Molecular profiling of endophytic Streptomyces cavourensis MH16 inhabiting Millingtonia hortensis Linn. and influence of different culture media on biosynthesis of antimicrobial metabolites.

Endophytic actinomycetes, a prolific source of natural products, are well known for their diverse metabolic versatility, and their association with medicinal plants and antimicrobial potential are well worth exploring. We isolated and identified the Streptomyces cavourensis strain MH16 inhabiting the tree Millingtonia hortensis Linn. using phylogenetic analysis based on a 16S rRNA molecular approach. We used the disc diffusion method to evaluate the impact of differences in the compositions of the media on the production of secondary metabolites from strain MH16. The production of antimicrobial metabolites was determined by the observation of inhibition zones on intensive bands when using a TLC-bioautography assay. Biosynthesis of secondary metabolites was optimal when the strain MH16 was cultured in ISP-2 medium as depicted by a zone of inhibition. Strain MH16 effectively inhibited methicillin-resistant Staphylococcus aureus, Escherichia coli, Candida albicans, and other multi drug-resistant pathogens. The minimum inhibitory concentration of the antimicrobial metabolites was 25-100 µg mL-1. The study manifests the optimization and utilization of different fermentation media which best suits for increased production of the secondary metabolites from Streptomyces cavourensis. This research suggests that the antimicrobial metabolites of strain MH16 found in M. hortensis has great potential for the biodiscovery of new anti-infective drugs against a wide range of multidrug-resistant pathogens.

Unraveling Multicopper [Cu3] and [Cu6] Clusters with Rare µ3-Sulfato and Linear µ2-Oxido-Bridges as Potent Antibiofilm Agents against Multidrug-Resistant Staphylococcus aureus.

In this research article, two multicopper [Cu3] and [Cu6] clusters, [Cu3(cpdp)(µ3-SO4)(Cl)(H2O)2]·3H2O (1) and [Cu6(cpdp)2(µ2-O)(Cl)2(H2O)4]·2Cl (2) (H3cpdp = N,N'-bis[2-carboxybenzomethyl]-N,N'-bis[2-pyridylmethyl]-1,3-diaminopropan-2-ol), have been explored as potent antibacterial and antibiofilm agents. Their molecular structures have been determined by a single-crystal X-ray diffraction study, and the compositions have been established by thermal and elemental analyses, including electrospray ionization mass spectrometry. Structural analysis shows that the metallic core of 1 is composed of a trinuclear [Cu3] assembly encapsulating a µ3-SO42- group, whereas the structure of 2 represents a hexanuclear [Cu6] assembly in which two trinuclear [Cu3] motifs are exclusively bridged by a linear µ2-O2- group. The most striking feature of the structure of 2 is the occurrence of an unusual linear oxido-bridge, with the Cu3-O6-Cu3' bridging angle being 180.00°. Whereas 1 can be viewed as an example of a copper(II)-based compound displaying a rare µ3:η1:η1:η1 bridging mode of the SO42- group, 2 is the first example of any copper(II)-based compound showing an unsupported linear Cu-O-Cu oxido-bridge. Employing variable-temperature SQUID magnetometry, the magnetic susceptibility data were measured and analyzed exemplarily for 1 in the temperature range of 2-300 K, revealing the occurrence of antiferromagnetic interactions among the paramagnetic copper centers. Both 1 and 2 exhibited potent antibacterial and antibiofilm activities against methicillin-resistant Staphylococcus aureus (MRSA BAA1717) and the clinically isolated culture of methicillin-resistant S. aureus (MRSA CI1). The mechanism of antibacterial and antibiofilm activities of these multicopper clusters was investigated by analyzing and determining the intracellular reactive oxygen species (ROS) generation, lipid peroxidation, microscopic observation of cell membrane disruption, membrane potential, and leakage of cellular components. Additionally, 1 and 2 showed a synergistic effect with commercially available antibiotics such as vancomycin with enhanced antibacterial activity. However, 1 possesses higher antibacterial, antibiofilm, and antivirulence actions, making it a potent therapeutic agent against both MRSA BAA1717 and MRSA CI1 strains.

Molecular profiling and anti-infective potency of endophytic actinomycetes inhabiting Madhuca insignis Radlk., from Western Ghats of India.

BACKGROUND: Endophytic actinomycetes are well known for their diverse bioactive entities and considered as an important source for drug development research. RESULTS: We isolated and identified four potential endophytic Streptomyces species, i.e., Streptomyces misionensis MI22, Streptomyces roietensis MI24, Streptomyces glaucescens MI29, and Streptomyces sp. MI04 inhabiting Madhuca insignis by its characteristic morphological features and 16S rRNA gene sequence analysis. S. misionensis MI22 exhibits a broad spectrum of anti-microbial activity against methicillin-resistant Staphylococcus aureus (25.00 ± 1.00 mm) followed by Bacillus subtilis (23.66 ± 0.57 mm), Escherichia coli (22.00 ± 0.00 mm), and Candida albicans (18.00 ± 0.00 mm). Minimum inhibitory concentrations of the ethyl acetate fraction of S. misionensis MI22 against test pathogens were ranged from 25 to 100 µg/mL. Indeed, strain MI22 also exhibited significant anti-proliferative activity against HeLa cell line with IC50 value 98 µg/mL and showed no cytotoxicity effect to the normal human embryonic kidney cell line in the MTT assay. The anti-microbial metabolites from strain MI22 were detected at Rf 0.55 as depicted by the inhibition zone on the intensive band in TLC-bioautography assay. CONCLUSION: The study indicates that, anti-microbial metabolites of these endophytic Streptomyces species, especially S. misionensis MI22 as a prolific source to discover novel bioactive metabolites to combat multidrug-resistant pathogens.

Direct observation of effect of crowding induced macromolecular hydration on molecular breathing in the stem of Fork-DNA by single-molecule FRET microspectroscopy.

The perpetually changing cellular conditions, nucleotide sequence, and environmental effects including osmotic stress have multiple effects on DNA, leading to several conformational alternations and subsequently influencing their activity, too. In this work, single-molecule FRET microspectroscopy has been employed to monitor the breathing dynamics as an effect of molecular crowding in the stem region of Fork-DNA. The structural integrity greatly alters with the presence or absence of nucleotide overhangs and on the nature and concentration of the crowding agent, thus affecting the stability of the stem region and hence the forked DNA. The multiple hydrogen bonds and hydrophobic interactions between the polynucleotide strands appear to be altered with osmotic crowding. This induces increased flexibility in the double helix and allows DNA to breath. The conformational alternation of the DNA happens in nanometer resolution, that is been monitored by the change in the FRET efficiency between the dyes attached to two different strands of the DNA. The nature and molecular weight of crowding agents control the degree of spatial breathing in the stem of Fork-DNA. These constant fluctuations between the entropically favorable partially folded structures to an enthalpically favorable folded structure are not only valuable for elucidating nucleic acid structure but might play an important role in enzyme kinetics.

In Silico Screening and Binding Characterization of Small Molecules toward a G-Quadruplex Structure Formed in the Promoter Region of c-MYC Oncogene.

Overexpression of c-MYC oncogene is associated with cancer pathology. Expression of c-MYC is regulated by the G-quadruplex structure formed in the G-rich segment of nuclease hypersensitive element (NHE III1), that is, "Pu27", which is localized in the promoter region. Ligand-induced stabilization of the Pu27 structure has been identified as a novel target for cancer therapeutics. Here, we have explored the library of synthetic compounds against the predefined binding site of Pu27. Three compounds were selected based on the docking analyses; they were further scrutinized using all atom molecular dynamics simulations in an explicit water model. Simulated trajectories were scrutinized for conformational stability and ligand binding free energy estimation; essential dynamic behavior was determined using principal component analysis. One of the molecules, "TPP (1-(3-(4-(1,2,3-thiadiazol-4-yl)phenoxy)-2-hydroxypropyl)-4-carbamoylpiperidinium)", with the best results was considered for further evaluation. The theoretical observations are supported well by biophysical analysis using circular dichroism, isothermal titration calorimetry, and high-resolution NMR spectroscopy indicating association of TPP with Pu27. The in vitro studies were then translated into c-MYC overexpression in the T47D breast cancer cell line. Biological evaluation through the MTT assay, flow cytometric assay, RT-PCR, and reporter luciferase assay suggests that TPP downregulates the expression of c-MYC oncogene by arresting its promoter region. In silico and in vitro observations cumulatively suggest that the novel skeleton of TPP could be a potential anticancer agent by stabilizing the G-quadruplex formed in the Pu27 and consequently downregulating the expression of c-MYC oncogene. Derivation of new molecules on its skeleton may confer anticancer therapeutics for the next generation.

Chelerythrine down regulates expression of VEGFA, BCL2 and KRAS by arresting G-Quadruplex structures at their promoter regions.

A putative anticancer plant alkaloid, Chelerythrine binds to G-quadruplexes at promoters of VEGFA, BCL2 and KRAS genes and down regulates their expression. The association of Chelerythrine to G-quadruplex at the promoters of these oncogenes were monitored using UV absorption spectroscopy, fluorescence anisotropy, circular dichroism spectroscopy, CD melting, isothermal titration calorimetry, molecular dynamics simulation and quantitative RT-PCR technique. The pronounced hypochromism accompanied by red shifts in UV absorption spectroscopy in conjunction with ethidium bromide displacement assay indicates end stacking mode of interaction of Chelerythrine with the corresponding G-quadruplex structures. An increase in fluorescence anisotropy and CD melting temperature of Chelerythrine-quadruplex complex revealed the formation of stable Chelerythrine-quadruplex complex. Isothermal titration calorimetry data confirmed that Chelerythrine-quadruplex complex formation is thermodynamically favourable. Results of quantative RT-PCR experiment in combination with luciferase assay showed that Chelerythrine treatment to MCF7 breast cancer cells effectively down regulated transcript level of all three genes, suggesting that Chelerythrine efficiently binds to in cellulo quadruplex motifs. MD simulation provides the molecular picture showing interaction between Chelerythrine and G-quadruplex. Binding of Chelerythrine with BCL2, VEGFA and KRAS genes involved in evasion, angiogenesis and self sufficiency of cancer cells provides a new insight for the development of future therapeutics against cancer.

LINCRNA00273 promotes cancer metastasis and its G-Quadruplex promoter can serve as a novel target to inhibit cancer invasiveness.

Discovery of anti-metastatic drugs is of immense clinical significance as metastasis is responsible for 90% of all cancer deaths. Here we report the inhibitory effect of a bis schiff base (M2) on cancer cell migration and invasion in vitro and in vivo. M2 has shown good solubility and permeability across the intestinal cell wall and hence can be classified as BCS (Biopharmaceutical classification system) class I. Microarray studies identified a long non coding intergenic RNA, LINC00273 as a novel molecular target of M2. We report that LINC00273 harbors a unique (4n-1) parallel G-Quadruplex structure in its promoter as validated by DMS footprint. M2 is proposed to stabilize this G-quadruplex structure resulting in the down-regulation of LINC00273 expression. Dual Luciferase reporter assay also suggests inhibition of LINC00273 promoter activity by M2. Involvement of this linc in metastasis is proven by siRNA and shRNA mediated knock down of LINC00273 in vitro and in vivo in nude mice which significantly decelerates cancer cell migration and invasion and also makes the cells unresponsive to TGF-ß's pro-metastatic effects. Furthermore, the real time expression of LINC00273 in thirty seven human clinical samples is found to be positively correlated with the histopathological staging of metastasis.

Myricetin arrests human telomeric G-quadruplex structure: a new mechanistic approach as an anticancer agent.

The use of small molecules to arrest G-quadruplex structure has become a potential strategy for the development and design of a new class of anticancer therapeutics. We have studied the interaction of myricetin, a plant flavonoid and a putative anticancer agent, with human telomeric G-quadruplex TTAGGG(TTAGGG)3 DNA. Reverse transcription PCR data revealed significant repression in hTERT expression in MCF-7 breast cancer cells upon increasing the concentration of myricetin. Further, we conducted a telomeric repeat amplification protocol assay to confirm the inhibition of telomerase by myricetin. Optical spectroscopic techniques like circular dichroism, UV spectroscopy and fluorescence spectroscopy revealed the formation of a stable myricetin-G-quadruplex complex. The thermodynamic parameters of myricetin-G-quadruplex complex formation, presented through isothermal titration calorimetry studies, indicate the binding process to be thermodynamically favorable. In addition, high resolution NMR spectroscopy in conjunction with molecular dynamics simulation is employed to provide detailed mechanistic insights into the binding in the myricetin-G-quadruplex complex at the atomic level. Our results thus propose a new mode of action of myricetin as an anticancer agent via arresting telomeric G-quadruplex structure.

Reverse Watson-Crick G-G base pair in G-quadruplex formation.

A stable intermediate dimeric G-rich form as a precursor of tetrameric G-quadruplex structures has been detected via MALDI-TOF spectrometry. Molecular dynamics simulation offered detailed insights at the atomic level, assigning reverse Watson-Crick G-G base pairing (not Hoogsteen) in the G-rich dimer. In support of this, cisplatin formed a stable adduct by binding to the dimeric G-rich structure, eliminating the possibility of G-G Hoogsteen hydrogen bond formation.

Expression of fascin, an actin-bundling protein, in astrocytomas of varying grades.

Malignant astrocytomas are highly infiltrative neoplasms that invade readily into regions of normal brain. On a cellular basis, the motility and invasiveness of human cancers can be ascribed in part to complex rearrangements of the actin cytoskeleton that are governed by several actinbinding proteins. One such actin-binding protein that has been linked to the invasive behavior of carcinomas is fascin, which serves to aggregate F actin into bundles. In this study, we examined the expression of fascin in a series of human malignant astrocytomas (WHO grades I-IV). Five grade I, 5 grade II, 10 grade III, and 26 grade IV human astrocytomas were examined for fascin and glial fibrillary acidic protein (GFAP) expression by double immunofluorescence confocal microscopy. Expression of fascin and GFAP was also determined by Western blot analysis. Fascin expression increased with increasing WHO grade of astrocytoma. This is in marked contrast to GFAP expression, which decreased with increasing WHO grade. In grades I and II neoplasms, and within non-neoplastic brain, fascin and GFAP were expressed diffusely within regions examined. However, in the higher-grade astrocytomas (grades III and IV), fascin and GFAP were expressed regionally in distinctly separate tumor cell populations. This is the first study to demonstrate the expression of fascin in human astrocytic neoplasms. The role that fascin plays in contributing to the invasive phenotype of anaplastic astrocytomas awaits further study and investigation.

Tigecycline activity against metallo-ß-lactamase-producing bacteria.

BACKGROUND: [corrected] Treatment of serious life-threatening multi-drug-resistant organisms poses a serious problem due to the limited therapeutic options. Tigecycline has been recently marketed as a broad-spectrum antibiotic with activity against both gram-positive and gram-negative bacteria. Even though many studies have demonstrated the activity of tigecycline against ESBL-producing Enterobacteriaceae, its activity is not well-defined against micro-organisms producing metallo-ß-lactamases (MBLs), as there are only a few reports and the number of isolates tested is limited. AIMS: The aim of the present study was to evaluate the activity of tigecycline against MBL-producing bacterial isolates. MATERIALS AND METHODS: The isolates were tested for MBL production by (i) combined-disk test, (ii) double disc synergy test (DDST), (iii) susceptibility to aztreonam (30 µg) disk. Minimum inhibitory concentration to tigecycline was determined according to agar dilution method as per Clinical Laboratory Standards Institute (CLSI) guidelines. Disc diffusion susceptibility testing was also performed for all these isolates using tigecycline (15 µg) discs. RESULTS: Among the total 308 isolates included in the study, 99 were found to be MBL producers. MBL production was observed mostly in isolates from pus samples (40.47%) followed by urine (27.4%) and blood (13.09%). MBL production was observed in E. coli (41.48%), K. pneumoniae (26.67%), Proteus mirabilis (27.78%), Citrobacter spp. (41.67%), Enterobacter spp. (25.08%), and Acinetobacter spp. (27.27%). The result showed that tigecycline activity was unaffected by MBL production and it was showed almost 100% activity against all MBL-producing isolates, with most of the isolates exhibiting an MIC ranging from 0.25-8 µg/ml, except 2 MBL-producing E. coli isolates who had an MIC of 8 µg/ml. CONCLUSION: To conclude, tigecycline was found to be highly effective against MBL-producing Enterobacteriaceae and acinetobacter isolates, but the presence of resistance among organisms, even before the mass usage of the drug, warrants the need of its usage as a reserve drug. The study also found that the interpretative criteria for the disc diffusion method, recommended by the FDA, correlates well with the MIC detection methods. So, the microbiology laboratories might use the relatively easier method of disc diffusion, as compared to the comparatively tedious method of MIC determination.

Immunolocalization of fascin, an actin-bundling protein and glial fibrillary acidic protein in human astrocytoma cells.

Fascin is a 55-kDa globular protein that functions to organize filamentous-actin into parallel bundles. A role for fascin in cell migration has led to its study in many tumor types. In this report, we investigate fascin in astrocytomas. We show that fascin is expressed in astrocytes and in a panel of human astrocytoma cell lines. Immunofluorescence analysis demonstrates that fascin and the intermediate filament protein, glial fibrillary acidic protein (GFAP), are both expressed in the perinuclear region and within cytoplasmic processes of astrocytes and astrocytoma cells. Amino acid residues within the NH2 terminus of GFAP can undergo phosphorylation; these modifications regulate intermediate filament disassembly and occur during cytokinesis. We show that fascin and specific phosphorylated species of GFAP colocalize within dividing cells. Finally, we demonstrate that fascin co-immunoprecipitates with GFAP and that immunocomplex formation is preferential for GFAP phosphorylated at serine residues 8 and 13. These data show that fascin and GFAP are immunolocalized regionally within cells and tumors of astrocytic origin and suggest that their binding may occur during dynamic reorganization of intermediate filaments.

p14ARF interacts with DAXX: effects on HDM2 and p53.

The p14(ARF) (ARF) tumour suppressor plays an important role in the cellular response to oncogene activation. In this report, we demonstrate an interaction between ARF and DAXX, a highly conserved protein with identified roles in the regulation of gene expression. HDM2 was shown to interact with each of ARF and DAXX upon upregulation of expression as well as at lower expression levels following transfection of ARF and DAXX. Through immunofluorescence analysis, we observed that endogenous ARF and DAXX colocalize both to nucleoli and to nuclear bodies in cell lines that co-express both proteins. Similar results were obtained upon co-transfection of ARF and DAXX. Co-expression of ARF and DAXX was further found to inhibit ARF-mediated HDM2 sumoylation and to induce sumoylation and ubiquitination of DAXX itself, implicating DAXX as a substrate of ARF-mediated post-translational events. We also observed induction of p53 sumoylation in the presence of ARF and DAXX, an effect that was inhibited by upregulation of HDM2 expression. In summary, we have identified DAXX as a novel ARF binding partner and substrate of ARF-mediated sumoylation and suggest that DAXX acts as a modifier of both p53-dependent and p53-independent ARF function.

Sloppy paired 1/2 regulate glial cell fates by inhibiting Gcm function.

Organization of the central nervous system during embryonic development is an intricate process involving a host of molecular players. The Drosophila segmentation genes, sloppy paired (slp) 1/2 have been shown to be necessary for development of a neuronal precursor cell subtype, the NB4-2 cells. Here, we show that slp1/2 also have roles in regulating glial cell fates. Using slp1/2 loss-of-function mutants, we show an increase in glial cell markers, glial cells missing (gcm) and reversed polarity. In contrast, misexpression of either slp1 or slp2 causes downregulation of glial cell-specific genes and alters the fate of glial and neuronal cells. Furthermore, we demonstrate that Slp1 and its mammalian ortholog, Foxg1, inhibit Gcm transcriptional activity as well as bind Gcm. Taken together, these data show that Slp1/Foxg1 regulate glial cell fates by inhibiting Gcm function.

In vitro effect of sex steroids on cytotoxic activity of splenic macrophages in wall lizard (Hemidactylus flaviviridis).

Sexual dimorphism was observed in nitrite release and IL-1-like molecule production by splenic macrophages of the wall lizard (Hemidactylus flaviviridis), with a higher level in females than in males. Gonadectomy in both males and females resulted in a considerable increase of nitrite and IL-1-like molecule secretion, suggesting that the sex hormones inhibit cytotoxic activity of macrophages. To verify this assumption, dose- and time-related in vitro experiments with male and female sex steroids, dihydrotestosterone (DHT) and 17beta-estradiol (E(2)), respectively, were carried out. E(2) and DHT both significantly reduced the nitrite release and IL-1-like molecule production with an increase of dose or duration of treatment.

Dose and time-related in vitro effects of glucocorticoid on phagocytosis and nitrite release by splenic macrophages of wall lizard Hemidactylus flaviviridis.

Glucocorticoids (GC) are usually considered to be immunosuppressive and anti-inflammatory. However, recent studies in mammals have demonstrated the diverse effects of GC on non-specific host-defense mechanism, depending on dose or duration of treatment. Hence, in the present study in vitro dose and time-related effects of glucocorticoid, i.e. hydrocortisone (HC) on phagocytosis and nitrite production by LPS-induced splenic macrophages in wall lizard Hemidactylus flaviviridis has been investigated. Hydrocortisone suppressed percentage phagocytosis, phagocytic index and nitrite production by splenic macrophages even at the lowest concentration (10(-13) M) for a short-term exposure (4 h). Hydrocortisone-induced suppression enhanced with the increase of concentration or duration of exposure time. The suppressive effect of hydrocortisone on phagocytic and cytotoxic activities of splenic macrophages was further corroborated since the pre-exposure of macrophages to glucocorticoid-receptor blocker (RU 486) considerably reduced the hydrocortisone-induced suppression of phagocytosis and nitrite production. The present study suggests that GC instead of diverse effects, has dose- and time-dependent immunosuppressive effect on non-specific host-defense immune responses in wall lizard H. flaviviridis.