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.

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.

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.

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.

Microvirga indica sp. nov., an arsenite-oxidizing Alphaproteobacterium, isolated from metal industry waste soil.

A novel Gram-stain-negative bacterium, strain S-MI1bT, belonging to the genus Microvirga was isolated from a metal industry waste soil sample in Pirangut village, Pune District, Maharashtra, India. Cells were non-spore-forming, small rod-shapes, motile and strictly aerobic with light-pink colonies. The strain grew in 0-7.0 % (w/v) NaCl and at 25-45 °C, with optimal growth at 40 °C. The predominant fatty acids detected were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and C19 : 0 cyclo ω8c. The predominant isoprenoid quinone was Q-10. The G+C content was 67.2 mol% and DNA-DNA relatedness values between strain S-MI1bTand Microvirga subterranea DSM 14364T and Microvirgaaerophila 5420S-12T were 53.9 and 54.8 %, respectively. Phylogenetic analysis, based on 16S rRNA gene sequences, indicated that strain S-MI1bT is a member of the genus Microvirga, with greatest sequence similarities of 97.7 and 97.4 % with M. subterranea DSM 14364T and M.aerophila 5420S-12T, respectively. Phylogenetic analysis showed that strain S-MI1bT forms a clade with the type strain of M. subterranea DSM 14364T, and was readily distinguishable from it due to various phenotypic characteristics. The combination of genotypic and phenotypic data suggests that the isolate represents a novel species of the genus Microvirga, for which the name Microvirga indica sp. nov. is proposed. The type strain is S-MI1bT (=NCIM-5595T=KACC 18792T=BCRC 80972T).

Enhanced Detoxification of Arsenic Under Carbon Starvation: A New Insight into Microbial Arsenic Physiology.

Nutrient availability in nature influenced the microbial ecology and behavior present in existing environment. In this study, we have focused on isolation of arsenic-oxidizing cultures from arsenic devoid environment and studied effect of carbon starvation on rate of arsenite oxidation. In spite of the absence of arsenic, a total of 40 heterotrophic, aerobic, arsenic-transforming bacterial strains representing 18 different genera were identified. Nineteen bacterial species were isolated from tannery effluent and twenty-one from tannery soil. A strong co-relation between the carbon starvation and arsenic oxidation potential of the isolates obtained from the said niche was observed. Interestingly, low carbon content enhanced the arsenic oxidation ability of the strains across different genera in Proteobacteria obtained. This represents the impact of physiological response of carbon metabolism under metal stress conditions. Enhanced arsenic-oxidizing ability of the strains was validated by the presence of aio gene and RT-PCR, where 0.5- to 26-fold up-regulation of arsenite oxidase gene in different genera was observed. The cultures isolated from tannery environment in this study show predominantly arsenic oxidation ability. This detoxification of arsenic in lack of carbon content can aid in effective in situ arsenic bioremediation.

The Penultimate Tyrosine Residues are Critical for the Genotoxic Effect of Human Hemoglobin.

Hemoglobin (Hb) is a potent oxidant outside the erythrocyte. The tyrosines α140 and ß145 play an important role in the structure and function of Hb by forming switch and hinge contacts. These carboxy-terminal residues of the alpha and beta chains, respectively, were replaced to phenylalanine and several different methods were used to characterize the obtained mutants including a comet and plasmid DNA cleavage assay. It was observed that the genotoxic effect was 40% higher for αY140F compared with the wildtype, the ßY145F and the double (αY140/ß145F) mutants as determined by the comet assay. Cleavage of purified plasmid DNA after Hb application also revealed that the αY140F mutant showed 2-fold higher activity, while the ßY145F and αY140/ß145F mutants reduced the activity compared to wildtype Hb. This study clearly indicates that the penultimate tyrosines are involved in the genotoxicity of Hb.

Biomarker responses in the earthworm, Dichogaster curgensis exposed to fly ash polluted soils.

Earthworms are globally accepted as a model organism in terrestrial ecotoxicology for assessment of environmental pollution. This study evaluated and compared effects of fly ash polluted soils collected from two geographically different thermal power plants on biomarker responses in the earthworm, Dichogaster curgensis. To evaluate relationship between distance sampling and biomarker responses in the earthworm D. curgensis, soil samples at 0.5, 1 and 3km from thermal plant were analyzed for physico-chemical properties and metal concentrations. Biochemical alterations, lysosomal membrane stability, genotoxic effects, and histological changes were examined on 1, 7, and 14 d of exposure to fly ash contaminated soils collected from different thermal power plants. The activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA) levels were significantly increased, while glutathione reductase (GR) activity was found to be decreased in treated animals. Catalase (CAT) and glutathione-S- transferase (GST) activities were found to be increased initially up to 7d exposure and further decreased on 14d exposure. D. curgensis exposed to fly ash contaminated soils showed significant lysosomal membrane destabilization and DNA damage. Extensive histopathological changes were observed in the tissues of the body wall and intestinal tract of the exposed D. curgensis along with accumulation of heavy metals. These results demonstrate that soil pollution around thermal power plants has adverse biological effects of on the indicator organism D. curgensis and no correlation was found between distance and extent of biological biochemical responses.

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.

Facile preparation of tetrahydro-5H-pyrido[1,2,3-de]-1,4-benzoxazines via reductive cyclization of 2-(8-quinolinyloxy)ethanones and their antioxidant activity.

Pd/C-catalyzed reductive cyclization of 1-aryl-2-(8-quinolinyloxy)ethanones opens a facile access to the title compounds in good yields. The scope of this reductive cyclization is explored and the antioxidant activities of the products are studied.

Effective bioremoval and detoxification of textile dye mixture by Alishewanella sp. KMK6.

Alishewanella sp. strain KMK6 was able to degrade mixture of textile dyes (0.5-2.0 g l(-1)) within 8 h. An initial 28 % reduction in COD was observed immediately after decolorization at static anoxic conditions which on further incubation at shaking conditions reduced by 90 %. Partially purified azoreductase was able to utilize different azo dyes as substrates. The HPLC profile of dye degradation showed formation of metabolic products. Further FTIR analysis showed significant changes in the peaks corresponding to functional groups present in dye mixture and its degradation products. The genotoxicity assessment showed that the dye degradation products were non-toxic compared to dye mixture.

Alishewanella solinquinati sp. nov., isolated from soil contaminated with textile dyes.

A novel Gram-negative, motile, rod-shaped, facultative anaerobic bacterial strain, KMK6(T), was isolated from soil contaminated with textile dyes from an industrial estate located at Ichalkaranji, Maharashtra, India, and its taxonomical position was established by using a polyphasic approach. The major cellular fatty acids included C17:1ω8c, summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH), C17:0, C16:0, and C18:1ω7c. The DNA G+C content of strain KMK6(T) was 48.8 mol %. 16S rRNA gene sequence analysis confirmed its placement in the genus Alishewanella, and exhibited sequence similarity levels of below 97 % to the type strains of validly published Alishewanella species. On the basis of genotypic and phenotypic evidence, strains KMK6(T) is considered to be a novel species of the genus Alishewanella, for which we propose that strain KMK6(T) (=NCIM 5295(T) =BCRC 17848(T)) is assigned to a novel species, Alishewanella solinquinati sp. nov.

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.

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.

Synthesis and biological evaluation of novel 2,4,6-triazine derivatives as antimicrobial agents.

A series of 2,4,6-trisubstituted [1,3,5]triazines were synthesized and evaluated for their antimicrobial activity against two representative Gram-positive, Gram-negative bacteria and two fungi. Biological data revealed that among all the compounds screened, compounds 3f, 3g, 3h, 3i, 3m, 3o and 3p found to have promising antimicrobial activity against all the selected pathogenic bacteria and fungi. Out of the synthesized compounds seven analogues have shown MIC in the range of 6.25-12.5 µg/mL. These compounds were generally nontoxic and may prove useful as antimicrobial agents.

Oxidation of arsenite by two ß-proteobacteria isolated from soil.

Two heterotrophic As(III)-oxidizing bacteria, SPB-24 and SPB-31 were isolated from garden soil. Based on 16S rRNA gene sequence analysis, strain SPB-24 was closely related to genus Bordetella, and strain SPB-31 was most closely related to genus Achromobacter. Both strains exhibited high As(III) (15 mM for SPB-24 and 40 mM for SPB-31) and As(V) (>300 mM for both strains) resistance. Both strains oxidized 5 mM As(III) in minimal medium with oxidation rate of 554 and 558 µM h(-1) for SPB-24 and SPB-31, respectively. Washed cells of both strains oxidized As(III) over broad pH and temperature range with optimum pH 6 and temperature 42°C for both strains. The As(III) oxidation kinetic by washed cells showed K (m) and V (max) values of 41.7 µM and 1,166 µM h(-1) for SPB-24, 52 µM and 1,186 µM h(-1) for SPB-31. In the presence of minimal amount of carbon source, the strains showed high As(III) oxidation rate and high specific arsenite oxidase activity. The ability of strains to resist high concentration of arsenic and oxidize As(III) with highest rates reported so far makes them potential candidates for bioremediation of arsenic-contaminated environment.

Decolorization of textile dyes by Alishewanella sp. KMK6.

Alishewanella sp. strain KMK6 was isolated from textile dye-contaminated soil. The strain was able to decolorize and degrade different azo dyes and displayed high dye degradation ability and tolerance. The bacterium could completely degrade 2.5 g l(-1) dye, Reactive Blue 59 within 6 h. The induction in the level of cytochrome P-450 and activities of azoreductase and NADH-dichlorophenolindophenol reductase were observed in the cells after dye decolorization indicating the role of these enzymes. The intermediates of Reactive Blue 59 degradation were identified by high-performance liquid chromatography, gas chromatography and mass spectroscopy, and Fourier transform infrared spectroscopy. The ecotoxicity has been evaluated for dye and its metabolites by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (a yellow tetrazole) and comet assay, and it revealed that the dye degradation products were nontoxic.

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.