Molecular,Physiological and Phenotypic Characterization of Paracoccus denitrificans ATCC 19367 Mutant Strain P-87 Producing Improved Coenzyme Q10

Coenzyme Q₁₀ (CoQ₁₀) is a blockbuster nutraceutical molecule which is often used as an oral supplement in the supportive therapy for cardiovascular diseases, cancer and neurodegenerative diseases. It is commercially produced by fermentation process, hence constructing the high yielding CoQ₁₀ producing strains is a pre-requisite for cost effective production. Paracoccus denitrificans ATCC 19367, a biochemically versatile organism was selected to carry out the studies on CoQ₁₀ yield improvement. The wild type strain was subjected to iterative rounds of mutagenesis using gamma rays and NTG, followed by selection on various inhibitors like CoQ₁₀ structural analogues and antibiotics. The screening of mutants were carried out using cane molasses based optimized medium with feeding strategies at shake flask level. In the course of study, the mutant P-87 having marked resistance to gentamicin showed 1.25-fold improvements in specific CoQ₁₀ content which was highest among all tested mutant strains. P-87 was phenotypically differentiated from the wild type strain on the basis of carbohydrate assimilation and FAME profile. Molecular differentiation technique based on AFLP profile showed intra specific polymorphism between wild type strain and P-87. This study demonstrated the beneficial outcome of induced mutations leading to gentamicin resistance for improvement of CoQ₁₀ production in P. denitrificans mutant strain P-87. To investigate the cause of gentamicin resistance, rpIF gene from P-87 and wild type was sequenced. No mutations were detected on the rpIF partial sequence of P-87; hence gentamicin resistance in P-87 could not be conferred with rpIF gene. However, detecting the mutations responsible for gentamicin resistance in P-87 and correlating its role in CoQ₁₀ overproduction is essential. Although only 1.25-fold improvement in specific CoQ₁₀ content was achieved through mutant P-87, this mutant showed very interesting characteristic, differentiating it from its wild type parent strain P. denitrificans ATCC 19367, which are presented in this paper.

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The online version of this article (doi:10.1007/s12088-014-0506-4) contains supplementary material, which is available to authorized users.     

Comparison of the levels of the growth factors in umbilical cord serum and human milk and its clinical significance

The process of the growth of the fetus begins in the uterus and gets further accelerated following the birth, especially during initial few months. The role of the growth factors in the physiology of the cellular growth is already well established. Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) seem to be imperative for angiogenesis, cell development and proliferation as well as maintenance of the tissues. The levels of these factors in the maternal serum during pregnancy as well as during postpartum period are insignificant. Consequently, we hypothesized that the fetus receives moderate supply of these growth factors from the placenta during its stay in the uterus. This supply gets further augmented during the postpartum period through the different source, i.e. mother's milk. To study this physiological transition of the source of the growth factors from the placenta to the breast milk, the concentrations of VEGF and HGF in the cord serum of full term neonates and that in the breast milk of the corresponding mothers were analyzed during ELISA. The human milk, especially the colostrum revealed significantly higher levels of VEGF and HGF (1541.759 ± 119.349 pg/ml and 7129.249 ± 273.472 pg/ml) than cord serum (16.632 ± 0.773 pg/ml and 2581.6 ± 108.275 pg/ml) respectively. The multifold higher levels of VEGF observed in colostrum probably correlates with its high neonatal requirement for the maturation of the gastrointestinal epithelium following birth. The higher levels of both the growth factors in the breast milk than those observed in the cord serum probably explain their higher needs by the neonates for immunological protection, protein synthesis and neurocognitive development. The observations of the present study strengthen the policy of the colostrum feeding, which is promoted by organizations like World Health Organization (WHO). This study further documents the fact that the commercial milk formulae cannot replace the human milk.     

In silico characterization of putative drug targets in Staphylococcus saprophyticus,causing bovine mastitis

The bovine mastitis caused by coagulase negative staphylococci (CNS) has increased in many herds of urban and rural areas of India. Emergence of multi drug resistant bacteria has further made its management more complex and serious. Therefore, innovation of novel specific drug for the treatment of disease caused by particular organism remained to be a challenge. Hence, in the present study a bacterium was isolated from milk of the cow with bovine mastitis and was identified as S. saprophyticus, 44 pathways of S. saprophyticus retrieved (KEGG) from web server were found to be non homologous to the host Bos taurus, out of which 39 pathways were found to be in cytoplasm, 2 in cell wall and 3 in the cell membrane. The knowledge of the present study could make the drug discovery easier which have high affinity to the target site of the causative organism.     

Arsenic ecotoxicology and innate immunity

Understanding the ecotoxicological effects of arsenic in theenvironment is paramount to mitigating its deleterious effectson ecological and human health, particularly on the immune response.Toxicological and long-term health effects of arsenic exposurehave been well studied. Its specific effects on immune function,however, are less well understood. Eukaryotic immune functionoften includes both general (innate) as well as specific (adaptive)responses to pathogens. Innate immunity is thought to be theprimary defense during early embryonic development, subsequentlypotentiating adaptive immunity in jawed vertebrates, whereasall other eukaryotes must rely solely on the innate immune responsethroughout their life cycle. Here, we review the known ecotoxicologicaleffects of arsenic on general health, including immune function,and propose the adoption of zebrafish as a vertebrate modelfor studying such effects on innate immunity.     

Principal-Component Analysis for Assessment of Population Stratification in Mitochondrial Medical Genetics

Although inherited mitochondrial genetic variation can cause human disease, no validated methods exist for control of confounding due to mitochondrial population stratification (PS). We sought to identify a reliable method for PS assessment in mitochondrial medical genetics. We analyzed mitochondrial SNP data from 1513 European American individuals concomitantly genotyped with the use of a previously validated panel of 144 mitochondrial markers as well as the Affymetrix 6.0 (n = 432), Illumina 610-Quad (n = 458), or Illumina 660 (n = 623) platforms. Additional analyses were performed in 938 participants in the Human Genome Diversity Panel (HGDP) (Illumina 650). We compared the following methods for controlling for PS: haplogroup-stratified analyses, mitochondrial principal-component analysis (PCA), and combined autosomal-mitochondrial PCA. We computed mitochondrial genomic inflation factors (mtGIFs) and test statistics for simulated case-control and continuous phenotypes (10,000 simulations each) with varying degrees of correlation with mitochondrial ancestry. Results were then compared across adjustment methods. We also calculated power for discovery of true associations under each method, using a simulation approach. Mitochondrial PCA recapitulated haplogroup information, but haplogroup-stratified analyses were inferior to mitochondrial PCA in controlling for PS. Correlation between nuclear and mitochondrial principal components (PCs) was very limited. Adjustment for nuclear PCs had no effect on mitochondrial analysis of simulated phenotypes. Mitochondrial PCA performed with the use of data from commercially available genome-wide arrays correlated strongly with PCA performed with the use of an exhaustive mitochondrial marker panel. Finally, we demonstrate, through simulation, no loss in power for detection of true associations with the use of mitochondrial PCA.     

Identification and analysis of putative promoter motifs in bovine herpes virus

The purpose of this study is to identify and analyse the putative promoter motifs in the bovine herpes virus which causes several diseases in cattle worldwide including bovine mastitis with large economic impact on dairy industry. Bovine mastitis caused due to virus is often neglected as bacterial infections are held mainly responsible for the disease. Therefore, in this in silico investigation with all the existing experimental data a total of 147 promoter were identified along with their sequences from three genome viz bovine herpes virus 1 (BHV), bovine herpes virus 4 and bovine herpes virus 5, out of which 39 promoters were from bovine herpes virus 4 (BHV 4), 95 from BHV1 and 13 from BHV5 and it was observed that BHV1 and BHV5 have a close evolutionary history. However, they belong to the same subfamily and size of the genome and GC% of BHV1 and BHV5 was almost equal and very high compare to that of BHV4. This analysis may help in designing the live attenuated vaccine against BHV causing bovine mastitis that reduces the incidence of bovine mastitis. Identification of promoters may also help in designing of expression vectors which help in better understanding of the regulation of gene expression. In the era of large genomics and proteomics prediction of promoters in the whole genome is crucial for the advancement of drug discovery and gene therapy.