Experimental design to optimize an <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> type b conjugate vaccine made with hydrazide-derivatized tetanus toxoid

The introduction of type b Haemophilus influenzae conjugate vaccines into routine vaccination schedules has significantly reduced the burden of this disease; however, widespread use in developing countries is constrained by vaccine costs, and there is a need for a simple and high-yielding manufacturing process. The vaccine is composed of purified capsular polysaccharide conjugated to an immunogenic carrier protein. To improve the yield and rate of the reductive amination conjugation reaction used to make this vaccine, some of the carboxyl groups of the carrier protein, tetanus toxoid, were modified to hydrazides, which are more reactive than the ε -amine of lysine. Other reaction parameters, including the ratio of the reactants, the size of the polysaccharide, the temperature and the salt concentration, were also investigated. Experimental design was used to minimize the number of experiments required to optimize all these parameters to obtain conjugate in high yield with target characteristics. It was found that increasing the reactant ratio and decreasing the size of the polysaccharide increased the polysaccharide:protein mass ratio in the product. Temperature and salt concentration did not improve this ratio. These results are consistent with a diffusion controlled rate limiting step in the conjugation reaction. Excessive modification of tetanus toxoid with hydrazide was correlated with reduced yield and lower free polysaccharide. This was attributed to a greater tendency for precipitation, possibly due to changes in the isoelectric point. Experimental design and multiple regression helped identify key parameters to control and thereby optimize this conjugation reaction.     

Ergosterol,an orphan fungal microbe‐associated molecular pattern (MAMP)

Fungal pathogens continue to pose a significant threat to crop production and food supply. The early stages of plant–fungus interactions are mostly mediated by microbe‐associated molecular pattern (MAMP) molecules, perceived by plant pattern recognition receptors (PRRs). Currently, the identified fungal MAMP molecules include chitin, chitosan, β‐glucans, elicitins and ergosterol. Although the molecular battles between host plants and infecting fungal phytopathogens have been studied extensively, many aspects still need to be investigated to obtain a holistic understanding of the intrinsic mechanisms, which is paramount in combating fungal plant diseases. Here, an overview is given of the most recent findings concerning an ‘orphan’ fungal MAMP molecule, ergosterol, and we present what is currently known from a synopsis of different genes, proteins and metabolites found to play key roles in induced immune responses in plant–fungus interactions. Clearly, integrative investigations are still needed to provide a comprehensive systems‐based understanding of the dynamics associated with molecular mechanisms in plant–ergosterol interactions and associated host responses.     

Multi-Platform Metabolomic Analyses of Ergosterol-Induced Dynamic Changes in Nicotiana tabacum Cells

Metabolomics is providing new dimensions into understanding the intracellular adaptive responses in plants to external stimuli. In this study, a multi-technology-metabolomic approach was used to investigate the effect of the fungal sterol, ergosterol, on the metabolome of cultured tobacco cells. Cell suspensions were treated with different concentrations (0–1000 nM) of ergosterol and incubated for different time periods (0–24 h). Intracellular metabolites were extracted with two methods: a selective dispersive liquid-liquid micro-extraction and a general methanol extraction. Chromatographic techniques (GC-FID, GC-MS, GC×GC-TOF-MS, UHPLC-MS) and ¹H NMR spectroscopy were used for quantitative and qualitative analyses. Multivariate data analyses (PCA and OPLS-DA models) were used to extract interpretable information from the multidimensional data generated from the analytical techniques. The results showed that ergosterol triggered differential changes in the metabolome of the cells, leading to variation in the biosynthesis of secondary metabolites. PCA scores plots revealed dose- and time-dependent metabolic variations, with optimal treatment conditions being found to be 300 nM ergosterol and an 18 h incubation period. The observed ergosterol-induced metabolic changes were correlated with changes in defence-related metabolites. The ‘defensome’ involved increases in terpenoid metabolites with five antimicrobial compounds (the bicyclic sesquiterpenoid phytoalexins: phytuberin, solavetivone, capsidiol, lubimin and rishitin) and other metabolites (abscisic acid and phytosterols) putatively identified. In addition, various phenylpropanoid precursors, cinnamic acid derivatives and - conjugates, coumarins and lignin monomers were annotated. These annotated metabolites revealed a dynamic reprogramming of metabolic networks that are functionally correlated, with a high complexity in their regulation.     

Epitope mapping of novel monoclonal antibodies to human angiotensin I‐converting enzyme

Angiotensin I‐converting enzyme (ACE, CD143) plays a crucial role in blood pressure regulation, vascular remodeling, and immunity. A wide spectrum of mAbs to different epitopes on the N and C domains of human ACE have been generated and used to study different aspects of ACE biology, including establishing a novel approach–conformational fingerprinting. Here we characterized a novel set of 14 mAbs, developed against human seminal fluid ACE. The epitopes for these novel mAbs were defined using recombinant ACE constructs with truncated N and C domains, species cross‐reactivity, ACE mutagenesis, and competition with the previously mapped anti‐ACE mAbs. Nine mAbs recognized regions on the N domain, and 5 mAbs–on the C domain of ACE. The epitopes for most of these novel mAbs partially overlap with epitopes mapped onto ACE by the previously generated mAbs, whereas mAb 8H1 recognized yet unmapped region on the C domain where three ACE mutations associated with Alzheimer''s disease are localized and is a marker for ACE mutation T877M. mAb 2H4 could be considered as a specific marker for ACE in dendritic cells. This novel set of mAbs can identify even subtle changes in human ACE conformation caused by tissue‐specific glycosylation of ACE or mutations, and can detect human somatic and testicular ACE in biological fluids and tissues. Furthermore, the high reactivity of these novel mAbs provides an opportunity to study changes in the pattern of ACE expression or glycosylation in different tissues, cells, and diseases, such as sarcoidosis and Alzheimer''s disease.