A Universal Method for Species Identification of Mammals Utilizing Next Generation Sequencing for the Analysis of DNA Mixtures

Species identification can be interesting in a wide range of areas, for example, in forensic applications, food monitoring and in archeology. The vast majority of existing DNA typing methods developed for species determination, mainly focuses on a single species source. There are, however, many instances where all species from mixed sources need to be determined, even when the species in minority constitutes less than 1 % of the sample. The introduction of next generation sequencing opens new possibilities for such challenging samples. In this study we present a universal deep sequencing method using 454 GS Junior sequencing of a target on the mitochondrial gene 16S rRNA. The method was designed through phylogenetic analyses of DNA reference sequences from more than 300 mammal species. Experiments were performed on artificial species-species mixture samples in order to verify the method’s robustness and its ability to detect all species within a mixture. The method was also tested on samples from authentic forensic casework. The results showed to be promising, discriminating over 99.9 % of mammal species and the ability to detect multiple donors within a mixture and also to detect minor components as low as 1 % of a mixed sample.     

Hypoxia may increase rat insulin mRNA levels by promoting binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich insulin mRNA 3'-untranslated region

BACKGROUND: Recent reports identify the 3'-UTR of insulin mRNA as crucial for control of insulin messenger stability. This region contains a pyrimidine-rich sequence, which is similar to the hypoxia-responsive mRNA-stabilizing element of tyrosine hydroxylase. This study aimed to determine whether hypoxia affects insulin mRNA levels. MATERIALS AND METHODS: Rat islets were incubated at normoxic or hypoxic conditions and with or without hydrogen peroxide and a nitric oxide donor. Insulin mRNA was determined by Northern hybridization. Islet homogenates were used for electrophoretic mobility shift assay with an RNA-oligonucleotide, corresponding to the pyrimidine-rich sequence of the 3'-UTR of rat insulin I mRNA. The expression of reporter gene mRNA, in islets transfected with reporter gene constructs containing the wild-type or mutated insulin mRNA pyrimidine-rich sequences, was measured by semiquantitive RT-PCR. RESULTS: Insulin mRNA was increased in response to hypoxia. This was paralleled by increased binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich sequence of the 3'-UTR of insulin mRNA, which was counteracted by hydrogen peroxide. The reporter gene mRNA level containing the wild-type binding site was not increased in response to hypoxia, but mutation of the site resulted in a destabilization of the mRNA. CONCLUSIONS: The complete understanding of different diabetic conditions requires the elucidation of mechanisms that control insulin gene expression. Our data show that hypoxia may increase insulin mRNA levels by promoting the binding of PTB to the insulin mRNA 3'-UTR. Hydrogen peroxide abolishes the hypoxic effect indicating involvement of reactive oxygen species and/or the redox potential in the oxygen-signaling pathway.     

Nicotinamide- and caspase-mediated inhibition of poly(ADP-ribose) polymerase are associated with p53-independent cell cycle (G2) arrest and apoptosis

Poly(ADP-ribose) polymerase (PARP), which is activated by DNA strand breaks, is involved in DNA repair and replication but, during apoptosis, undergoes early caspase-mediated cleavage. Activation of programmed cell death in response to DNA damage may rely on functional p53 protein. Tumor cells are commonly deficient in this oncogene product resulting in resistance to many cytostatic drugs. Here we report that nicotinamide-induced inhibition of poly(ADP-ribosyl)ation and cytokine-induced nitric oxide production both result in a transient increase in p53 levels in pancreatic tumor RINm5F cells. These treatments also induce disruption of the mitochondrial membrane potential (_m), as revealed using the mitochondrial probe JC-1, followed by PARP cleavage and apoptosis all of which are inhibited by the anti-apoptotic protein Bcl-2. Moreover, PARP-inhibition by nicotinamide or 3-aminobenzamide induces apoptosis and/or cell cycle arrest at the G2 checkpoint in all of four tested tumor cell lines of both mesenchymal and epithelial origin including mouse NIH-3T3 cells and p53 deficient human HeLa and Jurkat cells. Bcl-2 counteracts cytokine-, but not nicotinamide-induced G2 arrest. These findings indicate that both chemical and caspase-mediated inhibition of PARP activity, possibly by interfering with DNA replication and repair, may promote a p53-independent G2 arrest and apoptosis.     

Control of insulin mRNA stability in rat pancreatic islets. Regulatory role of a 3'-untranslated region pyrimidine-rich sequence.

Stabilization of insulin mRNA in response to glucose is a significant component of insulin production, but the mechanisms governing this process are unknown. We presently observe that insulin mRNA is a highly abundant messenger and that the content of this mRNA is mainly controlled by changes in messenger stability. We also demonstrate specific binding of the polypyrimidine tract-binding protein to a pyrimidine-rich sequence located in the 3'-untranslated region (3'-UTR) of insulin mRNA. This binding was increased in vitro by dithiothreitol and in vivo by glucose. Inhibition of polypyrimidine tract-binding protein binding to the pyrimidine-rich sequence by mutation of the core binding site resulted in a destabilization of a reporter gene mRNA. Thus, glucose-induced binding of polypyrimidine tract-binding protein to the 3'-UTR of insulin mRNA could be a necessary event in the control of insulin mRNA levels.     

Promoting islet cell function after transplantation

Engraftment (i.e., the adaptation of transplanted pancreatic islets to their new surroundings with regard to revascularization, reinnervation, and reorganization of other stromal compartments) is of crucial importance for the survival and function of the endocrine cells. Previous studies suggest that transplantation induces both vascular and stromal dysfunctions in the implanted islets when compared with endogenous islets. Thus the vascular density and the blood perfusion of islet grafts is decreased and accompanied with a capillary hypertension. This leads to hypoxic conditions, with an associated shift toward anaerobic metabolism in grafted islets. An improved engraftment will prevent or compensate for the vascular/stromal dysfunction seen in transplanted islets and thereby augment survival of the islet implant. By such means the number of islets needed to cure the recipient will be lessened. This will increase the number of patients that can be transplanted with the limited material available.