Silver Nanoparticles Induced Metabolic Perturbations in Pseudomonas aeruginosa: Evaluation Using the UPLC-QTof-MSE Platform.

Silver nanoparticles (AgNPs) have been widely utilized in various biomedical and antimicrobial technologies, displaying broad-spectrum activities against Gram-negative and Gram-positive bacteria including multidrug-resistant strains. However, the emergence of resistance to AgNPs upon repeated exposure and the survival of bacteria after initial exposure to antimicrobial agents pose a threat, as they may lead to the development of new resistant populations. To combat the early stages of antibacterial resistance, systematic analysis is essential to understand the immediate response of bacteria to antimicrobial agents. In this study, green-synthesized AgNPs with a diameter of approximately 14 nm were exposed toPseudomonas aeruginosaat three different inhibitory concentrations and at two different time intervals (1 and 4 h) to investigate the perturbations in the metabolome using liquid chromatography-high-resolution mass spectrometry. MetaboAnalyst 5.0 was employed for univariate and multivariate analysis, and the affected metabolic pathways were constructed using a variable important in projection scores above 1 from PLS-DA. The study revealed significant alterations in metabolites associated with cell wall synthesis, energy metabolism, nucleotide metabolism, the TCA cycle, and anaplerotic intermediates of the TCA cycle. Our investigation aimed to comprehensively understand the effects of green-synthesized AgNPs onP. aeruginosa metabolism, providing a more precise snapshot of the bacterium's physiological state through metabolomics approach.

Alterations in the pH of pancreatic juice are associated with chymotrypsin C inactivation and lithostathine precipitation in chronic pancreatitis patients: a proteomic approach.

BACKGROUND: The progression of chronic pancreatitis (CP), an inflammatory disease of the pancreas, causes pancreatic stones to form within the pancreatic ductal lumen/parenchyma, which occurs via protein plug formation. Pain is the most common symptom that necessitates clinical attention, and pain relief is the therapeutic goal for these patients. Endoscopic therapy and surgery are complimentary forms of therapy for pain relief. This study was envisaged to clarify the mechanism by which protein plug/soft stones form in pancreatic ducts prior to undergoing calcification. METHODS: Protein plugs were obtained from twenty CP patients undergoing therapeutic ERCP for stone removal. Pancreatic juice was obtained from five CP patients without stones. Proteins were isolated by TCA/acetone precipitation, SDS PAGE and 2-D gel electrophoresis to determine the protein profile. Protein spots from the 2-D gel were excised and subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) for identification. The effect of altered pH and elevated concentrations of trypsin on pancreatic juice protein was assessed by SDS‒PAGE to determine the protein profile. Differentially expressed protein bands were excised and subjected to MALDI-TOF. In silico analysis was performed by docking lithostathine with the calcite molecule using AutoDock Vina and PyMOL to clarify their interaction during stone formation. RESULTS: Twenty-three and twenty-nine spots from 2D gels of protein plugs and pancreatic juice, respectively, revealed that lithostathine (Reg1A) was the only protein in the protein plugs, whereas digestive enzymes and lithostathine were identified in pancreatic juice. Altered pH levels and increased trypsin concentrations in the pancreatic juice caused a protein to degrade via an unknown mechanism, and this protein was identified as chymotrypsin C (CTRC) by MALDI-TOF. Docking studies showed that the binding affinity of calcite was higher with the cleaved lithostathine, explaining the deposition of calcium that was observed around the protein plugs after calcified stones were formed through precipitation. CONCLUSION: Our results suggest that chymotrypsin C (CTRC) is degraded in an acidic environment, leading to the precipitation of lithostathine in the ductal lumen.

Bimetallic nanocomposite (Ag-Au, Ag-Pd, Au-Pd) synthesis using gum kondagogu a natural biopolymer and their catalytic potentials in the degradation of 4-nitrophenol.

Bimetallic nanoparticles (BNPs) constitute two different metal elements and exhibit relatively superior mechanistic and catalytic efficacies owing to their synergistic functions over monometallic nanoparticles. In the present study various bimetallic Ag-Au, Ag-Pd, Au-Pd nanoparticles were synthesized using a natural biopolymer gum kondagogu (GK) as a reducing and capping agent, by a simple and cost-effective method. The synthesized BNPs when characterized using UV-vis spectroscopy revealed a specific surface plasmon resonance band (SPR) of each nanocomposite. The average particle size of Ag-Au, Ag-Pd, and Au-Pd BNPs was found to be 23 ± 10.3, 21 ± 7.6, and 23 ± 9.4 nm respectively based on transmission electron microscopy analysis. Surface morphology and functional groups on the gum matrix of GK-BNPs were analyzed by XRD and FT-IR respectively. The bimetallic nanocomposites were evaluated for their catalytic reduction of 4-nitrophenol (4-NP) to 4-aminophenol in the presence of NaBH4. The kinetic studies performed, depicted rate constants for Ag-Au, Ag-Pd, and Au-PdNPs as 0.31, 0.39, and 0.28 min-1 respectively. The catalytic efficiencies of three bimetallic nanocomposites were of the following order Ag-Pd > Ag-Au > Au-Pd. This study establishes the catalytic potentials of the three different bimetallic nanocomposites in the reduction of 4-NP an environmental pollutant, and the impact of their synergistic property.

Synthesis and characterisation of novel biopolymer stabilised organic Pt-nanocomposite: assessment of its antioxidant and antitumour properties.

Green synthesis of organic Pt-nanocomposite was accomplished using carboplatin as a precursor and novel biopolymer - gum kondagogu (GK) as a reducing agent. The synthesised GK stabilised organic Pt-nanocomposite (GKCPt NC) was characterised by different analytical techniques such as ultraviolet-visible spectroscopy, nanoparticle analyser, scanning electron microscopy and energy dispersive X-ray analysis, X-ray diffraction (XRD), Fourier-transform infrared spectroscopy, transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and inductively coupled plasma optical emission spectrophotometer. The XRD pattern established the amorphous nature of GKCPt NC. TEM analysis revealed the homogeneous, monodisperse and spherical nature, with Pt metal size of 3.08 ± 0.62 nm. The binding energy at 71.2 and 74.6 eV show the presence of metallic platinum, Pt(0) confirmed by XPS studies. Further, in vitro radical scavenging and antitumour activity of GKCPt NC have been investigated. In comparison to GK and carboplatin, GKCPt NC showed superior 1, 1-diphenyl-2-picrylhydrazyle activity of 87.82%, whereas 2, 2-azinobis-(3-ethylbenzthinzoline-6-sulphonic acid) activity was 38.50%, respectively. In vitro studies of the antitumour property of GK, GKCPt NC and carboplatin were evaluated by potato disc tumour bioassay model. The efficacy of synthesised GKCPt NC concentration (IC50) on tumour inhibition was found to be 2.04-fold lower as compared to carboplatin. Overall, the synthesised GKCPt NC shows both antitumour and antioxidant properties when compared to the original drug - carboplatin and might have promising applications in cancer therapy.

Immuno-analytical method development for detection of transgenic Cry1Ac protein and its validation.

BACKGROUND: Bacillus thuringiensis (Bt) synthesizes Cry1Ac protein, which is toxic to many lepidopteran pests, and the cry1ac gene has been expressed in several transgenic crop plants. The Cry1Ac protein has been isolated from Bt kurstaki HD73 and purified to homogeneity. Polyclonal antibodies were raised against purified Cry1Ac in rabbits and goat. Sandwich ELISA was developed for Cry1Ac using goat IgG as a coating antibody, and affinity-purified rabbit IgG as the primary antibody. RESULTS: The sensitivity of the assay was in the range of 0.47-1000 ng. It was subsequently employed in validating biological samples. Fifteen different cotton-seed samples were screened: 12 were found to be Bt positive and 3 Bt negative. The CS7 seeds showed the highest Bt content of 8.51 ± 0.45 µg g-1 , followed by CS8 (6.0 ± 0.02 µg g-1 ), CS15 (5.9 ± 0.03 µg g-1 ), CS9 (5.5 ± 0.05 µg g-1 ), and CS10 (4.83 ± 0.013 µg g-1 ). The CS5 seeds showed Bt content of 3.6 ± 0.21 µg g-1 . The F2 generation, CS6 (Kaveri seeds) showed lower Bt content (2.9 ± 0.06 µg g-1 ). The CL5 samples showed Cry1Ac content of 0.99 ± 0.009 µg g-1 . The amount of Cry1Ac protein in leaves, stem, and roots of germinated Bt cotton plants (CS10 and CS4) were 1.76 ± 0.15 µg g-1 , 1.9 ± 0.01 µg g-1 , 2.0 ± 0.1 µg g-1 , and 1.6 ± 0.15 µg g-1 , 1.9 ± 0.01 µg g-1 , and 2.0 ± 0.01 µg g-1 dry tissue, respectively. CONCLUSION: The method developed can be used for screening the expression levels of Cry1Ac in different transgenic Bt cultivars and also spurious Bt cotton seeds procured by farmers. © 2019 Society of Chemical Industry.

Binding Studies of Andrographolide with Human Serum Albumin: Molecular Docking, Chromatographic and Spectroscopic Studies.

BACKGROUND: Human serum albumin acts as a carrier protein to a variety of drugs and aids their transport. Andrographis paniculata, a herbal plant has been used as a source of traditional medicine in the Asian countries. Among the various constituents of this plant, andrographolide is the most active and is being used from centuries in the treatment of many chronic and infectious diseases. OBJECTIVE: The present study was designed to evaluate the interaction and binding affinity of andrographolide with HSA, by molecular docking, chromatographic and spectral studies. METHODS: Andrographolide was docked with crystal structure of human serum albumin (1AO6) using Auto Dock Vina software and the interactions were analyzed by a visualizing software py- MOL. For further characterization and confirmation, andrographolide (3x10-5 M) and HSA (0.001, 0.005, 0.01, 0.02, 0.04 M) sample mixtures were incubated at 37°C for 3h in a metabolic shaker, followed by centrifugation. The supernatant and the filtrate were analyzed by UV spectroscopy, HPLC, CD and FTIR spectral analysis. RESULTS: The docking studies revealed that andrographolide interacted with HSA and formed hydrogen bonds with Trp 214, Arg 218 and Lys 444 amino acid residues. The UV spectral analysis revealed a decrease in the absorption peak of HSA due to its interaction with andrographolide. A new peak was observed at retention time 7.45 min by HPLC analysis and the Bmax was found to be 7.5 ± 0.4 mg protein with a Kd value of 1.89 mM, indicating interaction of andrographolide with HSA. The CD spectra results suggested, a marginal decrease in the negative ellipticity without any significant shift in peak, indicating the stabilization of the HSA-andrographolide complex. The FTIR analysis of the andrographolide-HSA mixture showed a peak at wave number 1637 cm-1 (a shift of amide I groups from 1646 cm-1) and 1016 cm-1 which corresponded to the ligand, confirming the complex formation. CONCLUSION: The molecular docking studies demonstrated the interactions of andrographolide to the crystal structure of HSA. The chromatographic and spectroscopic analysis confirmed the binding of andrographolide with HSA and their complex formation. Overall the present studies conclude the binding of andrographolide to HSA protein, favoring its pharmacokinetics.

Purification and characterization of mycoferritin from Fusarium verticillioides MRC 826.

The fungus Fusarium verticillioides MRC 826 (ascomycetes species), a toxigenic isolate is capable of synthesizing mycoferritin only upon induction with iron in yeast extract sucrose medium. The molecular mass, yield, iron and carbohydrate contents of the purified mycoferritin were 460 kDa, 0.010 mg/g of wet mycelia, 1.0 and 40.2%, respectively. Native gel electrophoresis of the mycoferritin revealed two bands possibly representing isoforms of ferritin. Subunit analysis by SDS-PAGE showed a single protein subunit of ~24 kDa suggesting similar sized subunits in the structure of apoferritin shell. Immunological cross reactivity was observed with the anti-fish liver ferritin. Transmission electron microscopy revealed an apparent particle size of 100 Å. N-terminal amino acid sequencing of mycoferritin showed identities with other eukaryotic ferritin sequences. The spectral characteristics were similar to equine spleen ferritin. However, circular dichroic spectra revealed a higher degree of helicity. Functionally, induction of mycoferritin minimizes the prooxidant role of iron.