Effects of different delivery modes on teaching biomedical science practical skills in higher education during the 2021 pandemic measures.

The COVID-19 pandemic related measures had augmented the rise of online education. While online teaching had mitigated the negative impacts from educational institutional closures, it was unable to displace hands-on biomedical laboratory practical lessons effectively. Without practical sessions, there was concern over the imparting of laboratory skills even with video demonstrations. To investigate the effectiveness of different delivery modes in imparting laboratory skills, theoretical and practical student assessments were analyzed alongside an anonymous survey on their motivation and prior experience. The undergraduate students were exposed to (1) instructor-live demonstration; (2) video demonstration or (3) no demonstration prior to the practical test which was a plasmid extraction. Significantly higher mini-prep yields and purity were found for both instructor-live and video demonstrations compared to no demonstration. Comparison with pre-pandemic theoretical assessment performance showed no significant differences despite longer contact hours during pre-pandemic times. Prior lab experience and motivation for selecting the course did not significantly affect student mini-prep yields. In conclusion, our findings suggest that video demonstrations were as effective as instructor-live demonstrations during the pandemic without noticeably compromising the teaching and learning of biomedical laboratory skills.

Augmenting recombinant antibody production in HEK293E cells: optimizing transfection and culture parameters.

BACKGROUND: Optimizing recombinant antibody production is important for cost-effective therapeutics and diagnostics. With impact on commercialization, higher productivity beyond laboratory scales is highly sought, where efficient production can also accelerate antibody characterizations and investigations. METHODS: Investigating HEK293E cells for mammalian antibody production, various transfection and culture parameters were systematically analyzed for antibody light chain production before evaluating them for whole antibody production. Transfection parameters investigated include seeding cell density, the concentration of the transfection reagent and DNA, complexation time, temperature, and volume, as well as culture parameters such as medium replacement, serum deprivation, use of cell maintenance antibiotic, incubation temperature, medium volume, post-transfection harvest day, and common nutrient supplements. RESULTS: Using 2 mL adherent HEK293E cell culture transfections with 25 kDa linear polyethylenimine in the most optimized parameters, we demonstrated a ~2-fold production increase for light chain alone and for whole antibody production reaching 536 and 49 µg, respectively, in a cost-effective manner. With the addition of peptone, κ light chain increased by ~4-fold to 1032 µg, whereas whole antibody increased to a lesser extent by ~2.5-fold to 51 µg, with benefits potentially for antibodies limited by their light chains in production. CONCLUSIONS: Our optimized findings show promise for a more efficient and convenient antibody production method through transfection and culture optimizations that can be incorporated to scale-up processes and with potential transferability to other mammalian-based recombinant protein production using HEK293E.

Engineering Ag43 Signal Peptides with Bacterial Display and Selection.

Protein display, secretion, and export in prokaryotes are essential for utilizing microbial systems as engineered living materials, medicines, biocatalysts, and protein factories. To select for improved signal peptides for Escherichia coli protein display, we utilized error-prone polymerase chain reaction (epPCR) coupled with single-cell sorting and microplate titer to generate, select, and detect improved Ag43 signal peptides. Through just three rounds of mutagenesis and selection using green fluorescence from the 56 kDa sfGFP-beta-lactamase, we isolated clones that modestly increased surface display from 1.4- to 3-fold as detected by the microplate plate-reader and native SDS-PAGE assays. To establish that the functional protein was displayed extracellularly, we trypsinized the bacterial cells to release the surface displayed proteins for analysis. This workflow demonstrated a fast and high-throughput method leveraging epPCR and single-cell sorting to augment bacterial surface display rapidly that could be applied to other bacterial proteins.

Yet Another Quick Assembly, Analysis and Trimming Tool (YAQAAT): A Server for the Automated Assembly and Analysis of Sanger Sequencing Data.

Even with the ubiquity of Sanger sequencing, automated assembly software are predominantly stand-alone software packages for desktop/laptop use with very few online equivalents, thus geospatially constraining sequence analysis and assembly. With increased data output worldwide, there is also a need for automated quality checks and trimming prior to large assemblies, along with automated detection of mutations. Through web servers with expanded automation and functionalities, even smartphones/phablets can be used to perform complex analysis previously limited to desktops, especially if they can upload files from cloud storage. To facilitate such online accessible sequence assembly and analysis, we created Yet Another Quick Assembly, Analysis and Trimming Tool web server for the automated assembly of multiple .ab1 and .FASTQ sequencing reads de novo with automated trimming and scanning of the assembled sequences for single nucleotide polymorphisms and insertions or deletions without installation of software, allowing it to be accessed from anywhere with Internet access and with minimal dependency on other software and web tools.

Yet Another Quick Assembly, Analysis and Trimming Tool (YAQAAT): A Server for the Automated Assembly and Analysis of Sanger Sequencing Data.

Even with the ubiquity of Sanger sequencing, automated assembly software are predominantly stand-alone software packages for desktop/laptop use with very few online equivalents, thus geospatially constraining sequence analysis and assembly. With increased data output worldwide, there is also a need for automated quality checks and trimming prior to large assemblies, along with automated detection of mutations. Through web servers with expanded automation and functionalities, even smartphones/phablets can be used to perform complex analysis previously limited to desktops, especially if they can upload files from cloud storage. To facilitate such online accessible sequence assembly and analysis, we created Yet Another Quick Assembly, Analysis and Trimming Tool web server for the automated assembly of multiple .ab1 and .FASTQ sequencing reads de novo with automated trimming and scanning of the assembled sequences for single nucleotide polymorphisms and insertions or deletions without installation of software, allowing it to be accessed from anywhere with Internet access and with minimal dependency on other software and web tools.

Probability of change in life: Amino acid changes in single nucleotide substitutions.

Mutations underpin the processes in life, be it beneficial or detrimental. While mutations are assumed to be random in the bereft of selection pressures, the genetic code has underlying computable probabilities in amino acid phenotypic changes. With a wide range of implications including drug resistance, understanding amino acid changes is important. In this study, we calculated the probabilities of substitutions mutations in the genetic code leading to the 20 amino acids and stop codons. Our calculations reveal an enigmatic in-built self-preserving organization of the genetic code that averts disruptive changes at the physicochemical properties level. These changes include changes to start, aromatic, negative charged amino acids and stop codons. Our findings thus reveal a statistical mechanism governing the relationship between amino acids and the universal genetic code.

Spontaneous Mutations in HIV-1 Gag, Protease, RT p66 in the First Replication Cycle and How They Appear: Insights from an In Vitro Assay on Mutation Rates and Types.

While drug resistant mutations in HIV-1 are largely credited to its error prone HIV-1 RT, the time point in the infection cycle that these mutations can arise and if they appear spontaneously without selection pressures both remained enigmatic. Many HIV-1 RT mutational in vitro studies utilized reporter genes (LacZ) as a template to investigate these questions, thereby not accounting for the possible contribution of viral codon usage. To address this gap, we investigated HIV-1 RT mutation rates and biases on its own Gag, protease, and RT p66 genes in an in vitro selection pressure free system. We found rare clinical mutations with a general avoidance of crucial functional sites in the background mutations rates for Gag, protease, and RT p66 at 4.71 × 10-5, 6.03 × 10-5, and 7.09 × 10-5 mutations/bp, respectively. Gag and p66 genes showed a large number of 'A to G' mutations. Comparisons with silently mutated p66 sequences showed an increase in mutation rates (1.88 × 10-4 mutations/bp) and that 'A to G' mutations occurred in regions reminiscent of ADAR neighbor sequence preferences. Mutational free energies of the 'A to G' mutations revealed an avoidance of destabilizing effects, with the natural p66 gene codon usage providing barriers to disruptive amino acid changes. Our study demonstrates the importance of studying mutation emergence in HIV genes in a RT-PCR in vitro selection pressure free system to understand how fast drug resistance can emerge, providing transferable applications to how new viral diseases and drug resistances can emerge.

Reviewing HIV-1 Gag Mutations in Protease Inhibitors Resistance: Insights for Possible Novel Gag Inhibitor Designs.

HIV protease inhibitors against the viral protease are often hampered by drug resistance mutations in protease and in the viral substrate Gag. To overcome this drug resistance and inhibit viral maturation, targeting Gag alongside protease rather than targeting protease alone may be more efficient. In order to successfully inhibit Gag, understanding of its drug resistance mutations and the elicited structural changes on protease binding needs to be investigated. While mutations on Gag have already been mapped to protease inhibitor resistance, there remain many mutations, particularly the non-cleavage mutations, that are not characterized. Through structural studies to unravel how Gag mutations contributes to protease drug resistance synergistically, it is thus possible to glean insights to design novel Gag inhibitors. In this review, we discuss the structural role of both novel and previously reported Gag mutations in PI resistance, and how new Gag inhibitors can be designed.