Identification and localization of polyketide synthase genes from cultures of diarrheic shellfish poisoning toxin producing dinoflagellates of the genus Prorocentrum

Aquatic toxins are responsible for a number of acute and chronic diseases in humans. Okadaic acid (OA) and other dinoflagellate derived polyketide toxins pose serious health risks on a global scale. Ingestion of OA contaminated shellfish causes diarrheic shellfish poisoning (DSP). Some evidence also suggests tumor promotion in the liver by OA. Microcystin-LR (MC-LR) is produced by cyanobacteria and is believed to be the most common freshwater toxin in the US. Humans may be exposed to this acute hepatotoxin through drinking or recreational use of contaminated waters. ^ OA producing dinoflagellates have not been cultured axenically. The presence of associated bacteria raises questions about the ultimate source of OA. Identification of the toxin-producing organism(s) is the first step in identifying the biosynthetic pathways involved in toxin production. Polyketide synthase (PKS) genes of toxic and non-toxic species were surveyed by construction of clonal libraries from PCR amplicons of various toxic and non-toxic species of Prorocentrum in an effort to identify genes, which may be part of the biosynthetic pathway of OA. Analysis of the PKS sequences revealed that toxic species shared identical PKS genes not present in non-toxic species. Interestingly, the same PKS genes were identified in a library constructed from associated bacteria. ^ Subsequent bacterial small subunit RNA (16S) clonal libraries identified several common bacterial species. The most frequent 16S sequences found were identified as species of the genus Roseobacter which has previously been implicated in the production of OA. Attempts to culture commonly occurring bacteria resulted in the isolation of Oceanicaulis alexandrii , a novel marine bacterium previously isolated from the dinoflagellate Alexandrium tamarense, from both P. lima, and P. hoffmanianum. ^ Metabolic studies of microcystin-LR, were conducted to probe the activity of the major human liver cytochromes (CYP) towards the toxin. CYPs may provide alternate routes of detoxification of toxins when the usual routes have been inhibited. For example, some research indicates that cyanobacterial xenobiotics, in particular, lipopolysaccharides may inhibit glutathione S-transferases allowing the toxin to persist long enough to be acted upon by other enzymes. These studies found that at least one human liver CYP was capable of metabolizing the toxin. ^

Variables affecting the collection and preservation of human scent components through instrumental and biological evaluations

In certain European countries and the United States of America, canines have been successfully used in human scent identification. There is however, limited scientific knowledge on the composition of human scent and the detection mechanism that produces an alert from canines. This lack of information has resulted in successful legal challenges to human scent evidence in the courts of law. ^ The main objective of this research was to utilize science to validate the current practices of using human scent evidence in criminal cases. The goals of this study were to utilize Headspace Solid Phase Micro Extraction Gas Chromatography Mass Spectrometry (HS-SPME-GC/MS) to determine the optimum collection and storage conditions for human scent samples, to investigate whether the amount of DNA deposited upon contact with an object affects the alerts produced by human scent identification canines, and to create a prototype pseudo human scent which could be used for training purposes. ^ Hand odor samples which were collected on different sorbent materials and exposed to various environmental conditions showed that human scent samples should be stored without prolonged exposure to UVA/UVB light to allow minimal changes to the overall scent profile. Various methods of collecting human scent from objects were also investigated and it was determined that passive collection methods yields ten times more VOCs by mass than active collection methods. ^ Through the use of polymerase chain reaction (PCR) no correlation was found between the amount of DNA that was deposited upon contact with an object and the alerts that were produced by human scent identification canines. Preliminary studies conducted to create a prototype pseudo human scent showed that it is possible to produce fractions of a human scent sample which can be presented to the canines to determine whether specific fractions or the entire sample is needed to produce alerts by the human scent identification canines. ^

A digital signal processing approach for affective sensing of a computer user through pupil diameter monitoring

Recent research has indicated that the pupil diameter (PD) in humans varies with their affective states. However, this signal has not been fully investigated for affective sensing purposes in human-computer interaction systems. This may be due to the dominant separate effect of the pupillary light reflex (PLR), which shrinks the pupil when light intensity increases. In this dissertation, an adaptive interference canceller (AIC) system using the H∞ time-varying (HITV) adaptive algorithm was developed to minimize the impact of the PLR on the measured pupil diameter signal. The modified pupil diameter (MPD) signal, obtained from the AIC was expected to reflect primarily the pupillary affective responses (PAR) of the subject. Additional manipulations of the AIC output resulted in a processed MPD (PMPD) signal, from which a classification feature, PMPDmean, was extracted. This feature was used to train and test a support vector machine (SVM), for the identification of stress states in the subject from whom the pupil diameter signal was recorded, achieving an accuracy rate of 77.78%. The advantages of affective recognition through the PD signal were verified by comparatively investigating the classification of stress and relaxation states through features derived from the simultaneously recorded galvanic skin response (GSR) and blood volume pulse (BVP) signals, with and without the PD feature. The discriminating potential of each individual feature extracted from GSR, BVP and PD was studied by analysis of its receiver operating characteristic (ROC) curve. The ROC curve found for the PMPDmean feature encompassed the largest area (0.8546) of all the single-feature ROCs investigated. The encouraging results seen in affective sensing based on pupil diameter monitoring were obtained in spite of intermittent illumination increases purposely introduced during the experiments. Therefore, these results confirmed the benefits of using the AIC implementation with the HITV adaptive algorithm to isolate the PAR and the potential of using PD monitoring to sense the evolving affective states of a computer user.

A theoretical analysis of eyewitness identification: Dual -process theory, signal detection theory and eyewitness confidence

Given the growing number of wrongful convictions involving faulty eyewitness evidence and the strong reliance by jurors on eyewitness testimony, researchers have sought to develop safeguards to decrease erroneous identifications. While decades of eyewitness research have led to numerous recommendations for the collection of eyewitness evidence, less is known regarding the psychological processes that govern identification responses. The purpose of the current research was to expand the theoretical knowledge of eyewitness identification decisions by exploring two separate memory theories: signal detection theory and dual-process theory. This was accomplished by examining both system and estimator variables in the context of a novel lineup recognition paradigm. Both theories were also examined in conjunction with confidence to determine whether it might add significantly to the understanding of eyewitness memory. ^ In two separate experiments, both an encoding and a retrieval-based manipulation were chosen to examine the application of theory to eyewitness identification decisions. Dual-process estimates were measured through the use of remember-know judgments (Gardiner & Richardson-Klavehn, 2000). In Experiment 1, the effects of divided attention and lineup presentation format (simultaneous vs. sequential) were examined. In Experiment 2, perceptual distance and lineup response deadline were examined. Overall, the results indicated that discrimination and remember judgments (recollection) were generally affected by variations in encoding quality and response criterion and know judgments (familiarity) were generally affected by variations in retrieval options. Specifically, as encoding quality improved, discrimination ability and judgments of recollection increased; and as the retrieval task became more difficult there was a shift toward lenient choosing and more reliance on familiarity. ^ The application of signal detection theory and dual-process theory in the current experiments produced predictable results on both system and estimator variables. These theories were also compared to measures of general confidence, calibration, and diagnosticity. The application of the additional confidence measures in conjunction with signal detection theory and dual-process theory gave a more in-depth explanation than either theory alone. Therefore, the general conclusion is that eyewitness identifications can be understood in a more complete manor by applying theory and examining confidence. Future directions and policy implications are discussed. ^

Identification of Conserved Splicing Motifs in Mutually Exclusive Exons of 15 Insect Species

Background: During alternative splicing, the inclusion of an exon in the final mRNA molecule is determined by nuclear proteins that bind cis-regulatory sequences in a target pre-mRNA molecule. A recent study suggested that the regulatory codes of individual RNA-binding proteins may be nearly immutable between very diverse species such as mammals and insects. The model system Drosophila melanogaster therefore presents an excellent opportunity for the study of alternative splicing due to the availability of quality EST annotations in FlyBase. Methods: In this paper, we describe an in silico analysis pipeline to extract putative exonic splicing regulatory sequences from a multiple alignment of 15 species of insects. Our method, ESTs-to-ESRs (E2E), uses graph analysis of EST splicing graphs to identify mutually exclusive (ME) exons and combines phylogenetic measures, a sliding window approach along the multiple alignment and the Welch’s t statistic to extract conserved ESR motifs. Results: The most frequent 100% conserved word of length 5 bp in different insect exons was “ATGGA”. We identified 799 statistically significant “spike” hexamers, 218 motifs with either a left or right FDR corrected spike magnitude p-value < 0.05 and 83 with both left and right uncorrected p < 0.01. 11 genes were identified with highly significant motifs in one ME exon but not in the other, suggesting regulation of ME exon splicing through these highly conserved hexamers. The majority of these genes have been shown to have regulated spatiotemporal expression. 10 elements were found to match three mammalian splicing regulator databases. A putative ESR motif, GATGCAG, was identified in the ME-13b but not in the ME-13a of Drosophila N-Cadherin, a gene that has been shown to have a distinct spatiotemporal expression pattern of spliced isoforms in a recent study. Conclusions: Analysis of phylogenetic relationships and variability of sequence conservation as implemented in the E2E spikes method may lead to improved identification of ESRs. We found that approximately half of the putative ESRs in common between insects and mammals have a high statistical support (p < 0.01). Several Drosophila genes with spatiotemporal expression patterns were identified to contain putative ESRs located in one exon of the ME exon pairs but not in the other.

Identification of genes downstream of endothelin-3 signaling: characterization of WSB1 and SPC12

Signaling of endothelin-3 (Edn3) through its receptor, endothelin receptor B (EdnrB), has been shown to be indispensable for the development of certain neural crest derivatives. Since no research has been directed to investigate what the downstream targets of this signaling pathway are, the purpose of this study was to identify and characterize genes that are transcriptionally regulated by Edn3 signaling. Data from Differential Display RT-PCR of Edn-3 induced cDNA vs. non-induced cDNA obtained from primary neural crest cultures was analyzed. Thirty bands that were differentially expressed were sequenced and submitted for a homology search (BLAST). Among the genes identified were WSB 1 (a member of the SOCS family of negative regulators) and SPC 12 (the smallest subunit, 12kDa, of mammalian signal peptidase). Using whole-mount in-situ hybridization, the expression patterns of EdnrB, WSB 1 and SPC 12 were characterized. WSB 1 and SPC 12 expression patterns were found to overlap with that of EdnrB, suggesting that Edn3 might regulate the transcription of these genes in specific neural crest derived lineages.

A Digital Signal Processing Approach for Affective Sensing of a Computer User through Pupil Diameter Monitoring

Recent research has indicated that the pupil diameter (PD) in humans varies with their affective states. However, this signal has not been fully investigated for affective sensing purposes in human-computer interaction systems. This may be due to the dominant separate effect of the pupillary light reflex (PLR), which shrinks the pupil when light intensity increases. In this dissertation, an adaptive interference canceller (AIC) system using the H∞ time-varying (HITV) adaptive algorithm was developed to minimize the impact of the PLR on the measured pupil diameter signal. The modified pupil diameter (MPD) signal, obtained from the AIC was expected to reflect primarily the pupillary affective responses (PAR) of the subject. Additional manipulations of the AIC output resulted in a processed MPD (PMPD) signal, from which a classification feature, PMPDmean, was extracted. This feature was used to train and test a support vector machine (SVM), for the identification of stress states in the subject from whom the pupil diameter signal was recorded, achieving an accuracy rate of 77.78%. The advantages of affective recognition through the PD signal were verified by comparatively investigating the classification of stress and relaxation states through features derived from the simultaneously recorded galvanic skin response (GSR) and blood volume pulse (BVP) signals, with and without the PD feature. The discriminating potential of each individual feature extracted from GSR, BVP and PD was studied by analysis of its receiver operating characteristic (ROC) curve. The ROC curve found for the PMPDmean feature encompassed the largest area (0.8546) of all the single-feature ROCs investigated. The encouraging results seen in affective sensing based on pupil diameter monitoring were obtained in spite of intermittent illumination increases purposely introduced during the experiments. Therefore, these results confirmed the benefits of using the AIC implementation with the HITV adaptive algorithm to isolate the PAR and the potential of using PD monitoring to sense the evolving affective states of a computer user.

Waveguide microgripper for identification, sensing and manipulation

A Waveguide Microgripper utilizes flexible optical waveguides as gripping arms, which provide the physical means for grasping a microobject, while simultaneously enabling light to be delivered and collected. This unique capability allows extensive optical characterization of the structure being held such as transmission, reflection or fluorescence. One of the simplest capabilities of the waveguide microgripper is to be able to detect the presence of a microobject between the microgripper facets by monitoring the transmitted intensity of light coupled through the facets. The intensity of coupled light is expected to drop when there is an object obstructing the path of light. The optical sensing and characterization function of the microgripper is a strong function of the optical power incident on the structure of interest. Hence it is important to understand the factors affecting the power distribution across the facet. The microgripper is also capable of detecting the fluorescence. This capability of microgripper is expected to have applications in medical, bio-medical and related fields.

Variables Affecting the Collection and Preservation of Human Scent Components through Instrumental and Biological Evaluations

In certain European countries and the United States of America, canines have been successfully used in human scent identification. There is however, limited scientific knowledge on the composition of human scent and the detection mechanism that produces an alert from canines. This lack of information has resulted in successful legal challenges to human scent evidence in the courts of law. The main objective of this research was to utilize science to validate the current practices of using human scent evidence in criminal cases. The goals of this study were to utilize Headspace Solid Phase Micro Extraction Gas Chromatography Mass Spectrometry (HS-SPME-GC/MS) to determine the optimum collection and storage conditions for human scent samples, to investigate whether the amount of DNA deposited upon contact with an object affects the alerts produced by human scent identification canines, and to create a prototype pseudo human scent which could be used for training purposes. Hand odor samples which were collected on different sorbent materials and exposed to various environmental conditions showed that human scent samples should be stored without prolonged exposure to UVA/UVB light to allow minimal changes to the overall scent profile. Various methods of collecting human scent from objects were also investigated and it was determined that passive collection methods yields ten times more VOCs by mass than active collection methods. Through the use of polymerase chain reaction (PCR) no correlation was found between the amount of DNA that was deposited upon contact with an object and the alerts that were produced by human scent identification canines. Preliminary studies conducted to create a prototype pseudo human scent showed that it is possible to produce fractions of a human scent sample which can be presented to the canines to determine whether specific fractions or the entire sample is needed to produce alerts by the human scent identification canines.