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.

Variable-heavy (VH) families influencing IgA1&2 engagement to the antigen, FcαRI and superantigen proteins G, A, and L.

Interest in IgA as an alternative antibody format has increased over the years with much remaining to be investigated in relation to interactions with immune cells. Considering the recent whole antibody investigations showing significant distal effects between the variable (V) and constant (C)- regions that can be mitigated by the hinge regions of both human IgA subtypes A1 and A2, we performed an in-depth mechanistic investigation using a panel of 28 IgA1s and A2s of both Trastuzumab and Pertuzumab models. FcαRI binding were found to be mitigated by the differing glycosylation patterns in IgA1 and 2 with contributions from the CDRs. On their interactions with antigen-Her2 and superantigens PpL, SpG and SpA, PpL was found to sterically hinder Her2 antigen binding with unexpected findings of IgAs binding SpG at the CH2-3 region alongside SpA interacting with IgAs at the CH1. Although the VH3 framework (FWR) is commonly used in CDR grafting, we found the VH1 framework (FWR) to be a possible alternative when grafting IgA1 and 2 owing to its stronger binding to antigen Her2 and weaker interactions to superantigen Protein L and A. These findings lay the foundation to understanding the interactions between IgAs and microbial superantigens, and also guide the engineering of IgAs for future antibody applications and targeting of superantigen-producing microbes.

Sagacious epitope selection for vaccines, and both antibody-based therapeutics and diagnostics: tips from virology and oncology.

The target of an antibody plays a significant role in the success of antibody-based therapeutics and diagnostics, and vaccine development. This importance is focused on the target binding site-epitope, where epitope selection as a part of design thinking beyond traditional antigen selection using whole cell or whole protein immunization can positively impact success. With purified recombinant protein production and peptide synthesis to display limited/selected epitopes, intrinsic factors that can affect the functioning of resulting antibodies can be more easily selected for. Many of these factors stem from the location of the epitope that can impact accessibility of the antibody to the epitope at a cellular or molecular level, direct inhibition of target antigen activity, conservation of function despite escape mutations, and even noncompetitive inhibition sites. By incorporating novel computational methods for predicting antigen changes to model-informed drug discovery and development, superior vaccines and antibody-based therapeutics or diagnostics can be easily designed to mitigate failures. With detailed examples, this review highlights the new opportunities, factors, and methods of predicting antigenic changes for consideration in sagacious epitope selection.

More Than Meets the Kappa for Antibody Superantigen Protein L (PpL).

Immunoglobulin superantigens play an important role in affinity purification of antibodies and the microbiota-immune axis at mucosal areas. Based on current understanding, Staphylococcal Protein A (SpA), Streptococcal Protein G (SpG) and Finegoldia Protein L (PpL) are thought to only bind specific regions of human antibodies, allowing for selective purification of antibody isotypes and chains. Clinically, these superantigens are often classified as toxins and increase the virulence of the producing pathogen through unspecific interactions with immune proteins. To perform an in-depth interaction study of these three superantigens with antibodies, bio-layer interferometry (BLI) measurements of their interactions with a permutation panel of 63 IgG1 variants of Pertuzumab and Trastuzumab CDRs grafted to the six human Vκ and seven human VH region families were tested. Through this holistic and systemic analysis of IgG1 variants with various antibody regions modified, comparisons revealed novel PpL-antibody interactions influenced by other non-canonical antibody known light-chain framework regions, whereas SpA and SpG showed relatively consistent interactions. These findings have implications on PpL-based affinity antibody purification and design that can guide the engineering and understanding of PpL-based microbiota-immune effects.

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.

Peering into Avian Influenza A(H5N8) for a Framework towards Pandemic Preparedness.

2014 marked the first emergence of avian influenza A(H5N8) in Jeonbuk Province, South Korea, which then quickly spread worldwide. In the midst of the 2020-2021 H5N8 outbreak, it spread to domestic poultry and wild waterfowl shorebirds, leading to the first human infection in Astrakhan Oblast, Russia. Despite being clinically asymptomatic and without direct human-to-human transmission, the World Health Organization stressed the need for continued risk assessment given the nature of Influenza to reassort and generate novel strains. Given its promiscuity and easy cross to humans, the urgency to understand the mechanisms of possible species jumping to avert disastrous pandemics is increasing. Addressing the epidemiology of H5N8, its mechanisms of species jumping and its implications, mutational and reassortment libraries can potentially be built, allowing them to be tested on various models complemented with deep-sequencing and automation. With knowledge on mutational patterns, cellular pathways, drug resistance mechanisms and effects of host proteins, we can be better prepared against H5N8 and other influenza A viruses.

Molecular Insights of Nickel Binding to Therapeutic Antibodies as a Possible New Antibody Superantigen.

The binding of nickel by immune proteins can manifest as Type IV contact dermatitis (Ni-specific T cells mediated) and less frequently as Type I hypersensitivity with both mechanisms remaining unknown to date. Since there are reports of patients co-manifesting the two hypersensitivities, a common mechanism may underlie both the TCR and IgE nickel binding. Focusing on Trastuzumab and Pertuzumab IgE variants as serendipitous investigation models, we found Ni-NTA interactions independent of Her2 binding to be due to glutamine stretches. These stretches are both Ni-inducible and in fixed pockets at the antibody complementarity-determining regions (CDRs) and framework regions (FWRs) of both the antibody heavy and light chains with influence from the heavy chain constant region. Comparisons with TCRs structures revealed similar interactions, demonstrating the possible underlying mechanism in selecting for Ni-binding IgEs and TCRs respectively. With the elucidation of the interaction, future therapeutic antibodies could also be sagaciously engineered to utilize such nickel binding for biotechnological purposes.

Method for Zero-Waste Circular Economy Using Worms for Plastic Agriculture: Augmenting Polystyrene Consumption and Plant Growth.

Polystyrene (PS) is one of the major plastics contributing to environmental pollution with its durability and resistance to natural biodegradation. Recent research showed that mealworms (Tenebrio molitor) and superworms (Zophobas morio) are naturally able to consume PS as a carbon food source and degrade them without observable toxic effects. In this study, we explored the effects of possible food additives and use of worm frass as potential plant fertilizers. We found that small amounts of sucrose and bran increased PS consumption and that the worm frass alone could support dragon fruit cacti (Hylocereus undatus) growth, with superworm frass in particular, supporting better growth and rooting than mealworm frass and control media over a fortnight. As known fish and poultry feed, these findings present worms as a natural solution to simultaneously tackle both the global plastic problem and urban farming issue in a zero-waste sustainable bioremediation cycle.

Design and Development of a Low Cost, Non-Contact Infrared Thermometer with Range Compensation.

Fever is a common symptom of many infections, e.g., in the ongoing COVID-19 pandemic, keeping monitoring devices such as thermometers in constant demand. Recent technological advancements have made infrared (IR) thermometers the choice for contactless screening of multiple individuals. Yet, even so, the measurement accuracy of such thermometers is affected by many factors including the distance from the volunteers' forehead, impurities (such as sweat), and the location measured on the volunteers' forehead. To overcome these factors, we describe the assembly of an Arduino-based digital IR thermometer with distance correction using the MLX90614 IR thermometer and HC-SR04 ultrasonic sensors. Coupled with some analysis of these factors, we also found ways to programme compensation methods for the final assembled digital IR thermometer to provide more accurate readings and measurements.

To Plate or to Simply Unfreeze, That Is the Question for Optimal Plasmid Extraction.

Many molecular biology applications require fast plasmid DNA extraction, spurring multiple studies on how to speed up the process. It is regularly instructed in standard laboratory protocols to plate out frozen glycerol bacterial stocks prior to bacteria incubation in liquid media and subsequent plasmid extraction, although the rationale for this is often unexplained (other than for the isolation of single colonies). Given the commonality and importance of this laboratory operation, such a practice is time-consuming and laborious. To study the impact of this practice and the alternative direct culturing method, we investigated the association between bacterial cell mass and its potential influence on plasmid yields from the 2 methods. Our results showed no difference with preplating for 7 out of 8 plasmid constructs used in the study, suggesting that direct glycerol recovery would not lead to poorer plasmid yields. The findings support the rationale for direct glycerol recovery for plasmid extraction, without the need of an intermediate preplating step.

An Alternative HIV-1 Non-Nucleoside Reverse Transcriptase Inhibition Mechanism: Targeting the p51 Subunit.

The ongoing development of drug resistance in HIV continues to push for the need of alternative drug targets in inhibiting HIV. One such target is the Reverse transcriptase (RT) enzyme which is unique and critical in the viral life cycle-a rational target that is likely to have less off-target effects in humans. Serendipitously, we found two chemical scaffolds from the National Cancer Institute (NCI) Diversity Set V that inhibited HIV-1 RT catalytic activity. Computational structural analyses and subsequent experimental testing demonstrated that one of the two chemical scaffolds binds to a novel location in the HIV-1 RT p51 subunit, interacting with residue Y183, which has no known association with previously reported drug resistance. This finding supports the possibility of a novel druggable site on p51 for a new class of non-nucleoside RT inhibitors that may inhibit HIV-1 RT allosterically. Although inhibitory activity was shown experimentally to only be in the micromolar range, the scaffolds serve as a proof-of-concept of targeting the HIV RT p51 subunit, with the possibility of medical chemistry methods being applied to improve inhibitory activity towards more effective drugs.

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.

Not all therapeutic antibody isotypes are equal: the case of IgM versus IgG in Pertuzumab and Trastuzumab.

The therapeutic potential of immunoglobulin M (IgM) is of considerable interest in immunotherapy due to its complement-activating and cell-agglutinating abilities. Pertuzumab and Trastuzumab are monoclonal antibodies used to treat human epidermal growth factor receptor 2 (HER2)-positive breast cancer but exhibit significantly different binding affinities as IgM when compared to its IgG isotype. Using integrative multiscale modelling and simulations of complete antibody assemblies, we show that Pertuzumab IgM is able to utilize all of its V-regions to bind multiple HER2 receptors simultaneously, while similar binding in Trastuzumab IgM is prohibited by steric clashes caused by the large globular domain of HER2. This is subsequently validated by confirming that Pertuzumab IgM inhibits proliferation in HER2 over-expressing live cells more effectively than its IgG counterpart and Trastuzumab IgM. Our study highlights the importance of understanding the molecular details of antibody-antigen interactions for the design and isotype selection of therapeutic antibodies.

The Determination of HIV-1 RT Mutation Rate, Its Possible Allosteric Effects, and Its Implications on Drug Resistance.

The high mutation rate of the human immunodeficiency virus type 1 (HIV-1) plays a major role in treatment resistance, from the development of vaccines to therapeutic drugs. In addressing the crux of the issue, various attempts to estimate the mutation rate of HIV-1 resulted in a large range of 10-5-10-3 errors/bp/cycle due to the use of different types of investigation methods. In this review, we discuss the different assay methods, their findings on the mutation rates of HIV-1 and how the locations of mutations can be further analyzed for their allosteric effects to allow for new inhibitor designs. Given that HIV is one of the fastest mutating viruses, it serves as a good model for the comprehensive study of viral mutations that can give rise to a more horizontal understanding towards overall viral drug resistance as well as emerging viral diseases.

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.

Role of the IgE variable heavy chain in FcεRIα and superantigen binding in allergy and immunotherapy.

BACKGROUND: Variable heavy chain (VH) family frameworks (FWRs) have been reported to affect antibody receptor and superantigen binding; however, such effects in IgE remain largely unknown. Given that VH family biases have been previously reported in IgE of certain allergies, there is a need to investigate this phenomenon for biotechnological and therapeutic purposes. OBJECTIVE: We sought to investigate the effects of VH families on IgE interaction with FcεRIα, anti-IgE omalizumab, antigen, and superantigen protein A (spA) by using the pertuzumab and trastuzumab IgE models. METHODS: Pertuzumab VH1-VH7 family variants of IgE with the same complementarity-determining regions were investigated with regard to their binding interactions to FcεRIα, Her2, omalizumab, and spA. Notable FcεRIα-IgE observations were cross-checked against appropriate trastuzumab IgE VH variants. Computational structural modeling and simulations were also performed for insight into the mechanism of interactions with various VH FWRs. RESULTS: The pertuzumab VH5 IgE variant, but not the trastuzumab VH5 IgE, was found to interact with FcεRIα significantly longer than the respective VH family variants within each model antibody. No significant differences in interaction were found between IgE and omalizumab for the pertuzumab VH variants. Although trastuzumab VH3 interacted with spA, none of our pertuzumab VH variants, including VH3, associated with spA. CONCLUSION: We found unexpected varying allosteric communications caused by the VH family FWRs to the FcεRIα-, Her2-, and spA-binding regions of pertuzumab IgE, with implications for use of IgE/anti-IgE therapeutics to treat allergy and IgE therapeutics in allergo-oncology.

Author Correction: The effects of Antibody Engineering CH and CL in Trastuzumab and Pertuzumab recombinant models: Impact on antibody production and antigen-binding.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Effect of VH-VL Families in Pertuzumab and Trastuzumab Recombinant Production, Her2 and FcγIIA Binding.

Many therapeutic antibodies are humanized from animal sources. In the humanization process, complementarity determining region grafting is tedious and highly prone to failure. With seven known VH families, and up to six known κ VL families, there are choices aplenty. However, the functions of these families remain largely enigmatic. To study the role of these V-region families, we made 84 recombinant combinations of the various VH and VL family whole IgG1 variants of both Trastuzumab and Pertuzumab. We managed to purify 66 of these to investigate the biophysical characteristics: recombinant protein production, and both Her2 and FcγIIA binding. Our findings revealed combinations that showed improved recombinant antibody production and both antigen and receptor binding kinetics. These findings show the need to rethink antibodies as a whole protein, relooking of the functions of the antibody domains, and the need to include immunoglobulin receptor investigations for effective antibody therapeutics development.

The effects of Antibody Engineering CH and CL in Trastuzumab and Pertuzumab recombinant models: Impact on antibody production and antigen-binding.

Current therapeutic antibodies such as Trastuzumab, are typically of the blood circulatory IgG1 class (Cκ/ CHγ1). Due to the binding to Her2 also present on normal cell surfaces, side effects such as cardiac failure can sometimes be associated with such targeted therapy. Using antibody isotype swapping, it may be possible to reduce systemic circulation through increased tissue localization, thereby minimising unwanted side effects. However, the effects of such modifications have yet to be fully characterized, particularly with regards to their biophysical properties in antigen binding. To do this, we produced all light and heavy chain human isotypes/subtypes recombinant versions of Trastuzumab and Pertuzumab, and studied them with respect to recombinant production and Her2 binding. Our findings show that while the light chain constant region changes have no major effects on production or Her2 binding, some heavy chain isotypes, in particularly, IgM and IgD isotypes, can modulate antigen binding. This study thus provides the groundwork for such isotype modifications to be performed in the future to yield therapeutics of higher efficacy and efficiency.

Discovery of a novel splice variant of Fcar (CD89) unravels sequence segments necessary for efficient secretion: A story of bad signal peptides and good ones that nevertheless do not make it.

The IgA receptor, Fcar (CD89) consists of 5 sequence segments: 2 segments (S1, S2) forming the potential signal peptide, 2 extracellular EC domains that include the IgA binding site, and the transmembrane and cytoplasmic tail (TM/C) region. Numerous Fcar splice variants have been reported with various combinations of the sequence segments mentioned above. Here, we report a novel splice variant termed variant APD isolated from a healthy volunteer that lacks only the IgA-binding EC1 domain. Despite possessing the complete signal peptide S1+S2, the variant APD is only found in the intracellular space whereas the wild-type variant 1 is efficiently secreted and variant 4 leaks to the extracellular space. Further mutational experiments involving signal peptide replacements, cleavage site modifications, and studies on alternative isoforms demonstrate that despite the completeness of the signal peptide motif, the presence of the EC1 domain is essential for efficient extracellular export.