Investigation of Substituted-Benzene Dopants for Charge Exchange Ionization of Nonpolar Compounds by Atmospheric Pressure Photoionization

Atmospheric pressure photoionization (APPI) using a dopant enables both polar and nonpolar compounds to be analyzed by LC/MS. To date, the charge exchange ionization pathway utilized for nonpolar compounds has only been efficient under restrictive conditions, mainly because the usual charge exchange reagent ions—the dopant photoions themselves—tend to be consumed in proton transfer reactions with solvent and/or dopant neutrals. This research aims to elucidate the factors affecting the reactivities of substituted-benzene dopant ions; another, overriding, objective is to discover new dopants for better implementing charge exchange ionization in reversed-phase LC/MS applications. The desirable properties for a charge exchange dopant include low reactivity of its photoions with solvent and dopant neutrals and high ionization energy (IE). Reactivity tests were performed for diverse substituted-benzene compounds, with substituents ranging from strongly electron withdrawing (EW) to strongly electron donating (ED). The results indicate that both the tendency of a dopant's photoions to be lost through proton transfer reactions and its IE depend on the electron donating/withdrawing properties of its substituent(s): ED groups decrease reactivity and IE, while EW groups increase reactivity and IE. Exceptions to the reactivity trend for dopants with ED groups occur when the substituent is itself acidic. All told, the desirable properties for a charge exchange dopant tend towards mutual exclusivity. Of the singly-substituted benzenes tested, chloro- and bromobenzene provide the best compromise between low reactivity and high IE. Several fluoroanisoles, with counteracting EW and ED groups, may also provide improved performance relative to the established dopants

Raman spectroscopic analysis of cell differentiation and death modes

Raman spectroscopy provides opportunities for non-invasive, non-destructive, label-free analysis of cell states based on changes in the biochemical composition of cells. We are investigating the suitability of Raman spectroscopy to assess the stages of human embryonic stem cell (hESC) differentiation towards pancreatic insulin-positive cells. Raman microspectrometry analysis has revealed macromolecular composition differences over time that distinguished cell populations differentiating to pancreatic cell types, such as by an increase in the protein-to-nucleic acid signal ratio and to distinguish the presence of insulin. Added insight into these macromolecular changes were provided by principal component analysis (PCA) of the data. However, the application of PCA can be difficult to interpret. The usefulness of non-negative matrix factorization was explored to improve the interpretability of overlapping Raman bands. We demonstrated the utility of this procedure by analyzing spectra to determine the cellular insulin or glucagon content. Thus, Raman spectroscopy can detect such differences in cells to detect the desired product as well as the potential to detect residual hESCs or the emergence of unwanted cells. We also investigated the suitability of Raman spectroscopy to detect the onset and types of cell death. Apoptotic, necrotic or autophagic Chinese Hamster Ovary cells were compared to uninduced cultures using Raman spectroscopy and PCA. Furthermore, uninduced cells were compared to cells sorted at different stages of apoptosis to determine how early the onset of apoptosis could be detected. Changes were observed in several peaks during the course of cell death, with repeated changes observed in nucleic acid- and lipid-associated peaks, enabling the distinction of cell death modes. Application of such death monitoring capabilities to cellular therapy cultures should be even more useful, given the need for more process analytical technologies to address the often more variable performance of these cultures, especially when adaptive control is needed for primary cell derived manufacturing

Process analytical utility of Raman spectroscopy for cell therapy manufacturing

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Acoustic cell washing and raman spectroscopy technologies To address cell therapy bioprocess challenges

Many organizations are confronting the challenges of economically ensuring the manufacture of safe and efficacious cell therapy products. These processes often depend on devices and methods that were developed for only related applications, such as blood cell processing or scientific research. Thus, we are in a window of opportunity to tailor innovative technologies to address the emerging specialized needs of cell therapy manufacturing. The most frequent unit operation is to wash cells between process stages, such as from DMSO containing cryopreservation medium to culture expansion medium. In particular for relatively small-scale autologous cell therapy processing, cell washing is imperfectly performed by closed system blood cell centrifuges or filters. We previously developed an acoustic cell separation device, widely used for over 15 years in CHO cell perfusion cultures. This technology acts as a non-fouling filter for months of operation, by using the forces generated in ultrasonic standing wave fields. These forces separate cells from medium based on differences in density and compressibility. Greater than 99.9% cell washing with 95% washed cell recovery efficiencies have been provided by our device. We also have recently enhanced the acoustic technology to perfuse 100 million cell/mL cultures, maintaining \u3e99% cell separation efficiencies. This provides an alternative high performance closed manufacturing system, to perfuse, concentrate and wash cells, with no physical filter barrier or mechanical moving parts. While many clinical trials have had few adverse events, the great promise of cellular therapies comes with grave risks, such as from potentially oncogenic pluripotent cells present in embryonic stem cell derived populations. There is an urgent need for process analytical technologies to non-invasively monitor mammalian cell populations and improve the reliability of manufactured cell products. This includes to monitor both the expected differentiation as well as to detect unexpected cells in the process. Recently, technological advances have led to an explosive growth in the capabilities of Raman spectroscopy, increasing the potential for novel applications. We are developing the use of this spectroscopic technique to track cell development, by measuring macromolecular changes in cell samples from cultures where stem cells are differentiated towards insulin-producing cells for the treatment of diabetes. Raman spectroscopy has great potential to provide continuous on-line assessment of cell quality during the manufacture of cell-derived therapeutic cells

Small Scale Structure at High Redshift: IV. Low Ionization Gas Intersecting Three Lines of Sight to Q2237+0305

We have obtained Keck HIRES spectra of three images of the gravitationally lensed QSO 2237+0305 to study low ionization absorption systems and their differences in terms of projected velocity and column density across the lines of sight. We detect CaII absorption from our Galaxy, and a system of High Velocity Clouds from the lensing galaxy (z=0.039). CaII components with total equivalent widths similar to those of Galactic intermediate and high velocity clouds are spread out over several hundred km/s at impact parameters of less than one kpc from the center of the lensing galaxy. We have also studied three low ionization MgII-FeII systems in detail. All three systems cover all three lines of sight, suggesting that the gaseous structures giving rise to MgII complexes are larger than about 0.5 kpc. However, in most cases it is difficult to trace individual MgII `cloudlets' over distances larger than 200-300 pc, indicating that typical sizes of the MgII cloudlets are smaller than the sizes inferred earlier for the individual CIV high ionization gas clouds. We tentatively interpret the absorption pattern of the strongest MgII system in terms of an expanding bubble or galactic wind and show that the possible loci occupied by the model bubble in radius-velocity space overlap with the observed characteristics of Galactic supershells. (abridged).Comment: 40 latex pages, 12 postscript figures. Accepted for publication by Ap

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Discovery and Fine-Mapping of Glycaemic and Obesity-Related Trait Loci Using High-Density Imputation

Reference panels from the 1000 Genomes (1000G) Project Consortium provide near complete coverage of common and low-frequency genetic variation with minor allele frequency ≥0.5% across European ancestry populations. Within the European Network for Genetic and Genomic Epidemiology (ENGAGE) Consortium, we have undertaken the fi

Comparison of Dopants for Charge Exchange Ionization of Nonpolar Polycyclic Aromatic Hydrocarbons with Reversed-Phase LC-APPI-MS

Atmospheric pressure photoionization (APPI) is capable of ionizing nonpolar compounds in LC/MS, through charge exchange reactions following photoionization of a dopant. Recently, several novel dopants—chlorobenzene, bromobenzene, 2,4-difluoroanisole, and 3-(trifluoromethyl)anisole—have been identified as having properties making them well-suited to serve as dopants for charge exchange ionization under reversed-phase LC conditions. Here, we report the results of experiments comparing their effectiveness to that of established dopants—toluene, anisole, and a toluene/anisole mixture, for the charge exchange ionization of model nonpolar compounds—the 16 polycyclic aromatic hydrocarbons (PAHs) identified by the US EPA as priority pollutants—when using a conventional reversed-phase LC method. Chloro- and bromobenzene were found to be much more effective than toluene for all the PAHs, due to the relatively low reactivity of their photoions with the solvent. Their overall performance was also better than that of anisole, due to anisole's ineffectiveness toward higher-IE compounds. Further, the experiments revealed that anisole's performance for higher-IE compounds can be dramatically improved by introducing it as a dilute solution in toluene, rather than neat. The two fluoroanisoles provided the highest overall sensitivity, by a slim margin, when introduced as dilute solutions in either chloro- or bromobenzene

The future of scholarly publishing : how open access is impacting the publishing landscape

Panel discussion exploring the impact of open access on the field of scholarly publishing, giving special consideration to academic libraries. The panel will feature experts from a variety of backgrounds including university presses and open education advocates, and begin to tie together diverse perspectives.Science, Faculty ofChemistry, Department ofEducation, Faculty ofLibrary, UBCOther UBCNon UBCUnreviewedFacultyOthe