus rhamnosus GG. CSP extract from the LAB (consisting of ~66 kDa, ~47 kDa, ~40 kDa and ~25 kDa protein) was mineralized to yield spindle-shaped HANPs having an average particle length of 371 nm as evidenced in FETEM analysis. CSP-mineralized HANPs (CSP-HANPs) were characterized by FTIR and BET analysis, while XRD and SAED analysis indicated their crystalline nature. Mechanistic studies suggested the key role of ~25 kDa CSP (F4SP) in mineralization. In contrast to CSP-HANPs, F4SP-mineralized crystalline HA was plate-shaped having an average length of 1.68 µm and breadth of 0.95 µm. HANP mineralization at the whole-cell (WC) level resulted in clusters of aggregated HANPs (WC-HANPs) adhering onto L. rhamnosus GG cells as evident in FETEM, FESEM and AFM analysis. FETEM analysis revealed that the desorbed WC-HANPs recovered by cell lysis were needle-shaped, with a particle size distribution of 70-110 nm. Given that CSP-HANPs were non-toxic to cultured HEK 293 cells and osteoblast-like MG-63 cells, chitosan-gelatin (CG) scaffold incorporated with 15% w/v CSP-HANP (H-CG) was generated and tested for bone cell growth. H-CG exhibited a favorable pore size distribution (160-230 µm), overall porosity (~84%) and biodegradation profile. H-CG scaffold was conducive to osteogenesis and rendered enhanced proliferation, alkaline phosphatase (ALP) activity, calcium mineralization and heightened marker gene expression (ALP, Col I, Runx2 and OCN) in seeded MG-63 cells. CSP sourced from a safe probiotic LAB is thus a viable and effective mineralization template for synthesis of biocompatible HANPs that can be leveraged for bone tissue engineering applications.

Generation of a Hydroxyapatite Nanocarrier through Biomineralization Using Cell-Free Extract of Lactic Acid Bacteria for Antibiofilm Application.

Nanoscale materials hold considerable promise in the mitigation of bacterial infections. In order to exploit nanomaterials as delivery systems in an antibacterial therapeutic paradigm, it is critical to ensure that the generated material is nontoxic. Based on the fundamental principle of biomineralization, we herein report the generation of biocompatible hydroxyapatite nanoparticles (HANPs) in the presence of proteins secreted by the lactic acid bacteria (LAB) Lactobacillus plantarum MTCC 1325, Lactobacillus plantarum CRA52, and Pediococcus pentosaceus CRA51. The biogenic HANPs were characterized by AFM, FETEM, powder XRD, DLS, and FTIR analysis. Interestingly, HANPs could also be synthesized using an ∼20 kDa protein purified from the secreted protein extract obtained from L. plantarum MTCC 1325, which suggested that this lower molecular weight protein fraction was perhaps significantly involved in biomineralization-based generation of HANPs. In order to develop a therapeutic bactericidal nanocomposite, HANPs were loaded with the antibiotic polymyxin B (PB). A Langmuir isotherm model was evident in the studies that measured adsorption of PB onto HANPs. A sustained release profile of PB from the nanocomposite was observed in buffers having varying pH and in simulated body fluid. The nanocomposite (PB-HNC) exhibited bactericidal as well as antibiofilm activity against Pseudomonas aeruginosa MTCC 2488 and was nontoxic to cultured human embryonic kidney cells.

Al3+ sensing through different turn-on emission signals vis-à-vis two different excitations: Applications in biological and environmental realms.

A rationally designed Schiff base chemosensor (L) could render specific detection of Al3+ ions with two distinct turn-on emission signals, separated by over 100 nm upon excitation at two different wavelengths. The utility of the probe lies in facilitating sensing in 80% aqueous medium with an emission close to 600 nm via an intramolecular charge transfer (ICT) mechanism. The biocompatible and cell permeable probe could readily sense Al3+ in live HeLa cells as well. The affinity of the probe for Al3+could be leveraged to specifically study DNA- Al3+ interaction in solution.