Bioactivity and Metabolomics Profiling of Endophytic Actinobacteria Isolated from Roots of the Medicinal Plants Dominant in South Asian Region.

Background: Plant-derived endophytic actinobacteria are the center of attention due to their capacity to produce diverse antimicrobial and anticancer compounds and their metabolites influence plant growth. Methods: In this study, 40 endophytic actinobacteria strains were isolated from the roots of eight medicinal plants used as folk medicine in South Asian region. The isolates were characterized morphologically, biochemically and physiologically and the genus level identification of the selected strains was done by 16SrRNA gene sequencing. In small scale cultivation (50ml broth), the isolates were grown in A-medium to prepare the crude extracts. These crude extracts were subsequently evaluated for their antimicrobial, anticancer and antioxidant activity and the metabolomics profile of each of the extract was determined by TLC and HPLC-UV/MS. Results: The taxonomic studies showed that the isolates belong to the group actinobacteria based on their morphological and physiological characteristics and the 16SrRNA gene sequencing of the selected strains identified the genera including Streptomyces, Micromonospora and Nocardia. Cumulatively,53% of extracts exhibited anti-Gram-(+) activity,47% exhibited anti-Gram-(-) activity,32% exhibited antifungal activity and 30% were cytotoxic to PC3 and A549 cancer cell lines and most of the extracts have shown antioxidant activity greater than 50%. The metabolomics analysis predicted the presence of an array of low molecular weight metabolites and indicated the promising isolates in collection for further studies for novel bioactive metabolite isolation and structure elucidation. Conclusion: Overall the study provides an overview of the endophytic actinobacteria residing in the roots of the selected medicinal plants prevalent in south Asian region and their potential to produce the medicinally and biotechnologically useful compounds.

Bioprospecting of desert actinobacteria with special emphases on griseoviridin, mitomycin C and a new bacterial metabolite producing Streptomyces sp. PU-KB10-4.

BACKGROUND: Bioprospecting of actinobacteria isolated from Kubuqi desert, China for antibacterial, antifungal and cytotoxic metabolites production and their structure elucidation. RESULTS: A total of 100 actinobacteria strains were selectively isolated from Kubuqi desert, Inner Mongolia, China. The taxonomic characterization revealed Streptomyces as the predominant genus comprising 37 different species, along with the rare actinobacterial genus Lentzea. The methanolic extracts of 60.8% of strains exhibited potent antimicrobial activities against Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli, Salmonella enterica, Saccharomyces cerevisiae and high to mild in vitro cytotoxicity against PC3 (prostate cancer) and A549 (lung carcinoma) cell lines. The metabolomics analysis by TLC, HPLC-UV/vis, HPLC-MS and NMR showed the presence of compounds with molecular weights ranging from 100 to 1000 Da. The scale-up fermentation of the prioritized anti-Gram-negative strain PU-KB10-4 (Streptomyces griseoviridis), yielded three pure compounds including; griseoviridin (1; 42.0 mgL- 1) with 20 fold increased production as compared to previous reports and its crystal structure as monohydrate form is herein reported for the first time, mitomycin C (2; 0.3 mgL- 1) and a new bacterial metabolite 4-hydroxycinnamide (3; 0.59 mgL- 1). CONCLUSIONS: This is the first report of the bioprospecting and exploration of actinobacteria from Kubuqi desert and the metabolite 4-hydroxycinnamide (3) is first time isolated from a bacterial source. This study demonstrated that actinobacteria from Kubuqi desert are a potential source of novel bioactive natural products. Underexplored harsh environments like the Kubuqi desert may harbor a wider diversity of actinobacteria, particularly Streptomyces, which produce unique metabolites and are an intriguing source to develop medicinally valuable natural products.

Morphogenetic variation and assorted biological activities in true branching Nostocales strains of Cholistan oasis, Pakistan.

From the oasis of Cholistan, true branching heterocystous cyanobacterial strains were studied for, the cell arrangement in primary branches being mono- or bi-seriate; the shape of cells in main filament large and irregular; profused secondary branching emerging on one or both sides and tapering along their length. In these observed traits, two clear morphological taxa were recognized, both well-assorted from the previously described species of the genus Westiellopsis. Both strains showed culturing responses and were studied for antibacterial, cytotoxic, and anticancer potentials. The strain derived from the site B-10 provenance exhibited antibacterial activity against Pseudomonas (18 mm), Klebsiella (15 mm), Staphylococcus (22 mm). On the contrary, the strain of site A-44 showed no activity against any of the above-mentioned bacterial strains. The cytotoxicity assay for the strain of the B-10 site showed 36% larval mortality, whereas strain A-44 showed 24% larval mortality. Performance of the strain B-10 in MTT assay (assessed on HCT-116 cell lines) revealed a dose-dependent activity: at 200, 100, 50, and 25 µg/ml; achieving a growth inhibition of 50.15%, 40.22%, 33.72%, and 10.21%, respectively; and the strain of A-44 could only exhibit a 30.06% growth inhibition at 200 µg/ml. The 16S rRNA sequencing revealed the sequence homology with Neowestiellopsis. Based on data presented here we report two diverse taxa of true branching Nostocales from Cholistan oasis, Pakistan.

Cytotoxic Evaluation and Molecular Docking Studies of Aminopyridine Derivatives as Potential Anticancer Agents.

BACKGROUND: The development of resistance to available anticancer drugs is increasingly becoming a major challenge and new chemical entities could be unveiled to compensate for this therapeutic failure. OBJECTIVES: The current study demonstrated whether N-protected and deprotected amino acid derivatives of 2- aminopyridine could attenuate tumor development using colorectal cancer cell lines. METHODS: Biological assays were performed to investigate the anticancer potential of synthesized compounds. The in silico ADME profiling and docking studies were also performed by docking the designed compounds against the active binding site of beta-catenin (CTNNB1) to analyze the binding mode of these compounds. Four derivatives 4a, 4b, 4c, and 4d were selected for investigation of in vitro anticancer potential using colorectal cancer cell line HCT 116. The anti-tumor activities of synthesized compounds were further validated by evaluating the inhibitory effects of these compounds on the target protein beta-catenin through in vitro enzyme inhibitory assay. RESULTS: The docking analysis revealed favorable binding energies and interactions with the target proteins. The in vitro MTT assay on colorectal cancer cell line HCT 116 and HT29 revealed potential anti-tumor activities with an IC50 range of 3.7-8.1µM and 3.27-7.7 µM, respectively. The inhibitory properties of these compounds on the concentration of beta-catenin by ELISA revealed significant percent inhibition of target protein at 100 µg/ml. CONCLUSION: In conclusion, the synthesized compounds showed significant anti-tumor activities both in silico and in vitro, having potential for further investigating its role in colorectal cancer.

Himalaquinones A-G, Angucyclinone-Derived Metabolites Produced by the Himalayan Isolate Streptomyces sp. PU-MM59.

Himalaquinones A-G, seven new anthraquinone-derived metabolites, were obtained from the Himalayan-based Streptomyces sp. PU-MM59. The chemical structures of the new compounds were identified based on cumulative analyses of HRESIMS and NMR spectra. Himalaquinones A-F were determined to be unique anthraquinones that contained unusual C-4a 3-methylbut-3-enoic acid aromatic substitutions, while himalaquinone G was identified as a new 5,6-dihydrodiol-bearing angucyclinone. Comparative bioactivity assessment (antimicrobial, cancer cell line cytotoxicity, impact on 4E-BP1 phosphorylation, and effect on axolotl embryo tail regeneration) revealed cytotoxic landomycin and saquayamycin analogues to inhibit 4E-BP1p and inhibit regeneration. In contrast, himalaquinone G, while also cytotoxic and a regeneration inhibitor, did not affect 4E-BP1p status at the doses tested. As such, this work implicates a unique mechanism for himalaquinone G and possibly other 5,6-dihydrodiol-bearing angucyclinones.

Taxonomic and Metabolomics Profiling of Actinobacteria Strains from Himalayan Collection Sites in Pakistan.

Actinobacteria have proven themselves as the major producers of bioactive compounds with wide applications. In this study, 35 actinobacteria strains were isolated from soil samples collected from the Himalayan mountains region in Pakistan. The isolated strains were identified by polyphasic taxonomy and were prioritized based on biological and chemical screening to identify the strains with ability to produce inimitable metabolites. The biological screening included antimicrobial activity against Staphylococcus aureus, Micrococcus luteus, Salmonella enterica, Escherichia coli, Mycobacterium aurum, and Bacillus subtilis and anticancer activity using human cancer cell lines PC3 and A549. For chemical screening, methanolic extracts were investigated using TLC, HPLC-UV/MS. The actinobacteria strain PU-MM93 was selected for scale-up fermentation based on its unique chemical profile and cytotoxicity (50-60% growth inhibition) against PC3 and A549 cell lines. The scale-up fermentation of PU-MM93, followed by purification and structure elucidation of compounds revealed this strain as a promising producer of the cytotoxic anthracycline aranciamycin and aglycone SM-173-B along with the potent neuroprotective carboxamide oxachelin C. Other interesting metabolites produced include taurocholic acid as first report herein from microbial origin, pactamycate and cyclo(L-Pro-L-Leu). The study suggested exploring more bioactive microorganisms from the untapped Himalayan region in Pakistan, which can produce commercially significant compounds.

Auraticoccus cholistanensis sp. nov., an actinomycete isolated from soil of the Cholistan Desert, and emended description of the genus Auraticoccus.

A Gram-stain-positive, aerobic, non-motile and non-spore-forming actinobacterium, designated as F435T, was isolated from soil sample collected from the Cholistan Desert, Pakistan. The taxonomic position of the strain was established by using a polyphasic taxonomic approach. The cells were coccoid-shaped and found in single or arrangement of pairs. The novel strain grew at 15‒37 °C (optimum, 25‒30 °C), pH 7‒11 (optimum, pH 7-8) and in the presence of 0‒8% (w/v) NaCl (optimum, 0 %). Results of blast analysis based on 16S rRNA gene sequences showed that Auraticoccus monumenti MON 2.2T was its closest relative with 97.4 % similarity followed by Desertihabitans aurantiacus CPCC 204711T (95.2 %). In phylogenetic trees, strain F435T formed a robust cluster with the only member of the genus Auraticoccus. The peptidoglycan isomer present in the cell wall was ll-diaminopimelic acid. The major fatty acid was determined to be anteiso-C15 : 0. Characteristic polar lipids of the strain were diphosphatidylglycerol, phosphatidylglycerol, phosphoglycolipids and glycolipids. The predominant menaquinone was MK-9(H4). The genomic G+C content was calculated as 73.5 mol%. The digital DNA-DNA hybridization (GGDC) and average nucleotide identity (ANI) values between strain F435T and A. monumenti MON 2.2T were 24.6 and 81.8 %, respectively. Based on the results of phenotypic, chemotaxonomic, phylogenetic and phylogenomic analyses, strain F435T represents a novel specie of the genus Auraticoccus, for which the name Auraticoccus cholistanensis sp. nov. is proposed. The type strain is F435T (=JCM 33648T=CGMCC 1.17443T). The description of the genus Auraticoccus has also been emended.

Green synthesis and biological evaluation of novel 5-fluorouracil derivatives as potent anticancer agents.

This study reports the formation of 5-FU co-crystals with four different pharmacologically safe co-formers; Urea, Thiourea, Acetanilide and Aspirin using methanol as a solvent. Two fabrication schemes were followed i.e., solid-state grinding protocol, in which API and co-formers were mixed through vigorous grinding while in the other method separate solutions of both the components were made and mixed together. The adopted approaches offer easy fabrication protocols, no temperature maintenance requirements, no need of expensive solvents, hardly available apparatus, isolation and purification of the desired products. In addition, there is no byproducts formation, In fact, a phenomenon embracing the requirements of green synthesis. Through FTIR analysis; for API the N-H absorption frequency was recorded at 3409.02 cm-1 and that of -C[bond, double bond]O was observed at 1647.77 cm-1. These characteristics peaks of 5-FU were significantly shifted and recorded at 3499.40 cm-1 and 1649.62 cm-1 for 5-FU-Ac (3B) and 3496.39 cm-1 and 1659.30 cm-1 for 5-FU-As (4B) co-crystals for N-H and -C[bond, double bond]O groups respectively. The structural differences between API and co-crystals were further confirmed through PXRD analysis. The characteristic peak of 5-FU at 2θ = 28.79918o was significantly shifted in the graphs of co-crystals not only in position but also with respect to intensity and FWHM values. In addition, new peaks were also recorded in all the spectra of co-formers confirming the structural differences between API and co-formers. In addition, percent growth inhibition was also observed by all the co-crystals through MTT assay against HCT 116 colorectal cell lines in vitro. At four different concentrations; 25, 50, 100 and 200 µg/mL, slightly different trends of the effectiveness of API and co-crystals were observed. However; among all the co-crystal forms, 5-FU-thiourea co-crystals obtained through solution method (2B) proved to be the most effective growth inhibitor at all the four above mentioned concentrations.

Spore forming Actinobacterial diversity of Cholistan Desert Pakistan: Polyphasic taxonomy, antimicrobial potential and chemical profiling.

BACKGROUND: Actinobacteria are famous for the production of unique secondary metabolites that help in controlling the continuously emerging drug resistance all over the globe. This study aimed at the investigation of an extreme environment the Cholistan desert, located in southern Punjab, Pakistan, for actinobacterial diversity and their activity against methicillin resistant Staphylococcus aureus (MRSA). The Cholistan desert is a sub-tropical and arid ecosystem with harsh environment, limited rainfall and low humidity. The 20 soil and sand samples were collected from different locations in the desert and the actinobacterial strains were selectively isolated. The isolated strains were identified using a polyphasic taxonomic approach including morphological, biochemical, physiological characterization, scanning electron microscopy (SEM) and by 16S rRNA gene sequencing. RESULTS: A total of 110 desert actinobacterial strains were recovered, which were found to be belonging to 3 different families of the order Actinomycetales, including the family Streptomycetaceae, family Pseudonocardiaceae and the family Micrococcaceae. The most frequently isolated genus was Streptomyces along with the genera Pseudonocardia and Arthrobacter. The isolated strains exhibited promising antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA) with zone of inhibition in the range of 9-32 mm in antimicrobial screening assays. The chemical profiling by thin layer chromatography, HPLC-UV/Vis and LC-MS analysis depicted the presence of different structural classes of antibiotics. CONCLUSION: The study revealed that Cholistan desert harbors immense actinobacterial diversity and most of the strains produce structurally diverse bioactive secondary metabolites, which are a promising source of novel antimicrobial drug candidates.

Puromycins B-E, Naturally Occurring Amino-Nucleosides Produced by the Himalayan Isolate Streptomyces sp. PU-14G.

The isolation and structure elucidation of four new naturally occurring amino-nucleoside [puromycins B-E (1-4)] metabolites from a Himalayan isolate ( Streptomyces sp. PU-14-G, isolated from the Bara Gali region of northern Pakistan) is reported. Consistent with prior reports, comparative antimicrobial assays revealed the need for the free 2″-amine for anti-Gram-positive bacteria and antimycobacterial activity. Similarly, comparative cancer cell line cytotoxicity assays highlighted the importance of the puromycin-free 2″-amine and the impact of 3'-nucleoside substitution. These studies extend the repertoire of known naturally occurring puromycins and their corresponding SAR. Notably, 1 represents the first reported naturally occurring bacterial puromycin-related metabolite with a 3'- N-amino acid substitution that differs from the 3'- N-tyrosinyl of classical puromycin-type natural products. This discovery suggests the biosynthesis of 1 in Streptomyces sp. PU-14G may invoke a uniquely permissive amino-nucleoside synthetase and/or multiple synthetases and sets the stage for further studies to elucidate, and potentially exploit, new biocatalysts for puromycin chemoenzymatic diversification.

Antitumor compounds from Streptomyces sp. KML-2, isolated from Khewra salt mines, Pakistan.

BACKGROUND: Actinomycetes are gram positive bacteria with high G + C content in their DNA and are capable of producing variety of secondary metabolites. Many of these metabolites possess different biological activities and have the potential to be developed as therapeutic agents. The aim of the present study was to screen actinomycetes inhabiting halophilic environment such as Khewra salt mines present in Pakistan for cytotoxic and antitumor compounds. RESULTS: An actiomycetes strain designated as Streptomyces sp. KML-2 was isolated from a saline soil of Khewra salt mines, Pakistan. The strain Streptomyces sp. KML-2 showed 84 % cytotoxic activity against larvae of Artemia salina. In the screening phase, the strain exhibited significant antitumor activity with IC50 values of 12, 48 and 56 µg/ml against Hela, MDBK and Vero cell lines, respectively. After that extract from 20 l fermentation was used to purify secondary metabolites by several chromatographic techniques. Structure elucidation of isolated compounds revealed that it is highly stable producer of Chromomycin SA (1) and 1-(1H-indol-3-yl)-propane-1,2,3-triol (2). Both of the isolated compounds showed significant antitumor activity against Hela and MCF-7 cancer cell lines (IC50 values 8.9 and 7.8 µg/ml against Hela; 12.6 and 0.97 µg/ml against MCF-7, respectively). The 16S rRNA gene sequence (1437 bp) of the strain confirm its identity (99 %) with Streptomyces griseus. CONCLUSIONS: From this research work we were successful in isolating two potent antitumor compounds, Chromomycin SA and 1-(1H-indol-3-yl)-propane-1,2,3-triol from Streptomyces KML-2 strain, isolated from Khewra salt mine. As such this is the second report which confirms that S. griseus can produce Chromomycin SA without introducing any mutagenesis in its biosynthesizing gene cluster and isolated indole derivative is being reported first time from any member of actinomycetes group with having novel antitumor activity against Hela and MCF-7 cells. Nucleotide sequences: Nucleotide sequence data reported are available in the GenBank database under the accession number: GenBank KJ009562.

Carpal tunnel syndrome: A rare manifestation of distal radius osteoid osteoma.

Osteoid Osteoma is a benign bone tumor that normally affects long bones and rarely affects distal radius. Because of its nonspecific presentation in the wrist, it remains a diagnostic challenge. We report an unusual case of Osteoid Osteoma at distal radius having symptoms resembling that of carpal tunnel syndrome. The diagnosis was confirmed preoperatively with X-rays; bone scintigraphy, CT, and MRI, later histological examination confirmed the diagnoses. Surgical excision lead to a dramatic improvement in the condition of the patient.

Antitumor compounds from Streptomyces sp. KML-2, isolated from Khewra salt mines, Pakistan

BACKGROUND: Actinomycetes are gram positive bacteria with high G + C content in their DNA and are capable of producing variety of secondary metabolites. Many of these metabolites possess different biological activities and have the potential to be developed as therapeutic agents. The aim of the present study was to screen actinomycetes inhabiting halophilic environment such as Khewra salt mines present in Pakistan for cytotoxic and antitumor compounds. RESULTS: An actiomycetes strain designated as Streptomyces sp. KML-2 was isolated from a saline soil of Khewra salt mines, Pakistan. The strain Streptomyces sp. KML-2 showed 84 % cytotoxic activity against larvae of Artemiasalina. In the screening phase, the strain exhibited significant antitumor activity with IC50 values of 12, 48 and 56 µg/ml against Hela, MDBK and Vero cell lines, respectively. After that extract from 20 l fermentation was used to purify secondary metabolites by several chromatographic techniques. Structure elucidation of isolated compounds revealed that it is highly stable producer of Chromomycin SA (1) and 1-(1H-indol-3-yl)-propane-1,2,3-triol (2). Both of the isolated compounds showed significant antitumor activity against Hela and MCF-7 cancer cell lines (IC50 values 8.9 and 7.8 µg/ml against Hela; 12.6 and 0.97 µg/ml against MCF-7, respectively). The 16S rRNA gene sequence (1437 bp) of the strain confirm its identity (99 %) with Streptomyces griseus. CONCLUSIONS: From this research work we were successful in isolating two potent antitumor compounds, Chromomycin SA and 1-(1H-indol-3-yl)-propane-1,2,3-triol from Streptomyces KML-2 strain, isolated from Khewra salt mine. As such this is the second report which confirms that S. griseus can produce Chromomycin SA without introducing any mutagenesis in its biosynthesizing gene cluster and isolated indole derivative is being reported first time from any member of actinomycetes group with having novel antitumor activity against Hela and MCF-7 cells Nucleotide sequences: Nucleotide sequence data reported are available in the GenBank database under the accession number: GenBank KJ009562.

Identification, isolation and optimization of antifungal metabolites from the Streptomyces Malachitofuscus ctf9

An indigenous Streptomyces isolate CTF9, exhibiting promising antifungal activity against Mucor miehei and Candida albicans in pre-screening studies, was investigated by cultivation in a 50-L fermenter and by subsequent isolation, purification, and structure elucidation of the active metabolites. Based on the morphological, biochemical, and physiological characterization, as well as the 16S rRNA gene sequence, the isolate CTF9 was identified as Streptomyces malachitofuscus. Using a series of chromatographic techniques, two pure compounds were isolated from the obtained extracts after the fermentation of the isolate CTF9. The isolated compounds were identified as phenylacetic acid and indolyl-3-lactic acid by mass spectrometry (MS) and NMR analysis. The culture optimization studies revealed that the isolate CTF9 can use a variety of low-cost carbon and nitrogen sources to generate the maximum quantity of industrially important metabolites at an elevated temperature of 35°C and at a pH 7.8.

Antitumour compounds from a saline soil isolate, Streptomyces griseoincarnatus CTF15.

A new actinomycete strain designated as Streptomyces sp. CTF15 was isolated from a saline soil using casein-KNO(3) agar medium. The strain Streptomyces sp. CTF15 exhibited promising antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Streptomyces viridochromogens Tu57 and high cytotoxicity (91.2% mortality) against Artimia salina in biological screening. The cultivation of this strain in a 50 L lab fermenter and subsequent isolation and purification by a series of chromatographic techniques and structure elucidation by MS and NMR analysis of the active metabolites revealed that it is a highly stable producer of resistomycin (1), tetracenomycin D (2) and actinomycin D (3), even under non-optimised culture conditions. The morphological, microscopic, biochemical and physiological characterisation suggested that the strain CTF15 belongs to the genus Streptomyces. A partial 16S rRNA gene sequence (1429 bp) from the strain CTF15 was determined and found to have high identity (99%) with Streptomyces griseoincarnatus. As such, this is the first report of a strain of S. griseoincarnatus capable of producing these three bioactive compounds simultaneously.