Assay, Purity, and Impurity Profile of Phosphorothioate Oligonucleotide Therapeutics by Ion Pair-HPLC-MS.

The relatively large molecular size, diastereoisomeric nature, and complex impurity profiles of therapeutic phosphorothioate oligonucleotides create significant analytical challenges for the quality control laboratory. To overcome the lack of selectivity inherent to traditional chromatographic approaches, an ion pair liquid chromatography-mass spectrometry (LCMS) method combining ultraviolet and mass spectrometry quantification was developed and validated for >35 different oligonucleotide drug substances and products, including several commercialized drugs. The selection of chromatographic and spectrometric conditions, data acquisition and processing, critical aspects of sample and buffer preparation and instrument maintenance, and results from method validation experiments are discussed.

Knowledge mobilization tool to promote, protect, and support breastfeeding during COVID-19.

SETTING: This knowledge mobilization project was conceptualized to increase awareness among breastfeeding mothers and the general public on safe infant feeding practices during the COVID-19 pandemic by addressing myths and misconceptions associated with breastfeeding practices, guiding breastfeeding mothers to make informed decisions around child feeding practices, and offering meaningful guidance in simple language through a short online animated video. INTERVENTION: This project was undertaken in four phases. During phase 1, an informal discussion was held with the breastfeeding mothers, service providers, and community partner in identifying issues surrounding lactation counselling facilities during the COVID-19 pandemic. During phase 2, recommendations from 23 organizations with regard to breastfeeding during COVID-19 were reviewed and analyzed. During phase 3, using evidence from reliable sources, a 5-minute animated e-resource on breastfeeding during COVID-19 was conceptualized and developed. During phase 4, the e-resource was disseminated to the breastfeeding mothers, general public, post-secondary institutions, and organizations providing services to breastfeeding mothers in Canada. OUTCOMES: This evidence-based e-resource facilitated addressing misconceptions around breastfeeding during COVID-19 and raising public awareness on safe infant feeding practices during this pandemic. Overall, the video was described as an informative, user-friendly, useful, and easily accessible resource by breastfeeding mothers who were in self-isolation with little access to healthcare services during the pandemic. IMPLICATIONS: This project highlighted the importance of patient engagement and collaboration with the community partner in protecting breastfeeding during the COVID-19 pandemic. It further illustrated how informational e-resources can protect breastfeeding in situations where breastfeeding mothers' access to healthcare services is compromised.

RéSUMé: LIEU: Notre projet de mobilisation des connaissances vise à sensibiliser les mères allaitantes et le grand public aux pratiques d'alimentation sûres pour les nourrissons durant la pandémie de COVID-19 en abordant les mythes et les idées fausses associés aux pratiques d'allaitement maternel, en aidant les mères allaitantes à prendre des décisions éclairées quant aux pratiques d'alimentation des bébés et en offrant des conseils utiles, en langage simple, dans une courte vidéo animée accessible en ligne. INTERVENTION: Le projet a été mené en quatre phases. Pendant la phase 1, nous avons eu une discussion informelle avec les mères allaitantes, les dispensateurs de services et le  partenaire associatif pour définir les problèmes entourant les services-conseils sur la lactation durant la pandémie de COVID-19. Pendant la phase 2, nous avons vu et analysé les recommandations de 23 organismes concernant l'allaitement durant la COVID-19. Pendant la phase 3, à l'aide de données probantes provenant de sources fiables, nous avons conceptualisé et créé une cyberressource animée de cinq minutes sur l'allaitement durant la COVID-19. Pendant la phase 4, nous avons diffusé cette cyberressource aux mères allaitantes, au grand public, à des établissements d'enseignement postsecondaires et à des organismes de services aux mères allaitantes au Canada. RéSULTATS: Cette cyberressource factuelle a permis d'aborder plus facilement les idées fausses entourant l'allaitement durant la COVID-19 et de sensibiliser le public aux pratiques sûres d'alimentation des nourrissons durant la pandémie. Dans l'ensemble, des mères allaitantes en isolement, qui avaient peu accès aux services de soins de santé durant la pandémie, ont trouvé notre vidéo informative, conviviale, utile et facilement accessible. CONSéQUENCES: Ce projet souligne l'importance du contact avec la patiente et de la collaboration avec le partenaire associatif pour protéger l'allaitement maternel durant la pandémie. Il montre aussi que des ressources informationnelles accessibles en ligne peuvent protéger l'allaitement dans les situations où les mères allaitantes ont moins accès aux services de soins de santé.

Small alkyl amines as ion-pair reagents for the separation of positional isomers of impurities in phosphate diester oligonucleotides.

In an effort to improve separation of impurities in oligonucleotide drugs, alkyl amines of different length and carbon content were evaluated as reagents in ion pair-reversed phase (IP-RP) HPLC with mass spectrometric detection. A range of columns was tested in combination with different buffers, ion-pair modifiers and varying pH adjustments. For phosphorothioate oligonucleotides, larger amines, like tributyl and hexyl amine provided the best chromatography, as small amines tended to broaden peaks due to the separation of diastereoisomers. For phosphate diester oligonucleotides, the best separations were obtained using small alkyl amines, like propyl-, isopropyl- and diethylamine. Conditions optimized for oligonucleotide sequence and type of impurity enabled full separation of the individual components of composite impurities, such as n-1, N3-(2-cyanoethyl)thymine (CNET), deaminated and 3-(2-oxopropyl)imidazopyrimidinone (OPC) impurities. The addition of long-chain alkyl acids like hexanoic acid to the IP buffer resulted in further improvements in peak separation.

The moderating effect of control over work scheduling and overtime on the relationship between workload demands and perceived job risk.

BACKGROUND: This study investigated the impact of workload demands on perceived job risk using the Job Demand-Control model as a research framework. OBJECTIVE: The primary objective was to test the hypothesis that employee control over work scheduling and overtime would moderate the relationship between workload demands and perceived job risk. METHOD: Ninety-six participants working in a variety of industries completed measures of workload demands, and of control over work scheduling and overtime, and a measure of perceived job risk. RESULTS: Workload demands predicted higher perceptions of job risk. However, the results also suggest that control over overtime moderated this relationship, where those with the combination of high workload demands and low control over overtime reported higher levels of perceived risk. The results indicate that the JDC model is applicable to safety research. CONCLUSIONS: The results suggest that employee control over workload demands is an important variable to consider in terms of managing workplace safety. The present study also points to important areas for future research to explore in order to further understand the connection between demands and safety.

Effect of dose and plasma concentration on liver uptake and pharmacologic activity of a 2'-methoxyethyl modified chimeric antisense oligonucleotide targeting PTEN.

The role of dose and plasma concentration on liver tissue uptake and resulting antisense pharmacology using a chemically modified antisense oligonucleotide (ASO) targeting PTEN was assessed in mice. A single bolus s.c. dose of 60 mg/kg in mice showed a time-dependent reduction in liver PTEN mRNA that was maximal at 48-72 h and returned to near control levels by 20 days after administration. These pharmacodynamics are in good agreement with liver concentrations of ASO and are consistent with slow elimination (t(1/2)=8 days) of the PTEN ASO from Balb/C mouse liver. As expected, highest ASO concentrations in liver resulted from the s.c. slow infusion at all doses tested. Unexpectedly, the liver EC(50) for the 24-h s.c. slow infusion was approximately twofold higher than the two bolus routes of administration. Based on plasma concentration analysis it appears that 1-2 microg/mL ASO plasma concentration is a threshold that, if exceeded, results in robust antisense effects and below which there is reduced or complete loss of antisense pharmacology in liver even though bulk uptake in the organ is improved. Co-administration of a nonsense ASO competed for liver uptake, but unexpectedly increased pharmacodynamic response for the active oligonucleotide (ISIS 116847) supporting inhibition of a nonproductive bulk uptake pathway while simultaneously improving productive uptake (pharmacodynamics). This competition effect was similar whether the nonsense oligonucleotide was co-administered with ASO or administered up to 24 h prior to active ASO injection.

Biotinylated biodegradable nanotemplated hydrogel networks for cell interactive applications.

We describe the synthesis of a novel biotinylated nanotextured degradable hydrogel that can be rapidly surface engineered with a diverse range of biotinylated moieties. The hydrogel is synthesized by reacting methacrylated biotin-PEG with dimethacrylated P LA-b- PEG-b-P LA (LPLDMA, PEG = poly(ethylene glycol), PLA = poly(lactic acid)),or dimethacrylated PEG-b-P LA-b- PEG (PLPDMA). Methacrylated biotin-PEG is prepared by reacting biotin-PEG-OH with methacrylic anhydride. Biotin-PEG-OH is prepared by reacting alpha-hydroxy-omega-amine PEG with N-hydroxysuccinimide-biotin. Confirmation of the final product is determined using (1)H NMR and Fourier transform infrared spectroscopy (FTIR). The integrity and surface presentation of the biotin units is observed spectrophotometrically using the HABA/avidin assay. To produce nanostructured polymer topography, a self-assembling lyotropic liquid crystalline mesophase is used as a polymerization template, generating biotinylated hydrogels with highly organized lamellar matrix geometry. Traditionally processed isotropic hydrogels are used for comparison. Scanning electron microscopy shows that isotropic hydrogels have a smooth glassy appearance while lamellar templated hydrogels have defined surface topographical features that enhance preosteoblast human palatal mesenchymal cell (HEPM) attachment. Engineering the surfaces of the hydrogels with cell adhesive Arg-Gly-Asp (RGD) peptide sequences using the biotin-avidin interaction significantly enhances cell attachment. Surface engineering of cell adhesive peptides in conjunction with the lamellar template induced surface topography generates additive enhancements in cell attachment.

Silicalite nanoparticles that promote transgene expression.

Here, we report on a new zeolite-based silicalite nanoparticle that can enhance the transfection efficiencies generated by poly ethylene imine-plasmid DNA (PEI-pDNA) complexes via a sedimentation mechanism and can enhance the transfection efficiencies of pDNA alone when surface functionalized with amine groups. The silicalite nanoparticles have a mean size of 55 nm. Functionalizing the silicalite nanoparticles with amine groups results in a clear transition in zeta potential from -25.9 ± 2.3 mV (pH 7.4) for unfunctionalized silicalite nanoparticles to 4.9 ± 0.7 mV (pH 7.4) for amine functionalized silicalite nanoparticles. We identify that silicalite nanoparticles used to promote non-viral vector acceleration to the cell surface are found in acidic vesicles or the cytoplasm but not the nucleus. An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay showed that the silicalite nanoparticles were non-toxic at the concentrations tested for transfection. We show that surface functionalization of silicalite nanoparticles with amine groups results in a significant (230%) increase in transfection efficiency of pDNA when compared to unfunctionalized silicalite nanoparticles. Silicalite nanoparticles enhanced pDNA-PEI induced transfection of human embryonic kidney (HEK-293) cells by over 150%.

Multifunctional nanorods for biomedical applications.

Multifunctional nanorods have shown significant potential in a wide range of biomedical applications. Nanorods can be synthesized by a top down or bottom-up approach. The bottom-up approach commonly utilizes a template deposition methodology. A variety of metal segments can easily be incorporated into the nanorods. This permits high degrees of chemical and dimensional control. High aspect-ratio nanorods have a large surface area for functionalization. By varying the metal segments in the nanorods, spatial control over the binding of functional biomolecules that correspond with the unique surface chemistry of the metal segment can be achieved. Functionalized multicomponent nanorods are utilized in applications ranging from multiplexing, protein sensing, glucose sensing, imaging, biomolecule-associated nanocircuits, gene delivery and vaccinations.

Microleakage of Class V resin composite restorations placed with self-etching adhesives.

STATEMENT OF PROBLEM: Although they are widely available, there is insufficient information about the capability of self-etching adhesives in sealing the margins of resin composite restorations. PURPOSE: The purpose of this study was to compare the in vitro microleakage of Class V resin composite restorations placed using a strong pH self-etching adhesive, an intermediate pH self-etching adhesive, and an adhesive with a separate etchant and primer. MATERIAL AND METHODS: Class V resin composite restorations (n = 8) were placed in prepared cavities in extracted human third molars using 2 self-etching dentin adhesives, Prompt L-Pop (Strong pH) or One-Up Bond F (Intermediate pH), and an adhesive with a separate etchant and primer, ScotchBond Multi-Purpose. The restored teeth received 1000 thermal cycles between 5 degrees C and 55 degrees C water baths with a 1-minute dwell time and were subsequently subjected to a methylene blue dye challenge and sectioned. The sectioned specimens were scored as demonstrating none, slight, or severe leakage (n=16). Ranked data were analyzed using a 1-way analysis of variance at a 5% confidence level. RESULTS: The only leakage observed was along gingival margins, with an incidence of 31% for Prompt L-Pop and One-Up Bond F, and 50% for ScotchBond Multi-Purpose. CONCLUSION: No significant differences in marginal leakage were found among the adhesives tested.