Synthetic gene design - The rationale for codon optimization and implications for molecular pharming in plants.

Degeneracy in the genetic code allows multiple codon sequences to encode the same protein. Codon usage bias in genes is the term given to the preferred use of particular synonymous codons. Synonymous codon substitutions had been regarded as "silent" as the primary structure of the protein was not affected; however, it is now accepted that synonymous substitutions can have a significant effect on heterologous protein expression. Codon optimization, the process of altering codons within the gene sequence to improve recombinant protein expression, has become widely practised. Multiple inter-linked factors affecting protein expression need to be taken into consideration when optimizing a gene sequence. Over the years, various computer programmes have been developed to aid in the gene sequence optimization process. However, as the rulebook for altering codon usage to affect protein expression is still not completely understood, it is difficult to predict which strategy, if any, will design the 'optimal' gene sequence. In this review, codon usage bias and factors affecting codon selection will be discussed and the evidence for codon optimization impact will be reviewed for recombinant protein expression using plants as a case study. These developments will be relevant to all recombinant expression systems, however, molecular pharming in plants is an area which has consistently encountered difficulties with low levels of recombinant protein expression, and should benefit from an evidence based rational approach to synthetic gene design

Bond-shear Behavior of FRP Rods as a Function of Attachment Configuration

The use of external reinforcement to improve or enhances the flexural capacity of a member depends on the transfer capacity, and the failure behavior of the composite between the reinforcement, the epoxy resin and the concrete. The most influencing factor is the bond-shear capacity between the rod and the epoxy, and the epoxy to the concrete. Fiber Reinforced Polymer (FRP) rods are the latest alternate for fulfilling the external reinforcement scheme. In the field, the mandated embedment depth as outlined by the ACI 440 code, could customary not be achieved since factors such as the depth of the concrete cover, and presence of stirrups limits the space. This study is aimed to evaluate the effect of FRP rod configurations with respect to the concrete surface, to the effectiveness of external reinforcement. The study looked into the bond-shear capacity as well as the mode of failure, influence by the rod attachment depth. It was shown that the embedment depth significantly influenced the failure mode, and therefore the strain transfer capacity from the concrete to the rods

On the Performance of a Multi Story Irregular Apartment Building Model Under Seismic Load in Indonesian Moderately High Seismicity Region

Purbalingga is regency with a potential moderately high seismicity requiring compliance of planning and implementation rules of the earthquake-resistant structural system. The purpose of this research is to evaluate the performance of a ten-story irregular apartment building model in Purbalingga due to the seismic load. The study is necessarily conducted to provide information on impacts and mitigation strategies that should be implemented. This research was conducted based on the seismic capacity of 2002 and 2012 Indonesian National Standard (SNI) including linear static analysis, dynamic response analysis, and pushover analysis. Based on the direct static review, it shows that the base shear is reduced and the drift ratio level decreases respectively for X and Y direction.Meanwhile, based on the dynamic response analysis, the drift ratio level also decreases respectively for X and Y direction. Also, the pushover analysis indicates that the performance of this apartment building model is still at Immediate Occupancy (IO) level as the post-earthquake damage state that remains safe to occupy, essentially retains the pre-earthquake design strength and stiffness of the structure. The risk of life-threatening injury as a result of structural damage is very low, and although some minor structural repairs may be appropriate, these would generally not be required before occupanc

High-level expression of the HIV entry inhibitor griffithsin from the plastid genome and retention of biological activity in dried tobacco leaves

The global HIV epidemic continues to grow, with 1.8 million new infections occurring per year. In the absence of a cure and an AIDS vaccine, there is a pressing need to prevent new infections in order to curb the disease. Topical microbicides that block viral entry into human cells can potentially prevent HIV infection. The antiviral lectin griffithsin has been identified as a highly potent inhibitor of HIV entry into human cells. Here we have explored the possibility to use transplastomic plants as an inexpensive production platform for griffithsin. We show that griffithsin accumulates in stably transformed tobacco chloroplasts to up to 5% of the total soluble protein of the plant. Griffithsin can be easily purified from leaf material and shows similarly high virus neutralization activity as griffithsin protein recombinantly expressed in bacteria. We also show that dried tobacco provides a storable source material for griffithsin purification, thus enabling quick scale-up of production on demand

Current progress on removal of recalcitrance coloured particles from anaerobically treated effluent using coagulation–flocculation

The palm oil industry is the most important agro industries in Malaysia and most of the mills adopt anaerobic digestion as their primary treatment for palm oil mill effluent (POME). Due to the public concern, decolourisation of anaerobically treated POME (AnPOME) is becoming a great concern. Presence of recalcitrant-coloured particles hinders biological processes and coagulation–flocculation may able to remove these coloured particles. Several types of inorganic and polymers-based coagulant/flocculant aids for coagulation–flocculation of AnPOME have been reviewed. Researchers are currently interested in using natural coagulant and flocculant aids. Modification of the properties of natural coagulant and flocculant aids enhanced coagulation–flocculation performance. Modelling and optimization of the coagulation–flocculation process have also been reviewed. Chemical sludge has the potential for plant growth that can be evaluated through pot trials and phytotoxicity test

Nonlinear 3D Model of Double Shear Lap Tests for the Bond of Near-surface Mounted FRP Rods in Concrete Considering Different Embedment Depth

The utilization of near-surface mounted Fiber Reinforced Polymer (FRP) reinforcement as a method of strengthening in reinforced concrete structures has increased considerably in recent years. Moreover, the application of double-shear lap tests for this rein-forcement method leads to the achievement of a local bond-slip behavior in a bonded joint. This research, therefore, focused on 3-D modeling of this type of test to suitably characterize the bond mechanics between FRP rods and concrete at various embedment depth. The use of different alternatives to represent the interface between the FRP rod and concrete were analyzed after which a comparison was drawn between the numerical finite element (FE) simulations and experimental measurements. The results showed the prediction of the load–slip corresponded with the data obtained from the experiment. Finally, the proposed model has the ability to express the relationship between the penalty stiffness parameters in shear direction Kss = (Ktt) and the embedment depth of FRP rods

Multiple gene expression in plants using MIDAS-P, a versatile type II restriction-based modular expression vector.

MIDAS-P is a plant expression vector with blue/white screening for iterative cloning of multiple, tandemly-arranged transcription units (TUs). We have used the MIDAS-P system to investigate expression of up to five genes encoding three anti-HIV proteins and the reporter gene DsRed in Nicotiana benthamiana plants. The anti-HIV cocktail was made up of a broadly neutralizing monoclonal antibody (VRC01), a lectin (Griffithsin), and a single-chain camelid nanobody (J3-VHH). Constructs containing different combinations of 3, 4 or 5 TUs encoding different components of the anti-HIV cocktail were assembled. mRNA levels of the genes of interest decreased beyond two TUs. Co-expression of the RNA silencing suppressor P19 dramatically increased overall mRNA and protein expression levels of each component. The position of individual TUs in 3 TU constructs did not affect mRNA or protein expression levels. However, their expression dropped to non-detectable levels in constructs with 4 or more TUs each containing the same promoter and terminator elements, with the exception of DsRed at the first or last position in 5 TU constructs. This drop was alleviated by co-expression of P19. In short, the MIDAS-P system is suitable for the simultaneous expression of multiple proteins in one construct. This article is protected by copyright. All rights reserved

Characterisation of a highly potent and near pan-neutralising anti-HIV monoclonal antibody expressed in tobacco plants.

BACKGROUND: HIV remains one of the most important health issues worldwide, with almost 40 million people living with HIV. Although patients develop antibodies against the virus, its high mutation rate allows evasion of immune responses. Some patients, however, produce antibodies that are able to bind to, and neutralise different strains of HIV. One such 'broadly neutralising' antibody is 'N6'. Identified in 2016, N6 can neutralise 98% of HIV-1 isolates with a median IC50 of 0.066 µg/mL. This neutralisation breadth makes N6 a very promising therapeutic candidate. RESULTS: N6 was expressed in a glycoengineered line of N. benthamiana plants (pN6) and compared to the mammalian cell-expressed equivalent (mN6). Expression at 49 mg/kg (fresh leaf tissue) was achieved in plants, although extraction and purification are more challenging than for most plant-expressed antibodies. N-glycoanalysis demonstrated the absence of xylosylation and a reduction in α(1,3)-fucosylation that are typically found in plant glycoproteins. The N6 light chain contains a potential N-glycosylation site, which was modified and displayed more α(1,3)-fucose than the heavy chain. The binding kinetics of pN6 and mN6, measured by surface plasmon resonance, were similar for HIV gp120. pN6 had a tenfold higher affinity for FcγRIIIa, which was reflected in an antibody-dependent cellular cytotoxicity assay, where pN6 induced a more potent response from effector cells than that of mN6. pN6 demonstrated the same potency and breadth of neutralisation as mN6, against a panel of HIV strains. CONCLUSIONS: The successful expression of N6 in tobacco supports the prospect of developing a low-cost, low-tech production platform for a monoclonal antibody cocktail to control HIV in low-to middle income countries

Engineering the N-glycosylation pathway of Nicotiana tabacum for molecular pharming using CRISPR/Cas9.

Molecular pharming in plants offers exciting possibilities to address global access to modern biologics. However, differences in the N-glycosylation pathway including the presence of β(1,2)-xylose and core α(1,3)-fucose can affect activity, potency and immunogenicity of plant-derived proteins. Successful glycoengineering approaches toward human-like structures with no changes in plant phenotype, growth, or recombinant protein expression levels have been reported for Arabidopsis thaliana and Nicotiana benthamiana. Such engineering of N-glycosylation would also be desirable for Nicotiana tabacum, which remains the crop of choice for recombinant protein pharmaceuticals required at massive scale and for manufacturing technology transfer to less developed countries. Here, we generated N. tabacum cv. SR-1 β(1,2)-xylosyltransferase (XylT) and α(1,3)-fucosyltransferase (FucT) knockout lines using CRISPR/Cas9 multiplex genome editing, targeting three conserved regions of the four FucT and two XylT genes. These two enzymes are responsible for generating non-human N-glycan structures. We confirmed full functional knockout of transformants by immunoblotting of total soluble protein by antibodies recognizing β(1,2)-xylose and core α(1,3)-fucose, mass spectrometry analysis of recombinantly produced VRC01, a broadly neutralizing anti-HIV-1 hIgG1 antibody, and Sanger sequencing of targeted regions of the putative transformants. These data represent an important step toward establishing Nicotiana tabacum as a biologics platform for Global Health

A Dual-Approach Strategy to Optimize the Safety and Efficacy of Anti-Zika Virus Monoclonal Antibody Therapeutics.

Antibody-dependent enhancement of infection (ADE) is clinically relevant to Dengue virus (DENV) infection and poses a major risk to the application of monoclonal antibody (mAb)-based therapeutics against related flaviviruses such as the Zika virus (ZIKV). Here, we tested a two-tier approach for selecting non-cross-reactive mAbs combined with modulating Fc glycosylation as a strategy to doubly secure the elimination of ADE while preserving Fc effector functions. To this end, we selected a ZIKV-specific mAb (ZV54) and generated three ZV54 variants using Chinese hamster ovary cells and wild-type (WT) and glycoengineered ΔXF Nicotiana benthamiana plants as production hosts (ZV54CHO, ZV54WT, and ZV54ΔXF). The three ZV54 variants shared an identical polypeptide backbone, but each exhibited a distinct Fc N-glycosylation profile. All three ZV54 variants showed similar neutralization potency against ZIKV but no ADE activity for DENV infection, validating the importance of selecting the virus/serotype-specific mAbs for avoiding ADE by related flaviviruses. For ZIKV infection, however, ZV54CHO and ZV54ΔXF showed significant ADE activity while ZV54WT completely forwent ADE, suggesting that Fc glycan modulation may yield mAb glycoforms that abrogate ADE even for homologous viruses. In contrast to the current strategies for Fc mutations that abrogate all effector functions along with ADE, our approach allowed the preservation of effector functions as all ZV54 glycovariants retained antibody-dependent cellular cytotoxicity (ADCC) against the ZIKV-infected cells. Furthermore, the ADE-free ZV54WT demonstrated in vivo efficacy in a ZIKV-infection mouse model. Collectively, our study provides further support for the hypothesis that antibody-viral surface antigen and Fc-mediated host cell interactions are both prerequisites for ADE, and that a dual-approach strategy, as shown herein, contributes to the development of highly safe and efficacious anti-ZIKV mAb therapeutics. Our findings may be impactful to other ADE-prone viruses, including SARS-CoV-2