In silico exploration of antioxidants as oxidation protectant for PITRM1 peptidase activity, an Alzheimer disease target.

To find efficient antioxidants to protect oxidation prone cysteine residues of the peptidase PITRM1 using molecular docking and simulation techniques. A total of 50 antioxidants were docked with PITRM1 at the oxidation prone region Cys89 and Cys96 using Autodock Vina software. The lowest socred compounds were predicted for its Blood brain barrier permeability using LightBBB. Molecular dynamic simulations of the PITRM1 and Ascorbic acid/Silymarin complex were performed using the GROMACS 2020.1 package and the free energy calculations were performed using gmx_MMPBSA. The RMSD, RMSF, Rg, Minimum distance and Hydrogen bonds were also evaluated. Silymarin, Ascorbic acid, Naringenin, Gallic acid, Chlorogenic acid, Rosmarinic acid, (-)-Epicatechin, Genistein showed a docking score of above -5.3 kcal/mol. Silymarin and Ascorbic acid were predicted to cross the Blood Brain Barrier. Molecular dynamic simulation and mmPBSA analysis revealed that Silymarin showed a positive free energy implying no affinity to PITRM1 and ascorbic acid has low ΔG with -13.13 kJ/mol. The stability of the ascorbic acid complex was high (RMSD: 0.160 ± 0.018 nm, Minimum Distance: 0.163 ± 0.001 nm and four hydrogen bonds) and fluctuation induced due to ascorbic acid was low. Ascorbic acid was found to effectively interact with the cysteine oxidation prone region and can have a potential role in reducing the oxidised cysteine in PITRM1 to modulate its peptidase activity.

Neurospecific fabrication and toxicity assessment of a PNIPAM nanogel encapsulated with trans-tephrostachin for blood-brain-barrier permeability in zebrafish model.

Biocompatible Poly(N-isopropylacrylamide) (PNIPAM) nanogels (NGs) were developed at 40-65 nm to deliver Trans-Tephrostachin (TT) in zebrafish brain. Neurospecific PNIPAM NGs are functionalized with polysorbate 80 (PS80) to overcome the Blood Brain Barrier (BBB). The TT loaded with NG (NG + TT) was confirmed in UV-spectroscopy and transmission electron microscopy (TEM) with 90% efficiency of controlled release at 37 °C. The neurospecificity of NG was confirmed in 144 hours post fertilization (hpf) larvae with PS80 surface-treated rhodamine-B (Rh-B) conjugated NG and visualized in the zebrafish CNS. Oral gavaging of TT loaded NG with PS80 surface treatment (NG + TT + PS80) was confirmed to cross the BBB in adult zebrafish at 37 °C. TT release was detected by RP-HPLC. LC50 was determined as 250 µg/ml for NG, 172 µg/ml for NG + TT, and 0.9 µg/ml for TT at 96 hpf and confirmed the lesser toxicity in TT bound NG. Delays in growth and malformations were observed at concentrations above the 96 hpf-LC50. The behavior outcomes were varied with phase - and concentration-dependent hypo- or hyperactivity. The altered expression of genes associated with Alzheimer's disease (AD) was found at 96 hpf of its LC50 concentration. The expression of appa was significantly increased for TT and supporting the TT to bind NG without altering the AD genes. Thus the study suggests the biocompatible potential of PNIPAM and its neurospecific delivery to the brain.

Cancer-Targeted Chitosan-Biotin-Conjugated Mesoporous Silica Nanoparticles as Carriers of Zinc Complexes to Achieve Enhanced Chemotherapy In Vitro and In Vivo.

Despite being the most common component of numerous metalloenzymes in the human body, zinc complexes are still under-rated as chemotherapeutic agents. Herein, the present study opens up a key route toward enhanced chemotherapy with the help of two ZnII complexes (ZnMBC) synthesized alongside Mannich base ligands to upsurge biological potency. Further, well-established mesoporous silica nanoparticles (MSNs) have been chosen as carriers of the titled metallodrugs in order to achieve anticancer drug delivery. A pH-sensitive additive, namely, chitosan (CTS) conjugated with biotin is tagged to MSNs for the targeted release of core agents inside tumors selectively. In general, CTS blocks ZnMBC inside the mesopores of MSNs, and biotin acts as a targeting ligand to improve tumor-specific cellular uptake. CTS-biotin surface decoration significantly enhanced the cellular uptake of ZnMBC through endocytosis. A panel of four human cancer cell lines has revealed that ZnMBC (1/2)@MSNs-CTS-biotin nanoparticles (NPs) exhibits unprecedented enhanced cytotoxicity toward cancer cells with IC50 values ranging from 6.5 to 28.8 µM through induction of apoptosis. NPs also possess great selectivity between normal and cancer cells despite this potency. Two-photon-excited in vitro imaging of normal (HEK) and cancer (HeLa) cells has been performed to confirm the biased drug delivery. Also, NP-induced apoptosis was found to be dependent on targeting DNA and ROS generation. Moreover, a lower range of LD50 values (153.6-335.5 µM) were observed upon treatment zebrafish embryos with NPs in vivo. Because of the anatomical similarity to the human heart, the heart rate of NP-treated zebrafish has been analyzed in assessing the cardiac functions, which is in favor of the early clinical trials of ZnMBC (1/2)@MSNs-CTS-biotin candidates for their further evaluation as a chemotherapeutic and chemopreventive agent toward human cancers, especially adenocarcinoma.

Zebrafish: A Promising Real-Time Model System for Nanotechnology-Mediated Neurospecific Drug Delivery.

Delivering drugs to the brain has always remained a challenge for the research community and physicians. The blood-brain barrier (BBB) acts as a major hurdle for delivering drugs to specific parts of the brain and the central nervous system. It is physiologically comprised of complex network of capillaries to protect the brain from any invasive agents or foreign particles. Therefore, there is an absolute need for understanding of the BBB for successful therapeutic interventions. Recent research indicates the strong emergence of zebrafish as a model for assessing the permeability of the BBB, which is highly conserved in its structure and function between the zebrafish and mammals. The zebrafish model system offers a plethora of advantages including easy maintenance, high fecundity and transparency of embryos and larvae. Therefore, it has the potential to be developed as a model for analysing and elucidating the permeability of BBB to novel permeation technologies with neurospecificity. Nanotechnology has now become a focus area within the industrial and research community for delivering drugs to the brain. Nanoparticles are being developed with increased efficiency and accuracy for overcoming the BBB and delivering neurospecific drugs to the brain. The zebrafish stands as an excellent model system to assess nanoparticle biocompatibility and toxicity. Hence, the zebrafish model is indispensable for the discovery or development of novel technologies for neurospecific drug delivery and potential therapies for brain diseases.

Bisphenol-A alters hematopoiesis through EGFR/ERK signaling to induce myeloblastic condition in zebrafish model.

Experimental evidence from the etiology of cancer studies suggests that a correlation between Bisphenol-A (BPA) exposure and alterations in hematopoiesis leads to blood cancer. In our study zebrafish were used to assess the lethality, developmental effect, embryonic apoptosis and changes in transcription factor of hematopoiesis through EGFR/ERK signaling pathways in response to BPA. The in silico interaction of EGFR and BPA was analysed by molecular dynamic simulation. According to our results, BPA induced a significant lethal effect in hatching retardation, reduction in heart rate and teratogenic effects on zebrafish embryos and larvae at three different concentrations 100, 500 and 2500 µg/L. The mortality of adult zebrafish exposed to the acute toxicity of BPA from 5 to 30 mg/L concentrations was determined for 96 h. The peripheral blood cells and vital organs such as kidney, liver and spleen from BPA exposed fish showed predominantly abnormal myeloid blast cells along with severe morphological changes in erythrocytes at sublethal concentration 245 µg/L. The BPA showed the highest binding affinity to zebrafish EGFR with a docking score of -7.5 kcal/mol with an RMSD of 3.0 nm during MD simulation. We found that EGFR/ERK overexpression leads to induce hematopoietic cell proliferation and impaired differentiation, which enhances the myeloid repopulating activity and the accumulation of immature myeloblast cells. BPA also caused a corresponding increase in expression of hematopoietic transcription factor c-MYB and RUNX-1 leading to polychromasia, poikilocytosis, acanthocytes and anisocytosis and promoted myeloblastosis by inhibiting GATA-1 expression. These morphological changes often resulted in the prior condition of acute myeloid leukemia (AML). Comprehensively, our data suggest that BPA can trigger the malignancy of AML cells by alteration of respective hematopoietic transcription factors via EGFR/ERK signaling in the zebrafish model.

ß-lactamase inhibitory potential of kalafungin from marine Streptomyces in Staphylococcus aureus infected zebrafish.

ß-lactamase inhibitors are potent synergistic drugs to deteriorate the multidrug-resistant bacteria. Here, we report the ß-lactamase inhibitory ability of kalafungin isolated from a marine sponge derived Streptomyces sp. SBRK1. The IC50 value of the kalafungin was calculated as 225.37 ± 1.95 µM against ß-lactamase. The enzyme kinetic analysis showed the Km value of 3.448 ± 0.7 µM and Vmax value of 215.356 ± 8 µM/min and the inhibition mechanism was identified as uncompetitive type. Along with the antibacterial activity, the cell surface analysis of kalafungin treated Staphylococcus aureus cells revealed destruction of cell membrane in response to ß-lactamase inhibition. Molecular docking studies have confirmed the binding property of kalafungin against ß-lactamase with two hydrogen bonds. In vivo efficacy studies in the zebrafish model by green fluorescent protein expressing S. aureus infection, survival, safety and behavioral profile were reported. The toxicity and anti-infection revealed that the compound was evidently active and safe to all organs. In conclusion, this is the first report on kalafungin with ß- lactamase inhibition and suggests that kalafungin may useful for synergic antibacterial therapy with ß-lactam drugs to overcome ß-lactamase-based resistance of any bacterial pathogens.

Cell wall distraction and biofilm inhibition of marine Streptomyces derived angucycline in methicillin resistant Staphylococcus aureus.

The emergence of life threatening antibiotic resistant pathogens and its associated mortality and morbidity necessitates many new antibiotics from diverse ecological habitats. Marine sponge associated microbes are promising to provide such antimicrobial compounds. In the present study, we report antibacterial and anti-biofilm potential of the angucycline antibiotic 8-O-metyltetrangomycin from Streptomyces sp. SBRK2 isolated from a marine sponge of Gulf of Mannar, Rameswaram, India. Our screening program to tackle methicillin-resistant Staphylococcus aureus (MRSA) drug resistance from marine sponge associated actinobacteria yielded the bioactive strain SBRK2. Based on 16S rRNA gene phylogenetic analysis the isolate was found to closely related with Streptomyces longispororuber NBRC 13488T. In vitro production by agar plate fermentation, solvent based extraction, TLC, HPLC purification and LC-MS based de-replication revealed the bioactive compound as 8-O-metyltetrangomycin. The antibacterial minimum inhibitory concentrations against MRSA was identified as 2 µg/mL. Sub-inhibitory concentration of the compound 8-O-metyltetrangomycin reduced the biofilm formation of S. aureus ATCC25923 and increased the cell surface hydrophobicity index. Scanning electron microscopic observation of the sub-inhibitory concentration exposure revealed a wrinkled membrane surface and slight cellular damage shows the cell wall distracting property of the compound. Zebrafish embryo based toxicity assays exhibited 100 µg/mL of compound as maximal non-lethal concentration which had demonstrated the positive relationship in safety index. The angucycline compound 8-O-metyltetrangomycin could be a potential candidate for the development of anti-biofilm agents against drug resistant pathogens.

Naringenin alleviates 6-hydroxydopamine induced Parkinsonism in SHSY5Y cells and zebrafish model.

6-Hydroxydopamine (6-OHDA) is a neurotoxin that inhibits the mitochondrial complex I causing mitochondrial impairment, aetiology of Parkinson's. Naringenin is a flavanone predominantly present in citrus fruits. Due to its high antioxidant and anti-inflammatory potential, it has been widely studied against various disorders. In this study, the neuroprotective effect of naringenin was determined against 6-OHDA induced toxicity with Levodopa (l-DOPA) as the standard. Naringenin reduced 6-OHDA induced oxidative stress biomarker levels such as CAT, GSH, SOD, and ROS. Naringenin rescued 6-OHDA induced reduction of the mitochondrial membrane potential. Treatment with naringenin improved the locomotion of the 6-OHDA treated zebrafish larvae which showed stagnant swimming patterns. Naringenin was also found to downregulate the expression of some Parkinsonian genes such as casp9, lrrk2, and polg and upregulate pink1. These studies attribute to naringenin as a viable molecule to study further for its neuroprotective effects against 6-OHDA induced neurotoxicity and neurodegeneration.

Streptomyces marianii sp. nov., a novel marine actinomycete from southern coast of India.

A novel marine actinomycete strain designated ICN19T was isolated from the subtidal sediment of Chinnamuttam coast of Kanyakumari, India and subjected to polyphasic taxonomic analysis. Neighbour-joining tree based on 16S rRNA gene sequences of validly described type strains had revealed the strain ICN19T formed distinct cluster with Streptomyces wuyuanensis CGMCC 4.7042T, Streptomyces tirandamycinicus HNM0039T and Streptomyces spongiicola HNM0071T. Morphological, physiological and chemotaxonomic characteristics were consistent with those of members of the genus Streptomyces. The strain possessed LL-diaminopimelic acid as the diagnostic diamino acid. The predominant isoprenoid quinone was identified as MK-9(H8) (70%), MK-9(H6) (20%) and MK-9(H2) (2%), with the major cellular fatty acids (>10%) being anteiso-C15:0, C16:0 and iso-C16:0. The main polar lipids were found to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannosides and three unidentified phospholipids. The dendrogram generated on the basis of MALDI-TOF mass spectra supports the strain differentiated from its neigbours. The genome sequence of strain ICN19T was 9,010,366 bp in size with a total of 7420 protein-coding genes and 98 RNA genes. The genomic G+C content of the novel strain was 71.27 mol%. The DNA-DNA relatedness between strain ICN19T and the reference strains with S. wuyuanensis CGMCC 4.7042T, S. tirandamycinicus HNM0039T and S. spongiicola HNM0071T were 42.8%, 39.5% and 38%, respectively. Based on differences in physiological, biochemical, chemotaxonomic differences and whole-genome characteristics the isolated strain represents a novel species of the genus Streptomyces, for which the name Streptomyces marianii sp. nov. is proposed. Type strain is ICN19T (=MCC 3599T = KCTC 39749T).

Hesperidin downregulates kinases lrrk2 and gsk3ß in a 6-OHDA induced Parkinson's disease model.

The depletion of dopamine in the striatum region and Lewy bodies are the hallmark characteristics of Parkinson's disease. The pathology also includes the upregulation of various Parkinson's disease (PARK) genes and kinases. Two such kinases, LRRK2 and GSK-3ß have been directly implicated in the formation of tau and alpha-synuclein proteins, causing PD. Hesperidin (HES) is a flavanone glycoside that has multiple therapeutic benefits including neuroprotective effects. In this study, we examined the neuroprotective effects of HES against 6-hydroxydopamine (6-OHDA) induced-neurotoxicity in the in-vitro and in-vivo model. Hesperidin significantly protected the SH-SY5Y cells' stress against 6-OHDA induced toxicity by downregulating biomarkers of oxidative stress. Furthermore, HES downregulated the kinases lrrk2 and gsk3ß along with casp3, casp9, and polg in the zebrafish model. The treatment with HES also improved the locomotor pattern of zebrafish that was affected by 6-OHDA. This study suggests that hesperidin could be a drug of choice in targeting kinases against a 6-OHDA model of PD.

Antiangiogenic molecules from marine actinomycetes and the importance of using zebrafish model in cancer research.

Blood vessel sprouting from pre-existing vessels or angiogenesis plays a significant role in tumour progression. Development of novel biomolecules from marine natural sources has a promising role in drug discovery specifically in the area of antiangiogenic chemotherapeutics. Symbiotic actinomycetes from marine origin proved to be potent and valuable sources of antiangiogenic compounds. Zebrafish represent a well-established model for small molecular screening and employed to study tumour angiogenesis over the last decade. Use of zebrafish has increased in the laboratory due to its various advantages like rapid embryo development, optically transparent embryos, large clutch size of embryos and most importantly high genetic conservation comparable to humans. Zebrafish also shares similar physiopathology of tumour angiogenesis with humans and with these advantages, zebrafish has become a popular model in the past decade to study on angiogenesis related disorders like diabetic retinopathy and cancer. This review focuses on the importance of antiangiogenic compounds from marine actinomycetes and utility of zebrafish in cancer angiogenesis research.

Exogenous human beta amyloid peptide interferes osteogenesis through Sox9a in embryonic zebrafish.

The two major hallmarks of Alzheimer's disease (AD) are beta-amyloid plaques and neurofibrillary tangles. Amyloid peptide aggregations in the brain cause loss of synaptic connections and subsequent neurotoxicity leading to neurodegeneration and memory deficits. However, the physiological effects of beta-amyloid on early embryonic development still remain unclear. Administration of human beta-amyloid peptide (1-42) through cerebrospinal ventricular injection was carried out at 24 hpf (hours post fertilization) and it was uptaken into the cellular layers of the early ventricular development without any plaque aggregation. Whole-mount Immunostaining of zebrafish embryos injected with the beta-amyloid at 60 hpf revealed the delay in Sox9a expression. Decreased level of cartilage to bone transformation rate in 15 dpf (days post fertilization) zebrafish was observed by differential staining. These results suggest the possible existence of a genetic relationship between extrinsic amyloid peptide and Sox9a expression. Thus, our results demonstrated that the human beta-amyloid influences bone development through Sox9a expression during osteogenesis in zebrafish.

Ala-geninthiocin, a new broad spectrum thiopeptide antibiotic, produced by a marine Streptomyces sp. ICN19.

Bioassay-guided screening of antibacterial compounds from the cultured marine Streptomyces sp. ICN19 provided Ala-geninthiocin (1), along with its known analogs geninthiocin (2) and Val-geninthiocin (3) and the indolocarbazole staurosporine (4). The structure of 1 was determined on the basis of 1D and 2D NMR spectra and ESI-HRMS. The absolute configurations of the amino acid residues were determined by enantioselective GC-MS analysis. Compound 1 exhibited potent activity against Gram-positive bacteria including Staphylococcus aureus, Bacillus subtilis, Mycobacterium smegmatis, and Micrococcus luteus, as well as cytotoxicity against A549 human lung carcinoma cells with an IC50 value of 6 nM.

Zebrafish: an emerging real-time model system to study Alzheimer's disease and neurospecific drug discovery.

Zebrafish (Danio rerio) is emerging as an increasingly successful model for translational research on human neurological disorders. In this review, we appraise the high degree of neurological and behavioural resemblance of zebrafish with humans. It is highly validated as a powerful vertebrate model for investigating human neurodegenerative diseases. The neuroanatomic and neurochemical pathways of zebrafish brain exhibit a profound resemblance with the human brain. Physiological, emotional and social behavioural pattern similarities between them have also been well established. Interestingly, zebrafish models have been used successfully to simulate the pathology of Alzheimer's disease (AD) as well as Tauopathy. Their relatively simple nervous system and the optical transparency of the embryos permit real-time neurological imaging. Here, we further elaborate on the use of recent real-time imaging techniques to obtain vital insights into the neurodegeneration that occurs in AD. Zebrafish is adeptly suitable for Ca2+ imaging, which provides a better understanding of neuronal activity and axonal dystrophy in a non-invasive manner. Three-dimensional imaging in zebrafish is a rapidly evolving technique, which allows the visualisation of the whole organism for an elaborate in vivo functional and neurophysiological analysis in disease condition. Suitability to high-throughput screening and similarity with humans makes zebrafish an excellent model for screening neurospecific compounds. Thus, the zebrafish model can be pivotal in bridging the gap from the bench to the bedside. This fish is becoming an increasingly successful model to understand AD with further scope for investigation in neurodevelopment and neurodegeneration, which promises exciting research opportunities in the future.

Exploring the functional impact of mutational drift in LRRK2 gene and identification of specific inhibitors for the treatment of Parkinson disease.

Parkinson's disease (PD) is a disorder of the central nervous system that is caused due to the death of the dopaminergic neurons in the region of the brain called substantia nigra. Mutations in LRRK2 genes are associated with disease condition and it's been reported as crucial factor for drug resistance. Identification of deleterious mutations and studying the structural and functional impact of such mutations may lead to the identification of potential selective inhibitors. In this study, we analyzed 52 PD associated mutations, among that 20 were identified as highly deleterious. The deleterious mutations G2019S and I2020T in the kinase domain were playing a key role in causing resistance to drug levedopa. Molecular docking analyses have been performed to understand the binding affinity of levodapa with LRRK2 in wild and mutant condition. Molecular docking results show that levedopa binds differentially and obtained less number of hydrogen bonds in compared with wild type LRRK2. In addition, molecular dynamics simulations were performed to study the efficacy of docked complexes and it was observed that the efficacy of the mutant complexes (G2019S-Levodopa and I2020T-Levodopa) affected in the presence of mutation. Finally, through virtual screening approach specific inhibitors SCHEMBL6473053 and SCHEMBL1278779 have been identified that could potentially inhibit LLRK2 mutants G2019S and I2020T respectively. Over all this computational investigation correlates the impact of genotypic modulation in structure and function of drug target which enhanced in the identification of precision medicine to treat PD.

In vivo safety evaluation of antibacterial silver chloride nanoparticles from Streptomyces exfoliatus ICN25 in zebrafish embryos.

Silver chloride nanoparticles were synthesized from the cell-free culture supernatant of Streptomyces strain using green synthesis approach with good yield. The nanoparticles were characterized by UV-Vis, IR, SEM, AFM and XRD techniques. These nanoparticles exhibited broad spectrum of antibacterial activity towards Methicillin-resistant Staphylococcus aureus, Methicillin sensitive S. aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumonia at ≤ 2 µg/ml minimal inhibitory concentrations. In vivo bioassay in nanoparticles treated zebrafish embryos exhibited 16 µg/ml dose as maximal cardiac safety concentration and further increases in concentration revealed adverse effects such as pericardial bulging, mouth protrudation, hemorrhage and yolk sac elongation. The less toxicity of nanoparticles treated embryos in terms of cardiac assessment and lethality analysis was observed. The dose below 5 µg/ml is concluded as an in vitro and in vivo therapeutic dose. The properties of this biosynthesized nanoparticle suggest a path towards developing antibiotic nanoparticles that are likely to avoid development of multidrug resistance.

Chemical genetic effects of Sargassum wightii during embryonic development in zebrafish.

OBJECTIVE: Phenotype based small molecule discovery is a category of chemical genetic study. The aim of this study was to observe the phytochemical based genetic effects of Sargassum wightii during organogenesis in embryonic zebrafish. MATERIALS AND METHODS: The phytomolecules from S. wightii were extracted using organic solvents and treated with the 24 h old developing zebrafish embryos. The active extract was partially purified by column chromatography, C18 Sep-Pak column and reversed-phase high-performance liquid chromatography. RESULTS: Initially, cardiac bulging was found in 2 dpf to 3 dpf (days post fertilization), then bradycardia and tubular heart were observed in the next 8 h, which also showed the reduction in the heart beat rates. The phenotypic mutation effects of bre, has, dou yan, heg and you were observed in the 3 dpf and 4 dpf of the extract treated zebrafish embryos. CONCLUSIONS: This study demonstrated that the phytomolecules from S. wightii exhibited potential molecular switches on the developmental process, which might have significant role in understanding the development based chemical genetic studies in zebrafish.

Production of a compound against methicillin resistant Staphylococcus aureus (MRSA) from Streptomyces rubrolavendulae ICN3 & its evaluation in zebrafish embryos.

BACKGROUND & OBJECTIVES: Antibiotic resistance in pathogens has become a serious problem worldwide. Therefore, the search for new antibiotics for drug resistanct pathogens is an important endeavor. The present study deals with the production of anti-methicillin resistant Staphylococcus aureus (MRSA) potential of Streptomyces rubrolavendulae ICN3 and evaluation of anti-MRSA compound in zebrafish embryos. METHODS: The antibiotic production from S. rubrolavendulae ICN3 was optimized in solid state fermentation and extracted. The antagonistic activity was confirmed against MRSA and purified in silica gel column and reverse phase--HPLC with an absorption maximum at 215 nm. Minimal inhibitory concentration of the compound was determined by broth microdilution method. Zebrafish embryos were used to evaluate the extract/compound for its minimal inhibition studies, influences on heart beat rates, haematopoietic blood cell count and lethal dose values. RESULTS: Streptomyces rubrolavendulae ICN3 showed potent antagonistic activity against MRSA with a zone of 42 mm. The minimum inhibitory concentration was calculated as 500 µg/ml of the crude extract and the purified C23 exhibited 2.5 µg/ml in in vitro assay. The LC 50 value of the anti MRSA compound C23 was calculated as 60.49 µg/ml and the MRSA treated embryos survived in the presence of purified compound C23 at a dose of 10 µg/ml. INTERPRETATION & CONCLUSIONS: Our results suggested that the compound was potent with less toxic effects in zebrafish embryonic model system for MRSA infection. Further structural evaluation and analysis in higher mammalian model system may lead to a novel drug candidate for drug resistant Staphylococcus aureus.

Cynodon dactylon and Sida acuta extracts impact on the function of the cardiovascular system in zebrafish embryos.

The aim of the present study was to screen cardioactive herbs from Western Ghats of India. The heart beat rate (HBR) and blood flow during systole and diastole were tested in zebrafish embryos. We found that Cynodon dactylon (C. dactylon) induced increases in the HBR in zebrafish embryos with a HBR of (3.968±0.344) beats/s, which was significantly higher than that caused by betamethosone [(3.770±0.344) beats/s]. The EC50 value of C. dactylon was 3.738 µg/mL. The methanolic extract of Sida acuta (S. acuta) led to decreases in the HBR in zebrafish embryos [(1.877±0.079) beats/s], which was greater than that caused by nebivolol (positive control). The EC50 value of Sida acuta was 1.195 µg/mL. The untreated embryos had a HBR of (2.685±0.160) beats/s at 3 d post fertilization (dpf). The velocities of blood flow during the cardiac cycle were (2,291.667±72.169) µm/s for the control, (4,250±125.000) µm/s for C. dactylon and (1,083.333±72.169) µm/s for S. acuta. The LC50 values were 32.6 µg/mL for C. dactylon and 20.9 µg/mL for S. acuta. In addition, the extracts exhibited no chemical genetic effects in the drug dosage range tested. In conclusion, we developed an assay that can measure changes in cardiac function in response to herbal small molecules and determine the cardiogenic effects by microvideography.

Isolation of a small molecule with anti-MRSA activity from a mangrove symbiont Streptomyces sp. PVRK-1 and its biomedical studies in Zebrafish embryos.

OBJECTIVE: The aim of the present study was to isolate the anti-MRSA (Methicillin Resistant Staphylococcus aureus) molecule from the Mangrove symbiont Streptomyces and its biomedical studies in Zebrafish embryos. METHODS: MRSA was isolated from the pus samples of Colachal hospitals and confirmed by amplification of mecA gene. Anti-MRSA molecule producing strain was identified by 16s rRNA gene sequencing. Anti-MRSA compound production was optimized by Solid State Fermentation (SSF) and the purification of the active molecule was carried out by TLC and RP-HPLC. The inhibitory concentration and LC50 were calculated using Statistical software SPSS. The Biomedical studies including the cardiac assay and organ toxicity assessment were carried out in Zebrafish. RESULTS: The bioactive anti-MRSA small molecule A2 was purified by TLC with Rf value of 0.37 with 1.389 retention time at RP-HPLC. The Inhibitory Concentration of the purified molecule A2 was 30 µg/mL but, the inhibitory concentration of the MRSA in the infected embryo was 32-34 µg/mL for TLC purified molecule A2 with LC50 mean value was 61.504 µg/mL. Zebrafish toxicity was assessed in 48-60 µg/mL by observing the physiological deformities and the heart beat rates (HBR) of embryos for anti MRSA molecule showed the mean of 41.33-41.67 HBR/15 seconds for 40 µg/mL and control was 42.33-42.67 for 15 seconds which significantly showed that the anti-MRSA molecule A2 did not affected the HBR. CONCLUSIONS: Anti-MRSA molecule from Streptomyces sp PVRK-1 was isolated and biomedical studies in Zebrafish model assessed that the molecule was non toxic at the minimal inhibitory concentration of MRSA.