1,720,967 research outputs found
Patterns of Degradation in Prm1 and Semg1 Transcripts in Semen During 6 Months of Aging
No full textThe purpose of this study was to study the degradation rates of mRNA molecules in semen stains as a possible approach to estimating the age of semen samples found at crime scenes. Specific primers directing amplicons of different lengths from transcripts from the sperm specific PRM1 gene and the seminal fluid specific SEMG1 gene were designed and synthesized. Using two fluid-specific primers allowed for the investigation of the difference in degradation rates between sperm and seminal fluid. No previous study had a large enough sample size to investigate if there is a difference in degradation rate among individuals. In this study, ten young adult males provided semen samples which were dried in 50�l aliquots and stored under controlled conditions. Aliquots were extracted using TRizol every 2 weeks for 6 months to obtain RNA for analysis. The degradation of 18S rRNA and GAPDH transcripts was analyzed as possible stable control transcripts along with PRM1 and SEMG1. 18S started showing increased degradation at 14 weeks while GAPDH seemed to having an increase in degradation over 6 months of aging. Degradation in control genes prevented meaningful statistical analysis from being completed. Looking at raw CT values there was great variation of mRNA concentration between individuals and time-points, but overall PRM1 and SEMG1 showed little to no indication of degradation from 0 to 6 months of aging. It is possible PRM1 and SEMG1 could still be useful in determining the age of semen samples if the samples were over 6 month of age. Additional research with a longer time course needs to be conducted to determine at what time PRM1 and SEMG1 start degrading quickly enough to be used to determine that age of semen samples.Forensic Scienc
Evaluation of Collection Methods for Extraction of Trace Amounts of DNA from Cloth Substrates
No full textDNA analysis has become the golden standard in many crime laboratories around the world. As technology advances, new possibilities arise in using evidence left at crime scenes, touch DNA being one of these possibilities. Touch DNA, available in trace amounts, is often available on surfaces handled at a crime scene. Due to the limited amount of DNA in these samples, recovery efficiency is crucial if the samples are to be used as evidence in an investigation. Fabric was chosen as a substrate for its high prevalence in everyday life. A common technique of recovery from these materials is cutting extraction, however several additional methods are available. A total of 5 methods (cutting, tape lifting, and 3 swab types) were evaluated for their efficiency in recovery of DNA from these fabric substrates. Known amounts of DNA were spotted onto marked fabric during the first portion of the study in order to estimate the percentage of DNA recovered. In the second portion, volunteers were asked to wear garments to put this information into real world perspective. While cutting was the best option for most of the substrates in the laboratory portion, no one method showed greater efficiency among all the garments in the real world portion. The majority of samples from the garments were sufficient for STR typing, according to the limit of detection given by the PowerPlex multi-locus STR kit.Forensic Scienc
Detection of RNA methylation patterns in forensically relevant transcripts in dried bloodstains
No full textRNA degradation kinetics can be used to estimate the age of a biological sample found at a crime scene. RNA sequencing of transcripts from various tissue types shows that degradation occurs faster at the 5’ end than the 3’ end. This discovery led to the development of the 5’-3’ assay, which quantifies and compares each end of a transcript to estimate sample age. This assay has been validated on dried bloodstains, however why the 5’ end of the transcript degrades faster than the 3’ end remains unknown. As this phenomenon is being observed in dried bloodstains, we hypothesize that chemical modifications of RNA molecules may be playing a role. Increasing research shows that methylation of RNA molecules can alter the kinetics of RNA degradation, leading to either increased or decreased transcript stability, dependent on the RNA binding proteins (RBPs) present. This study aims to investigate the methylation patterns in forensically relevant transcripts in dried bloodstains via next generation sequencing (NGS) using Oxford Nanopore Technologies’ (ONT) native RNA sequencing protocol followed by bioinformatic analysis. We also developed a novel RNA enrichment technique that utilizes 120 nucleotide DNA probes designed to hybridize to the 5’ or 3’ end of a transcript for selected target enrichment. We detected possible modifications on twelve transcripts sequenced from dried bloodstains, five of which were identified on more than one sample. Although the results of this study are preliminary due to the lack of controls for statistical comparison, the duplicate modifications identified strengthens the overall findings. The results provided here can serve as the basis for future studies aiming to interrogate the epitranscriptome of forensically relevant samples to further optimize RNA-based assays for implementation into the field of forensic science
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Estimation and analysis of allelic drop-out of sequenced autosomal STRs
No full textIntroduction: Sequencing of autosomal STRs through massively parallel sequencing (MPS) is being adopted by laboratories world-wide. With new technologies, proper interpretation and statistical analysis methods must be updated. As the majority of samples processed in forensic labs consist of low-level DNA, data loss must be appropriately considered during these processes. The purpose of this study was to explore allelic and locus drop-out in data produced through MPS methods and evaluate possible probabilistic models for allelic drop-out prediction. Methods: Three data sets produced using the ForenSeqᵀᴹ DNA Signature Prep Kit on the MiSeq FGxᵀᴹ were procured from two laboratories. Samples consisted of mostly cell line single source genetic profiles. Both Primer Mix A and B were included using manufacturer protocols and a modified protocol using Primer Mix B. Library or flow cell, sample, and locus specific parameters were investigated for correlation with drop-out of allelic data. Correlation statistics were performed, and top predictor variables were used to generate potential models using both logistic regression analysis and a logistic or exponential relationship with drop-out rates. Top models were further evaluated for accuracy and robustness. Results: Library and flow cell specific parameters influenced drop-out, however, they were not considered in modeling due to run-to-run inconsistencies. Total read depth of a sample was found to be highly correlated with drop-out rates of an entire sample. Locus specific variables including amplification efficiency, template, and total locus coverage were found to be promising predictors. Further model evaluation suggested total locus coverage combined with total sample reads was useful in predicting allelic drop-out. Total read count was found to have a positive Brier score when using a logistic or exponential relationship with drop-out rates. Evaluation of the predictive capabilities of the locus dependent model showed favorable prediction when true allelic drop-out did not occur and intermediate support for when drop-out occurred. Conclusion: While these models both have their limitations, this information can be used by software developers or laboratories that are working to adopt massively parallel sequencing. Proper protocols or analyses software can be built for interpretation of low-level DNA samples that exhibit allelic drop-out
Archaea in mammalian gut microbiomes
No full textArchaea are the most enigmatic of the three domains of life. Most archaea are extremophiles found in highly acidic, high-salt, or high-temperature environments. However, members of the archaea also have been discovered in animal and human intestines, albeit their functional roles in host health or disease are poorly understood. Interestingly, archaea are now considered as indigenous microorganisms of the human gut microbiota and their biological importance has recently been reevaluated. Targeted sequencing of the 16S rRNA gene, metagenomic shotgun sequencing, or other metaomic approaches are used to define the composition, activity, and dynamics of the microbial communities. Archaea themselves are not known to be pathogenic; however. the presence of methanogens, a group of archaea in the human gut, has been linked to several digestive disorders such as IBD and IBS and metabolic diseases such as obesity and diabetes. Our study aimed to mine rat 16S rRNA gene amplicon and shotgun metagenomic sequencing datasets in our laboratory for the presence of archaeal sequence reads. Additionally, we investigated whether these sequences are present in 16S sequencing data from pig fecal DNA samples. Taxonomic profiling workflows in the Qiagen CLC Genomics Workbench were used to elucidate the relative abundances of archaeal and bacterial reads in the sequence datasets. Our results showed that archaeal sequences were either absent or present in extremely low abundance in the investigated rat metagenomes. In contrast, we found evidence that methanogens were abundant in sow and piglet fecal microbiotas. The correlation of sow and offspring archaea profiles was evaluated using longitudinal sequence data and quantitative PCR with group-specific primers. The genus Methanobrevibacter was dominant in sows and piglets, while Methanosphaera and Methanomethylophilaceae showed disparate abundances. Future studies will include investigation of archaeome inheritance and development as well as study of archaea-bacteria co-occurrence networks and correlations with host health status
Apoptotic and bioenergetic changes in pancreatic cells after exposure to individual toxicants and their mixtures
No full textPancreatic cancer has one of the worst fatality rates in oncology. Despite advances in treatment and recovery rates for other cancers, pancreatic cancer's lack of early symptomology culminates in a dismal five-year survival rate of 3-9% that has remained unchanged for decades. Cadmium has been implicated in the initiation of pancreatic cancer. Its prevalence in the environment make it a candidate for interactions with pesticides that have gone relatively unexplored. In this study, we examined the role of cadmium, pesticides, and their mixtures on the p53 apoptotic pathway and in the adaptive bioenergetics that foretell transition to a cancer state. We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to establish a No Observable Adverse Effect Limit (NOAEL) which allowed us to identify sub-toxic chronic exposure concentrations to emulate realistic combination exposures for molecular evaluation of biological endpoints. These concentrations were used to examine p53 recruitment and downstream apoptotic markers phosphatidylserine (PS) and caspase 3/7 activity. Bioenergetic shifts were assessed by mitochondrial toxicity assays in the presence of glucose or galactose and evaluated in conjunction with mitochondrial membrane potential (MMP), oxygen consumption rates, and reactive oxygen species (ROS) production. Non-linear regression analysis led to the employment of 500nM Cd and 1uM Atrazine and Glyphosate test concentrations. Cadmium drove p53 response in HPNE cells while glyphosate and both mixtures increased p53 expression in tumor cells. Interestingly, only cadmium exhibited increased PS by annexin labeling, but mixtures showed significantly less PS, indicating a potential reinforcement of membrane integrity. No treatment groups in either cell line responded to treatment by increases in caspase activity. Taken together, we can surmise that the p53 apoptotic pathway is not initiated by exposure to these toxicants. Although ROS production was elevated in all groups and glutathione response was unaffected, it is unlikely the mild increase is responsible for differences in mitochondrial health. Summation of mitochondrial health using ATP production with cell membrane analysis is the lone parameter where mixtures behaved differently from their parts. Both mixtures distinctly presented as mitochondrial toxins and remodeled metabolism in a manner similar to that found in tumorigenic cells, indicating a potential pathway to carcinogenicity
- …
