Characterization of the complex locus of bean encoding polygalacturonase-inhibiting proteins reveals subfunctionalization for defense against fungi and insects.

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant inhibitors of fungal endopolygalacturonases (PGs) that belong to the superfamily of Leu-rich repeat proteins. We have characterized the full complement of pgip genes in the bean (Phaseolus vulgaris) genotype BAT93. This comprises four clustered members that span a 50-kb region and, based on their similarity, form two pairs (Pvpgip1/Pvpgip2 and Pvpgip3/Pvpgip4). Characterization of the encoded products revealed both partial redundancy and subfunctionalization against fungal-derived PGs. Notably, the pair PvPGIP3/PvPGIP4 also inhibited PGs of two mirid bugs (Lygus rugulipennis and Adelphocoris lineolatus). Characterization of Pvpgip genes of Pinto bean showed variations limited to single synonymous substitutions or small deletions. A three-amino acid deletion encompassing a residue previously identified as crucial for recognition of PG of Fusarium moniliforme was responsible for the inability of BAT93 PvPGIP2 to inhibit this enzyme. Consistent with the large variations observed in the promoter sequences, reverse transcription-PCR expression analysis revealed that the different family members differentially respond to elicitors, wounding, and salicylic acid. We conclude that both biochemical and regulatory redundancy and subfunctionalization of pgip genes are important for the adaptation of plants to pathogenic fungi and phytophagous insects

Plant antibodies for human antifungal therapy

There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting β-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the β-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially β1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases

An overview of molecular mechanisms in fabry disease

Fabry disease (FD) (OMIM #301500) is a rare genetic lysosomal storage disorder (LSD). LSDs are characterized by inappropriate lipid accumulation in lysosomes due to specific enzyme deficiencies. In FD, the defective enzyme is alpha-galactosidase A (alpha-Gal A), which is due to a mutation in the GLA gene on the X chromosome. The enzyme deficiency leads to a continuous deposition of neutral glycosphingolipids (globotriaosylceramide) in the lysosomes of numerous tissues and organs, including endothelial cells, smooth muscle cells, corneal epithelial cells, renal glomeruli and tubules, cardiac muscle and ganglion cells of the nervous system. This condition leads to progressive organ failure and premature death. The increasing understanding of FD, and LSD in general, has led in recent years to the introduction of enzyme replacement therapy (ERT), which aims to slow, if not halt, the progression of the metabolic disorder. In this review, we provide an overview of the main features of FD, focusing on its molecular mechanism and the role of biomarkers

Design of a Diagnostic Immunoassay for Aflatoxin M1 Based on a Plant-Produced Antibody

A new green competitive ELISA for aflatoxin M1 quantification in raw milk was developed. This diagnostic tool is based on an anti AFM1 mAb produced by plant molecular farming in alternative to classical systems. Our assay, showing an IC50 below 25 ng/L, fits with the requirements of EU legislation limits for AFM1 (50 ng/L). Optimal accuracy was achieved in correspondence of the decision levels (25 and 50 ng/L), and the assay enabled AFM1 quantification in the range 5–110 ng/L, with limit of detection 3 ng/L. Moreover, to evaluate a real applicability in diagnostics, raw milk-spiked samples were analysed, achieving satisfactory recovery rates of AFM1. In conclusion, an efficient and ready-to-use diagnostic assay for the quantification of aflatoxin M1 in milk, based on a plant-produced recombinant mAb, has been successfully developed

Design of a Diagnostic Immunoassay for Aflatoxin M1 Based on a Plant-Produced Antibody

A new green competitive ELISA for aflatoxin M1 quantification in raw milk was developed. This diagnostic tool is based on an anti AFM1 mAb produced by plant molecular farming in alternative to classical systems. Our assay, showing an IC50 below 25 ng/L, fits with the requirements of EU legislation limits for AFM1 (50 ng/L). Optimal accuracy was achieved in correspondence of the decision levels (25 and 50 ng/L), and the assay enabled AFM1 quantification in the range 5–110 ng/L, with limit of detection 3 ng/L. Moreover, to evaluate a real applicability in diagnostics, raw milk-spiked samples were analysed, achieving satisfactory recovery rates of AFM1. In conclusion, an efficient and ready-to-use diagnostic assay for the quantification of aflatoxin M1 in milk, based on a plant-produced recombinant mAb, has been successfully developed

LA DIAGNOSI DI IPOTIROIDISMO CONGENITO AI TEMPI DELLA PANDEMIA DA SARS-CoV-2: L’ESPERIENZA DELLA CLINICA PEDIATRICA DI PALERMO

Obiettivi La pandemia da SARS-CoV-2 ha severamente compromesso i programmi di assistenza sanitaria, specie in casi in cui l’accesso alle cure ha richiesto tempi brevi, non programmabili. Lo screening neonatale per l’ipotiroidismo congenito (IC) rientra fra queste necessità assistenziali, con cooperazione fra componenti di un team multi-specialistico. E’ indispensabile l’integrazione fra medici e infermieri professionali, con competenze ed esperienza in ambito neonatologico. Metodi Abbiamo valutato l’attività integrata diagnostico-terapeutica del nostro centro di Endocrinologia Pediatrica, nel periodo gennaio 2020–aprile 2021, corrispondente alla diffusione del SARS-CoV-2 in Italia. Risultati Su un totale di 21300 neonati sottoposti a screening neonatale, sono stati screenati 1122 neonati con un TSH > 6. Fra questi, 75 neonati (7%) avevano un incremento del TSH sul secondo spot e/o su siero (48 M, 27 F, età gestazionale: 38.3 ± 1.3 w; p.c. neonatale: 3154 ± 121 gr). Il TSH al primo screening era 12.5 ± 21; il TSH su siero all’accesso presso il nostro centro, prima di un eventuale terapia con L-tiroxina, era 37.1 ± 77.5. Fra questi, 25 (33%) hanno presentato la normalizzazione di TSH, fT3 e fT4, valutati su siero al momento della valutazione presso il nostro centro e, pertanto, non hanno iniziato la terapia con L-tiroxina. I neonati ai quali è stato confermato un livello di TSH, fT3, fT4 patologico, avevano un’età all’inizio della terapia sostitutiva con L-tiroxina di 17 ± 3 gg. Fra questi pazienti, 2 con agenesia tiroidea; 3 con ipoplasia tiroidea, 25 con tiroide in situ. 4 hanno iniziato terapia oltre 22 gg ma non in relazione al lockdown: 1 proveniva da altra provincia, tutti e 4, comunque, con TSH < 10 al primo screening, e solo successivamente hanno presentato livelli di TSH francamente patologici. Conclusioni Il follow-up terapeutico è stato realizzabile, nonostante le limitazioni numeriche relative agli accessi in ospedale, grazie ad un programma di telemedicina, coordinato con i pediatri di famiglia. La strategia del team multi-specialistico, costruito attorno alle esigenze del piccolo paziente sottoposto a screening neonatale, ha garantito tempistica, coordinazione dei ruoli e affidabilità di una presa in carico efficace ai fini terapeutici e del follow-up

Targeted Modification of Homogalacturonan by Transgenic Expression of a Fungal Polygalacturonase Alters Plant Growth

Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis