Fluorosensor-controlled Fed-batch Production Of Cyclosporin A From Beauveria Nivea

Cyclosporin-A (CyA), a secondary endocellular metabolite of the fungus Beauveria nivea ATCC 34921, is an immunosuppressant peptide used worldwide in human organ transplant operations. CyA has also been successfully used to treat a number of autoimmune diseases and parasitic infections. While a great number of papers are published each year about medical applications and mode of action of CyA, however, limited information is available regarding the production of CyA.;This work was divided into two sections: the first dealt with the optimization of medium in shake flasks and the second with the development of fermentation strategies to further increase the volumetric productivity of Cyclosporin-A in a 14-L bioreactor unit.;A new medium was developed that resulted in 222 mg of CyA per litre of fermentation broth in shake flasks. This corresponded to the yield of 14.8 mg CyA/g mycelial dry weight. Fructose concentration higher than 30 g/L inhibited the production of CyA.;Different fed-batch strategics were applied to increase the volumetric productivity of CyA in a 14-L bioreactor. A nicotinamide adenine dinucleotide (NADH) fluorosensor, interfaced with a computer, was used to monitor the growth of the fungus B. nivea. Mathematical models were developed to relate the fluorescence intensity with biomass and fructose concentrations in the bioreactor. The most successful fed-batch fermentation strategy involved a continuous feeding of substrate at the rate of substrate consumption for 48 h, after the fructose concentration in batch mode had dropped down to 5 g/L. The substrate feed rate was then switched to the maintenance level for the next 72 h of fermentation. This strategy gave the highest CyA concentration of 504 mg CyA/L with a yield of 15.14 mg CyA/g mycelial dry weight. This is the highest concentration of CyA reported in the literature using the wild strain of B. nivea ATCC 34921.;This study also demonstrated that by monitoring in situ fluorescence of NADH, a fed-batch strategy can be developed by correlating it to the biomass and the substrate concentrations in the fermentation broth

Genetic Variation and Structure of the People of Uttarakhand, Central Himalayas, India

The Indian Himalayas, being semi-isolated geographically, provide ideal conditions for population genetics investigations. The main aim of this study is to genetically characterize and analyze the genetic structure of the people of Uttarakhand, a newly created North Indian hill state in the Central Himalayas, using original phenotype and allele-frequency data on a battery of seven red cell enzyme polymorphisms. For this analysis, blood samples were collected from 3,222 unrelated subjects belonging to various endogamous caste populations (Brahmin, Rajput, and Shilpkar) and tribal Bhotia inhabiting seven different districts in the Garhwal (northern) and Kumaon (southern) regions of Uttarakhand. Hemolysates were typed for isozymes of ESD, PGM1, ADA, AK1, GLO1, ACP1, and GPI using standard electrophoretic techniques. The genetic structure of these regional caste and tribal population groups was investigated with the help of different statistical measures. The present biochemical marker results show that the overall genetic constitution of the different populations of Uttarakhand is rather heterogeneous but similar to that of various caste and tribal populations of the neighboring hill state of Himachal Pradesh, situated on Uttarakhand’s western border. The extent of genic differentiation observed in different contemporary populations of Garhwal was twice as high as that of Kumaon. Interestingly, in genetic distance dendrograms of both the regions and of all of Uttarakhand, all the Shilpkar groups are differentiated from the remaining groups of Brahmin, Rajput, and Bhotia. The genetic constitution of the Shilpkar (a scheduled caste population of Uttarakhand) and to a lesser extent that of the Bhotia (a scheduled tribe population of Uttarakhand) are rather different from both the Brahmin and Rajput high-caste populations, which tend to show genetic similarities between them. These observations are corroborated by the known ethnohistory of different populations of Uttarakhand

Inter-specific gene flow from herbicide-tolerant crops to their wild relatives

The global policy agenda of agriculture is based around producing sufficient food, feed, and fiber to meet the needs of the world’s growing population (Anonymous 2009). Additionally, agricultural cropping systems have been challenged to create oil-based fuels and plant-based bio-products, reduce use of pesticides, and develop crops tolerant to biotic and abiotic stresses. Therefore, crop cultivars with higher yields and resistance to important pests are required to meet food requirements in a sustainable manner. Technological advances for developing bio-products, improved crop cultivars, and pest and/or herbicide-tolerant crops are predicated on the use of plant biotechnology

Antigenic reactivity of recombinant structural proteins.

<p>Lysates of cells transfected with pTT5-P12A3C, co-transfected with pTT5-VP0, pTT5-VP3 and pTT5-VP1, mock transfected cells or purified iFMDV were analyzed by ELISA with four MAbs (1–5,2–4,3–2,3–3) directed against conformational epitopes of FMDV. Error bars are standard deviation from three independent experiments.</p

Schematic representation of FMDV genomic fragments cloned into the pTT5-vector.

<p>Schematic representation of FMDV genomic fragments cloned into the pTT5-vector.</p

Assembly of recombinant structural proteins into subviral particles.

<p>Lysates of cells transfected with pTT5-P12A3C, mock transfected cells or iFMDV were loaded onto 45-15% sucrose gradients. Fractions were collected and analyzed by solid phase ELISA. Fractions of iFMDV were measured at 260 nm for RNA detection. The known positions of the FMDV virions (146S), empty capsids (75S), pentamers (12S) and protomers (5S) are indicated.</p

Western blot analysis of recombinant proteins produced after transfection with pTT5-P12A3C or with pTT5-P12A.

<p>Lysates of cells transfected with pTT5-P12A3C, pTT5-P12A or mock transfected were analyzed by SDS-PAGE and Western blot using anti-FMDV guinea pig serum (1:500). iFMDV was used as positive control. The expected migration positions of P12A, VP0, VP3 and VP1 are indicated by arrows.</p

Recombinant empty capsids recognition by sera from vaccinated bovines.

<p>Percentage of positivity of bovine sera analyzed by ELISA for the recognition of recombinant empty capsids and mock transfected cell lysates. Percentage of positivity was calculated as (A₄₉₂ from recombinant protein or mock lysate/A₄₉₂ from iFMDV) × 100. Each pair of bars represents the sera from an individual animal.</p

Analysis of humoral response of BALB/c mice immunized with recombinant proteins.

<p>The results were obtained from the average of seven sera in each group. Mean ± SD sample is shown.</p

Western blot analysis of recombinant structural proteins.

<p>Lysates of cells transfected with pTT5-P12A3C, co-transfected with pTT5-VP0, pTT5-VP3 and pTT5-VP1 or mock transfected were analyzed by SDS-PAGE and Western blot using anti-FMDV guinea pig serum (1:500). iFMDV was used as positive control. The expected migration positions of VP0, VP3 and VP1 are indicated by arrows.</p