Selection of single-chain antibodies against the VP8* subunit of rotavirus VP4 outer capsid protein and their expression in Lactobacillus casei.
ABSTRACTSingle-chain antibodies (scFv) recognizing the VP8* fraction of rotavirus outer capsid and blocking rotavirus infection in vitro were isolated by phage display. Vectors for the extracellular expression inLactobacillus caseiof one of the scFv were constructed.L. caseiwas able to secrete active scFv to the growth medium, showing the potential of probiotic bacteria to be engineered to express molecules suitable for in vivo antirotavirus therapies.
Secretion of the rotavirus VP8* protein inLactococcus lactis
Secretion of the VP8* subunit of the VP4 capsid protein of rotavirus by Lactococcus lactis has been achieved. For this purpose, a secretion vector has been constructed with the lactococcal signal sequence AL9 and the VP8*-encoding gene fragment. The amount of VP8* secreted by L. lactis in the culture supernatant was quantified and visualised by Western blot. Furthermore, it was shown to retain its hemagglutination capability, indicating that the conformation of the secreted peptide may be retaining its biological activity.
Screening and Construction of Probiotic Strains with Enhanced Protective Properties against Intestinal Disorders
Within the scope of the DEPROHEALTH project, a range of wild-type strains of Lactobacillus were analysed for their ability to interact with the host immune system. While the studied isolates interacted in a strain-specific way with immune cells, they seemed to have little and non-discriminative effect on epithelial cells. However, they were shown to facilitate the cross-talk between intestinal and immune cells. Studies conducted in mouse colitis models confirmed that specific strains possess higher intrinsic anti-inflammatory properties. Two of these were further engineered to produce murine IL10 and are presently being evaluated for their protective effect in a TNBS colitis model. Cell wal…
Single-chain variable fragment (scFv) antibodies against rotavirus NSP4 enterotoxin generated by phage display.
The rotavirus non-structural NSP4 protein causes membrane destabilization as well as an increase in intracellular calcium levels in eukaryotic cells and induces diarrhea in young mice, acting as a viral enterotoxin. In this study the phage display technique was used to generate a panel of single-chain variable fragment (scFv) antibodies specific for the NSP4 protein of the human rotavirus strain Wa from a human semi-synthetic scFv library. After several rounds of panning and selection on NSP4 adsorbed to polystyrene tubes, individual scFv were isolated and characterised by fingerprinting and by sequencing the VH and VL genes. The isolated scFv antibodies specifically recognize NSP4 in enzym…
Genome-Wide Expression Profiles in Very Low Birth Weight Infants With Neonatal Sepsis
BACKGROUND: Bacterial sepsis is associated with high morbidity and mortality in preterm infants. However, diagnosis of sepsis and identification of the causative agent remains challenging. Our aim was to determine genome-wide expression profiles of very low birth weight (VLBW) infants with and without bacterial sepsis and assess differences. METHODS: This was a prospective observational double-cohort study conducted in VLBW (<1500 g) infants with culture-positive bacterial sepsis and non-septic matched controls. Blood samples were collected as soon as clinical signs of sepsis were identified and before antibiotics were initiated. Total RNA was processed for genome-wide expression an…
Ultrasonically-Assisted and Conventional Extraction from Erodium Glaucophyllum Roots Using Ethanol:Water Mixtures: Phenolic Characterization, Antioxidant, and Anti-Inflammatory Activities
The paper presents experimental results concerning the ultrasonically-assisted extraction of bioactive compounds from Erodium glaucophyllum roots. A comparison with conventional methodology is presented, and thereby the phytochemical composition and the antioxidant and anti-inflammatory activities of extracts are evaluated. The phenolic profile of Erodium extracts was analyzed by TOF&ndash
Untargeted metabolomics of fresh and heat treatment Tiger nut ( Cyperus esculentus L. ) milks reveals further insight into food quality and nutrition
Tiger nut (Cyperus esculentus L.) is a crop traditionally grown in Valencia Region (Spain) and other temperate and tropical regions in the world, where its tubers are commonly consumed as tiger nut milk (horchata). Because of their nutritive potential and original taste, these products are beginning to spread internationally and, as consequence, analytical procedures to assess nutritional profiles, quality control issues are acquiring increasing relevance. The main objective of this study was to use an advance analytical method and chemometrics tools to determine if the ultra-high temperature (UHT) treatment necessary to extend the shelf life of tiger nut milk would affect the profile of nu…
Noroviral P-Particles as an In Vitro Model to Assess the Interactions of Noroviruses with Probiotics
Noroviruses (NoVs) are the main etiologic agents of acute epidemic gastroenteritis and probiotic bacteria have been reported to exert a positive effect on viral diarrhea. The protruding (P) domain from NoVs VP1 capsid protein has the ability to assemble into the so-called P-particles, which retain the binding ability to host receptors. We purified the P-domains from NoVs genotypes GI.1 and GII.4 as 6X(His)-tagged proteins and determined that, similar to native domains, they were structured into P-particles that were functional in the recognition of the specific glycoconjugated receptors, as established by surface plasmon resonance experiments. We showed that several lactic acid bacteria (pr…
The lactose operon from Lactobacillus casei is involved in the transport and metabolism of the human milk oligosaccharide core-2 N-acetyllactosamine
The lactose operon (lacTEGF) from Lactobacillus casei strain BL23 has been previously studied. The lacT gene codes for a transcriptional antiterminator, lacE and lacF for the lactose-specific phosphoenolpyruvate: phosphotransferase system (PTS) EIICB and EIIA domains, respectively, and lacG for the phospho-β-galactosidase. In this work, we have shown that L. casei is able to metabolize N-acetyllactosamine (LacNAc), a disaccharide present at human milk and intestinal mucosa. The mutant strains BL153 (lacE) and BL155 (lacF) were defective in LacNAc utilization, indicating that the EIICB and EIIA of the PTS are involved in the uptake of LacNAc in addition to lactose. Inactivation of lacG aboli…