Stability of Diluted Adenosine Solutions in Polyolefin Infusion Bags

Background Intravenous or intracoronary adenosine is used in the cardiac catherization lab to achieve maximal coronary blood flow and determine fractional flow reserve. Objective To determine the stability of adenosine 10 and 50 µg/mL in either 0.9% sodium chloride injection or 5% dextrose injection in polyolefin infusion bags stored at 2 temperatures, refrigeration (2°C-8°C) or controlled room temperature (20°C-25°C). Methods Adenosine 10 µg/mL and 50 µg/mL solutions were prepared in 50 mL polyolefin infusion bags containing 0.9% sodium chloride injection or 5% dextrose injection and stored at controlled room temperature or under refrigeration. Each combination of concentration, diluent, and storage was prepared in triplicate. Samples were assayed using stability-indicating, reversed-phase high-performance liquid chromatography immediately at time 0 and at 24 hours, 48 hours, 7 days, and 14 days. Stability was defined as retaining 90% to 110% of the initial adenosine concentration. The samples were also visually inspected against a light background for clarity, color, and the presence of particulate matter. Results After 14 days, all samples retained 99% to 101% of the initial adenosine concentration. No considerable change in pH or visual appearance was noted. The stability data indicated no significant loss of drug due to chemical degradation or physical interactions during storage. Conclusion Adenosine solutions of 10 and 50 µg/mL were stable for at least 14 days in 50 mL polyolefin infusion bags of 0.9% sodium chloride injection or 5% dextrose injection stored at controlled room temperature and refrigerated conditions

Investigating USP2 as a mediator of therapy resistance in lethal prostate cancer

The growth of prostate cancer (PCa) is dependent on male sex hormones, termed androgens, and the androgen receptor (AR). Therefore, implementing strategies to inhibit AR activity, collectively referred to as androgen deprivation therapy (ADT), is the key therapeutic strategy for metastatic prostate cancer. Unfortunately, ADT is never curative, and patients eventually develop a lethal therapy resistant form of the disease termed castration-resistant prostate cancer (CRPC). The AR signalling pathway is altered in CRPC and a subset of CRPC tumours may evade inhibition by ADT by progressing to a state in which tumour growth is independent of this pathway. One such AR-independent CRPC subtype is termed neuroendocrine PCa (NEPC). Understanding how tumours transition to therapy-resistant, AR-independent states is crucial for the development of new and more effective therapies. We recently undertook transcriptomic profiling of patient tumours treated ex vivo with a clinical AR antagonist, enzalutamide, as a strategy to identify therapy-mediated adaptive changes. This study identified Ubiquitin specific protease 2 (USP2) as being increased in response to enzalutamide. The role of USP2 is to remove ubiquitin groups from proteins that causes therapy resistance and cancer progression, thereby preventing these proteins from degradation and increasing their stability in cancer cells. Thus, we hypothesised that USP2 can mediate resistance to ADT by stabilising key oncoproteins, a concept that was tested in my PhD project. Increased USP2 expression in response to ADT and in therapy-resistant states was validated in multiple prostate cancer cell line models, clinical transcriptomic cohorts, and additional patient tumours. Growing androgen-dependent cell lines in the presence of enzalutamide resulted in increased USP2 expression; conversely, androgen treatment resulted in the repression of USP2 expression. Clinical datasets also revealed that USP2 expression is elevated in AR-low/negative CRPC tumours, particularly those classified as neuroendocrine PCa. These observations suggest that USP2 is consistently upregulated in response to AR-targeted therapies and may represent a previously unknown resistance factor. Supporting this, targeting USP2 in multiple castrate-resistant prostate cancer models, either pharmacologically with a USP2-specific inhibitor (ML364) or by genetic knockdown, resulted in reduced cell viability and increased cell death. In contrast, overexpression of USP2 drove the development of an aggressive, therapy-resistant neuroendocrine phenotype and conferred partial resistance to enzalutamide and a growth advantage in androgen-depleted growth conditions. Importantly, ML364 was also potently active in an in vivo model of aggressive, AR-negative prostate cancer. Mechanistically, we found that USP2 stabilises the levels of oncogenic proteins including Aurora kinase A (AURKA), Cyclin D1 and Fatty acid synthase (FAS). Interrogation of the proteome and transcriptome of PCa cells overexpressing USP2 revealed positive enrichment of neuroendocrine-associated signalling, lipid metabolism and cell cycle, and negative enrichment of interferon signalling. All these enriched pathways are important in driving growth, survival, and progression of cancer. Overall, the findings herein revealed that USP2 can promote the development of neuroendocrine prostate cancer and acts to confer resistance to standard-of-care therapies, revealing it as a bona fide therapeutic target.Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 202

Ablation of elongation factor 2 kinase enhances heat-shock protein 90 chaperone expression and protects cells under proteotoxic stress

Eukaryotic elongation factor 2 kinase (eEF2K) negatively regulates the elongation stage of mRNA translation and is activated under different stress conditions to slow down protein synthesis. One effect of eEF2K is to alter the repertoire of expressed proteins, perhaps to aid survival of stressed cells. Here, we applied pulsed stable isotope labeling with amino acids in cell culture (SILAC) to study changes in the synthesis of specific proteins in human lung adenocarcinoma (A549) cells in which eEF2K had been depleted by an inducible shRNA. We discovered that levels of heat-shock protein 90 (HSP90) are increased in eEF2K-depleted human cells as well as in eEF2K-knockout (eEF2K-/-) mouse embryonic fibroblasts (MEFs). This rise in HSP90 coincided with an increase in the fraction of HSP90 mRNAs associated with translationally active polysomes, irrespective of unchanged total HSP90 levels. These results indicate that blocking eEF2K function can enhance expression of HSP90 chaperones. In eEF2K-/- mouse embryonic fibroblasts (MEFs), inhibition of HSP90 by its specific inhibitor AUY922 promoted the accumulation of ubiquitinated proteins. Notably, HSP90 inhibition promoted apoptosis of eEF2K-/- MEFs under proteostatic stress induced by the proteasome inhibitor MG132. Up-regulation of HSP90 likely protects cells from protein folding stress, arising, for example, from faster rates of polypeptide synthesis due to the lack of eEF2K. Our findings indicate that eEF2K and HSPs closely cooperate to maintain proper proteostasis and suggest that concomitant inhibition of HSP90 and eEF2K could be a strategy to decrease cancer cell survival

PROCESS SCALE-UP AND OPTIMIZATION FOR PRODUCTION OF HIGH EFFICACY ORAL RABIES VACCINE

Rabies is an important causative agent of disease resulting in an acute infection of the nervous system and death of the individual. Rabies remains an important public health program in developing countries, and the indigenous threat of rabies continues in developed countries because of wildlife reservoirs. Globally, there are about 55,000 fatal human cases of rabies each year [WHO, 2007]. Control of rabies in wildlife remains an important challenge for government offices. There are numbers of rabies vaccines commercially available for controls of wildlife rabies. However, these vaccines currently distributed to wildlife do not effectively immunize all at-risk species, especially skunks. Alternative efficacious vaccines are needed. A human adenovirus rabies glycoprotein recombinant vaccine candidate (AdRG1.3), developed by the Rabies Research and Development Unit at Ontario Ministry of Natural Resources, Canada, has shown the most promising result in laboratory trials. The adenovirus used in rabies vaccine laden bait is produced using HEK 293 cell culture process. This presentation will focus on demonstrating the successful scale-up of AdRG1.3 adenovirus production from 1 liter to 500 liter to manufacture large quantities of bulk material required to support field trials and demonstrate efficacy of this new vaccine. The robustness of production process was improved through elimination of medium replacement operation at the time of virus infection, and culture titer was increased by over 3 folds through optimization of cell culture medium. The elimination of medium replacement step reduced the risk of culture contamination, and resulted in significant saving in material expenses and reduction in labor costs. Over 10,000 liters of active AdRG1.3 adenovirus cultures were manufactured so far to support field trials. AdRG1.3 adenovirus is formulated and packaged in baits using Artemis Technologies Inc. proprietary technology prior to aerial-baiting. AdRG1.3 rabies vaccine has been distributed by several provincial agencies to testing areas located in Ontario, Quebec and New Brunswick provinces, Canada, for field trials in yearly campaigns from 2006 to 2009. The field results showed that the new vaccine was more efficient than the existing ones in immunizing animals that were previously difficult to vaccinate

Optimization and scale-up of cell culture and purification processes for production of an adenovirus-vectored tuberculosis vaccine candidate

Tuberculosis (TB) is the second leading cause of death by infectious disease worldwide. The only available TB vaccine is the Bacille Calmette-Guerin (BCG). However, parenterally administered Mycobacterium bovis BCG vaccine confers only limited immune protection from pulmonary tuberculosis in humans. There is a need for developing effective boosting vaccination strategies. AdAg85A, an adenoviral vector expressing the mycobacterial protein Ag85A, is a new tuberculosis vaccine candidate, and has shown promising results in pre-clinical studies and phase I trial. This adenovirus vectored vaccine is produced using HEK 293 cell culture. Here we report on the optimization of cell culture conditions, scale-up of production and purification of the AdAg85A at different scales. Four commercial serum-free media were evaluated under various conditions for supporting the growth of HEK293 cell and production of AdAg85A. A culturing strategy was employed to take advantages of two culture media with respective strengths in supporting the cell growth and virus production, which enabled to maintain virus productivity at higher cell densities and resulted in more than two folds of increases in culture titer. The production of AdAg85A was successfully scaled up and validated at 60L bioreactor under the optimal conditions. The AdAg85A generated from the 3L and 60L bioreactor runs was purified through several purification steps. More than 98% of total cellular proteins was removed, over 60% of viral particles was recovered after the purification process, and purity of AdAg85A was similar to that of the ATCC VR-1516 Ad5 standard. Vaccination of mice with the purified AdAg85A demonstrated a very good level of Ag85A-specific antibody responses. The optimized production and purification conditions were transferred to a GMP facility for manufacturing of AdAg85A for generation of clinical grade material to support clinical trials

Methodologies for Housing Justice Resource Guide

This Resource Guide is the outcome of a Summer Institute on Methodologies for Housing Justice convened by the Institute on Inequality and Democracy at UCLA Luskin as part of the Housing Justice in Unequal Cities Network, which is supported by the National Science Foundation (BCS 1758774). Held in Los Angeles in August 2019, the Summer Institute brought together participants from cities around the world. As is the case with the overall scope and purpose of the Housing Justice in Unequal Cities Network, it created a shared terrain of scholarship for movement-based and university-based scholars. Dissatisfied with the canonical methods that are in use in housing studies and guided by housing justice movements that are active research communities, the Summer Institute was premised on the assertion that methodology is political. Methodology is rooted in arguments about the world and involves relations of power and knowledge. The method itself – be it countermapping or people’s diaries – does not ensure an ethics of solidarity and a purpose of justice. Such goals require methodologies for liberation. Thus, as is evident in this Resource Guide, our endeavor foregrounds innovative methods that are being used by researchers across academia and activism and explicitly situates such methods in an orientation towards housing justice

The Size and Culturability of Patient-Generated SARS-CoV-2 Aerosol

BACKGROUND: Aerosol transmission of COVID-19 is the subject of ongoing policy debate. Characterizing aerosol produced by people with COVID-19 is critical to understanding the role of aerosols in transmission. OBJECTIVE: We investigated the presence of virus in size-fractioned aerosols from six COVID-19 patients admitted into mixed acuity wards in April of 2020. METHODS: Size-fractionated aerosol samples and aerosol size distributions were collected from COVID-19 positive patients. Aerosol samples were analyzed for viral RNA, positive samples were cultured in Vero E6 cells. Serial RT-PCR of cells indicated samples where viral replication was likely occurring. Viral presence was also investigated by western blot and transmission electron microscopy (TEM). RESULTS: SARS-CoV-2 RNA was detected by rRT-PCR in all samples. Three samples confidently indicated the presence of viral replication, all of which were from collected sub-micron aerosol. Western blot indicated the presence of viral proteins in all but one of these samples, and intact virions were observed by TEM in one sample. SIGNIFICANCE: Observations of viral replication in the culture of submicron aerosol samples provides additional evidence that airborne transmission of COVID-19 is possible. These results support the use of efficient respiratory protection in both healthcare and by the public to limit transmission

Genome-Wide Association Study of Exercise Behavior in Dutch and American Adults

The objective of this study was to identify genetic variants that are associated with adult leisure-time exercise behavior using genome-wide association in two independent samples

Association studies of up to 1.2 million individuals yield new insights into the genetic etiology of tobacco and alcohol use.

Tobacco and alcohol use are leading causes of mortality that influence risk for many complex diseases and disorders1. They are heritable2,3 and etiologically related4,5 behaviors that have been resistant to gene discovery efforts6-11. In sample sizes up to 1.2 million individuals, we discovered 566 genetic variants in 406 loci associated with multiple stages of tobacco use (initiation, cessation, and heaviness) as well as alcohol use, with 150 loci evidencing pleiotropic association. Smoking phenotypes were positively genetically correlated with many health conditions, whereas alcohol use was negatively correlated with these conditions, such that increased genetic risk for alcohol use is associated with lower disease risk. We report evidence for the involvement of many systems in tobacco and alcohol use, including genes involved in nicotinic, dopaminergic, and glutamatergic neurotransmission. The results provide a solid starting point to evaluate the effects of these loci in model organisms and more precise substance use measures