Rhinosporidium seeberi proven as a fungus for the first time after a century since its discovery

The 18S rRNA gene sequencing of a pure microorganism isolated in pure culture from human rhinosporidiosis cases coded UMH.48 and preserved at 4oC, and, the fungal extracts of biopsy from new cases of nasal rhinosporidiosis were done. Both the sequences were compared for the presence any identical regions by BLAST tool. Astonishingly both the sequences showed 100% identity with each other. The sequences were further compared with the sequences present in NCBI database, followed by sequences of specific organisms like Mesomycetozoa sp and Synchytrium sp. Based on the morphological features, life cycle and BLAST analysis the organism UMH.48 was categorized as a Fungus. The sequences of UMH.48 and sequences from the fungus extracts from new tissue biopsies were deposited in Genbank with accession numbers JN807465 and JN807466 respectively. This paper reports the identity of 18S rRNA sequences between the pure, preserved, isolate with those obtained from biopsies of nasal rhinosporidiosis obtained from totally new cases. Our isolate has been tentatively identified as a lower aquatic fungus with 100% alignment with Colletotrichum truncatum and Glomerulla sps and lesser score similarity with Synchytrium minutum. Yet the absence of a perfect sexual phase or any asexual fungal spores, very rare  microscopic morphology, life cycle and remarkable resemblance with members of lower aquatic fungi led us to surmise (also through personal communication with NCBI, Taxonomy expert) that the isolate is a Fungus (unknown) and not an Ascomycete

IN VITRO STUDIES ON THE SYNERGISTIC EFFECT OF MORINDA CITRIFOLIA L. (NONI) AND METHOTREXATE ON CYTOTOXICITY OF HELA CELL LINES

Objective: Cancer remains to be one of the leading causes of death around the world. Modern targeted therapies have undeniably improved cancer patients’ survival, but search still continues for safer and more effective drugs. This work is an attempt to assess the effectiveness of cotreatment of aqueous fruit extract of Morinda citrifolia L. (Noni) on methotrexate (MTX)-induced cytotoxicity on HeLa cancer cell line. Methods: HeLa cells were treated with different concentrations of MTX and Noni alone and in combination for 24 and 48 h and studied for the cytotoxic effects by various approaches. Results: There was a dose-dependent inhibition of growth when treated with MTX in a dose range of 0.045–45.4 μg/ml and Noni for a dose range of 0.3–30 mg/ml for 48 h. The IC50 for MTX was 4.45 μg/ml (1 μM/ml), and for Noni, it was 8 mg/ml while the combination exhibited a more intensive growth inhibitory effect. Our results were confirmed with phase-contrast microscopy, whereby the number of viable cells decreased with an increase in the concentration of Noni. Cells were found to have rounded up and detached from the flask indicating apoptosis. Apoptosis in HeLa cell lines was further confirmed by DNA fragmentation and flow cytometry. Conclusion: It can be concluded that Noni may enhance MTX cytotoxicity by inducing apoptosis and cell cycle arrest. Hence, cotreatment with Noni may reduce the dosage of MTX necessary for inhibiting proliferation of malignant cells and hence decrease the toxic effects of MTX on the system

Optimization and cultural characterization of alkalophilic protease producing Bacillus sp. GPA4

A new bacterial isolate belonging to Genus Bacillus coded as GPA4 was analyzed for protease activity. The strain GPA4 had the ability to produce extracellular protease. Nutrient media supplemented with 0.1% of casein showed maximum production of protease of 305 U/ml during the  stationary phase. Maximum growth of the organism was observed at pH 7 but alkaline pH favored protease production. An increased level of 390  U/ml of enzyme was produced at pH 8. At 30ºC under mild shaking conditions 100 rpm it  produced 399.4 U/ml of enzyme compared to static and shaking incubation at 37º C. Among different metals, addition of Zn2+ induced more enzyme  production. Addition of carbon source in the form of glucose and nitrogen source as soy bean meal increased protease production to 451.4 U/ ml and 465 U/ml respectively

Bacillus isolates VTGP. A-D. 30808 Alcaligenes sp., Exiguobacterium sp., B. pumilus and B. fusiformis producing extracellular alkaline proteases, amylases and cellulases - a preliminary report

Garden soil samples collected from Angamali, Kerala, India were screened for potent bacteria capable of synthesizing extracellular hydrolytic enzymes. Four bacteria were obtained in pure culture. The isolates were systematically identified by microscopy, Gram and special staining techniques for capsule and spores, biochemical reactions and phylogeny by molecular techniques like 16 S rRNA ene sequencing followed by Blast analysis. Production of protease, cellulase and amylase were detected by inoculating nutrient agar containing casein/ skim milkagar, carboxy methyl cellulose and soluble starch respectively.  Alkalophilic and thermophilic properties were investigated by inoculation and incubation of the isolates on specific nutrient media at pH 7-12 and at a wide range of temperatures 28-30, 37, 50 and 650.C. The isolates were coded VTGP. A-D 30808. All the four expressed significant alkalophilic growth at pH 7-12. With respect to protease activity all  except A showed marked protease activity over a high pH range pH 7-12(A-115, B-1119, C-1500, D-1350 Units / ml of liquid culture   supernatant). Both C & D secreted protease as early as 8-12 hours on nutrient agar with 0.1% skim milk forming a clear wide zone of casein hydrolysis. Hence the proteases produced were highly alkalophilic. Amylase activity was marked in all (A-37.38, B-27.58, C-27.92, D-34.82 units per ml culture supernatant). On CMC agar, all the four isolates showed CMCase activity indicated by pale yellow zone of hydrolysis of carboxy methyl cellulose agar when tested with Congo red reagent. A, B and C were strongly positive with minimal visible activity in D. But when tested in CMC broth culture the activities were A-6.71, B-4.30, C-6.56 and D 0.58 units/ ml of culture supernatant). 16S r RNA gene  sequencing of isolates A to D showed maximum alignment with Alcaligenes sp., Exiguobacterium sp., Bacillus pumilus and B. fusiformis. The sequences have been deposited in GenBank with Accession  numbers HQ 848384, HQ 848385, HQ 848386, and HQ 848387

Biological characterization of a fast growing non-sporing alkalophilic lignin degrading fungus MVI.2011

MVI.2011 a rapidly multiplying alkalophilic non sporing fungus was isolated in 1990 and preliminarily identified as a Deuteromycete. The isolate was characterized in detail. The original isolate produced highly fluffy, cottony, fragile aerial mycelia on SDA and a similar growth in liquid SDB also. The organism grew out even on the surface of the conical flask containing the liquid medium inoculated indicating the high aerobic nature. With frequent sub culturing over 20 years the colony morphology on the same media appeared very confined with regular margin and dry surface. Yet there were no reproductive structures. LP staining showed dimorphism with apical fragmentation and no conidia, spores sexual or asexual etc. The pH range was very wide 5-11. The optimum cultural conditions for lignin degradation were pH 8.5, temperature 25-28oC, 12-18 hours and medium- 1% glucose, 0.5% peptone in basal mineral medium. The isolate could breakdown and decolourise commercial lignin (0.1-5%) and alkaline wood extract (1-50%) within 12-18 hours in static cultures evidenced by a clear reduction in absorption at 380 nm (lignin) and a marked shift to increased absorption at 360 nm and between 180 and 300 nm indicating appearance of lignin breakdown products. In optimised  media containing commercial lignin (0.1%) and alkaline wood extract (10%), MVI.2011 secreted Lignin peroxidase (9.39 units/ml), Manganese peroxidase (2.093 units/ml) and laccase (3.5 units/ml) enzymes. The  above data led us to conclude that the isolate was novel being highly alkalophilic, capable of rapid growth, decolourisation of lignins and  secretion of lignin degrading enzymes. Based on microscopic morphology and colony features, the isolate coded MVI.2011 has been identified as “Uncultured Fungus†with NCBI Accession No JN606084. It has been  deduced to be a member of Mycelia sterilia group

UMH.48 (NCBI JN807465) the Fungus causing Rhinosporidiosis is sensitive to anti fungal

UMH.48, JN807465, a fungus causing Rhinosporidiosis was isolated in pure culture from biopsies from patients with nasal Rhinosporidiosis. It was  identified as a lower aquatic fungus by 18S rRNA gene sequencing which compared 100% similar to the sequences from fungal extract of the tissue, thus establishing the etiologic role of UMH.48 in Rhinosporidiosis. UMH.48 18S rRNA sequence showed significant similarity with Synchytridium minutum and very low varying percentages of similarity with Mycobacterium sps., Corynebactrium sps., and Actinomycetales. The organism was tested for susceptibility and sensitivity to antibiotics and antifungal drugs such as  Norfloxacin, Dapsone, Rifampicin and Amphotericin B. UMH.48 was highly sensitive to Amphotericin B and Rifampicin. It was resistant to Dapsone at the concentrations tested

Screening of alkalophilic thermophilic protease isolated from Bacillus RV.B2.90 for Industrial applications

Protease enzyme from Bacillus RV.B2.90 was purified, fractionated and tested for various applications. This enzyme could degrade the natural proteins like coagulated egg and blood clot. Blood and natural pigment stain (carrot, beetroot, green leaves, coffee and tea) were removed by this enzyme easily. It exhibited good compatibility with the commercial detergents such as Surf Excel, Tide, Ujala, Ariel and Rin. The keratinolyticactivity of the enzyme was evidenced by the complete degradation of  feathers. X-ray films treated with the enzyme showed release of protein from the gelatin coating, a pre requisite for recovery of silver. Also showed better stability with surfactants and solvents, which will be advantageous as detergent formulations containing chelating agents like EDTA and in peptide synthesis. The partially purified enzyme showed direct hydrolysis of various natural proteins (BSA, casein and azocasein) applied to clean glass slides followed by denaturation by boiling. Bacillus RV.B2.90 immobilization with 3% alginate showed maximum production (2002 U/ml). In continuous production, the beads were stable over 24 hrs X 9 cycles. RV.B2.90 protease highly thermostable and alkalophilic with a variety of activities, has great potential for application in a wide range of industry

An integrated process for Industrial effluent treatment and Biodiesel production using Microalgae

The present day necessities and requirements have emphasized the need for a renewable and alternate energy source is very high. Besides, the present day energy resources posse potential threat to the environment by emitting Greenhouse gases (GHGs) etc. An integrated process which involves a model of the wastewater High Rate Algal Ponds (HRAPs) near the industries and establishment of Biodiesel plants nearer to these ponds to produce algal biodiesel along with other byproducts is elucidated.  Wastewater HRAPs also help in sequestering the CO2 emitted by the industries and is used by the microalga for their photosynthesis in turn providing oxygen to the bacterial population which accumulates and degrades the toxic compounds present in the industrial effluents. This integrated process involving cheaper treatment of industrial effluents, production of algal biodiesel, accumulation of toxic  compounds, sequestration of CO2 and various other non-fuel applications contribute to an effective energy management system

IMPACT OF ANTIOXIDANT SUPPLEMENTATION ON TOXICITY OF METHOTREXATE-AN IN VITRO STUDY ON ERYTHROCYTES USING VITAMIN E

 Objective: Methotrexate (MTX) is an antimetabolite used widely in cancer and autoimmune diseases as an inhibitor of the enzyme dihydrofolatereductase. Treatment regimens with MTX lead to the formation of reactive oxygen species and are involved in drug-induced toxicity presenting severeside effects. In the present investigation, red blood cells were used as a model to understand drug-mediated hemolysis and alterations in antioxidantdefense system stimulated by MTX. This effort attempts to evaluate, whether pre-supplementation of vitamin E can modulate drug induced toxicity.Methods: Blood samples from healthy subjects were collected and processed to obtain 10% RBC solution in saline. This solution was further treatedwith 80μM MTX in presence and absence of 90μM vitamin E respectively. Treated and control RBC solutions were used to assess percent hemolysis,levels of lipid peroxides, GSH, lactate dehydrogenase and activities of specific antioxidant enzymes.Results: Our study reveals a significant increase in lipid peroxides, reduced glutathione GSH reductase activity after incubation with MTX, lactatedehydrogenase, and a considerable decline in catalase, superoxide dismutase, GSH peroxidase, GSH S-transferase, and GSH reductase after incubationwith MTX.Conclusion: Ameliorative effect of vitamin E supplementation reduces oxidative stress and restores the activities of these antioxidant enzymes,thereby demonstrating the protection rendered by vitamin E. Our data indicates that vitamin E administration during chemotherapy is effective inmodulating the chemotherapy-induced side effects thus stressing on the importance of vitamin E supplementation in combination with chemotherapyduring cancer treatments.Keywords: Antioxidant, Lipid peroxides, Superoxide dismutase, Glutathione, Catalase, Red blood cell