In silico screening, molecular dynamic simulations, and in vitro activity of selected natural compounds as an inhibitor of Leishmania donovani 3-mercaptopyruvate sulfurtransferase.

In Leishmania sp., the enzymes of de novo cysteine biosynthesis pathway require sulfide. Other organisms utilize sulfide through the sulfide reduction pathway, but Leishmania lacks the gene that encodes these enzymes. Hence, the major source of sulfide for Leishmania is believed to be from the action of 3-mercaptopyruvate sulfurtransferase (3MST) on 3-mercapto-pyruvate (3MP). There has been no effort reported in the past to screen inhibitors against L. donovani 3MST (Ld3MST). As a result, this study examines natural compounds that are potent against Ld3MST and validates it by in vitro activity and cytotoxicity tests. Initially, a library of ~ 5000 natural compounds was subjected to molecular docking approach for screening, and the best hit was validated using a long-term molecular dynamic simulation (MD). Among the docking results, quercetine-3-rutinoside (Rutin) was deemed the best hit. The results of the MD indicated that Rutin was highly capable of interacting with the varied active site segments, possibly blocking substrate access. Additionally, promastigotes and amastigotes were tested for Rutin activity and the IC50 was found to be 40.95 and 90.09 µM, respectively. Similarly, the cytotoxicity assay revealed that Rutin was not toxic even at a concentration of 819.00 µM to THP-1 cell lines. Additionally, the Ld3MST was cloned, purified, and evaluated for enzyme activity in the presence of Rutin. Reduction in the enzyme activity (~ 85%) was observed in the presence of ~ 40 µM Rutin. Thus, this study suggests that Rutin may act as a potent inhibitor of Ld3MST. With further in vivo investigations, Rutin could be a small molecule of choice for combating leishmaniasis.

Carbon material produced by hydrothermal carbonisation of food waste as an electrode material for supercapacitor application: A circular economy approach.

This study aims to use landfill leachate (LL) as an aqueous medium during hydrothermal carbonisation (HTC) of food waste to produce hydrochar (FWH-LL-C), which could be used as an electrode material in energy storage devices. The structural properties and electrochemical performance of the hydrochar were compared to that obtained using distilled water as a reaction medium (FWH-DW-C). The results showed that there is a difference in Brunauer-Emmett-Teller (BET) surface area of FWH-LL-C (220 m2 gm-1) and FWH-DW-C (319 m2 gm-1). The electrochemical properties were comparable, with FWH-LL-C having 227 F g-1 specific capacitance at 1 A g-1 current density and FWH-DW-C having 235 F g-1 specific capacitance at 1 A g-1 current density. Furthermore, at a power density of 634 W kg-1, FWH-DW-C achieved the highest energy density of 14.4 Wh kg-1. The energy retention capacity of the electrode was 98% which indicate that the material has an excellent energy storage capacity. The findings suggested that LL could be used as an alternative source of aqueous media during the HTC of food waste to produce hydrochar which could be used as an effective electrode material in supercapacitors.

Hierarchical cage-frame type nanostructure of CeO2 for bio sensing applications: from glucose to protein detection.

Self-assembled hierarchical nanostructures are slowly superseding their conventional counterparts for use in biosensors. These morphologies show high surface area with tunable porosity and packing density. Modulating the interfacial interactions and subsequent particle assembly occurring at the water-and-oil interface in inverse miniemulsions, are amongst the best strategies to stabilize various type of hollow nanostructures. The paper presents a successful protocol to obtain CeO2 hollow structures based biosensors that are useful for glucose to protein sensing. The fabricated glucose sensor is able to deliver high sensitivity (0.495 µA cm-2 nM-1), low detection limit (6.46 nM) and wide linear range (0 nM to 600 nM). CeO2 based bioelectrode can also be considered as a suitable candidate for protein sensors. It can detect protein concentrations varying from 0 to 30 µM, which is similar or higher than most reports in the literature. The limit of detection (LOD) for protein was ∼0.04 µM. Therefore, the hollow CeO2 electrodes, with excellent reproducibility, stability and repeatability, open a new area of application for cage-frame type particles.

Time-dependent emission stokes shift in Au, Ag and Au/Ag fluorescent nanoclusters: evidence of multiple emissive states.

Au, Ag and bimetallic Au/Ag nanoclusters were prepared under similar microwave-irradiated synthesis conditions in aqueous solutions, using BSA as a stabilizer. The nanoclusters exhibited strong fluorescence with characteristic emission spectral peak features that suggested the presence of multiple emissive states in each case. Time-resolved emission studies revealed the very rare incidence of the time-dependent emission Stokes shift in metal nanoclusters over a time-scale of ∼10 ns. The shifts were of varying degrees: ∼200 cm-1 for Ag, ∼750 cm-1 for Au and ∼1400 cm-1 for Au/Ag nanoclusters. Application of the Time-Resolved Area Normalized Emission Spectra (TRANES) method confirmed the existence of multiple emissive species in each of the nanoclusters, which also helped to explain the time-dependent emission Stokes shift.

Fluorescence response from the surface states of nitrogen-doped carbon nanodots: evidence of a heterogeneous population of molecular-sized fluorophores.

Fluorescent Nitrogen-doped Carbon Nanodots (NCDs) of ∼4 nm diameter were prepared by acid-driven microwave irradiation of DMF solvent. Spectroscopic studies of the NCDs demonstrated that excitation of the carbon core did not contribute any fluorescence emission. Instead, the emission originated exclusively from the surface states. The fluorescence featured a prominent red edge excitation shift (REES), while changing the excitation wavelength over ∼0.5 eV indicated the emergence of different emitter species in the temporal evolution of fluorescence. These results combined to indicate a large degree of heterogeneity in the population of these surface-localized emitters, so that working with different excitation energies produced different sets of excited surface fluorophores that evolved independently of each other. Fluorescence anisotropy dynamics in the NCDs was attributable to the reorientational motion of the surface fluorophores which was decoupled from the rotational diffusion of the carbon core of the NCD. The anisotropy decay rates revealed that the fluorophores had size comparable to typical organic fluorophores, irrespective of excitation energy.

DNA supported graphene quantum dots for Ag ion sensing.

The use of graphene quantum dots can be extended for bio-sensing and metal ion detection. Synergistic combination of graphene quantum dots (GQDs) with DNA leads to high performance Ag-ion detection system. The thoroughly characterized GQDs were found to have spherical morphology, with dimensions in the range of 5-10 nm. The atomic force microscopy studies proved that the synthesized GQDs were mostly comprised of two to four graphene layers. To make the system biocompatible, GQDs/NGQDs were combined with DNA. Two properties of DNA were exploited, capacity to provide nitrogen to GQDs; and to synergistically contribute to Ag+ detection. In addition to Ag+, the strong green photoluminescence (PL) of GQDs showed significant quenching, owing to the appearance of associated Förster resonance energy transfer processes. This led to high sensing efficiencies.

Quantification of protein aggregation rates and quenching effects of amylin-inhibitor complexes.

The formation of amyloid aggregates is the hallmark of many protein misfolding diseases, including Type-II diabetes mellitus, which is caused by the fibrillation of amylin protein. It is established that nano-sized ligands such as curcumin, resveratrol and graphene quantum dots can modify protein aggregation rates. In this article, we report a comparative study of these ligands to estimate their protein aggregation rates and fluorescence quenching using various experimental techniques. Through light scattering experiments, the RH of bare amylin was found to increase at a rate of 43% per day, whereas in the presence of the ligands in different molar ratios (A1C10, A1R10 and A1GQDs20), the sizes of the complexes were found to grow at rates of 7%, 8% and 13% per day, respectively. We observed fluorescence quenching using photoluminescence experiments for all three protein-ligand complexes. The protein aggregation rate and fluorescence quenching exhibited a concentration-dependent competitive role in the inhibition process. Interestingly, for graphene quantum dots, the protein aggregation rate is more affected at lower concentrations, while fluorescence quenching dominates at higher concentrations; this is in contrast to curcumin and resveratrol, where fluorescence quenching dominates at all concentrations of the ligands in the complex. The FTIR data showed appreciable conversion of ß-sheets into less aggregation-prone secondary structures for all three amylin-ligand ratios; however, the inhibition performance of curcumin overshadowed those of the other two inhibitors. The inhibition behavior of these three ligands was corroborated by analysis of analytical and high-resolution TEM images of the fibrils.

Nonsense suppression in archaea.

Bacterial strains carrying nonsense suppressor tRNA genes played a crucial role in early work on bacterial and bacterial viral genetics. In eukaryotes as well, suppressor tRNAs have played important roles in the genetic analysis of yeast and worms. Surprisingly, little is known about genetic suppression in archaea, and there has been no characterization of suppressor tRNAs or identification of nonsense mutations in any of the archaeal genes. Here, we show, using the ß-gal gene as a reporter, that amber, ochre, and opal suppressors derived from the serine and tyrosine tRNAs of the archaeon Haloferax volcanii are active in suppression of their corresponding stop codons. Using a promoter for tRNA expression regulated by tryptophan, we also show inducible and regulatable suppression of all three stop codons in H. volcanii. Additionally, transformation of a ΔpyrE2 H. volcanii strain with plasmids carrying the genes for a pyrE2 amber mutant and the serine amber suppressor tRNA yielded transformants that grow on agar plates lacking uracil. Thus, an auxotrophic amber mutation in the pyrE2 gene can be complemented by expression of the amber suppressor tRNA. These results pave the way for generating archaeal strains carrying inducible suppressor tRNA genes on the chromosome and their use in archaeal and archaeviral genetics. We also provide possible explanations for why suppressor tRNAs have not been identified in archaea.

Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs.

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA2(Ile)) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA2(Ile) binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.

Unusually slow intramolecular proton transfer dynamics of 4'-N,N-dimethylamino-3-hydroxyflavone in high n-alcohols: involvement of solvent relaxation.

Excited state intramolecular proton transfer (ESIPT) time-constants of 4'-N,N-dimethylamino-3-hydroxyflavone (DMA3HF) in high n-alcohols--1-butanol, 1-hexanol and 1-decanol--were measured to be 90 ps, 130 ps and 190 ps, respectively, which are unusually slow. At the same time, the solvation time-constants of the DMA3HF enol in the same set of solvents were measured as 100 ps, 150 ps and >300 ps, respectively. Thus, both the ESIPT and enol solvation time-constants in high n-alcohols increase monotonically with the alkyl chain-length of the solvent, although the increase is not strictly proportional. It appears that the H-bonding capacity of the solvent is the single major factor influencing both processes, causing them to become closely correlated. Solvation causes a drastic change in the solvent molecular configuration around the excited enol, E*, inducing the breakage of DMA3HF···solvent inter-molecular H-bonding, which in turn promotes ESIPT. Following previously reported theoretical work on ESIPT, a qualitative description of the S1 potential energy surface can be formulated, where the involvement of solvent relaxation with the ESIPT process is explained.

Fluorescent Ag nanoclusters prepared in aqueous poly(acrylic acid-co-maleic acid) solutions: a spectroscopic study of their excited state dynamics, size and local environment.

Stable, fluorescent Ag nanoclusters were prepared in aqueous solutions of Na(+) salt of the carboxylate-rich polymer poly(acrylic acid-co-maleic acid) under brief spells of UV irradiation. The nanoclusters were nearly spherical, with diameters within 1.90 ± 0.50 nm, but displayed a prominent red edge excitation shift (REES) of fluorescence upon exciting within the visible absorption band, indicating heterogeneity of energy level distributions. Spectroscopic studies revealed that irrespective of whether the nanoclusters are excited in their UV or visible absorption bands, their fluorescence always ensues from the same manifold of emissive states, with a broad range of fluorescence lifetimes from ∼150 fs to 1 ns.

Effect of an Electron-Donating Substituent at the 3',4'-position of 3-Hydroxyflavone: Photophysics in Bulk Solvents.

Introduction of the methylenedioxy substituent group in the 3',4'-position of 3-hydroxyflavone produced a significant impact on its proton-transfer response, much like the well-known 4'-N,N-dialkylamino group. The potential electron-donating property of the substituent helped sustain a high degree of charge separation in the excited enolic form of the molecule, which was stabilized in relatively polar solvents, whereupon the enol → tautomer excited state intramolecular proton-transfer (ESIPT) rate decreased. Hydrogen-bonding solvents caused further retardation by interfering with the intramolecular hydrogen bond that promotes ESIPT. Among these solvents, hydrogen bond donors appear to be more efficient ESIPT inhibitors than hydrogen bond acceptors. Femtosecond fluorescence experiments revealed that even among the latter the ESIPT time-constants become steadily longer as the hydrogen bond basicity of the solvent increases.

Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile.

Translation of the isoleucine codon AUA in most prokaryotes requires a modified C (lysidine or agmatidine) at the wobble position of tRNA2(Ile) to base pair specifically with the A of the AUA codon but not with the G of AUG. Recently, a Bacillus subtilis strain was isolated in which the essential gene encoding tRNA(Ile)-lysidine synthetase was deleted for the first time. In such a strain, C34 at the wobble position of tRNA2(Ile) is expected to remain unmodified and cells depend on a mutant suppressor tRNA derived from tRNA1(Ile), in which G34 has been changed to U34. An important question, therefore, is how U34 base pairs with A without also base pairing with G. Here, we show (i) that unlike U34 at the wobble position of all B. subtilis tRNAs of known sequence, U34 in the mutant tRNA is not modified, and (ii) that the mutant tRNA binds strongly to the AUA codon on B. subtilis ribosomes but only weakly to AUG. These in vitro data explain why the suppressor strain displays only a low level of misreading AUG codons in vivo and, as shown here, grows at a rate comparable to that of the wild-type strain.

Photoisomerization and reorientational dynamics of DTDCI in AOT/alkane reverse micelles containing non-aqueous polar liquids.

Molecular mobility of the symmetric carbocyanine fluorophore DTDCI was studied in AOT/alkane reverse micelles containing non-aqueous polar liquids DMF, formamide, ethylene glycol and glycerol by monitoring both the torsional photoisomerization and rotational reorientation, both of which were sensitive to microviscosity of the local environment. The DTDCI fluorophore resides completely within the AOT-polar liquid reverse micelle nano-droplets, where its dynamics were found to be significantly retarded irrespective of the polar liquid taken, due to a combination of electrostatic and hydrophobic forces that induce the guest DTDC(+) cation to attach to the AOT molecules of the host droplet. The addition of strong hydrogen-bond donating polar liquids like formamide, ethylene glycol and glycerol causes a systematic enlargement of the droplets. Rotational dynamics of the fluorophore inside the nano-droplets was characterized by a diffusion coefficient comparable to that in highly viscous solvents like ethylene glycol.

Proton transfer reactions of 4'-chloro substituted 3-hydroxyflavone in solvents and aqueous micelle solutions.

Flavonol 4'-chloro,3-hydroxyflavone (Cl-3HF) has been investigated in solvents of varying polarity and hydrogen-bonding capacity as well as in aqueous micelle solutions. Quantum chemical calculations indicate that although the Cl-atom at the 4'-position of the 2-phenyl ring weakly perturbs the electron distribution of the parent 3-hydroxyflavone, the nuclear framework remains largely intact, and excited state intra-molecular proton-transfer (ESIPT) is feasible. The ESIPT process in both polar solvents and micelles was found to be fast and irreversible, with remarkably long time-constants of several tens of picoseconds. This dramatic inhibition of the ESIPT rate (which is intrinsically a sub-picosecond event) could be rationalized in terms of the emergence of complexes between the solvent and the enol form of Cl-3HF, whose dynamics is coupled to the relatively slow dynamics of inter-molecular hydrogen bonds. In the micelle solutions, spectroscopic data establish that the guest Cl-3HF molecules localized almost exclusively at the polar exterior shell, where they experienced a nearly uniform local environment similar to that in moderately polar solvents. Thus, the Cl-3HF molecules tend to avoid the non-polar core of the micelles, in spite of being strongly hydrophobic themselves. This apparently unusual observation is explained by the formation of inter-molecularly hydrogen-bonded complexes between the guest Cl-3HF and the water molecules tethered to the polar shells of the micelles.

A new Eosin Y-based 'turnon' fluorescent sensor for ratiometric sensing of toxic mercury ion (Hg2+) offering unaided eye detection and its antibacterial activity.

A unique fluorescent molecule (ND-S) was obtained from Eosin Y in two simple yet high yielding steps (1). ND-S has special metal ion sensing ability, such that it can selectively detect toxic Hg2+ present in very low concentration in aqueous solutions in the presence of other competing metal ions. The host-guest complexation is ratiometric and is associated with significant increase in fluorescence during the process. Isothermal titration calorimetry (ITC) experiments provided thermodynamic parameters related to interaction between ND-S and Hg2+. Using inductively coupled plasma mass spectrometry (ICP-MS), the Hg2+(aq) removal efficiency of ND-S was estimated to be 99.88%. Appreciable limit of detection (LOD = 7.4 nM) was observed. Other competing ions did not interfere with the sensing of Hg2+ by ND-S. The effects of external stimuli (temperature and pH) were studied. Besides, the complex (ND-M), formed by 1:1 coordination of ND-S and Hg2+ was found to be effective against the survival of Gram-positive bacteria (S. aureus and B. subtilis) with a high selectivity index. Moreover, bacterial cell death mechanism was studied systematically. Overall, we have shown the transformation of a toxic species (Hg2+), extracted from polluted water by a biocompatible sensor (ND-S), into an effective and potent antibacterial agent (ND-M).

Design, Synthesis, and Biological Studies of C-5-Substituted Diazenyl Derivatives of Uracil as Potent and Selective Antileishmanial Agents Targeting Uridine Biosynthesis Pathway Enzymes.

Herein, we describe the design and synthesis of a series of C-5-substituted diazenyl derivatives of uracil, exhibiting selective and potent antileishmanial but not antibacterial or antifungal activity. The formation of the substituted derivatives was confirmed by using FTIR, 1H, 13C NMR, and HRMS analysis. Among all of the sets of tested compounds, only three [4a, 6b, and 8b] showed the highest activity against Leishmania donovani (LD) promastigote and amastigote models of LD infections. Further, the cytotoxicity assays performed using three different cell lines, Vero cells, J774 cells, and THP1 cells, along with erythrocyte hemolysis assay showed the highest biocompatibility for the 4a, making it a lead compound for further biological assays. The LD cell death associated with 4a was not linked with ergosterol depletion, a common mechanism of action of antileishmanial drugs like amphotericin B (AmB). However, the LD cell death in the presence of 4a was reversed significantly through supplementation of uridine monophosphate (UMP), indicating the specific role of uridine biosynthesis pathway as the target of 4a. Furthermore, the in silico studies predicted orotidine monophosphate decarboxylase enzyme (OMPDCase) from LD as the plausible target for 4a. The proteomics analysis showed stronger downregulation of the aforementioned OMPDCase and also for a few other enzymes that are involved in the UMP biosynthesis pathway. This indicates that OMPDCase and other enzymes that regulate the UMP biosynthesis may be the target of 4a. Overall, the C-5-substituted diazenyl derivatives of uracil are presented here as novel and potent antileishmanial agents that can be used for treating visceral leishmaniasis (VL) wherein at present drug resistance and side effects of existing drugs demand a look for safer alternatives.

Agmatidine, a modified cytidine in the anticodon of archaeal tRNA(Ile), base pairs with adenosine but not with guanosine.

Modification of the cytidine in the first anticodon position of the AUA decoding tRNA(Ile) (tRNA2(Ile)) of bacteria and archaea is essential for this tRNA to read the isoleucine codon AUA and to differentiate between AUA and the methionine codon AUG. To identify the modified cytidine in archaea, we have purified this tRNA species from Haloarcula marismortui, established its codon reading properties, used liquid chromatography-mass spectrometry (LC-MS) to map RNase A and T1 digestion products onto the tRNA, and used LC-MS/MS to sequence the oligonucleotides in RNase A digests. These analyses revealed that the modification of cytidine in the anticodon of tRNA2(Ile) adds 112 mass units to its molecular mass and makes the glycosidic bond unusually labile during mass spectral analyses. Accurate mass LC-MS and LC-MS/MS analysis of total nucleoside digests of the tRNA2(Ile) demonstrated the absence in the modified cytidine of the C2-oxo group and its replacement by agmatine (decarboxy-arginine) through a secondary amine linkage. We propose the name agmatidine, abbreviation C(+), for this modified cytidine. Agmatidine is also present in Methanococcus maripaludis tRNA2(Ile) and in Sulfolobus solfataricus total tRNA, indicating its probable occurrence in the AUA decoding tRNA(Ile) of euryarchaea and crenarchaea. The identification of agmatidine shows that bacteria and archaea have developed very similar strategies for reading the isoleucine codon AUA while discriminating against the methionine codon AUG.

Time-dependent exfoliation study of MoS2 for its use as a cathode material in high-performance hybrid supercapacitors.

Quick and precise exfoliation of bulk molybdenum sulphide into few layers can bring a quantum leap in the electrochemical performance of this material. Such a cost-effective exfoliation route to obtain few layers of MoS2 nanosheets with a high mass yield of ∼75% is presented in this study. The electrochemical behaviours of three types of samples, namely pristine MoS2 and MoS2 exfoliated for 3 h and 5 h, were compared and the reasons leading to their performance modulation are explained. The performance could be tuned by changing the nature of the electrolytes, as shown using three different electrolytes, i.e. H2SO4, Na2SO4, and KOH. The electrochemical performance of a supercapacitor device fabricated using the 5 h-exfoliated sample showed many fold improvement. The strategy of combining with a 2D material-based anode is an interesting way forward for such devices. In addition, the anode material has to be carefully chosen so that high performance can be ensured. The usefulness of 2D flake-like WO3 as an anode was investigated first before establishing its worthiness in a hybrid device. The hybrid device was able to deliver an excellent energy density of 33.74 W h kg-1 with long-term cycling stability and coulombic efficiency, thus proving its applicability for high-performance energy-storage devices.

Rationally Designed Ionic Covalent Organic Networks (iCONs) with Efficient Antimicrobial Activities.

Two unique ionic covalent organic networks (iCONs) incorporated with guanidinium motifs were obtained and characterized by various techniques. Upon 8 h of treatment with iCON-HCCP (250 µg/mL), >97% killing of Staphylococcus aureus, Candida albicans, and Candida glabrata strains was observed. Antimicrobial efficacies against bacteria and fungi were also evident from FE-SEM studies. High antifungal efficacies also correlated well with >60% reduction of ergosterol content, high lipid peroxidation, and membrane damage leading to necrosis.