Genetic and Environmental Factors that Mediate Survival of Prolonged Oxygen Deprivation in the Nematode Caenorhabditis Elegans

Ischemic events of even a very short duration are not tolerated Ill in humans. The human cost of ischemia, when looked at as combined cardiovascular disease, dwarfs all other causes of death in the United States. Annually, CVD kills as many people in the US as does cancer, chronic lower respiratory disease, accidents, and diabetes mellitus combined. In 2005 (the latest year for which final statistics are available), CVD was responsible for 864,480 deaths or 35.3 percent of total deaths for the year. In my study, I have used the nematode Caenorhabditis elegans to determine genetic and environmental modulators of oxygen deprivation a key component of ischemia. I have found that animals with mutations in insulin like signaling pathways, neuronal function, electron transport chain components, germline function, and animals that are preconditioned by being raised on a diet of E. coli HT115 bacteria at 25°C have an enhanced ability to survive long-term (>72 hours) anoxia (<.005 kPa O2) at 20°C. The enhanced anoxia survival phenotype partially correlates with increased levels of carbohydrate stores in the nematodes. Suppression of this enhanced anoxia survival phenotype is possible by altering expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, the FOXO transcription factor DAF-16, and 5’-AMP kinase

Collection and direct amplification methods using the GlobalFiler™ kit for DNA recovered from common pipe bomb substrates

When analyzing DNA from exploded pipe bombs, quantities are often in trace amounts, making DNA typing extremely difficult. Amplifying minute amounts of DNA can cause stochastic effects resulting in partial or uninterpretable profiles. Therefore, the initial DNA collection from “touch” evidence must be optimized to maximize the amount of DNA available for analysis. This proof-of-concept study evaluated two different swab types with two direct amplification strategies to identify the most effective method for recovering DNA from common pipe bomb substrates. PVC and steel pipes, electrical tape, and copper wire spiked with epithelial cells were swabbed with cotton or microFLOQ® Direct Swabs and amplified directly or via a pre-treatment prior to STR amplification. Not only was the microFLOQ® Direct Swab protocol the quickest method with the least risk of contamination, but in combination with direct amplification, the microFLOQ® Direct Swabs also generated the most complete STR profiles

Underlying Data for Sequencing the Mitochondrial Genome with the Massively Parallel Sequencing Platform Ion Torrent (TM) PGM (TM)

Background: Massively parallel sequencing (MPS) technologies have the capacity to sequence targeted regions or whole genomes of multiple nucleic acid samples with high coverage by sequencing millions of DNA fragments simultaneously. Compared with Sanger sequencing, MPS also can reduce labor and cost on a per nucleotide basis and indeed on a per sample basis. In this study, whole genomes of human mitochondria (mtGenome) were sequenced on the Personal Genome Machine (PGM (TM)) (Life Technologies, San Francisco, CA), the out data were assessed, and the results were compared with data previously generated on the MiSeq (TM) (Illumina, San Diego, CA). The objectives of this paper were to determine the feasibility, accuracy, and reliability of sequence data obtained from the PGM. Results: 24 samples were multiplexed (in groups of six) and sequenced on the at least 10 megabase throughput 314 chip. The depth of coverage pattern was similar among all 24 samples; however the coverage across the genome varied. For strand bias, the average ratio of coverage between the forward and reverse strands at each nucleotide position indicated that two-thirds of the positions of the genome had ratios that were greater than 0.5. A few sites had more extreme strand bias. Another observation was that 156 positions had a false deletion rate greater than 0.15 in one or more individuals. There were 31-98 (SNP) mtGenome variants observed per sample for the 24 samples analyzed. The total 1237 (SNP) variants were concordant between the results from the PGM and MiSeq. The quality scores for haplogroup assignment for all 24 samples ranged between 88.8%-100%. Conclusions: In this study, mtDNA sequence data generated from the PGM were analyzed and the output evaluated. Depth of coverage variation and strand bias were identified but generally were infrequent and did not impact reliability of variant calls. Multiplexing of samples was demonstrated which can improve throughput and reduce cost per sample analyzed. Overall, the results of this study, based on orthogonal concordance testing and phylogenetic scrutiny, supported that whole mtGenome sequence data with high accuracy can be obtained using the PGM platform.Peer reviewe

Environmental and Genetic Preconditioning for Long-Term Anoxia Responses Requires AMPK in Caenorhabditis elegans

Article on environmental and genetic preconditioning for long-term anoxia responses requires AMPK in Caenorhabditis elegans

Glyceraldehyde-3-Phosphate Dehydrogenase Mediates Anoxia Response and Survival in Caenorhabditis elegans

Oxygen deprivation has a role in the pathology of many human diseases. Thus it is of interest in understanding the genetic and cellular responses to hypoxia or anoxia in oxygen-deprivation-tolerant organisms such as Caenorhabditis elegans. In C. elegans the DAF-2/DAF-16 pathway, an IGF-1/insulin-like signaling pathway, is involved with dauer formation, longevity, and stress resistance. In this report we compared the response of wild-type and daf-2(e1370) animals to anoxia. Unlike wild-type animals, the daf-2(e1370) animals have an enhanced anoxia-survival phenotype in that they survive long-term anoxia and high-temperature anoxia, do not accumulate significant tissue damage in either of these conditions, and are motile after 24 hr of anoxia. RNA interference was used to screen DAF-16-regulated genes that suppress the daf-2(e1370)-enhanced anoxia-survival phenotype. We identified gpd-2 and gpd-3, two nearly identical genes in an operon that encode the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. We found that not only is the daf-2(e1370)-enhanced anoxia phenotype dependent upon gpd-2 and gpd-3, but also the motility of animals exposed to brief periods of anoxia is prematurely arrested in gpd-2/3(RNAi) and daf-2(e1370);gpd-2/3(RNAi) animals. These data suggest that gpd-2 and gpd-3 may serve a protective role in tissue exposed to oxygen deprivation

Correction to: Nuclear, chloroplast, and mitochondrial data of a US cannabis DNA database (International journal of legal medicine (2018) 132 3 (713-725))

The original version of this article contained a mistake. In page 10 of the original article, the significant level (p\ua0> 0.01) is incorrect. The correct significant level is (p\ua

Analysis of DNA from post-blast pipe bomb fragments for identification and determination of ancestry

Improvised explosive devices (IEDs) such as pipe bombs are weapons used to detrimentally affect people and communities. A readily accessible brand of exploding targets called Tannerite (R) has been identified as a potential material for abuse as an explosive in pipe bombs. The ability to recover and genotype DNA from such weapons may be vital in the effort to identify suspects associated with these devices. While it is possible to recover DNA from post-blast fragments using short tandem repeat markers (STRs), genotyping success can be negatively affected by low quantities of DNA, degradation, and/or PCR inhibitors. Alternative markers such as insertion/null (INNULs) and single nucleotide polymorphisms (SNPs) are bi-allelic genetic markers that are shorter genomic targets than STRs for amplification, which are more likely to resist degradation.In this study, we constructed pipe bombs that were spiked with known amounts of biological material to: 1) recover "touch" DNA from the surface of the device, and 2) recover traces of blood from the ends of wires (simulated finger prick). The bombs were detonated with the binary explosive Tannerite (R) using double-base smokeless powder to initiate the reaction.DNA extracted from the post-blast fragments was quantified with the Quantifiler (R) Trio DNA Quantification Kit. STR analysis was conducted using the GlobalFiler (R) Amplification Kit, INNULs were amplified using an early-access version of the InnoTyper (TM) 21 Kit, and SNP analysis via massively parallel sequencing (MPS) was performed using the HID-Ion Ampliseq (TM) Identity and Ancestry panels using the Ion Chef and Ion PGM sequencing system.The results of this study showed that INNUL markers resulted in the most complete genetic profiles when compared to STR and SNP profiles. The random match probabilities calculated for samples using INNULs were lower than with STRs when less than 14 STR alleles were reported. These results suggest that INNUL analysis may be well suited for low-template and/or degraded DNA samples, and may be used to supplement incomplete or failed STR analysis. Human identification using SNP analysis via MPS showed variable success with low-level post-blast samples in this study

The stability and persistence of blood and semen mRNA and miRNA targets for body fluid identification in environmentally challenged and laundered samples

The identification of body fluids in evidentiary stains may provide investigators with probative information during an investigation. In this study, quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays were performed to detect the presence of mRNA and miRNA in fresh and environmentally challenged samples. Blood, semen, and reference markers were chosen for both mRNA/miRNA testing. Samples of blood and semen were exposed to heat, humidity, and sunlight, and controlled conditions (room temperature, low humidity, and darkness) for 6 months. All mRNA targets were observed through six months under controlled conditions, but were undetected after 30 days in experimental conditions. However, miRNA targets persisted under all test conditions for the duration of the study. Additionally, cotton stained with blood or semen was laundered using a liquid detergent in various washing and drying conditions. An unstained cutting was evaluated for potential transfer. Both miRNA targets were observed in all stained samples regardless of the wash protocol used. Of the mRNA markers, HBB was detected in all bloodstained samples and PRM1 persisted in all but one semen stained sample. The unstained samples showed transfer of at least one body fluid specific miRNA marker in all samples and at least one body fluid specific mRNA in approximately half of the samples. These results support that RNA markers can be used for body fluid identification in challenging samples, and that miRNA markers may be more persistent than mRNA for blood and semen stains. However, some caution is warranted with laundered items due to possible transfer