High-Throughput Mining of Novel Compounds from Known Microbes: A Boost to Natural Product Screening.

Advanced techniques can accelerate the pace of natural product discovery from microbes, which has been lagging behind the drug discovery era. Therefore, the present review article discusses the various interdisciplinary and cutting-edge techniques to present a concrete strategy that enables the high-throughput screening of novel natural compounds (NCs) from known microbes. Recent bioinformatics methods revealed that the microbial genome contains a huge untapped reservoir of silent biosynthetic gene clusters (BGC). This article describes several methods to identify the microbial strains with hidden mines of silent BGCs. Moreover, antiSMASH 5.0 is a free, accurate, and highly reliable bioinformatics tool discussed in detail to identify silent BGCs in the microbial genome. Further, the latest microbial culture technique, HiTES (high-throughput elicitor screening), has been detailed for the expression of silent BGCs using 500-1000 different growth conditions at a time. Following the expression of silent BGCs, the latest mass spectrometry methods are highlighted to identify the NCs. The recently emerged LAESI-IMS (laser ablation electrospray ionization-imaging mass spectrometry) technique, which enables the rapid identification of novel NCs directly from microtiter plates, is presented in detail. Finally, various trending 'dereplication' strategies are emphasized to increase the effectiveness of NC screening.

Wheat bran as an efficient agro-process waste for enhanced yellow laccase production by Lentinus tigrinus SSB_W2 and its application in anthraquinone dye degradation.

Lentinus tigrinus SSB_W2, isolated from Mahabaleshwar in the Western Ghats of Maharashtra, India, was employed to enhance laccase production in solid-state fermentation (SSF). The spectral analysis indicated that the laccase produced by L. tigrinus is a typical yellow laccase, exhibiting no absorption at 600 nm. Notably, this yellow laccase demonstrated exceptional catalytic activity, as confirmed by electrochemical analysis. Four agricultural processing wastes were evaluated as substrates for SSF, and the results showed that L. tigrinus effectively utilized wheat bran. Initial testing by one-factor-at-a-time method showed 3.79-fold increase in yellow laccase production, which subsequently increased to 6.51-fold after Plackett-Burman design. Moreover, employing response surface methodology resulted in 11.87-fold increase (108,472 IU gds-1) in laccase production. The utilization of yellow laccase for the biotransformation of various textile dyes was investigated, and it exhibited the highest degradation efficiency toward Reactive blue 4, a recalcitrant anthraquinone dye, with a rate of 18.36 mg L-1 h-1, for an initial concentration of 1000 mg L-1. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03881-9.

Effect of trifluoroacetic acid on the antigenicity of capsular polysaccharides obtained from various Streptococcus pneumoniae serotypes.

Determining the safety, antigenicity, and immunogenicity by in vitro and in vivo studies is a prerequisite for the development of new vaccines. And this study investigated it for a vaccine made from Streptococcus pneumoniae serotypes 2, 5, 12F, 18C, and 22F. The crude CPS was purified and partially depolymerized by conventional and trifluoroacetic acid methods. 1H NMR analysis confirmed the identity of the depolymerized CPS which gave similar profiles to reference polysaccharides, except for serotype 18C which was de-O-acetylated during TFA treatment. The antigenicity of the depolymerized CPS prepared by either method was comparable to that of the native CPS for serotypes 2, 5, 18C, and 22F based on multiplex bead based competitive inhibition assay. This study demonstrated a relationship between antigenicity and immunogenicity, which offers more suitable candidates for conjugation. It was found that after partial depolymerization process, the CPS with optimal molecular size resulted in higher antigenicity. The immunogenicity of S. pneumoniae serotype 2 conjugates in mice was evaluated by opsonophagocytic assay and a multiplex bead-based assay, wherein on day 42 after immunization, the total and functional IgG titer was found to be increased by 32-fold.

In situ treatment of real textile effluent in constructed furrows using consortium of Canna indica and Saccharomyces cerevisiae and subsequent biochemical and toxicity evaluation.

Emerging contaminants removals like dyes and heavy metals from the textile effluent have an immense challenge. The present study focuses on the biotransformation and detoxification of dyes and in situ textile effluent treatment by plants and microbes efficiently. A mixed consortium of perennial herbaceous plant Canna indica and fungi Saccharomyces cerevisiae showed decolorization of di-azo dye Congo red (CR, 100 mg/L) up to 97% within 72 h. Root tissues and Saccharomyces cerevisiae cells revealed induction of various dye-degrading oxidoreductase enzymes such as lignin peroxidase, laccase, veratryl alcohol oxidase and azo reductase during CR decolorization. Chlorophyll a, Chlorophyll b and carotenoid pigments were notably elevated in the leaves of a plant during the treatment. Phytotransformation of CR into its metabolic constituents was detected by using several analytical techniques, including FTIR, HPLC, and GC-MS and its non-toxic nature was confirmed by cyto-toxicological evaluation on Allium cepa and on freshwater bivalves. Mix consortium of plant Canna indica and fungi Saccharomyces cerevisiae efficiently treated textile wastewater (500 L) and reduced ADMI, COD, BOD, TSS and TDS (74, 68, 68, 78, and 66%) within 96 h. In situ textile wastewater treatment for in furrows constructed and planted with Canna indica, Saccharomyces cerevisiae and consortium-CS within 4 days reveals reduced ADMI, COD, BOD, TDS and TSS (74, 73, 75, 78, and 77%). Comprehensive observations recommend this is an intelligent tactic to exploit this consortium in the furrows for textile wastewater treatment.

Photodissociation of nitric oxide from designed ruthenium nitrosyl complex: Studies on wound healing and antibacterial activity.

A photoactivable NO releasing complex [Ru(L1-2)(PPh3)(NO)Cl2](PF6)(1a) have been synthesized by complex [RuL1-2(PPh3)2Cl2](1). Newly designed bidentate ligands, i.e., 4-methoxy-N'-phenyl-N'-(pyridin-2-ylmethyl)benzohydrazide(L1) and 4-nitro-N'-phenyl-N'-(pyridin-2-ylmethyl)benzohydrazide (L2) were utilized to synthesize complex (1). Complex (1) was characterized by ESI-MS, and the solid structure of the complex [1a](PF6) was acquired by X-ray crystallography. Different spectroscopic techniques were employed for the identification of ligands (L1 and L2) and complexes (1 and [1a](PF6)). Calculations employing DFT and TD-DFT were made better to understand the electronic properties of the complex [1a](PF6). The photo liberation experiments were screened in the presence of visible light lamp. Griess assay experiment was used to quantify the photo released amount to NO. The photo liberated NO was successfully transferred to reduced myoglobin (Mb). The complex [1a](PF6) at 50 µg/mL concentration was used for wound healing and antimicrobial activity on B16F1 mouse skin cells and Escherichia coli bacteria, respectively. In results, we observed a considerable wound healing activity of [1a](PF6) complex after 36 h of incubation in the light-treated cells compared to the control medium, and also it shows more than 99% inhibition of bacterial cells after 1.5 h of treatment in the presence of light. These study suggested that this complex 1a](PF6) could be utilized for topical delivery of NO for combating several dermatological infections.

Biosynthesis of vanillic acid by Ochrobactrum anthropi and its applications.

Vanillic acid has always been in high-demand in pharmaceutical, cosmetic, food, flavor, alcohol and polymer industries. Present study achieved highly pure synthesis of vanillic acid from vanillin using whole cells of Ochrobactrum anthropi strain T5_1. The complete biotransformation of vanillin (2 g/L) in to vanillic acid (2.2 g/L) with 95 % yield was achieved in single step in 7 h, whereas 5 g/L vanillin was converted to vanillic acid in 31 h. The vanillic acid thus produced was validated using LC-MS, GC-MS, FTIR and NMR. Further, vanillic acid was evaluated for in vitro anti-tyrosinase and cytotoxic properties on B16F1 skin cell line in dose dependent manner with IC50 values of 15.84 mM and 9.24 mM respectively. The in silico Swiss target study predicted carbonic acid anhydrase IX and XII as key targets of vanillic acid inside the B16F1 skin cell line and revealed the possible mechanism underlying cell toxicity. Molecular docking indicated a strong linkage between vanillic acid and tyrosinase through four hydrogen and several hydrophobic bonds, with ΔG of -3.36 kJ/mol and Ki of 3.46 mM. The bioavailability of vanillic acid was confirmed by the Swiss ADME study with no violation of Lipinski's five rules.

Purification of capsular polysaccharides isolated from S. pneumoniae serotype 2 by hydrogen peroxide and endonuclease.

A high-quality and cost-effective purification procedure is one of the most important requirements for manufacturing glycoconjugate vaccines. The goal of the present work was to devise a method for removing impurities such as protein and nucleic acid from Streptococcus pneumoniae serotype 2 capsular polysaccharides (CPS). The use of hydrogen peroxide for the reduction of impurities of crude CPS was investigated. Centrifugation followed by filtration decreased protein contaminant of the hydrogen peroxide-treated CPS to meet the limit specified by WHO. The nucleic acid impurity remaining was removed by a further step of endonuclease treatment to yield the purified CPS. Characterization of purified CPS was evaluated by various analytical techniques including 1H NMR and antigenicity by competitive inhibition assay. Various hydrogen peroxide concentrations have significant impact on the antigenic property of CPS. Whereas, optimum process conditions can preserve the native characteristics of CPS.

Partial depolymerization of capsular polysaccharides isolated from Streptococcus pneumoniae serotype 2 by various methods.

Partial depolymerization of bacterial capsular polysaccharides (CPS) is an essential process carried out before its use as an antigenic preparation in a vaccine industry. Choice of CPS depolymerization methods depends on the process robustness, reproducibility, yield, retention of CPS bioactivity, etc. Partial depolymerization methods based on chemicals, enzymes, mechanical, thermal, etc. have been subject of many investigations before. Partial depolymerization of Streptococcus pneumoniae serotype 2 purified CPS was conducted by methods such as acid hydrolysis, microfluidization, ultrasonication, thermal and microwave. Partial depolymerization of the CPS was evaluated by size exclusion high performance liquid chromatography, whereas structural identity and conformity of CPS was ensured by 1H NMR spectroscopy. The antigenicity of CPS was assessed by bead based competitive inhibition assay. Microwave and thermal methods effectively depolymerized CPS and reduced the concentration of cell wall polysaccharide (CWPS) impurity, but both methods have a negative impact on the antigenicity of CPS. Whereas the trifluoroacetic acid treatment not only depolymerized the CPS but completely removed the CWPS while retaining the antigenicity of 92 ± 4% and this method is advantageous over other methods.

Siderophore mediated mineralization of struvite: A novel greener route of sustainable phosphate management.

Efficient and sustainable removal of phosphate ions from an aqueous solution is of great challenge. Herein we demonstrated a greener route for phosphate recovery through struvite formation by using bacterial siderophore. This method was efficient for removal of phosphate as low as 1.3 mM with 99% recovery efficiency. The siderophore produced by Pseudomonas taiwanensis R-12-2 act as template for the nucleation of struvite crystals and was found sustainable for recycling the phosphorous efficiently after twenty cycles. The formation of struvite crystals is driven by surrounding pH (9.0) and presence of Mg2+ and NH4+ ions along with PO43- and siderophore which was further validated by computational studies. The morphology of struvite was characterized by scanning electron microscopy, followed by elemental analysis. Furthermore, our results revealed that the siderophore plays an important role in struvite biomineralization. We have successfully demonstrated the phosphate sequestration by using industrial waste samples, as possible application for environmental sustainability and phosphate conservation. For the first time electrochemical super-capacitance performance of the struvite was studied. The specific capacitance value for the struvite was found to be 320 F g-1 at 1.87 A g-1 and retained 92 % capacitance after 250 cycles. The study revealed the potential implications of siderophore for the phosphate recycling and the new mechanism for biomineralization by sequestering into struvite.

Photophysical and NMR studies of encapsulation of 2-cyano-6-hydroxy benzothiazole in p-sulfonatocalix[6]arene and its biological applications.

This work deals with the study of the interaction between 2-cyano-6-hydroxy benzothiazole (CHBT) and p-sulfonatocalix[6]arene (SCX6) at different pH values in aqueous medium by UV-visible absorption spectroscopy and steady-state fluorescence spectroscopy. The results demonstrate the strong influence of SCX6 on the fluorescence properties of CHBT. The steady-state emission of CHBT shows strong sensitivity to its environment. The mode of inclusion complexation of CHBT and SCX6 has also been investigated using HR-MS, FT-IR, NMR, 2D NMR, and FESEM analysis. With the increase in SCX6 concentration, absorbance decreased with an isosbestic point at 305 nm. The binding constant is calculated by a spectrofluorimetric method and stoichiometry by Job's method. The formation of an inclusion complex has been confirmed by 2D NMR NOESY, COSY, ROESY, HMBC, and HSQC spectroscopic methods. The complex is seen to be stabilized by electrostatic interactions between CHBT and the nanocavity of SCX6. Studies with cellular systems support that the CHBT-SCX6 complex is more effective in killing cancerous cells and hence, SCX6 may prove to be an effective carrier for drug molecules like CHBT.

Thermoresponsive Pluronic based microgels for controlled release of curcumin against breast cancer cell line.

We developed here stimuli responsive curcumin loaded microgels based on Pluronic F-127. These microgels were prepared using coupling reaction between the amine modified Pluronic and EDTA. The microgel exhibited the affinity for hydrophobic drug, curcumin and showed pH as well as temperature-dependent release. Furthermore, the cytotoxicity study demonstrated dose-dependent inhibition of MDA-MB-231 cell growth with the most effective IC50 value (3.8 ±â€¯0.2 µg mL-1 after 24 h). Based on these findings, the fabricated curcumin loaded microgels offered additional advantages over conventional drug therapies for treatment of cancer.

Simultaneous purification and depolymerization of Streptococcus pneumoniae serotype 2 capsular polysaccharides by trifluoroacetic acid.

Development of an effective purification process in order to provide low cost and high-quality vaccine is the necessity of glycoconjugate vaccine manufacturing industries. In the present study, we have attempted to develop a method for simultaneous purification and depolymerization process for capsular polysaccharides (CPS) derived from Streptococcus pneumoniae serotype 2. Trifluoroacetic acid (TFA) was used to precipitate impurities which were then removed by centrifugation. It was observed that the TFA treatment could simultaneously depolymerize the CPS and purify it. The purified and depolymerized CPS was analyzed for its purity, structural identity and conformity, molecular size, antigenicity to meet desired quality specifications. The obtained results showed that the purification and depolymerization of S. pneumoniae serotype 2 CPS did not affect the antigenicity of CPS.

Synthesis and Evaluation of Cytotoxicity of Novel Coumarin Peptide Alcohol Derivatives.

BACKGROUND: Coumarins are naturally occurring biologically active heterocyclic molecules endowed with a wide range of biological properties, including antibacterial, antifungal, and antitumor activities. OBJECTIVE: The present work was aimed to synthesize new coumarin-containing compounds and to investigate their cytotoxic activity. METHODS: Coumarin peptide and coumarin amino alcohols were prepared by treating epoxidecontaining coumarin derivatives with suitable aromatic amines and peptides in trifluoroethanol as a solvent at 50°C. These derivatives were evaluated for their cytotoxic activity on three different cell lines: HeLa, MDA-MB-231 and L-132. Cell viability was determined by MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RESULTS: A new protocol was developed for the synthesis of thirteen novel coumarin peptide and coumarin amino alcohol derivatives. Among the tested compounds, three derivatives showed significant activity against all the tested cell lines. Docking studies indicated favorable interactions of the disubstituted peptide coumarin derivatives with the Asp 351 and Thr 347 amino acids at the active site of the human estrogen receptor. CONCLUSIONS: The results suggest that the synthesized compounds may be promising candidates in the research of new antitumor compounds.

Doxorubicin-Conjugated Innovative 16-mer DNA Aptamer-Based Annexin A1 Targeted Anti-Cancer Drug Delivery.

Aptamers are small, functional single-stranded DNA or RNA oligonucleotides that bind to their targets with high affinity and specificity. Experimentally, aptamers are selected by the systematic evolution of ligands by exponential enrichment (SELEX) method. Here, we have used rational drug designing and bioinformatics methods to design the aptamers, which involves three different steps. First, finding a probable aptamer-binding site, and second, designing the recognition and structural parts of the aptamers by generating a virtual library of sequences, selection of specific sequence via molecular docking, molecular dynamics (MD) simulation, binding energy calculations, and finally evaluating the experimental affinity. Following this strategy, a 16-mer DNA aptamer was designed for Annexin A1 (ANXA1). In a direct binding assay, DNA1 aptamer bound to the ANXA1 with dissociation constants value of 83 nM. Flow cytometry and fluorescence microscopy results also showed that DNA1 aptamer binds specifically to A549, HepG2, U-87 MG cancer cells that overexpress ANXA1 protein, but not to MCF7 and L-02, which are ANXA1 negative cells. We further developed a novel system by conjugating DNA1 aptamer with doxorubicin and its efficacy was studied by cellular uptake and cell viability assay. Also, anti-tumor analysis showed that conjugation of doxorubicin with aptamer significantly enhances targeted therapy against tumors while minimizing overall adverse effects on mice health.

NTO Sensing by Fluorescence Quenching of a Pyoverdine Siderophore-A Mechanistic Approach.

In this study, a siderophore, pyoverdine (PVD), has been isolated from Pseudomonas sp. and used to develop a fluorescence quenching-based sensor for efficient detection of nitrotriazolone (NTO) in aqueous media, in contrast to other explosives such as research department explosive (RDX), picric acid, and trinitrotoulene (TNT). The siderophore PVD exhibited enhanced fluorescence quenching above 50% at 470 nm for a minimal concentration (38 nM) of NTO. The limit of detection estimated from interpolating the graph of fluorescence intensity (at 470 nm) versus NTO concentration is found to be 12 nM corresponding to 18% quenching. The time delay fluorescence spectroscopy of the PVD-NTO solution showed a negligible change of 0.09 ns between the minimum and maximum NTO concentrations. The in silico absorption at the emission peak of static fluorescence remains invariant upon the addition of NTO. The computational studies revealed the formation of inter- and intramolecular hydrogen-bonding interactions between the energetically stable complexes of PVD and NTO. Although the analysis of Stern-Volmer plots and computational studies imply that the quenching mechanism is a combination of both dynamic and static quenching, the latter is dominant over the earlier. The static quenching is attributed to ground-state complex formation, as supported by the computational analysis.

Characterisation of hyper tolerant Bacillus firmus L-148 for arsenic oxidation.

Groundwater arsenic pollution causes millions of deaths worldwide. Long term natural and anthropogenic activities have increased arsenic levels in groundwater causing higher threats of arsenic exposure. Arsenic hyper-tolerant Firmicute Bacillus firmus L-148 was isolated from arsenic limiting Lonar lake soil, which tolerated more than 3 M arsenic and could oxidize 75 mM arsenite [As(III)] in 14 days. It oxidized As(III) in presence of heavy metals and had unusual pH optima at 9.2. B. firmus L-148 was studied at the biochemical, protein, genomic and transcript level for understanding its arsenic oxidizing machinery. The proteomic and transcript analysis exhibited the presence of ars and aio operon and supported the inducible nature of ars operon. Robust, hyper-tolerant, fast As(III) oxidizing, least nutrient requiring and multi-metal resistance qualities of the strain were used in microcosm studies for bioremediation. Artificial groundwater mimicking microcosm with 75 mM As(III) was developed. Modulation of carbon source, iron and multi metals affected growth and As(III) oxidation rate. The As(III) oxidation was recorded to be 77% in 15 days in presence of sodium acetate and Fe ions. This microcosm study can be explored for bioremediation of arsenic contaminated water and followed by precipitation using other methods.

In vitro toxicological evaluation of ionic liquids and development of effective bioremediation process for their removal.

The present study deals with the cyto-genotoxicological impact of ionic liquids, 1-butyl-3-methylimidazolium bromide, trihexyl tetradecylphosphonium dicyanamide, 1-decyl-3-methylimidazolium tetrafluoroborate, benzyldimethyltetradecylammonium chloride, and 1-butyl-4-methylpyridinium chloride, on animal cells and their biodegradation. The long alkyl chain containing ionic liquids were found to be more toxic whereas benzene functional group in benzyldimethyltetradecylammonium chloride enhances its toxicity. Aerobic bacterial granules, a bacterial consortium, were developed that have promising ability to break down these organic pollutants. These aerobic bacterial granules have been applied for the biodegradation of ionic liquids. The biological oxygen demand (5 days) and chemical oxygen demand parameters confirmed that the biodegradation was solely due to aerobic bacterial granules which further decreased the time period needed for regular biodegradation by biological oxygen demand (28 days). The high resolution mass spectrometry analysis further approved that the degradation of ionic liquids was mainly via removal of the methyl group. Elevated N-demethylase enzyme activity supports the ionic liquids degradation which may be occurring through demethylation mechanism. The amplicon sequencing of aerobic bacterial granules gives insight into the involvement of the bacterial community in the biodegradation process.

Click chemistry based multicomponent approach in the synthesis of spirochromenocarbazole tethered 1,2,3-triazoles as potential anticancer agents.

A series of spirochromenocarbazole tethered 1,2,3-triazoles were synthesized via click chemistry based one-pot, five component reaction between N-propargyl isatins, malononitrile, 4-hydroxycarbazole, aralkyl halides and sodium azide using cellulose supported CuI nanoparticles (Cell-CuI NPs) as the heterogeneous catalyst. Antiproliferative activity of all the synthesized compounds was investigated against panel of cancer cell lines such as MCF-7, MDA-MB-231, HeLa, PANC-1, A-549, and THP-1. Many of the synthesized compounds exhibited good anti-proliferative activity against breast (MCF-7 and MDA-MB-231) and cervical (HeLa) cancer cells with IC50 values less than 10 µM. In case of MCF-7 cells, among the nine compounds that showed good anti-proliferative activity, compounds 6f and 6j were found to be highly potent (IC50 = 2.13 µM and 4.80 µM, respectively). In case of MDA-MB-231, three compounds (6k, 6j and 6s) showed antiproliferative activity amongst which 6k was the most potent one (IC50 = 3.78 µM). On the other hand, in cervical cancer HeLa cells, compounds 6b, 6g, 6s and 6u showed excellent antiproliferative activity (IC50 = 4.05, 3.54, 3.83, 3.35 µM, respectively). All the compounds were found to be nontoxic to the human umbilical vein endothelial cells (HUVECs). AO and EtBr staining and fluorescence microscopy studies of the active compounds (IC50 < 5 µM) suggested that these compounds induce cell death by apoptosis.

Assessment of arsenic oxidation potential of Microvirga indica S-MI1b sp. nov. in heavy metal polluted environment.

Arsenic oxidizing α-proteobacterial strain Microvirga indica S-MI1b sp. nov. was isolated from metal industry soil and has the ability to oxidize 15 mM of arsenite [As(III)] completely in 39 h. The strain S-MI1b resists to different heavy metals and it oxidizes arsenite in presence of Li, Pb, Hg, Sb(III), Cd, Cr(VI), Ni, and exhibited growth inhibitory effect in presence of Hg, Cu, and Cd at higher concentration. The morphology of Microvirga indica S-MI1b changed in presence of heavy metals however there was no accumulation of As(III) in the cells. The study showed that Microvirga indica S-MI1b can oxidize arsenite at broad pH ranges from 4.0 to 9.0 with optimum at pH 7.0. The kinetic studies of arsenite oxidation by strain S-MI1b signified that it has greater affinity towards As(III). The arsenite oxidase activity of cells grown in presence of Li and Cr(VI) supported the cell culture studies. This is first report on biotransformation of arsenite by Microvirga genus and also arsenite oxidation in presence of heavy metals.