High fidelity one-pot DNA assembly using orthogonal serine integrases.

BACKGROUND: Large serine integrases (LSIs, derived from temperate phages) have been adapted for use in a multipart DNA assembly process in vitro, called serine integrase recombinational assembly (SIRA). The versatility, efficiency, and fidelity of SIRA is limited by lack of a sufficient number of LSIs whose activities have been characterized in vitro. METHODS AND MAJOR RESULTS: In this report, we compared the activities in vitro of 10 orthogonal LSIs to explore their suitability for multiplex SIRA reactions. We found that Bxb1, ϕR4, and TG1 integrases were the most active among the set we studied, but several others were also usable. As proof of principle, we demonstrated high-efficiency one-pot assembly of six DNA fragments (made by PCR) into a 7.5 kb plasmid that expresses the enzymes of the ß-carotenoid pathway in Escherichia coli, using six different LSIs. We further showed that a combined approach using a few highly active LSIs, each acting on multiple pairs of att sites with distinct central dinucleotides, can be used to scale up "poly-part" gene assembly and editing. CONCLUSIONS AND IMPLICATIONS: We conclude that use of multiple orthogonal integrases may be the most predictable, efficient, and programmable approach for SIRA and other in vitro applications.

Preclinical evaluation of a SARS-CoV-2 mRNA vaccine PTX-COVID19-B.

Safe and effective vaccines are needed to end the COVID-19 pandemic. Here, we report the preclinical development of a lipid nanoparticle­formulated SARS-CoV-2 mRNA vaccine, PTX-COVID19-B. PTX-COVID19-B was chosen among three candidates after the initial mouse vaccination results showed that it elicited the strongest neutralizing antibody response against SARS-CoV-2. Further tests in mice and hamsters indicated that PTX-COVID19-B induced robust humoral and cellular immune responses and completely protected the vaccinated animals from SARS-CoV-2 infection in the lung. Studies in hamsters also showed that PTX-COVID19-B protected the upper respiratory tract from SARS-CoV-2 infection. Mouse immune sera elicited by PTX-COVID19-B vaccination were able to neutralize SARS-CoV-2 variants of concern, including the Alpha, Beta, Gamma, and Delta lineages. No adverse effects were induced by PTX-COVID19-B in either mice or hamsters. Based on these results, PTX-COVID19-B was authorized by Health Canada to enter clinical trials in December 2020 with a phase 2 clinical trial ongoing.

Susceptibility of Selected Multi-Drug Resistant Clinical Isolates to Leaves of Carpolobia lutea.

BACKGROUND: The decline in the production of new effective antibiotics coupled with the constantly evolving antimicrobial resistance remains a public health concern. This study thus evaluated the antibacterial and antifungal effects of the ethanolic, n-hexane and hot aqueous extracts of Carpolobialutea leaves. METHODS: The extracts were tested using agar well diffusion method against selected clinical isolates: Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli, Staphylococcus aureus and Candida albicans. Antibiogram profile of the isolates were deduced by disc diffusion method. RESULTS: Multi-drug resistance was confirmed in all bacteria with a notable pandrug resistance in Pseudomonas aeruginosa. Ofloxacin, Erythromycin and Gentamicin were effective on two or three organisms, notably on Salmonella typhi and Escherichia coli. The preliminary antibacterial assay marked the efficacy of the ethanol and n-hexane extracts except on E. coli, with zero activity for hot water extracts at the stock concentration (200 mg/ml). Pseudomonas aeruginosaand Candida albicans were susceptible to lesser concentrations of the ethanol extracts at 5 mg/ml and 25 mg/ml respectively. None of the isolates showed sensitivity to lesser concentrations of n-hexane extract. Carpolobia lutea leaves proved to be effective over the use of antibiotics in inhibiting the activity of Pseudomonas aeruginosa which was resistant to the latter. The Minimum Inhibitory concentration of the ethanolic extract was considerably low (≤5mg/ml for P. aeruginosa and 25mg/ml for C. albicans). However, there was no Minimum Bactericidal concentration for the extracts against the clinical isolates. CONCLUSION: Carpolobia lutea shelters bioactive components with pharmacological potentials that could show efficiency in the treatment of bacterial infections.