Mining of transcriptome identifies CD109 and LRP12 as possible biomarkers and deregulation mechanism of T cell receptor pathway in Acute Myeloid Leukemia.

Acute Myeloid Leukemia (AML) is a heterogeneous disease with highest mortality compared to other types of leukemia. There is a need to find the gene abnormalities and mechanisms behind them due to their heterogenic nature. The present study is aimed to understand genes, pathways and biomarker proteins influenced by transcriptomic deregulation due to AML. Differentially expressed gene (DEG), protein-protein interaction network, gene ontology, KEGG pathway, variant analysis and secretome analyses were performed using different AML RNAseq datasets. A total of 655 DEGs including 291 up-regulated and 364 down-regulated genes, which were satisfied with a fold change of 1.5 were identified. Top hub genes for AML were identified as TP53, PTPRC and AKT1. This integrative bioinformatics approach revealed the deregulation of T Cell Receptor (TCR) pathway and altered immune response related genes. The survival analysis revealed the associated deregulation of multiple TCR pathway related genes. Variant analysis identified the benign and likely benign nature of many important target genes and markers screened, which were found to have an important role in the progression of AML. DEGs and secretome analysis found out a set of seven molecules represents potential biomarkers for AML. In vitro analytical validation showed overexpression pattern of CD109 and LRP12 in AML cell line and HL-60 cells than the normal human bone marrow-derived stromal cell line HS-5. Here we report first time for CD109 and LRP12 as a possible biomarkers for the diagnostic significance. Amino acid substitutions detected by variant analysis and deregulation of immune checkpoint molecules revealed their role in reducing immune response and inability to fight cancer cells. In conclusion, this study highlights the possibility of new biomarkers for AML and the mechanism of decrease in immune response due to the downregulation of co-stimulatory immune molecules, which needs further clinical validation investigations.

Mutation in MCL1 predicted loop to helix structural transition stabilizes MCL1-Bax binding interaction favoring cancer cell survival.

Myeloid cell leukemia-1 (MCL1), an anti-apoptotic BCL-2 family protein plays a major role in the control of apoptosis as the regulator of mitochondrial permeability which is deregulated in various solid and hematological malignancies. Interaction of the executioner proteins Bak/Bax with anti-apoptotic MCL1 and its cellular composition determines the apoptotic or survival pathway. Mutations act at various levels in the apoptotic process and can contribute to disease. Single nucleotide polymorphism (SNP) in MCL1 gene was focused as they result in changes in the amino acid sequence and have been associated with tumorigenesis. This study highlighted the deleterious MCL1-Bax stabilizing effect of the mutation V220F on MCL1 structure through computational protein-protein interaction predictions and molecular dynamics simulations. The single point mutation at V220F was selected as it is residing at the hydrophobic core region of BH3 conserved domain, the site of Bax binding. The molecular dynamics simulation studies showed increase in stability of the mutated MCL1 before and after Bax binding comparable with the native MCL1. The clusters from free energy landscape found out structural variation in folding pattern with additional helix near the BH3 domain in the mutated structure. This loop to helix structural change in the mutated complex favored stable interaction of the complex and also induced Bax conformational change. Moreover, molecular mechanics-based binding free energy calculations confirmed increased affinity of Bax toward mutated MCL1. Residue-wise interaction network analysis showed the individual residues in Bax binding responsible for the change in stability and interaction due to the protein mutation. In conclusion, the overall findings from the study reveal that the presence of V220F mutation on MCL1 is responsible for the structural confirmational change leading to disruption of its biological functions which might be responsible for tumorigenesis. The mutation could possibly be used as future diagnostic markers in treating cancers.

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.

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).

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.

HR-LC-MS based analysis of two antibacterial metabolites from a marine sponge symbiont Streptomyces pharmamarensis ICN40.

On the effort to screen antibiotics against Methicillin resistant Staphylococcus aureus (MRSA), an actinomycete strain which can produce bactericidal compound was isolated from a marine sponge of Kanyakumari Coast, India. Two anti-MRSA compounds (PVI401 and PVI402) were isolated from the fermentation plates of Streptomyces pharmamarensis ICN40. TLC bioautography analysis yielded two active spots with Rf value of 0.75 (PVI401) and 0.8 (PVI402) from the crude extract. Both the compounds were characterized by HR-LC-MS analysis. LC-MS based de-replication analysis found out the compound PVI401 with an exact mass of 376.09435 Da and PVI402 with an exact mass of 273.26795 Da were found to be unidentified. Antibacterial spectrum showed significant minimal inhibitory concentration as 0.5 µg/ml of PVI401 and 2 µg/ml of PVI402 against MRSA. The whole organism zebrafish safety evaluation exhibited the compound PVI402 is safe upto 1 mg/ml 40 µg/ml of PVI401 exhibited thrombosis in cardiac chamber and this compound exhibited 44 µg/ml of LC50 against HepG2 hepatic carcinoma cell line. Both the compounds may be identified further for its structural novelty and clinical studies.

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.

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.