Alternative Ionization Techniques for Analysis of Perfluoroalkyl Substances (PFAS) at Low Levels

Perfluoroalkyl substances (PFAS) are a group of compounds commonly used in industrial and consumer goods. As a result of their widespread use, these compounds have been found in all types of environmental samples. Current guidelines focus on PFAS contamination in water and require parts per trillion (ppt) detection. Liquid chromatography with mass spectrometry (LC-MS/MS) using electrospray ionization (ESI) is widely accepted as the standard technique for PFAS analysis in environmental samples. The growing concern for exposure to these persistent chemicals dictates that analytical testing push for even lower detection limits and methodology as new PFAS compounds are added to regulations. A strategy to reach lower detection limits would be to improve the ionization efficiency as compounds are introduced to the MS. For the legacy and emerging PFAS compounds, we have found that an alternate ionization, UniSpray, produced a boost in signal and an increased response in the MS. As new compounds are added to regulations, the standard LC-MS/MS with ESI will not be suitable. Neutral PFAS such as volatile fluorotelomer alcohols are analyzed with gas chromatography (GC)-MS and we have found that using an atmospheric pressure source coupled with GC allows us to detect these compounds at ppt levels.Ope

Mass spectrometry-based identification and quantitation of endogenous peptides in complex biological matrices

In the brain, chemical signaling molecules transfer information from cell-to-cell to modulate mood, behavior, and physiological functions like metabolism and circadian rhythm. The research presented in this dissertation is focused on characterizing a specific class of signaling molecules, endogenous signaling peptides. Despite being gene products, this class of signaling molecules is complex and diverse. Cell to cell signaling peptides are differentially processed, modified, and distributed depending on time and spatial location. The detection of the peptide complement in a biological sample is an analytical challenge due to the broad range of concentrations and diverse chemical properties of endogenous peptides. The presence of other classes of molecules, like salts and lipids, add to sample complexity and further complicates the characterization of signaling peptides. The ability to characterize endogenous peptides is dependent on the selection of appropriate, information-rich tools. In this dissertation, a set of mass spectrometry approaches coupled with optimized liquid chromatography separations and appropriate sample preparation were successfully utilized to measure chemical information for peptides in biological samples. Multiple MS platforms, each with distinct advantages and limitations, were utilized for identification and quantitation. To gain insight into the role of signaling peptides in a specific behavior, relative quantitation measurements with MS were performed in a non-targeted approach. Specifically, measurements were made for peptide level changes in the hippocampus and amygdala of mice to study the extinction of conditioned place preference (CPP) for cocaine in mice that have access to a running wheel after CPP training. MS measurements have yielded both known and novel candidate peptides that show relative abundance changes with exercise and context re-exposure, which yields insight into the biochemical mechanisms driving the observed extinguishment of CPP with running. Successful MS measurements of endogenous peptide measurements are dependent on appropriate and optimized sample preparation procedures. An optimized sampling procedure was developed and applied to detect the novel peptide alarin in the supernatant of a transfected cell line. While a synthetic version of the peptide spiked into the medium was detected by MS, the endogenous form of the peptide expressed by the cell line could not be unambiguously identified. Additionally, MS measurements were employed to evaluate the effects of different sample handling treatments on the detection of peptides in the mouse striatum and hypothalamus. The results of this preliminary study indicate for the sample treatments used, each has its limitations and the selection of an appropriate sample treatment in biological studies is dependent on the study goals and resources. This demonstrates the capabilities of mass spectrometric approaches to investigate peptide identities and dynamics, which can be utilized to enhance our understanding of the role of peptides in cell-to-cell signaling in the nervous system

Visitation to Cottonseed Storage Sites by Feral Swine and Evidence of Gossypol Exposure

Texas ranks first in U.S. cotton production, and southern Texas is a major region of production within the state. Within Kleberg County, for example, approximately 16,147 ha are planted in cotton annually, yielding approximately 68,200 bales, or 15,467 metric tons, of cotton (U.S. Department of Agriculture [USDA] 2009). Cotton producers have discovered new uses for cotton ginned by-products, such as hydro-mulch (Holt et al. 2005) used as a protein supplement for range livestock (DelCurto et al. 2000) and white-tailed deer (Odocoileus virginianus; Cooper 2006). Because of this, much of the materials are temporarily stored for later use

Growth factor therapy for Parkinson's disease: alternative delivery systems

Despite decades of research and billions in global investment, there remains no preventative or curative treatment for any neurodegenerative condition, including Parkinson’s disease (PD). Arguably, the most promising approach for neuroprotection and neurorestoration in PD is using growth factors which can promote the growth and survival of degenerating neurons. However, although neurotrophin therapy may seem like the ideal approach for neurodegenerative disease, the use of growth factors as drugs presents major challenges because of their protein structure which creates serious hurdles related to accessing the brain and specific targeting of affected brain regions. To address these challenges, several different delivery systems have been developed, and two major approaches—direct infusion of the growth factor protein into the target brain region and in vivo gene therapy—have progressed to clinical trials in patients with PD. In addition to these clinically evaluated approaches, a range of other delivery methods are in various degrees of development, each with their own unique potential. This review will give a short overview of some of these alternative delivery systems, with a focus on ex vivo gene therapy and biomaterial-aided protein and gene delivery, and will provide some perspectives on their potential for clinical development and translation

Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies

The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUC5B. ROA indicated that the protein backbone of MUC5B is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUC5B O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUC5B (D1-D2-D′-D3 domains, NT5B), C-terminal region of MUC5B (D4-B-C-CK domains, CT5B) and the Cys-domain (within the central mucin domain of MUC5B) were found to be dominated by the β-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUC5B molecule. Interestingly, the molecule was found to contain a large amount of α-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin

Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies

The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUC5B. ROA indicated that the protein backbone of MUC5B is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUC5B O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUC5B (D1-D2-D′-D3 domains, NT5B), C-terminal region of MUC5B (D4-B-C-CK domains, CT5B) and the Cys-domain (within the central mucin domain of MUC5B) were found to be dominated by the β-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUC5B molecule. Interestingly, the molecule was found to contain a large amount of α-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin

Childhood trauma as a mediator of the association between autistic traits and psychotic experiences evidence from the ALSPAC birth cohort

BACKGROUND: Little is known on whether associations between childhood autistic traits and psychotic experiences persist into adulthood and whether genetic confounding and childhood trauma influence them. Here we investigate the associations between childhood autistic traits and psychotic experiences until young adulthood and assess the influence of schizophrenia polygenic risk and childhood traumatic experiences, using the Avon Longitudinal Study of Parents and Children (ALSPAC) population-based birth cohort. STUDY DESIGN: We used a measure of broad autistic traits (autism factor mean score), and four dichotomised measures of autistic traits capturing social communication difficulties (age 7), repetitive behaviours (age 5), sociability (age 3), and pragmatic language (age 9). Psychotic experiences were assessed at ages 18 and 24 using the semi-structured Psychosis-Like Symptoms interview (PLIKSi). Traumatic experiences between ages 5 and 11 were assessed with questionnaires and interviews administered to children and parents at multiple ages. STUDY RESULTS: Broad autistic traits, as well as social communication difficulties, were associated with psychotic experiences that were distressing and/or frequent until age 24 (autism factor mean score, n = 3707: OR 1.19, 95%CI 1.01-1.39; social communication difficulties, n = 3384: OR 1.54, 95%CI 0.97-2.45). Childhood trauma mediated a substantial proportion of the identified associations (~28% and 36% respectively, maximum n = 3577). Schizophrenia polygenic risk did not appear to confound the associations. Multiple imputation analyses (maximum n = 13 105) yielded comparable results. CONCLUSIONS: Childhood trauma may be an important, potentially modifiable pathway between autistic features and later onset of psychotic psychopathology