Major progress has been made in the past decade towards understanding of the biosynthesis of red carotenoid astaxanthin and its roles in stress response while exploiting microalgae-based astaxanthin as a potent antioxidant for human health and as a coloring agent for aquaculture applications. In this review, astaxanthin-producing green microalgae are briefly summarized with Haematococcus pluvialis and Chlorella zofingiensis recognized to be the most popular astaxanthin-producers. Two distinct pathways for astaxanthin synthesis along with associated cellular, physiological, and biochemical changes are elucidated using H. pluvialis and C. zofingiensis as the model systems. Interactions between astaxanthin biosynthesis and photosynthesis, fatty acid biosynthesis and enzymatic defense systems are described in the context of multiple lines of defense mechanisms working in concert against photooxidative stress. Major pros and cons of mass cultivation of H. pluvialis and C. zofingiensis in phototrophic, heterotrophic, and mixotrophic culture modes are analyzed. Recent progress in genetic engineering of plants and microalgae for astaxanthin production is presented. Future advancement in microalgal astaxanthin research will depend largely on genome sequencing of H pluvialis and C. zofingiensis and genetic toolbox development. Continuous effort along the heterotrophic-phototrophic culture mode could lead to major expansion of the micro algal astaxanthin industry.

Nitrogen availability and cell density each affects growth and cellular astaxanthin content of Haematococcus pluvialis, but possible combined effects of these two factors on the content and productivity of astaxanthin, especially under outdoor culture conditions, is less understood. In this study, the effects of the initial biomass densities IBDs of 0.1, 0.5, 0.8, 1.5, 2.7, 3.5, and 5.0 g L-1 DW and initial nitrogen concentrations of 0, 4.4, 8.8, and 17.6 mM nitrate on growth and cellular astaxanthin content of H. pluvialis Flotow K-0084 were investigated in outdoor glass column photobioreactors in a batch culture mode. A low IBD of 0.1 g L-1 DW led to photo-bleaching of the culture within 1-2 days. When the IBD was 0.5 g L-1 and above, the rate at which the increase in biomass density and the astaxanthin content on a per cell basis was higher at lower IBD. When the IBD was optimal (i.e., 0.8 g L-1), the maximum astaxanthin content of 3.8% of DW was obtained in the absence of nitrogen, whereas the maximum astaxanthin productivity of 16.0 mg L-1 d(-1) was obtained in the same IBD culture containing 4.4 mM nitrogen. The strategies for achieving maximum Haematococcus biomass productivity and for maximum cellular astaxanthin content are discussed.

Recent outbreaks of Zika virus (ZIKV) highlight the urgent need to develop efficacious interventions against flaviviruses, many of which cause devastating epidemics around the world. Monoclonal antibodies (mAb) have been at the forefront of treatment for cancer and a wide array of other diseases due to their specificity and potency. While mammalian cell-produced mAbs have shown promise as therapeutic candidates against several flaviviruses, their eventual approval for human application still faces several challenges including their potential risk of predisposing treated patients to more severe secondary infection by a heterologous flavivirus through antibody-dependent enhancement (ADE). The high cost associated with mAb production in mammalian cell cultures also poses a challenge for the feasible application of these drugs to the developing world where the majority of flavivirus infection occurs. Here, we review the current therapeutic mAb candidates against various flaviviruses including West Nile (WNV), Dengue virus (DENV), and ZIKV. The progress of using plants for developing safer and more economical mAb therapeutics against flaviviruses is discussed within the context of their expression, characterization, downstream processing, neutralization, and in vivo efficacy. The progress of using plant glycoengineering to address ADE, the major impediment of flavivirus therapeutic development, is highlighted. These advancements suggest that plant-based systems are excellent alternatives for addressing the remaining challenges of mAb therapeutic development against flavivirus and may facilitate the eventual commercialization of these drug candidates.

Several Zika virus (ZIKV) vaccine candidates have recently been described which use inactivated whole virus, DNA or RNA that express the virus’ Envelope (E) glycoprotein as the antigen. These were successful in stimulating production of virus-targeted antibodies that protected animals against ZIKV challenges, but their use potentially will predispose vaccinated individuals to infection by the related Dengue virus (DENV). We have devised a virus like particle (VLP) carrier based on the hepatitis B core antigen (HBcAg) that displays the ZIKV E protein domain III (zDIII), and shown that it can be produced quickly and easily purified in large quantities from Nicotiana benthamiana plants. HBcAg-zDIII VLPs are shown to be highly immunogenic, as two doses elicited potent humoral and cellular responses in mice that exceed the threshold correlated with protective immunity against multiple strains of Zika virus. Notably, HBcAg-zDIII VLPs-elicited antibodies did not enhance the infection of DENV in Fc gamma receptor-expressing cells, offsetting the concern of ZIKV vaccines inducing cross-reactive antibodies and sensitizing people to subsequent DENV infection. Thus, our zDIII-based vaccine offers improved safety and lower cost production than other current alternatives, with equivalent effectiveness.

The lack of lipidome analytical tools has limited our ability to gain new knowledge about lipid metabolism in microalgae, especially for membrane glycerolipids. An electrospray ionization mass spectrometry-based lipidomics method was developed for Nannochloropsis oceanica IMET1, which resolved 41 membrane glycerolipids molecular species belonging to eight classes. Changes in membrane glycerolipids under nitrogen deprivation and high-light (HL) conditions were uncovered. The results showed that the amount of plastidial membrane lipids including monogalactosyldiacylglycerol, phosphatidylglycerol, and the extraplastidic lipids diacylglyceryl-O-4′-(N, N, N,-trimethyl) homoserine and phosphatidylcholine decreased drastically under HL and nitrogen deprivation stresses. Algal cells accumulated considerably more digalactosyldiacylglycerol and sulfoquinovosyldiacylglycerols under stresses. The genes encoding enzymes responsible for biosynthesis, modification and degradation of glycerolipids were identified by mining a time-course global RNA-seq data set. It suggested that reduction in lipid contents under nitrogen deprivation is not attributable to the retarded biosynthesis processes, at least at the gene expression level, as most genes involved in their biosynthesis were unaffected by nitrogen supply, yet several genes were significantly up-regulated. Additionally, a conceptual eicosapentaenoic acid (EPA) biosynthesis network is proposed based on the lipidomic and transcriptomic data, which underlined import of EPA from cytosolic glycerolipids to the plastid for synthesizing EPA-containing chloroplast membrane lipids.

In vitro models that mimic in vivo host-pathogen interactions are needed to evaluate candidate drugs that inhibit bacterial virulence traits. We established a new approach to study Pseudomonas aeruginosa biofilm susceptibility on biotic surfaces, using a three-dimensional (3-D) lung epithelial cell model. P. aeruginosa formed antibiotic resistant biofilms on 3-D cells without affecting cell viability. The biofilm-inhibitory activity of antibiotics and/or the anti-biofilm peptide DJK-5 were evaluated on 3-D cells compared to a plastic surface, in medium with and without fetal bovine serum (FBS). In both media, aminoglycosides were more efficacious in the 3-D cell model. In serum-free medium, most antibiotics (except polymyxins) showed enhanced efficacy when 3-D cells were present. In medium with FBS, colistin was less efficacious in the 3-D cell model. DJK-5 exerted potent inhibition of P. aeruginosa association with both substrates, only in serum-free medium. DJK-5 showed stronger inhibitory activity against P. aeruginosa associated with plastic compared to 3-D cells. The combined addition of tobramycin and DJK-5 exhibited more potent ability to inhibit P. aeruginosa association with both substrates. In conclusion, lung epithelial cells influence the efficacy of most antimicrobials against P. aeruginosa biofilm formation, which in turn depends on the presence or absence of FBS.

We described the rapid production of the domain III (DIII) of the envelope (E) protein in plants as a vaccine candidate for West Nile Virus (WNV). Using various combinations of vector modules of a deconstructed viral vector expression system, DIII was produced in three subcellular compartments in leaves of Nicotiana benthamiana by transient expression. DIII expressed at much higher levels when targeted to the endoplasmic reticulum (ER) than that targeted to the chloroplast or the cytosol, with accumulation level up to 73 μg DIII per gram of leaf fresh weight within 4 days after infiltration. Plant ER-derived DIII was soluble and readily purified to > 95% homogeneity without the time-consuming process of denaturing and refolding. Further analysis revealed that plant-produced DIII was processed properly and demonstrated specific binding to an anti-DIII monoclonal antibody that recognizes a conformational epitope. Furthermore, subcutaneous immunization of mice with 5 and 25 μg of purified DIII elicited a potent systemic response. This study provided the proof of principle for rapidly producing immunogenic vaccine candidates against WNV in plants with low cost and scalability.

The increasing world demand for human biologics cannot be met by current production platforms based primarily on mammalian cell culture due to prohibitive cost and limited scalability [1]. Recent progress in plant expression vector development, downstream processing, and glycoengineering has established plants as a superior alternative to biologic production [2–4]. Plants not only offer the traditional advantages of proper eukaryotic protein modification, potential low cost, high scalability, and increased safety but also allow the production of biologics at unprecedented speed to control potential pandemics or with specific glycoforms for better efficacy or safety (biobetters) [5, 6]. The approval of the first plant-made biologic (PMB) by the United States Food and Drug Administration (FDA) for treating Gaucher’s disease heralds a new era for PMBs and sparks new innovations in this field [7, 8].

Strategies are needed to improve repopulation of decellularized lung scaffolds with stromal and functional epithelial cells. We demonstrate that decellularized mouse lungs recellularized in a dynamic low fluid shear suspension bioreactor, termed the rotating wall vessel (RWV), contained more cells with decreased apoptosis, increased proliferation and enhanced levels of total RNA compared to static recellularization conditions. These results were observed with two relevant mouse cell types: bone marrow-derived mesenchymal stromal (stem) cells (MSCs) and alveolar type II cells (C10). In addition, MSCs cultured in decellularized lungs under static but not bioreactor conditions formed multilayered aggregates. Gene expression and immunohistochemical analyses suggested differentiation of MSCs into collagen I-producing fibroblast-like cells in the bioreactor, indicating enhanced potential for remodeling of the decellularized scaffold matrix. In conclusion, dynamic suspension culture is promising for enhancing repopulation of decellularized lungs, and could contribute to remodeling the extracellular matrix of the scaffolds with subsequent effects on differentiation and functionality of inoculated cells.

Extra-intestinal pathogenic E. coli (ExPEC), including avian pathogenic E. coli (APEC), pose a considerable threat to both human and animal health, with illness causing substantial economic loss. APEC strain χ7122 (O78∶K80∶H9), containing three large plasmids [pChi7122-1 (IncFIB/FIIA-FIC), pChi7122-2 (IncFII), and pChi7122-3 (IncI₂)]; and a small plasmid pChi7122-4 (ColE2-like), has been used for many years as a model strain to study the molecular mechanisms of ExPEC pathogenicity and zoonotic potential. We previously sequenced and characterized the plasmid pChi7122-1 and determined its importance in systemic APEC infection; however the roles of the other pChi7122 plasmids were still ambiguous. Herein we present the sequence of the remaining pChi7122 plasmids, confirming that pChi7122-2 and pChi7122-3 encode an ABC iron transport system (eitABCD) and a putative type IV fimbriae respectively, whereas pChi7122-4 is a cryptic plasmid. New features were also identified, including a gene cluster on pChi7122-2 that is not present in other E. coli strains but is found in Salmonella serovars and is predicted to encode the sugars catabolic pathways. In vitro evaluation of the APEC χ7122 derivative strains with the three large plasmids, either individually or in combinations, provided new insights into the role of plasmids in biofilm formation, bile and acid tolerance, and the interaction of E. coli strains with 3-D cultures of intestinal epithelial cells. In this study, we show that the nature and combinations of plasmids, as well as the background of the host strains, have an effect on these phenomena. Our data reveal new insights into the role of extra-chromosomal sequences in fitness and diversity of ExPEC in their phenotypes.