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.

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.

Nature and bioprospecting of haloalkaliphilics: a review.

The haloalkaliphilics are an important subset of extremophiles that grow in salt [upto 33% (wt/vol) NaCl] and alkaline pH (> 9). They are found in hypersaline environments especially in the brines in arid, coastal and deep sea locations, and in alkaline environments, such as soda soils, lakes and deserts. Some authors have described haloalkaliphilic bacteria as moderate halophilic bacteria, but the molecular and classical studies revealed that they belong to moderately to extremely halophilic bacteria and archaea. Organic solutes, such as glycine, betaine and other amino acid derivatives, sugars such as, sucrose and trehalose, and sugar alcohols present in the haloalkaliphilics help for their osmoadaptation, and also serve as stabilizers. Haloalkalphilics secrete exoenzymes like proteases, amylases, xylanases, cellulases and peroxidases which have potential industrial applications. They also produce bacteriorhodopsin, compatible solutes, pigments, biopolymers, secondary metabolites like biosurfactants, polyhydroxyalkanoate (PHA) and exopolysaccharides and antimicrobial/anticancer compounds. They have unique metabolic pathways which can be used to treat industrial pollutants, heavy metals and waste water.

Novel Sequential Screening and Enhanced Production of Fibrinolytic Enzyme by Bacillus sp. IND12 Using Response Surface Methodology in Solid-State Fermentation.

Fibrinolytic enzymes have wide applications in clinical and waste treatment. Bacterial isolates were screened for fibrinolytic enzyme producing ability by skimmed milk agar plate using bromocresol green dye, fibrin plate method, zymography analysis, and goat blood clot lysis. After these sequential screenings, Bacillus sp. IND12 was selected for fibrinolytic enzyme production. Bacillus sp. IND12 effectively used cow dung for its growth and enzyme production (687 ± 6.5 U/g substrate). Further, the optimum bioprocess parameters were found out for maximum fibrinolytic enzyme production using cow dung as a low cost substrate under solid-state fermentation. Two-level full-factorial experiments revealed that moisture, pH, sucrose, peptone, and MgSO4 were the vital parameters with statistical significance (p < 0.001). Three factors (moisture, sucrose, and MgSO4) were further studied through experiments of central composite rotational design and response surface methodology. Enzyme production of optimized medium showed 4143 ± 12.31 U/g material, which was more than fourfold the initial enzyme production (978 ± 36.4 U/g). The analysis of variance showed that the developed response surface model was highly significant (p < 0.001). The fibrinolytic enzyme digested goat blood clot (100%), chicken skin (83 ± 3.6%), egg white (100%), and bovine serum albumin (29 ± 4.9%).

Medium optimization for the production of fibrinolytic enzyme by Paenibacillus sp. IND8 using response surface methodology.

Production of fibrinolytic enzyme by a newly isolated Paenibacillus sp. IND8 was optimized using wheat bran in solid state fermentation. A 2(5) full factorial design (first-order model) was applied to elucidate the key factors as moisture, pH, sucrose, yeast extract, and sodium dihydrogen phosphate. Statistical analysis of the results has shown that moisture, sucrose, and sodium dihydrogen phosphate have the most significant effects on fibrinolytic enzymes production (P < 0.05). Central composite design (CCD) was used to determine the optimal concentrations of these three components and the experimental results were fitted with a second-order polynomial model at 95% level (P < 0.05). Overall, 4.5-fold increase in fibrinolytic enzyme production was achieved in the optimized medium as compared with the unoptimized medium.

Thermostable Alkaline Phytase from Alcaligenes sp. in Improving Bioavailability of Phosphorus in Animal Feed: In Vitro Analysis.

A bacterial isolate, Alcaligenes sp. secreting phytase (EC 3.1.3.8), was isolated and characterized. The optimum conditions for the production of phytase included a fermentation period of 96 h, pH 8.0, and the addition of 1% (w/v) maltose and 1% (w/v) beef extract to the culture medium. This enzyme was purified to homogeneity and had an apparent molecular mass of 41 kDa. The optimum pH range and temperature for the activity of phytase were found to be 7.0-8.0 and 60°C, respectively. This enzyme was strongly inhibited by 0.005 M of Mn(2+), Mg(2+), and Zn(2+). In vitro studies revealed that the phytase from Alcaligenes sp. released inorganic phosphate from plant phytates. Phytase released 1930 ± 28, 1740 ± 13, 1050 ± 31, 845 ± 7, 1935 ± 32, and 1655 ± 21 mg inorganic phosphate/kg plant phytates, namely, chick pea, corn, green pea, groundnut, pearl pea, and chick feed, respectively.

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.