Immuno-potentiating activity of surfactant vesicles in DNA and subunit vaccination

Enhanced immune responses for DNA and subunit vaccines potentiated by surfactant vesicle based delivery systems outlined in the present study, provides proof of principle for the beneficial aspects of vesicle mediated vaccination. The dehydration-rehydration technique was used to entrap plasmid DNA or subunit antigens into lipid-based (liposomes) or non-ionic surfactant-based (niosomes) dehydration-rehydration vesicles (DRV). Using this procedure, it was shown that both these types of antigens can be effectively entrapped in DRV liposomes and DRV niosomes. The vesicle size of DRV niosomes was shown to be twice the diameter (~2µm) of that of their liposome counterparts. Incorporation of cryoprotectants such as sucrose in the DRV procedure resulted in reduced vesicle sizes while retaining high DNA incorporation efficiency (~95%). Transfection studies in COS 7 cells demonstrated that the choice of cationic lipid, the helper lipid, and the method of preparation, all influenced transfection efficiency indicating a strong interdependency of these factors. This phenomenon has been further reinforced when 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): cholesteryl 3b- [N-(N’ ,N’ -dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol)/DNA complexes were supplemented with non-ionic surfactants. Morphological analysis of these complexes using transmission electron microscopy and environmental scanning electron microscopy (ESEM) revealed the presence of heterogeneous structures which may be essential for an efficient transfection in addition to the fusogenic properties of DOPE. In vivo evaluation of these DNA incorporated vesicle systems in BALB/c mice showed weak antibody and cell-mediated immune (CMI) responses. Subsequent mock challenge with hepatitis B antigen demonstrated that, 1-monopalmitoyl glycerol (MP) based DRV, is a more promising DNA vaccine adjuvant. Studying these DRV systems as adjuvants for the Hepatitis B subunit antigen (HBsAg) revealed a balanced antibody/CMI response profile on the basis of the HBsAg specific antibody and cytokine responses which were higher than unadjuvated antigen. The effect of addition of MP, cholesterol and trehalose 6,6’-dibehenate (TDB) on the stability and immuno-efficacy of dimethyldioctadecylammonium bromide (DDA) vesicles was investigated. Differential scanning calorimetry showed a reduction in transition temperature of DDA vesicles by ~12°C when incorporated with surfactants. ESEM of MP based DRV system indicated an increased vesicle stability upon incorporation of antigen. Adjuvant activity of these systems tested in C57BL/6j mice against three subunit antigens i.e., mycobacterial fusion protein- Ag85B-ESAT-6, and two malarial antigens - merozoite surface protein-1, (MSP1), and glutamate rich protein, (GLURP) revealed that while MP and DDA based systems induced comparable antibody responses, DDA based systems induced powerful CMI responses.

Prenatal environmental exposure and neurodevelopmentally important gene expression in malformed brain tissue from pediatric intractable epilepsy patients

The primary objective of this proposal was to determine whether mitochondrial oxidative stress and variation in a particular mtDNA lineage contribute to the risk of developing cortical dysplasia and are potential contributing factors in epileptogenesis in children. The occurrence of epilepsy in children is highly associated with malformations of cortical development (MCD). It appears that MCD might arise from developmental errors due to environmental exposures in combination with inherited variation in response to environmental exposures and mitochondrial function. Therefore, it is postulated that variation in a particular mtDNA lineage of children contributes to the effects of mitochondrial DNA damage on MCD phenotype. Quantitative PCR and dot blot were used to examine mitochondrial oxidative damage and single nucleotide polymorphism (SNP) in the mitochondrial genome in brain tissue from 48 pediatric intractable epilepsy patients from Miami Children’s Hospital and 11 control samples from NICHD Brain and Tissue Bank for Developmental Disorders. Epilepsy patients showed higher mtDNA copy number compared to normal health subjects (controls). Oxidative mtDNA damage was lower in non-neoplastic but higher in neoplastic epilepsy patients compared to controls. There was a trend of lower mtDNA oxidative damage in the non-neoplastic (MCD) patients compared to controls, yet, the reverse was observed in neoplastic (MCD and Non-MCD) epilepsy patients. The presence of mtDNA SNP and haplogroups did not show any statistically significant relationships with epilepsy phenotypes. However, SNPs G9804A and G9952A were found in higher frequencies in epilepsy samples. Logistic regression analysis showed no relationship between mtDNA oxidative stress, mtDNA copy number, mitochondrial haplogroups and SNP variations with epilepsy in pediatric patients. The levels of mtDNA copy number and oxidative mtDNA damage and the SNPs G9952A and T10010C predicted neoplastic epilepsy, however, this was not significant due to a small sample size of pediatric subjects. Findings of this study indicate that an increase in mtDNA content may be compensatory mechanisms for defective mitochondria in intractable epilepsy and brain tumor. Further validation of these findings related to mitochondrial genotypes and mitochondrial dysfunction in pediatric epilepsy and MCD may lay the ground for the development of new therapies and prevention strategies during embryogenesis.

Linking Old Librarianship to New: Aligning 5-Steps of The Innovator's DNA in Creating Thematic Discovery Systems for the Everglades

This poster presentation from the May 2015 Florida Library Association Conference, along with the Everglades Explorer discovery portal at http://ee.fiu.edu, demonstrates how traditional bibliographic and curatorial principles can be applied to: 1) selection, cross-walking and aggregation of metadata linking end-users to wide-spread digital resources from multiple silos; 2) harvesting of select PDFs, HTML and media for web archiving and access; 3) selection of CMS domains, sub-domains and folders for targeted searching using an API. Choosing content for this discovery portal is comparable to past scholarly practice of creating and publishing subject bibliographies, except metadata and data are housed in relational databases. This new and yet traditional capacity coincides with: Growth of bibliographic utilities (MarcEdit); Evolution of open-source discovery systems (eXtensible Catalog); Development of target-capable web crawling and archiving systems (Archive-it); and specialized search APIs (Google). At the same time, historical and technical changes – specifically the increasing fluidity and re-purposing of syndicated metadata – make this possible. It equally stems from the expansion of freely accessible digitized legacy and born-digital resources. Innovation principles helped frame the process by which the thematic Everglades discovery portal was created at Florida International University. The path -- to providing for more effective searching and co-location of digital scientific, educational and historical material related to the Everglades -- is contextualized through five concepts found within Dyer and Christensen’s “The Innovator’s DNA: Mastering the five skills of disruptive innovators (2011). The project also aligns with Ranganathan’s Laws of Library Science, especially the 4th Law -- to "save the time of the user.”

The Effect of Sample and Sample Matrix on DNA Processing: Mechanisms for the Detection and Management of Inhibition in Forensic Samples

The presence of inhibitory substances in biological forensic samples has, and continues to affect the quality of the data generated following DNA typing processes. Although the chemistries used during the procedures have been enhanced to mitigate the effects of these deleterious compounds, some challenges remain. Inhibitors can be components of the samples, the substrate where samples were deposited or chemical(s) associated to the DNA purification step. Therefore, a thorough understanding of the extraction processes and their ability to handle the various types of inhibitory substances can help define the best analytical processing for any given sample. A series of experiments were conducted to establish the inhibition tolerance of quantification and amplification kits using common inhibitory substances in order to determine if current laboratory practices are optimal for identifying potential problems associated with inhibition. DART mass spectrometry was used to determine the amount of inhibitor carryover after sample purification, its correlation to the initial inhibitor input in the sample and the overall effect in the results. Finally, a novel alternative at gathering investigative leads from samples that would otherwise be ineffective for DNA typing due to the large amounts of inhibitory substances and/or environmental degradation was tested. This included generating data associated with microbial peak signatures to identify locations of clandestine human graves. Results demonstrate that the current methods for assessing inhibition are not necessarily accurate, as samples that appear inhibited in the quantification process can yield full DNA profiles, while those that do not indicate inhibition may suffer from lowered amplification efficiency or PCR artifacts. The extraction methods tested were able to remove >90% of the inhibitors from all samples with the exception of phenol, which was present in variable amounts whenever the organic extraction approach was utilized. Although the results attained suggested that most inhibitors produce minimal effect on downstream applications, analysts should practice caution when selecting the best extraction method for particular samples, as casework DNA samples are often present in small quantities and can contain an overwhelming amount of inhibitory substances.

Isolation and Characterization of Microsatellite DNA Markers in the Deep-Sea Amphipod Paralicella tenuipes by Illumina MiSeq Sequencing

Funding This work was supported by the HADEEP projects, funded by the Nippon Foundation, Japan (2009765188), the Natural Environmental Research Council, UK (NE/E007171/1) and the Total Foundation, France. We acknowledge additional support from the Marine Alliance for Science and Technology for Scotland (MASTS) funded by the Scottish Funding Council (Ref: HR09011) and contributing institutions. We also acknowledge support from the Leverhulme Trust to SBP. Additional sea time was supported by NIWA’s ‘Impact of Resource Use on Vulnerable Deep-Sea Communities’ project (CO1_0906)

ENVIRONMENTAL INFLUENCES AND EPIGENETIC MECHANISMS IN RISK FOR DEPRESSION

Genetic and environmental factors interact to influence vulnerability for internalizing psychopathology, including Major Depressive Disorder (MDD). The mechanisms that account for how environmental stress can alter biological systems are not yet well understood yet are critical to develop more accurate models of vulnerability and targeted interventions. Epigenetic influences, and more specifically, DNA methylation, may provide a mechanism by which stress could program gene expression, thereby altering key systems implicated in depression, such as frontal-limbic circuitry and its critical role in emotion regulation. This thesis investigated the role of environmental factors from infancy and throughout the lifespan affecting the serotonergic (5-HT) system in the vulnerability to and treatment of depression and anxiety and potential underlying DNA methylation processes. First, we investigated the contributions of additive genetic vs. environmental factors on an early trait phenotype for depression (negative emotionality) in infants and their stability over time in the first 2 years of life. We provided evidence of the substantial contributions of both genetic and shared environmental factors to this trait, as well as genetically- and environmentally- mediated stability and innovation. Second, we studied how childhood environmental stress is associated with peripheral DNA methylation of the serotonin transporter gene, SLC6A4, as well as long-term trajectories of internalizing behaviours. There was a relationship between childhood psychosocial adversity and SLC6A4 methylation in males, as well as between SLC6A4 methylation and internalizing trajectory in both sexes. Third, we investigated changes in emotion processing and epigenetic modification of the SLC6A4 gene in depressed adolescents before and after Mindfulness-Based Cognitive Therapy (MBCT). The alterations from pre- to post-treatment in connectivity between the ACC and other network regions and SLC6A4 methylation suggested that MBCT may work to optimize the connectivity of brain networks involved in cognitive control of emotion as well as also normalize the relationship between SLC6A4 methylation and activation patterns in frontal-limbic circuitry. Our results from these three studies strengthen the theory that environmental influences are critical in establishing early vulnerability factors for MDD, driving epigenetic processes, and altering brain processes as an individual undergoes treatment, or experiences relapse.

Endonuclease Controlled Aggregation of Gold Nanoparticles for the Ultrasensitive Detection of Pathogenic Bacterial DNA

The development of an ultrasensitive biosensor for the low-cost and on-site detection of pathogenic DNA could transform detection capabilities within food safety, environmental monitoring and clinical diagnosis. Herein, we present an innovative approach exploiting endonuclease-controlled aggregation of plasmonic gold nanoparticles (AuNPs) for label-free and ultrasensitive detection of bacterial DNA. The method utilizes RNA-functionalized AuNPs which form DNA-RNA heteroduplex structures through specific hybridization with target DNA. Once formed, the DNA-RNA heteroduplex is susceptible to RNAse H enzymatic cleavage of the RNA probe, allowing the target DNA to liberate and hybridize with another RNA probe. This continuously happens until all of the RNA probes are cleaved, leaving the nanoparticles unprotected and thus aggregated upon exposure to a high electrolytic medium. The assay is ultrasensitive, allowing the detection of target DNA at femtomolar level by simple spectroscopic analysis (40.7 fM and 2.45 fM as measured by UV-vis and dynamic light scattering (DLS), respectively). The target DNA spiked food matrix (chicken meat) is also successfully detected at a concentration of 1.2 pM (by UV-vis) or 18.0 fM (by DLS). In addition to the ultra-high sensitivity, the total analysis time of the assay is less than 3 hours, thus demonstrating its practicality for food analysis.

Parental exposure to the herbicide diuron results in oxidative DNA damage to germinal cells of the Pacific oyster Crassostrea gigas

Chemical pollution by pesticides has been identified as a possible contributing factor to the massive mortality outbreaks observed in Crassostrea gigas for several years. A previous study demonstrated the vertical transmission of DNA damage by subjecting oyster genitors to the herbicide diuron at environmental concentrations during gametogenesis. This trans-generational effect occurs through damage to genitor-exposed gametes, as measured by the comet-assay. The presence of DNA damage in gametes could be linked to the formation of DNA damage in other germ cells. In order to explore this question, the levels and cell distribution of the oxidized base lesion 8-oxodGuo were studied in the gonads of exposed genitors. High-performance liquid chromatography coupled with UV and electrochemical detection analysis showed an increase in 8-oxodGuo levels in both male and female gonads after exposure to diuron. Immunohistochemistry analysis showed the presence of 8-oxodGuo at all stages of male germ cells, from early to mature stages. Conversely, the oxidized base was only present in early germ cell stages in female gonads. These results indicate that male and female genitors underwent oxidative stress following exposure to diuron, resulting in DNA oxidation in both early germ cells and gametes, such as spermatozoa, which could explain the transmission of diuron-induced DNA damage to offspring. Furthermore, immunostaining of early germ cells seems indicates that damages caused by exposure to diuron on germ line not only affect the current sexual cycle but also could affect future gametogenesis.

Neuropsychiatric disease (NPD) clustering in families with Autism Spectrum Disorder (ASD): genetic, epigenetic and environmental issues

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication/interaction and by unusual repetitive and restricted behaviors and interests. ASD often co-occurs in the same families with other neuropsychiatric diseases (NPD), such as intellectual disability, schizophrenia, epilepsy, depression and attention deficit hyperactivity disorder. Genetic factors have an important role in ASD etiology. Multiple copy number variants (CNVs) and single nucleotide variants (SNVs) in candidate genes have been associated with an increased risk to develop ASD. Nevertheless, recent heritability estimates and the high genotypic and phenotypic heterogeneity characteristic of ASD indicate a role of environmental and epigenetic factors, such as long noncoding RNA (lncRNA) and microRNA (miRNA), as modulators of genetic expression and further clinical presentation. Both miRNA and lncRNA are functional RNA molecules that are transcribed from DNA but not translated into proteins, instead they act as powerful regulators of gene expression. While miRNA are small noncoding RNAs with 22-25 nucleotides in length that act at the post-transcriptional level of gene expression, the lncRNA are bigger molecules (>200 nucleotides in length) that are capped, spliced, and polyadenylated, similar to messenger RNA. Although few lncRNA were well characterized until date, there is a great evidence that they are implicated in several levels of gene expression (transcription/post-transcription/post-translation, organization of protein complexes, cell– cell signaling as well as recombination) as shown in figure 1.

PCR-Internal Transcribed Spacer (ITS) genes sequencing and phylogenetic analysis of clinical and environmental Aspergillus species associated with HIV-TB co infected patients in a hospital in Abeokuta, southwestern Nigeria.

Background: Aspergillosis has been identified as one of the hospital acquired infections but the contribution of water and inhouse air as possible sources of Aspergillus infection in immunocompromised individuals like HIV-TB patients have not been studied in any hospital setting in Nigeria. Objective: To identify and investigate genetic relationship between clinical and environmental Aspergillus species associated with HIV-TB co infected patients. Methods: DNA extraction, purification, amplification and sequencing of Internal Transcribed Spacer (ITS) genes were performed using standard protocols. Similarity search using BLAST on NCBI was used for species identification and MEGA 5.0 was used for phylogenetic analysis. Results: Analyses of sequenced ITS genes of selected fourteen (14) Aspergillus isolates identified in the GenBank database revealed Aspergillus niger (28.57%), Aspergillus tubingensis (7.14%), Aspergillus flavus (7.14%) and Aspergillus fumigatus (57.14%). Aspergillus in sputum of HIV patients were Aspergillus niger, A. fumigatus, A. tubingensis and A. flavus. Also, A. niger and A. fumigatus were identified from water and open-air. Phylogenetic analysis of sequences yielded genetic relatedness between clinical and environmental isolates. Conclusion: Water and air in health care settings in Nigeria are important sources of Aspergillus sp. for HIV-TB patients.

Environmental variables, habitat discontinuity and life history shaping the genetic structure of Pomatoschistus marmoratus

Coastal lagoons are semi-isolated ecosystems exposed to wide fluctuations of environmental conditions and showing habitat fragmentation. These features may play an important role in separating species into different populations, even at small spatial scales. In this study, we evaluate the concordance between mitochondrial (previous published data) and nuclear data analyzing the genetic variability of Pomatoschistus marmoratus in five localities, inside and outside the Mar Menor coastal lagoon (SE Spain) using eight microsatellites. High genetic diversity and similar levels of allele richness were observed across all loci and localities, although significant genic and genotypic differentiation was found between populations inside and outside the lagoon. In contrast to the FST values obtained from previous mitochondrial DNA analyses (control region), the microsatellite data exhibited significant differentiation among samples inside the Mar Menor and between lagoonal and marine samples. This pattern was corroborated using Cavalli-Sforza genetic distances. The habitat fragmentation inside the coastal lagoon and among lagoon and marine localities could be acting as a barrier to gene flow and contributing to the observed genetic structure. Our results from generalized additive models point a significant link between extreme lagoonal environmental conditions (mainly maximum salinity) and P. marmoratus genetic composition. Thereby, these environmental features could be also acting on genetic structure of coastal lagoon populations of P. marmoratus favoring their genetic divergence. The mating strategy of P. marmoratus could be also influencing our results obtained from mitochondrial and nuclear DNA. Therefore, a special consideration must be done in the selection of the DNA markers depending on the reproductive strategy of the species.

Genetic diversity and connectivity remain high in Holothuria polii (Delle Chiaje 1823) across a coastal lagoon-open sea environmental gradient

Coastal lagoons represent habitats with widely heterogeneous environmental conditions, particularly as regards salinity and temperature,which fluctuate in both space and time. These characteristics suggest that physical and ecological factors could contribute to the genetic divergence among populations occurring in coastal lagoon and opencoast environments. This study investigates the genetic structure of Holothuria polii at a micro-geographic scale across theMar Menor coastal lagoon and nearbymarine areas, estimating the mitochondrial DNA variation in two gene fragments, cytochrome oxidase I (COI) and 16S rRNA (16S). Dataset of mitochondrial sequences was also used to test the influence of environmental differences between coastal lagoon andmarine waters on population genetic structure. All sampled locations exhibited high levels of haplotype diversity and low values of nucleotide diversity. Both genes showed contrasting signals of genetic differentiation (non-significant differences using COI and slight differences using 16S, which could due to different mutation rates or to differential number of exclusive haplotypes. We detected an excess of recent mutations and exclusive haplotypes, which can be generated as a result of population growth. However, selective processes can be also acting on the gene markers used; highly significant generalized additive models have been obtained considering genetic data from16S gene and independent variables such as temperature and salinity.

The effect of sample and sample matrix on DNA processing: Mechanisms for the detection and management of inhibition in forensic samples

The presence of inhibitory substances in biological forensic samples has, and continues to affect the quality of the data generated following DNA typing processes. Although the chemistries used during the procedures have been enhanced to mitigate the effects of these deleterious compounds, some challenges remain. Inhibitors can be components of the samples, the substrate where samples were deposited or chemical(s) associated to the DNA purification step. Therefore, a thorough understanding of the extraction processes and their ability to handle the various types of inhibitory substances can help define the best analytical processing for any given sample. A series of experiments were conducted to establish the inhibition tolerance of quantification and amplification kits using common inhibitory substances in order to determine if current laboratory practices are optimal for identifying potential problems associated with inhibition. DART mass spectrometry was used to determine the amount of inhibitor carryover after sample purification, its correlation to the initial inhibitor input in the sample and the overall effect in the results. Finally, a novel alternative at gathering investigative leads from samples that would otherwise be ineffective for DNA typing due to the large amounts of inhibitory substances and/or environmental degradation was tested. This included generating data associated with microbial peak signatures to identify locations of clandestine human graves. Results demonstrate that the current methods for assessing inhibition are not necessarily accurate, as samples that appear inhibited in the quantification process can yield full DNA profiles, while those that do not indicate inhibition may suffer from lowered amplification efficiency or PCR artifacts. The extraction methods tested were able to remove >90% of the inhibitors from all samples with the exception of phenol, which was present in variable amounts whenever the organic extraction approach was utilized. Although the results attained suggested that most inhibitors produce minimal effect on downstream applications, analysts should practice caution when selecting the best extraction method for particular samples, as casework DNA samples are often present in small quantities and can contain an overwhelming amount of inhibitory substances.^

ENVIRONMENTAL INFLUENCES AND EPIGENETIC MECHANISMS IN RISK FOR DEPRESSION

Thesis (Ph.D, Psychology) -- Queen's University, 2016-10-04 17:37:07.888

Life history & environmental effects on telomere dynamics in Atlantic salmon

While much of the study of molecular biology inevitably focuses on the parts of the genome that contain active genes, there are also non-coding regions that nonetheless play an essential role in maintaining genome integrity. One such region are telomeres, which cap the ends of all eukaryotic chromosomes and play an important role in chromosome protection. Telomere loss occurs at each cell division as a result of the ‘end replication problem’ and a relatively short telomere length is indicative of poor biological state. Thus far, the majority of studies on the dynamics and role of telomeres have been biased towards certain taxa. Research to date has mostly focussed on humans, other mammals and birds. There has been far less research on the telomere dynamics of ectotherms. It is important that we do so, especially since ectothermic vertebrates do not seem to down-regulate telomerase expression in the same way as endotherms, suggesting that their telomere dynamics may be less predictable in the later life stages. The main objective of this thesis was therefore to investigate how life history and environmental effects may influence telomere dynamics in Atlantic salmon Salmo salar. I carried out carefully designed experiments, both in the laboratory and in the wild, using a longitudinal approach where possible, in order to address a number of specific questions that are connected to this central theme. In chapter 2, I demonstrate that there can be significant links between parental life history and offspring telomere dynamics. Maternal life history traits, in particular egg size, were most strongly related to offspring telomere length at the embryonic stages. Paternal life history traits, such as early life growth rate, had a greater association with offspring telomere dynamics in the later stages of development. In chapter 3, using a wild Atlantic salmon population, I found that most individuals experienced a reduction in telomere length during the migratory phase of their life cycle; however the relative rate of telomere loss was dependent on sex, with males experiencing a relatively greater loss. Unexpectedly, I also found that juvenile salmon that had the shortest telomeres at the time of outward migration, had the greatest probability of surviving through to the return migration. In chapter 4, again using a wild system involving experimental manipulations of juvenile Atlantic salmon in Scottish streams, I found that telomere length in juvenile fish was influenced by parental traits and by direct environmental effects. Faster-growing fish had shorter telomeres and there was a greater cost (in terms of reduced telomere length) if the growth occurred in a harsher environment. I also found a positive association between offspring telomere length and the growth history of their fathers (but not mothers), represented by the number of years that fathers had spent at sea. Chapter 5 explored the hypotheses that oxidative DNA damage, catalase (CAT) antioxidant activity and cell proliferation rate are underlying mechanisms linking incubation temperature and telomere dynamics in salmon embryos. No evidence was found for any such effects, but telomere lengths in salmon embryos were found to be significantly affected by the temperature of the water in which they were living. There is also evidence that telomere length significantly increases during embryonic development. In summary, this thesis has shown that a complex mix of environmental and parental effects appear to influence telomere dynamics in Atlantic salmon, with parental effects especially evident during early life stages. It also demonstrated that telomeres lengthen through the embryo stages of development before reducing once the fry begin feeding, indicating that the patterns of telomere loss commonly found in endotherms may differ in ectotherms. Reasons for this variation in telomere dynamics are presented in the final Discussion chapter of the thesis.