Investigative Genetics

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    (2014-03) Kreutzer, McKensie; Howard, Taylor; Curtis, Mary; Allen, Michael; Planz, John
    Purpose (a): In the areas of conservation genetics and wildlife forensics, it is important to be able to accurately identify an individual and relate that individual back to the population from whence it came. With the low levels of genetic diversity possessed by some isolated North American bear populations, an identification method is needed that can provide higher resolution in short tandem repeat (STR) regions than current capillary electrophoresis assays. High resolution sequencing methods, such as those provided by next-generation sequencing (NGS) technologies, allow sequence motif changes within STR regions to be detected, whereas capillary electrophoresis only detects STR size. In this study, I hypothesized that when compared to capillary electrophoresis, NGS of STR loci would attain better genetic resolution among bear populations and thereby improve the accuracy of assigning an individual to its true population. Methods (b): Polymerase chain reaction (PCR) conditions were optimized for ten Ursid STR loci. An amplicon pool was generated and used to develop a library for NGS on the Ion Torrent™ Personal Genome Machine™ (PGM™) Sequencer (Life Technologies™, Carlsbad, CA). Deoxyribonucleic acid (DNA) barcode adaptors were ligated to the amplicons, thus allowing multiple samples to be sequenced in one run. Sequencing reads were aligned to a virtual ladder, first by sample via barcode, then by locus via primer. Sequence analysis was then performed using NextGENe® (SoftGenetics®, State College, PA) software. Results (c): Successful sequencing of ten loci for seven black bear (Ursus americanus) samples was carried out in one run. Allele call concordance was shown between capillary electrophoresis and NGS technologies. Variants within alleles (base changes) were evaluated and showed that NGS provided higher genetic resolution. Conclusions (d): In conclusion, NGS provided better genetic resolution than current capillary electrophoresis assays. Higher resolution has implications for increasing the accuracy of assigning an individual to its true population. Better assignment could improve genetic monitoring for conservation officers, as well as help wildlife forensic analysts improve evidentiary weight in wildlife crime cases that go to court.
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    (2014-03) Howard, Taylor E.; Kreutzer, McKensie; Curtis, Mary; Allen, Michael; Planz, John
    Purpose (a): The purpose of this study was to characterize by deep sequencing fifteen short tandem repeat (STR) loci in North American bald and golden eagles currently utilized by the US Fish and Wildlife Service. These STR loci are used for eagle identification in North America, however they were all developed from related species of European eagles. We hypothesized that using next-generation sequencing techniques we would be able to not only show concordance between allele calls using capillary electrophoresis (CE) methods and our new sequencing method, but also have an increased discrimination power of individuals by using this technique. Methods (b): Five samples of both bald and golden eagles were provided by the US Fish and Wildlife Service and deoxyribonucleic acid (DNA) was extracted from each using organic extraction. The current method of analysis through polymerase chain reaction (PCR) using fluorescent CE size based detection was adapted to create a protocol with increased specificity for the 15 sets of primers. Once the amplicon pool was developed, the amplicons were combined into a library using Library Prep Set for Ion Torrent™ kit for sequencing on the Ion Torrent™ Personal Genome Machine (PGM™) Sequencer® (Life Technologies™, Carlsbad, CA) with individual samples identified through barcodes embedded in the sequencing adaptors of the kit. After sequencing, the alleles were analyzed to identify the allele classification and determine the repeat structure of the STR motifs. The sequence data was used to determine if the sequences of North American population differed from existing reference sequences in GenBank®. Data was evaluated with the NextGENe® software using a virtual allelic ladder developed for each locus and contigs were assembled by anchoring the reads with the primer sequence. Results (c): Variants of alleles and allele distributions were recorded. The data revealed that the presence of sequence motif variation can increase the power of discrimination when using sequence analysis as compared to CE. Concordance was observed between the allele calls made using CE and with the sequence determined alleles. Conclusions (d): The method developed can be used to identify individual eagles with a higher discrimination power, which will be helpful in determining identity of individuals when needed for law enforcement investigations of harmed protected species such as bald and golden eagles.
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    (2014-03) Maji, Sayantan; Vishwanatha, Jamboor K.; Valapala, Mallika
    Neurodegenerative diseases like age related macular degeneration (AMD) and Retinitis Pigmentosa (RP) are major causes of blindness affecting millions of people around the world. One of the major reasons of cell death observed in these diseases is the increased accumulation of glutamate, an excitatory amino acid. Unfortunately, the mechanisms behind glutamate induced toxicity are not yet known. Here we are investigating a possible role of a protein Annexin A2 (AnxA2) in glutamate induced toxicity. We found that glutamate causes increased membrane translocation of AnxA2. Increased membrane localization of AnxA2 and thereby its function can lead to the death of the eye cells leading to degenerative diseases like AMD and RP. The present study shows one of the possible mechanisms that can lead to glutamate induced cell death of the eye. Thus, using AnxA2 targeted therapy as an adjunctive therapy can lead to better and more efficient outcomes. Purpose (a): Glutamate-induced intracellular increase in Ca2+ levels leads to the hyper-activation of several normal Ca2+-mediated physiological processes including the activation of intracellular kinases, phosphatases, phospholipases and proteases which contribute to the degeneration of the retinal neurons as seen in many diseases including age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Despite intensive research, the mechanisms that contribute to glutamate-induced cellular loss are yet to be elucidated. AnxA2, a Ca2+-dependent phospholipid binding protein serves as an extracellular proteolytic center by recruiting tissue plasminogen activator and plasminogen, and mediating localized generation of plasmin. We investigated whether AnxA2 plays a major role in glutamate induced neuronal excitotoxicity in a cone-photoreceptor cell line, 661W. Understanding the molecular mechanisms of glutamate-induced retinal degeneration can lead to the development of better therapeutic approaches for neurodegenerative diseases including AMD and RP. Our study provides new insights into one of the mechanisms that might contribute to glutamate-induced loss of photoreceptors in the retina. Methods (b): Ratiometric Ca2+ imaging and time lapse confocal microscopy were used to study glutamate-induced Ca2+ influx. EDTA eluates of 661W cells were immunoblotted to study the membrane translocation of endogenous as well as AnxA2-GFP in the presence or absence of different treatments. To determine whether glutamate induced membrane translocation of AnxA2 is dependent on the phosphorylation of the 23rd tyrosine residue or not, phosphomimetic and non-phosphomimetic variants were studied. Results (c): Glutamate translocated both endogenous and AnxA2-GFP to the cell surface in a process dependent on the activity of the NMDA receptor. Glutamate-induced translocation of AnxA2 is dependent on the phosphorylation of tyrosine 23 at the N-terminus and mutation of tyrosine 23 to a non-phosphomimetic variant inhibits the translocation process. The cell surface translocated AnxA2 forms an active plasmin-generating complex and this activity can be neutralized by a hexapeptide directed against the N-terminus. Conclusions (d): These results suggest an involvement of AnxA2 in potentiating glutamate-induced cell death processes. Thereby, targeting AnxA2 can be used as an adjunctive therapy in neurodegenerative diseases like AMD and RP.