Adeno-Associated Virus 5 Protein Particles Produced by E. coli Cell-Free Protein Synthesis

Recombinant adeno-associated viruses (rAAVs) have emerged as important tools for gene therapy and, more recently, vaccine development. Nonetheless, manufacturing can be costly and time-consuming, emphasizing the importance of alternative production platforms. We investigate the potential of E. coli-based cell-free protein synthesis (CFPS) to produce recombinant AAV5 virus-like particles (VLPs). AAV5 virus protein 3 (VP3) constructs, both with and without Strep-tag II, were expressed with CFPS. Lower reaction temperatures resulted in increased solubility, with the untagged variant containing nearly 90% more soluble VLP VP3 protein at 18 °C than at 37 °C. Affinity chromatography of N-terminally Strep(II)-tagged VP3 enabled successful isolation with minimal processing. DLS and TEM confirmed the presence of ∼20 nm particles. Furthermore, the N-terminally tagged AAV5 VP3 VLPs were biologically active, successfully internalizing into HeLa cells. This study describes an innovative approach to AAV VLP production using E. coli-based CFPS, demonstrating its potential for rapid and biologically active AAV VLP synthesis

Epidemiology, treatment patterns, and clinical outcomes in de novo oligometastatic hormone-sensitive prostate cancer.

92 Background: Oligometastatic hormone-sensitive prostate cancer (omHSPC) represents an advanced prostate cancer subset where metastasis-directed therapy (MDT) and prostate radiation therapy (RT) may improve clinical response and outcomes; however, there is a lack of published data on the epidemiology, clinical outcomes, and current treatment patterns. As such, we conducted a study to better characterize de novo omHSPC in the United States Veterans Affairs Health Care System (VA). Methods: This observational retrospective cohort study utilized chart abstracted data from the VA electronic medical record, as well as data from the VA Corporate Data Warehouse, a central repository of VA patient medical records. We randomly selected 400 men diagnosed with de novo mHSPC from 1/2015-12/2020. omHSPC was defined as up to 5 bone, lymph node, and/or visceral (excluding liver) metastases in total, identified by conventional imaging (bone scan, CT, and/or MRI). We estimated prevalence, described treatment patterns and used Kaplan-Meier methods to estimate overall survival (OS) and time to castration resistance from date of mHSPC diagnosis. The log rank test was used to compare differences in outcomes between omHSPC and non-omHSPC groups. Results: Of the 400 men with de novo mHSPC, 76 (19%) had omHSPC by conventional imaging. Men with omHSPC and non-omHSPC were similar in age, race, Gleason grade group, comorbidities, and metastatic site (bone and lymph node being most common). Men with non-omHSPC had a higher median PSA at mHSPC diagnosis (147.0) than omHSPC (38.3). The percentage of men on first-line (1L) novel hormonal therapy (NHT) use (most commonly abiraterone or enzalutamide) was similar between groups in the 1L setting (22.4% (omHSPC) vs 20.4% (non-omHSPC)), but the percentage of men on a 1L chemotherapy regimen was lower in omHSPC (5.3%) vs. non-omHSPC (13.6%). Overall, there was a higher percentage of men treated with MDT or prostate RT in omHSPC (13.2%) vs non-omHSPC cases (2.5%). Median OS in months (mos) was higher in men with omHSPC (55.3 mos, 95% CI 35.9-79.0) vs. non-omHSPC (25.9 mos, 95% CI 20.5-31.7, p=0.002). Median time to castration resistance was also longer in omHSPC (not reached [NR], 95% CI 42.2-NR) vs. non-omHSPC (29.3 mos, 95% CI 23.7-36.1, p=0.0014). Conclusions: Our study provides real-world insight into the prevalence, treatment patterns and clinical outcomes for omHSPC using a nationally representative VA sample. Approximately 1 in 5 men with de novo mHSPC were oligometastatic, and OS in men with omHSPC was more than double that of non-omHSPC. Although more men with omHSPC compared to non-omHSPC received potential curative therapy, the percentage was still relatively low. Future studies are warranted as several clinical trials are investigating the potential for prolonged responses with aggressive, multimodal therapy inclusive of systemic and local therapies. </jats:p

Quantitative PCR study on the mode of action of oligosaccharide elicitors on penicillin G production by Penicillium chrysogenum

Aim: To investigate the effects of single and multiple additions of the oligosaccharide elicitors, obtained from alginate and locust bean gum, on penicillin G production and the transcript level of penicillin G biosynthetic genes. Methods and Results: The transcript copy numbers and penicillin G concentration in liquid cultures of Penicillium chrysogenum grown under control and elicited conditions were compared using quantitative PCR and HPLC assay respectively. An increase in the penicillin G production rate and transcript copy numbers of the three major penicillin G biosynthetic genes pcbAB, pcbC and penDE was observed in the elicited cultures compared to control cultures. The effects were observed to be higher in multiple elicitor added cultures compared to single elicitor supplemented and control cultures. Conclusions: The results show, for the first time in bioreactor cultures, the enhancement of penicillin G transcript copy number of the penicillin biosynthetic genes using qPCR with a corresponding increase in the penicillin G production upon multiple elicitor addition of two different types of elicitors. Significance and Impact of the Study: Establishment of the effect of multiple elicitor addition on penicillin G production and investigating the role of oligosaccharide elicitors as transcriptional activators has wide spread impact for antibiotic industry

Enhanced intracellular Ca(2+) concentrations in Escherichia coli and Bacillus subtilis after addition of oligosaccharide elicitors

Elicitation can lead to overproduction of secondary metabolites in plants and microbes. Potential changes in cytosolic Ca(2+) levels in bacteria were studied in response to elicitation. We report, for the first time, the effect of oligosaccharide elicitors on intracellular Ca(2+) levels. The apoaequorin gene was cloned into Escherichia coli DH5α and Bacillus subtilis 1604 cultures. Addition of elicitors, oligoguluronate and mannan oligosaccharides, to the cultures caused up to 11-fold increase in cytosolic Ca(2+) in E. coli and tenfold increase in B. subtilis. These increases in Ca(2+) levels could therefore contribute to the enhancement of secondary metabolite levels

Evidence for the involvement of intracellular Ca 2+ ions in the elicitation mechanism of Bacillus Licheniformis

Treatment of Bacillus licheniformis cultures with biotic oligosaccharide elicitors is known to increase the production of the antibiotic bacitracin A. The mechanism of the elicitation is currently under investigation and in this paper we provide evidence on the modulatory role of Ca 2+ ions during this process. Addition of elicitors, mannan oligosaccharides, oligoguluronate and oligomannuronate to the liquid cultures resulted in 9.0, 5.2 and 5.0% increase in cytosolic Ca 2+ levels in B. licheniformis , while the presence of verapamil (Ca 2+ channel blocker) resulted in 74% decrease in bacitracin A levels, as compared to the control culture. We propose that Ca 2+ ions may acts as a secondary messenger in the regulation of the bacitracin A synthesis in the elicited B. licheniformis cultures