Assessment of Synthesis Machinery of Two Antimicrobial Peptides from Paenibacillus alvei NP75

Paenibacillus alvei NP75, a Gram-positive bacterium, produces two different antimicrobial peptides, paenibacillin N and P, which has potent antimicrobial activity against many clinical pathogens. The synthesis pattern of these antimicrobial peptides by P. alvei NP75 was studied extensively. The results were outstanding in a way that the paenibacillin N was synthesized irrespective of the growth of bacteria (non-ribosomal mediated), whereas paenibacillin P production was carried out by ribosomal mediated. In addition to the antimicrobial peptides, P. alvei NP75 also produces an immunogenic extracellular protease to defend itself from its own antimicrobial peptide, paenibacillin P. Furthermore, this immunogenic protease production was impaired by the addition of protease inhibitor, phenylmethylsulfonyl fluoride (PMSF). The sodium dodecyl sulfate (SDS) treated strain (mutant) failed to produce paenibacillin P, whereas the production of neither paenibacillin N nor the protease was affected by the plasmid curing. The plasmid curing studies that divulge the genes responsible for the synthesis of paenibacillin N and protease were found to be genome encoded, and paenibacillin P was plasmid encoded. We are reporting, first of its kind, the co-production of two different antimicrobial peptides from P. alvei NP75 through non-ribosomal and ribosomal pathways that could be used as effective antibiotics.

     

Development of selective and reversible pyrazoline based MAO-A inhibitors: Synthesis,biological evaluation and docking studies

3,5-Diaryl pyrazolines analogs were synthesized and evaluated for their monoamine oxidase (MAO) inhibitory activity. The compounds were found reversible and selective towards MAO-A with selectivity index in the magnitude of 10³–10⁵. The docking studies were carried out to gain further structural insights of the binding mode and possible interactions with the active site of MAO-A. Interestingly, the theoretical (K_i) values obtained by molecular docking studies were in congruence with their experimental (K_i) values.     

Electrocatalysis and simultaneous determination of catechol and quinol by poly(malachite green) coated multiwalled carbon nanotube film

Electrochemically active composite film that contains multiwalled carbon nanotubes (MWCNTs), Nafion (NF), and poly(malachite green) (PMG) has been synthesized on glassy carbon electrode (GCE), gold, and indium tin oxide (ITO) electrodes by potentiodynamic method. The presence of MWCNTs in the composite film (MWCNT–NF–PMG) enhances the surface coverage concentration (Γ) of PMG by fivefold. Similarly, an electrochemical quartz crystal microbalance study revealed enhancement in the deposition of PMG at MWCNT–NF film when compared with bare and only NF modified electrodes. The surface morphology of the composite film was studied using atomic force microscopy, which revealed that the PMG incorporated on MWCNT–NF film. The composite film exhibited enhanced electrocatalytic activity toward the mixture of biochemical compounds catechol and quinol. The electrocatalytic responses of analytes at MWCNT–NF–PMG composite film were measured using both cyclic voltammetry (CV) and differential pulse voltammetry (DPV). From electrocatalysis studies, well-separated voltammetric peaks were obtained at the composite film for catechol and quinol with a peak separation of 147 mV. The sensitivity values of the composite film toward catechol and quinol by the DPV technique were 0.4 and 3.2 mA mM⁻¹ cm⁻², respectively, which are higher than the values obtained by the CV technique. Similarly, the above-mentioned values are better than the previously reported electroanalytical values for the same analytes.     

Vitamin B(12) incorporated with multiwalled carbon nanotube composite film for the determination of hydrazine

Electrochemically active composite film containing multiwalled carbon nanotubes (MWCNTs) and vitamin B₁₂ was synthesized on glassy carbon, gold, and indium tin oxide electrodes by the potentiodynamic method. The presence of MWCNTs in the composite film (MWCNT–B₁₂) modified electrode mediates vitamin B₁₂’s redox reaction, whereas vitamin B₁₂’s redox reaction does not occur at bare electrode. The electrochemical impedance spectroscopy studies reveal that MWCNTs present in MWCNT–B₁₂ film enhance electron shuttling between the reactant and electrode surface. The surface morphology of bare electrode, MWCNT film. and MWCNT–B₁₂ composite film was studied using atomic force microscopy, which reveals vitamin B₁₂ incorporated with MWCNTs. The MWCNT–B₁₂ composite film exhibits promising enhanced electrocatalysis toward hydrazine. The electrocatalysis response of hydrazine at MWCNT film and MWCNT–B₁₂ composite film was measured using cyclic voltammetry and amperometric current–time (i–t) curve techniques. The linear concentration range of hydrazine obtained at MWCNT–B₁₂ composite film using the i–t curve technique is 2.0 μM–1.95 mM. Similarly, the sensitivity of MWCNT–B₁₂ composite film for hydrazine determination using the i–t curve technique is 1.32 mA mM⁻¹ cm⁻², and the hydrazine’s limit of detection at MWCNT–B₁₂ composite film is 0.7 μM.     

Three Dimensional Carbon Nanosheets as a Novel Catalyst Support for Enzymatic Bioelectrodes

A high performance laccase‐based biofuel cell cathode is developed using carbon nanosheets (CNS) as the catalyst support and buckypaper (BP) as the substrate electrode. Compared to multiwalled carbon nanotube (MWNT)‐based electrodes, CNS‐based electrodes exhibit better electrochemical properties for the oxygen reduction reaction (ORR) under biologically relevant conditions. It is shown that CNSs are conformally coated on the nanotubule bundles within the BP and that laccase is intimately attached to the CNS‐BP. Electrochemical characterization is carried out to derive the kinetic parameters of the ORR at the laccase‐CNS‐BP cathode. The laccase‐CNS‐BP exhibits a steep ORR cathodic wave with a Tafel slope of 19 mV decade^‐1. The onset potential obtained for laccase ORR at CNS‐BP is 20 mV higher than that of the MWNT‐based electrodes, and the laccase‐CNS‐BP cathode has a higher current density than MWNT electrodes.     

Structural Requirements for PACSIN/Syndapin Operation during Zebrafish Embryonic Notochord Development

PACSIN/Syndapin proteins are membrane-active scaffolds that participate in endocytosis. The structure of the Drosophila Syndapin N-terminal EFC domain reveals a crescent shaped antiparallel dimer with a high affinity for phosphoinositides and a unique membrane-inserting prong upon the concave surface. Combined structural, biochemical and reverse genetic approaches in zebrafish define an important role for Syndapin orthologue, Pacsin3, in the early formation of the notochord during embryonic development. In pacsin3-morphant embryos, midline convergence of notochord precursors is defective as axial mesodermal cells fail to polarize, migrate and differentiate properly. The pacsin3 morphant phenotype of a stunted body axis and contorted trunk is rescued by ectopic expression of Drosophila Syndapin, and depends critically on both the prong that protrudes from the surface of the bowed Syndapin EFC domain and the ability of the antiparallel dimer to bind tightly to phosphoinositides. Our data confirm linkage between directional migration, endocytosis and cell specification during embryonic morphogenesis and highlight a key role for Pacsin3 in this coupling in the notochord.     

Nanocomposite of functionalized multiwall carbon nanotubes with nafion, nano platinum, and nano gold biosensing film for simultaneous determination of ascorbic acid, epinephrine, and uric acid

A unique bimetallic, nano platinum (Pt) with nano gold (Au) on nafion (NF) incorporated with functionalized multiwall carbon nanotubes (f-MWCNTs) composite film (f-MWCNTs-NF-PtAu) was developed by the potentiostatic method. The composite film exhibits promising efficient catalytic activity towards the oxidation of mixture of biochemical compounds and simultaneous measurement of ascorbate anion, epinephrine and urate anion in aqueous buffer solution (pH 6.75). Both, the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used for the measurement of electroanalytical properties of neurotransmitters by means of composite film modified electrodes. Well-separated voltammetric peaks were obtained for ascorbate, epinephrine and urate anions with the peak separations of 0.222 and 0.131V. The composite film can also be produced on gold and transparent semiconductor indium tin oxide electrodes for different kinds of studies such as electrochemical quartz crystal microbalance (EQCM), scanning electron microscopy (SEM), and atomic force microscopy (AFM). The incorporation of Pt and Au onto the f-MWCNTs-NF was revealed by the EQCM technique and the morphology of the film was studied using SEM, AFM and scanning electrochemical microscopy (SECM) techniques. Further, extensive studies were carried out using SECM for obtaining the surface current topographic images of composite film modified electrodes, and these indicated the presence of f-MWCNTs-NF-PtAu composite film on the electrode.     

A Phosphotyrosine Switch for Cargo Sequestration at Clathrin-coated Buds

The AP-2 clathrin adaptor complex oversees endocytic cargo selection in two parallel but independent manners. First, by physically engaging peptide-based endocytic sorting signals, a subset of clathrin-dependent transmembrane cargo is directly collected into assembling buds. Synchronously, by interacting with an assortment of clathrin-associated sorting proteins (CLASPs) that independently select different integral membrane cargo for inclusion within the incipient bud, AP-2 handles additional cargo capture indirectly. The distal platform subdomain of the AP-2 β2 subunit appendage is a privileged CLASP-binding surface that recognizes a cognate, short α-helical interaction motif. This signal, found in the CLASPs β-arrestin and the autosomal recessive hypercholesterolemia (ARH) protein, docks into an elongated groove on the β2 appendage platform. Tyr-888 is a critical constituent of this spatially confined β2 appendage contact interface and is phosphorylated in numerous high-throughput proteomic studies. We find that a phosphomimetic Y888E substitution does not interfere with incorporation of expressed β2-YFP subunit into AP-2 or alter AP-2 deposition at surface clathrin-coated structures. The Y888E mutation does not affect interactions involving the sandwich subdomain of the β2 appendage, indicating that the mutated appendage is folded and operational. However, the Y888E, but not Y888F, switch selectively uncouples interactions with ARH and β-arrestin. Phyogenetic conservation of Tyr-888 suggests that this residue can reversibly control occupancy of the β2 platform-binding site and, hence, cargo sorting.     

Molecular docking studies and in vitro screening of new dihydropyridine derivatives as human MRP1 inhibitors

The overexpression of multidrug resistance protein 1 (MRP1) by tumor cells results in multidrug resistance (MDR) to structurally unrelated anticancer drugs. Circumvention of MDR by combination of chemosensitizers with antitumor compounds is a new field of investigation in cancer chemotherapy. Much effort has been put-in recently to identify the modulators/inhibitors of MRP1 to overcome the MDR. 1,4-Dihydropyridine (DHP) derivatives are indicated to be a new class of MRP1 inhibitors in cancer treatment. Molecular docking studies were carried out on 48 newly synthesized DHP derivatives with the crystal structure of MRP1 to gain some structural insights on the binding mode and possible interactions with the active site of MRP1 (NBD1). The 10 top-ranked molecules were selectively evaluated, experimentally for their MRP1 inhibitory effect using the insect cell membrane MRP1 ATPase assay. The inhibitory capacity (IC(50) concentrations) of the test compounds was compared with the reported IC(50)- or the K(i)-concentrations for benzbromarone, a standard MRP1 inhibitor. Amongst the compounds tested, compounds IA(1) and IIA(5) were found to exhibit a potent MRP1 inhibitory action with IC(50) values of 20±4 and 14±2 μM (mean±SD), respectively as compared to benzbromarone (IC(50)=4 μM). The compound IIA(5), in particular was found to be more potent than IA(1) in accordance with the docking results. These new DHP derivatives possess promising characteristics for their development as MDR reversal agents.