Genetics and Evolution of Abiotic Stress Tolerance in Plants.

Now more than ever, the understanding of the genetics and evolution of the gene mechanisms and the networks of different molecular pathways acting on plant abiotic stress tolerance has an important role in the finding of new solutions and approaches mitigating the effects of global climate changes, thus contributing to a correct equilibrium among human needs, food security and human health and wellbeing [...

Expression Analysis of the Gene in Field-Grown Durum Wheat under Full and Reduced Irrigation.

Some of the key genes and regulatory mechanisms controlling drought response in durum wheat have been identified. One of the major challenges for breeders is how to use this knowledge for the achievement of drought stress tolerance. In the present study, we report the expression profiles of the gene, at consecutive plant growth stages, from different durum wheat genotypes evaluated in two different field environments.

Phenotyping of Different Italian Durum Wheat Varieties in Early Growth Stage With the Addition of Pure or Digestate-Activated Biochars.

This study aims to highlight the major effects of biochar incorporation into potting soil substrate on plant growth and performance in early growth stages of five elite Italian varieties of durum wheat (). The biochars used were obtained from two contrasting feedstocks, namely wood chips and wheat straw, by gasification under high temperature conditions, and were applied in a greenhouse experiment either as pure or as nutrient-activated biochar obtained by incubation with digestate. The results of the experiment showed that specific genotypes as well as different treatments with biochar have significant effects on plant response when looking at shoot traits related to growth.

B genome specific polymorphism in the TdDRF1 gene is in relationship with grain yield.

A and B genome copies of DRF1 gene in durum wheat were isolated and sequenced using gene variability. B genome specific polymorphism resulted, in a RIL population, in relationship with grain yield mainly in drought condition. Drought tolerance is one of the main components of yield potential and stability, and its improvement is a major challenge to breeders.

Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat.

Phenylalanine Ammonia Lyase (PAL) gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp.

Evaluation of triticale as energy crop in Italy.

The promotion of renewable energy represents a target of the European 2020 strategy for economical growth and sustainable competitiveness. Cereals are considered a promising biomass producing crop in temperate regions of Europe to be used for both fuel alcohol and biogas production. Among cereals, triticale represents a good candidate for this kind of application, showing a number of advantages such as high grain yield even in marginal environments, tolerance to drought, tolerance to more acid soils, lower production costs and lower susceptibility to biotic stresses.

Dependence of nanoparticle-cell recognition efficiency on the surface orientation of scFv targeting ligands.

The surface activation of multifunctional nanoparticles (MNPs) with peptide ligands directing their targeting to cancer cells is an emerging research area in nanobiotechnology. In this paper, water-soluble MNPs have been synthesized and functionalized with an scFv antibody variant specific toward the HER2 receptor overexpressed in several breast cancer cell lines. The scFv was genetically engineered to introduce a cysteine residue inside the loop sequence bridging the V and V lobes of the molecule and a histidine tag at the C-terminus in the V fragment.

Variability and expression profile of the DRF1 gene in four cultivars of durum wheat and one triticale under moderate water stress conditions.

The dehydration responsive element binding (DREB) proteins are important transcription factors that contribute to stress endurance in plants triggering the expression of a set of abiotic stress-related genes. A DREB2-related gene, previously referred to as dehydration responsive factor 1 (DRF1) was originally isolated and characterized in durum wheat. The aim of this study was to monitor the expression profiles of three alternatively spliced TdDRF1 transcripts during dehydration experiments and to evaluate the effects of genetic diversity on the molecular response, using experimental conditions reflecting as closely as possible water stress perceived by cereals in open field.

Site-specific conjugation of ScFvs antibodies to nanoparticles by bioorthogonal strain-promoted alkyne-nitrone cycloaddition.

Particularly suitable: An N-terminal serine mutant of anti-HER2 scFv antibody was conjugated to polymer-coated magnetofluorescent nanoparticles by strain-promoted alkyne-nitrone cycloaddition. The resulting nanoparticles (see scheme) proved effective in targeting and labeling HER2-positive breast cancer cells.

Bacterial cytoplasm production of an EGFP-labeled single-chain Fv antibody specific for the HER2 human receptor.

The human epidermal growth factor receptor 2 (HER2) is the main diagnostic marker of breast and ovary cancers. Here, to obtain a rapid and sensitive immunodiagnostic tool a single-chain antibody (scFv800E6) specific for the HER2 was fused to the N-terminus of the enhanced green fluorescent protein (EGFP) by a flexible linker. The soluble production of the novel scFv800E6-EGFP protein in the cytoplasm of Escherichia coli was investigated at different induction temperatures (25, 30 and 37°C); the intrinsic fluorescent properties and the binding activity to HER2 positive tumour cells of the fusion protein were analysed.

Multiple presentation of Scfv800E6 on silica nanospheres enhances targeting efficiency toward HER-2 receptor in breast cancer cells.

Spherical silica nanoparticles (SNP) have been synthesized and functionalized with anti-HER-2 scFv800E6 antibody by both localized histidine-tag recognition, leading to an oriented protein ligation, and glutaraldehyde cross-linking, exploiting a statistical reactivity of lysine amine groups in the primary sequence of the molecule. The targeting efficiency of nanocomplexes in comparison with free scFv was evaluated by flow cytometry using a HER-2 antigen-positive MCF-7 breast cancer cell line, exhibiting a 4-fold increase in scFv binding efficacy, close to the affinity of intact anti-HER-2 monoclonal antibody, which suggests the effectiveness of presenting multiple scFv molecules on nanoparticles in improving antigen recognition. Unexpectedly, the conjugation method did not affect the binding efficacy of scFv, suggesting a structural role of lysines in the scFv molecule.

Highly efficient production of anti-HER2 scFv antibody variant for targeting breast cancer cells.

The human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor overexpressed in 30% of human breast cancers. One of the mechanisms by which tumor cell proliferation can be inhibited consists in hampering HER2 dimerization by targeting its extracellular domain with specific antibodies. In recent clinical practice, a valuable alternative to entire IgGs resides in the use of smaller molecules, such as single-chain variable fragments (scFv), developed for selective molecular targeting.

Fully "Recombinant Enzyme-Linked Immunosorbent Assays" Using Genetically Engineered Single-Chain Antibody Fusion Proteins for Detection of Citrus tristeza virus.

ABSTRACT Recombinant single-chain variable fragment antibodies (scFv) that bind specifically to Citrus tristeza virus (CTV), which cause the most detrimental viral disease in the citrus industry worldwide, were obtained from the hybridoma cell lines 3DF1 and 3CA5. These scFv were genetically fused with dimerization domains as well as with alkaline phosphatase, respectively, and diagnostic reagents were produced by expressing these fusion proteins in bacterial cultures. The engineered antibodies were successfully used for CTV diagnosis in plants by tissue print enzyme-linked immunosorbent assay (ELISA) and double antibody sandwich-ELISA.

Structure and dynamics of the anti-AMCV scFv(F8): effects of selected mutations on the antigen combining site.

The recombinant antibody fragment scFv(F8), which recognizes the coat protein of the plant virus AMCV, is characterized by peculiar high in vitro stability and functional folding even in reducing environments, making it fit for designing stable antibodies with desired properties. Mutagenesis and functional analysis evidenced two residues, at positions 47 and 58 of the V(H) chain, playing a crucial role in the antigen binding recognition. Here, we used a computational procedure to assess the effects of these mutations on the stability, structure and dynamics of the antigen-binding site.

Understanding structural/functional properties of immunoconjugates for cancer therapy by computational approaches.

Monoclonal antibodies coupled to highly toxic molecules (immunoconjugates) are currently being developed for cancer therapy. We have used an in silico procedure for evaluating some physicochemical properties of two tumor-targeting anti-HER2 immunoconjugates: (a) the single-chain antibody scFv(FRP5) linked to a bacterial toxin, that has been recently progressed to phase I clinical trial in human cancer; (b) the putative molecule formed by the intrinsically stable scFv(800E6), which has been proposed as toxin carrier to cancer cells in human therapy, joined to the same toxin of (a). Structural models of the immunoconjugates have been built by homology modeling and assessed by molecular dynamics simulations.

Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems.

Background: Aberrant signaling by ErbB-2 (HER 2, Neu), a member of the human Epidermal Growth Factor (EGF) receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen.

Methods: Herein, we describe a multi-platform approach for the production of recombinant Single chain Fragments of antibody variable regions (ScFvs) to ErbB-2 that involves their functional expression in (a) bacteria, (b) transient as well as stable transgenic tobacco plants, and (c) a newly developed cell-free transcription-translation system.

Functional expression of a single-chain antibody specific for the HER2 human oncogene in a bacterial reducing environment.

Recombinant antibody fragments represent useful tools for cancer diagnosis and therapy. Aberrant expression of the HER2 receptor is implicated in metastatic breast and ovary cancers, two malignancies with a high prevalence in young women. In this study, we focussed on a single-chain fragment of variable antibody regions specific for HER2 (scFv800E6) that can be expressed in a functional form in the cytoplasm of Escherichia coli.

Expression of single-chain antibodies in transgenic plants.

There is an ever-growing interest in plant molecular farming as a system for producing valuable recombinant pharmaceutical molecules, such as single-chain variable fragments, on an industrial/agricultural scale and it appears that it is going to become a reality. Human epidermal growth factor receptor-2 (HER2) is an oncogene involved in abnormal cell growth in breast cancer and is considered for the development of new cancer therapies. We describe here the cloning and expression of a scFv-alpha HER2 that has been produced in Escherichia coli and in plants using both stable and transient systems in tobacco and Nicotiana benthamiana.

Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack.

Expression of viral genes in transgenic plants is a very effective tool for attenuating plant viral infection. Nevertheless, the lack of generality and risk issues related to the expression of viral genes in plants might limit the exploitation of this strategy. Expression in plants of antibodies against essential viral proteins could provide an alternative approach to engineer viral resistance.

'Phytoantibodies': a general vector for the expression of immunoglobulin domains in transgenic plants.

Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new general purpose vector to transform plants via Agrobacterium. The expression of an isolated VH domain (IVD) after introduction into the plant genome has been monitored by northern, western and immunohistochemical analysis. Immunoblotting showed that the polypeptide was stably expressed and accounted for up to 1% of the soluble protein fraction.

The propeptide region of clotting factor IX is a signal for a vitamin K dependent carboxylase: evidence from protein engineering of amino acid -4.

Homologous "propeptide" regions are present in a family of vitamin K-dependent mammalian proteins, including clotting factors II, VII, IX, X, protein C, protein S and bone "gla" proteins. To test the hypothesis that the propeptide is a signal for the correct gamma-carboxylation of the adjacent gamma-carboxy region, we have mutated amino acid -4 of human factor IX from an arginine to a glutamine residue, by M13-directed site-specific mutagenesis of a cDNA clone. After expression of mutant factor IX in dog kidney cells, we find that it is secreted into the medium in a precursor form containing the propeptide, and is inefficiently gamma-carboxylated compared to the control, wild-type, recombinant factor IX.

Molecular studies on DNA sequences coding for factor VII and factor XII of human coagulation.

In this paper are described the immunological and molecular procedures that have allowed the identification and the nucleotide sequence characterization of recombinant cDNA coding for factor XII of human coagulation and have suggested the possible identification of other cDNA clones as coding for factor VII of human coagulation.

Identification and primary sequence of an unspliced human urokinase poly(A)+ RNA.

Human urokinase cDNA clones have been identified from a cDNA library prepared from total RNA of human fibroblasts transformed by simian virus 40 [Okayama, H. & Berg, P. (1983) Mol.