A Mass Spectrometric Study for Comparative Analysis and Evaluation of Metabolite Recovery from Plasma by Various Solvent Systems

A solvent system that extracts a maximum number of metabolites belonging to diverse chemical classes from complex biofluids, such as plasma, may offer useful inputs to understand the metabolic and physiological state of an individual. The present study compared seven solvent systems for extraction of metabolites from plasma. The extracts were analyzed by mass spectrometry (MS) and MS/MS (MS2) using a quadrupole time-of-flight liquid chromatography/MS system in positive and negative modes of ionization. Metabolites with molecular mass below 400 were identified using Human Metabolome Database MS2 and MS search interfaces. The acetone/isopropanol (2:1) system yielded promising results in positive ionization mode, as the maximum number of MS and MS2 features was detected in the extract. It was found to be superior in extraction of various classes of metabolites, especially organic acids, nucleosides and nucleoside derivatives, and heterocyclic molecules. Glycerophosphocholines in the mass range of 400–700 were found to be efficiently extracted by the methanol/chloroform/water (8:1:1) system. In negative mode as well, the maximum number of MS2 features was detected in methanol/chloroform/water and acetone/isopropanol extracts. The fingerprints of molecular features obtained in the negative and positive modes differed from each other to a significant extent.     

Mapping of genetic modifiers affecting the eye phenotype of ocular retardation (Chx10 or-J ) mice

Ocular retardation is a recessive murine mutation whose phenotypic expression is greatly affected by genetic background effects. Mice of the inbred 129/SvJ background that are homozygous for the Chx10^or-J mutation are blind and have a thin, poorly differentiated retina and no optic nerve. A backcross between 129/SvJ and Mus musculus castaneus (CASA/Rk) produced animals that were homozygous for the Chx10^or-J mutation, yet showed a much milder phenotype. Such animals, when brother-sister mated and selected for mild phenotype for several generations, resulted in partial recovery of visual function, including presence of an optic nerve and pupillary response. In this article we report a genome scan of phenotypic extremes of the backcross to identify the genetic loci affecting this phenotype modification. Our scan revealed significant loci on Chromosomes 6 and 14 where the CASA/Rk alleles are maintained selectively. Markers were developed near candidate genes, but no candidate gene could be identified unequivocally. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Long Term Liver Engraftment of Functional Hepatocytes Obtained from Germline Cell-Derived Pluripotent Stem Cells

One of the major hurdles in liver gene and cell therapy is availability of ex vivo-expanded hepatocytes. Pluripotent stem cells are an attractive alternative. Here, we show that hepatocyte precursors can be isolated from male germline cell-derived pluripotent stem cells (GPSCs) using the hepatoblast marker, Liv2, and induced to differentiate into hepatocytes in vitro. These cells expressed hepatic-specific genes and were functional as demonstrated by their ability to secrete albumin and produce urea. When transplanted in the liver parenchyma of partially hepatectomised mice, Liv2-sorted cells showed regional and heterogeneous engraftment in the injected lobe. Moreover, approximately 50% of Y chromosome-positive, GPSC-derived cells were found in the female livers, in the region of engraftment, even one month after cell injection. This is the first study showing that Liv2-sorted GPSCs-derived hepatocytes can undergo long lasting engraftment in the mouse liver. Thus, GPSCs might offer promise for regenerative medicine.     

Regulation of Selenocysteine Incorporation into the Selenium Transport Protein,Selenoprotein P

Selenoproteins are unique as they contain selenium in their active site in the form of the 21st amino acid selenocysteine (Sec), which is encoded by an in-frame UGA stop codon. Sec incorporation requires both cis- and trans-acting factors, which are known to be sufficient for Sec incorporation in vitro, albeit with low efficiency. However, the abundance of the naturally occurring selenoprotein that contains 10 Sec residues (SEPP1) suggests that processive and efficient Sec incorporation occurs in vivo. Here, we set out to study native SEPP1 synthesis in vitro to identify factors that regulate processivity and efficiency. Deletion analysis of the long and conserved 3′-UTR has revealed that the incorporation of multiple Sec residues is inherently processive requiring only the SECIS elements but surprisingly responsive to the selenium concentration. We provide evidence that processive Sec incorporation is linked to selenium utilization and that reconstitution of known Sec incorporation factors in a wheat germ lysate does not permit multiple Sec incorporation events, thus suggesting a role for yet unidentified mammalian-specific processes or factors. The relationship between our findings and the channeling theory of translational efficiency is discussed.     

Genome Sequence of Janibacter hoylei MTCC8307, Isolated from the Stratospheric Air

Janibacter hoylei MTCC8307 was isolated from stratospheric air at an altitude of 41.4 km over Hyderabad, India. Here, we present the draft genome of Janibacter hoylei MTCC8307, which contains 3,139,099 bp with a G+C content of 72.8 mol%, 2,972 protein-coding genes, and 57 structural RNAs.