Plant Defensin PgD1 a Biotechnological Alternative Against Plant Pathogens.

Plant defensins are small antimicrobial proteins (AMP) that participate in the immune defense of plants through their antibacterial, antiviral and antifungal activities. PgD1 is a defensin from Picea glauca (Canadian Pine) and has antifungal activity against plant pathogens. This activity positions it as an alternative biotechnological agent to pesticides commonly used against these plant fungi diseases. The present study aimed to recombinantly produce PgD1 in Escherichia coli to characterize its in vitro antifungal potential against different phytopathogens. To achieve this, the coding gene was amplified and cloned into pET30a( +). Recombinant plasmid was subsequently introduced into E. coli for the soluble expression of defensin PgD1. To evaluate the antifungal activity of the expressed protein, the growth inhibition test was used in solid and liquid media for approximately 7 days against significant plant pathogens, that cause significant crop damage including: Botrytis cinerea, Colletotrichum gloeosporioides, Colletotrichum musae, Colletotrichum graminicola and Fusarium oxysporum. Additionally, stability assessments included temperature variation experiments and inhibition tests using dithiothreitol (DTT). The results showed that there was significant inhibition of the fungal species tested when in the presence of PgD1. Furthermore, defensin proved to be resistant to temperature variations and demonstrated that part of its stability is due to its primary structure rich in cysteine ​​residues through the denaturation test with dithiothreitol (DTT) where the antifungal activity of PgD1 defensin was inhibited. These data indicate that recombinant PgD1 could be utilized as a plant protection technology in agriculture.

Roasted yerba mate (Ilex paraguariensis) infusions in bovine milk model before and after in vitro digestion: Bioaccessibility of phenolic compounds, antioxidant activity, protein-polyphenol interactions and bioactive peptides.

Yerba mate is increasingly acknowledged for its bioactive properties and is currently being incorporated into various food and pharmaceutical products. When roasted, yerba mate transforms into mate tea, consumed as a hot aqueous infusion, and has gained popularity. This study investigated the bioaccessibility of phenolic compounds, protein-polyphenol interactions, antioxidant activity, and bioactive peptides in roasted yerba mate infusions, utilizing whole, semi-skimmed, and skimmed bovine milk models. The phytochemical profile of roasted yerba mate was analyzed in infusions with water and milk (whole, semi-skimmed, and skimmed), before and after in vitro digestion, identifying 18 compounds that exhibited variations in composition and presence among the samples. Bioavailability varied across different milk matrices, with milk being four times more efficient as a solvent for extraction. Gastric digestion significantly impacted (p < 0.05) the release of phenolic compounds, such as chlorogenic acid and rutin, with only chlorogenic acid remaining 100 % bioavailable in the infusion prepared with skimmed milk. Protein-polyphenol interaction did not influence protein digestion in different infusions, as there was a similarity in the hydrolysis pattern during the digestive process. Changes in antioxidant activity during digestion phases, especially after intestinal digestion in milk infusions, were related to alterations in protein structures and digestive interactions. The evaluation of total phenolic compounds highlighted that skimmed milk infusion notably preserved these compounds during digestion. Peptidomic analysis identified 253, 221, and 191 potentially bioactive peptides for whole, semi-skimmed, and skimmed milk-digested infusions, respectively, with a focus on anti-inflammatory and anticancer activities, presenting a synergistic approach to promote health benefits. The selection of milk type is crucial for comprehending the effects of digestion and interactions in bioactive compound-rich foods, highlighting the advantages of consuming plant infusions prepared with milk.

Validation and optimization of the loop-mediated isothermal amplification (LAMP) technique for rapid detection of wheat stripe mosaic virus, a wheat-infecting pathogen.

BACKGROUND: Wheat stripe mosaic virus (WhSMV) is a significant wheat pathogen that causes substantial yield losses in Brazil and other countries. Although several detection methods are available, reliable and efficient tools for on-site WhSMV detection are currently lacking. In this study, a Loop-Mediated Isothermal Amplification (LAMP) method was developed for rapid and reliable field detection of WhSMV. We designed WhSMV-specific primers for the LAMP assay and optimized reaction conditions for increased sensitivity and specificity using infected plant samples. RESULTS: We have developed a diagnostic method utilizing the Loop-Mediated Isothermal Amplification (LAMP) technique capable of rapidly and reliably detecting WhSMV. The LAMP assay has been optimized to enhance sensitivity, specificity, and cost-effectiveness. CONCLUSION: The LAMP assay described here represents a valuable tool for early WhSMV detection, serving to mitigate the adverse economic and social impacts of this viral pathogen. By enabling swift and accurate identification, this assay can significantly improve the sustainability of cereal production systems, safeguarding crop yields against the detrimental effects of WhSMV.

TRIM21 chimeric protein as a new molecular tool for multispecies IgG detection.

BACKGROUND: The production of monoclonal antibodies for immunoglobulin detection is not cost-effective, while polyclonal antibody production depends on laboratory animals, raising concerns on animal welfare. The widespread use of immunoglobulins in the pharmaceutical industry and the increasing number and variety of new antibodies entering the market require new detection and purification strategies. The Tripartite motif-containing protein 21 is a soluble intracellular immunoglobulin G receptor that binds to the constant region of immunoglobulin G from various species with high affinity. We hypothesized that using this protein as an antibody-binding module to create immunoglobulin detection probes will improve the portfolio of antibody affinity ligands for diagnostic or therapeutic purposes. RESULTS: We created a chimeric protein containing a mutated form of the C-terminal domain of mouse Tripartite motif-containing protein 21 linked to streptavidin to detect immunoglobulin G from various species of mammals. The protein is produced by heterologous expression and consists of an improved molecular tool, expanding the portfolio of antibody-affinity ligands for immunoassays. We also demonstrate that this affinity ligand may be used for purification purposes since imidazole elution of antibodies can be achieved instead of acidic elution conditions of current antibody purification methods. CONCLUSION: Data reported here provides an additional and superior alternative to the use of secondary antibodies, expanding the portfolio of antibodies affinity ligands for detection and purification purposes.

Engineering defensin α-helix to produce high-affinity SARS-CoV-2 spike protein binding ligands.

The binding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein to the angiotensin-converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high-affinity and cost-effective ACE2 mimetic ligands that disrupt this protein-protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2-5 kDa) and highly stable proteins containing solvent-exposed alpha-helix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha-helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS-CoV-2 spike protein. The engineered proteins (h-deface2, p-deface2, and p-deface2-MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7°C), high-affinity binding to the spike protein with apparent Kd values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h-deface2, p-deface2, and p-deface2-MUT, respectively, and were used in a diagnostic assay that detected SARS-CoV-2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha-helices in a constrained form for designing of high-affinity ligands.

Targeting Trypanosoma evansi with disulphide-rich peptides derived from a phage display library.

A phage-display library was generated using a Bus thalamus scorpion toxin (BTK-2) as a peptide scaffold. BTK-2 belongs to the disulfide-rich family of proteins with pronounced structural stability due to the presence of three disulfide bridges that connects antiparallel beta-sheets and one alpha helix. Using BTK-2 as a phage display scaffold, we introduced mutations in five residues located in the alpha-helix and two residues located in the smaller loop, keeping intact the disulfide bridges to create a peptide phage-displayed library with disulfide-rich family properties. The library was subjected to in vivo and in vitro phage display selections against Trypanosoma evansi, the etiological agent of "Surra", a disease that affects a wide range of mammals. The development of T. evansi specific biomarkers is essential to improve diagnostic methods and epidemiological studies leading to a more accurate clinical decision for the treatment of this disease of economic impact for commercial livestock production. In this study, we identified two disulfide-rich peptides targeting T. evansi parasites. Further specificity studies are necessary to investigate the potential of selected peptides as new biomarkers to aid diagnostic and treatment procedures of T. evansi infections.

Prevalence of Eimeria spp. in Broilers by Multiplex PCR in the Southern Region of Brazil on Two Hundred and Fifty Farms.

Parasitic infections caused by Eimeria species are responsible for most economic losses in poultry production. Prevalence studies can adequately assist the design of prophylaxis strategies for disease control. Therefore, stool samples from 251 flocks of broilers from 28 to 48 days old were collected in 21 municipalities in the state of Santa Catarina, Brazil, to detect and examine the prevalence of Eimeria acervulina, Eimeria maxima, Eimeria tenella, Eimeria mitis, Eimeria praecox, Eimeria necatrix, and Eimeria brunetti. The oocysts were recovered and quantified, and the species were identified by a multiplex PCR technique. Amplicons of seven Eimeria species originating from the PCR-positive samples were cloned. Microscopy studies demonstrated that 96% of the farms were positive for the Eimeria. Seven species were identified, as follows: E. maxima (63.7%) and E. acervulina (63.3%) were the most prevalent species, followed by E. tenella (54.6%), E. mitis (38.6%), E. praecox (25.1%), E. necatrix (24.3%), and E. brunetti (13.1%). The average number of species detected per farm was 2.96, and the most common were E. acervulina, E. maxima, and E. tenella (9.16%). The sequencing of the clones confirmed the specificity and effectiveness of multiplex PCR for the identification of seven species of Eimeria, so this tool can be useful in studying circulating species in poultry farms, thereby assisting prophylactic measures against coccidiosis.

An RNA alternative to human transferrin: a new tool for targeting human cells.

The transferrin receptor, CD71, is an attractive target for drug development because of its high expression on a number of cancer cell lines and the blood brain barrier. To generate serum-stabilized aptamers that recognize the human transferrin receptor, we have modified the traditional aptamer selection protocol by employing a functional selection step that enriches for RNA molecules which bind the target receptor and are internalized by cells. Selected aptamers were specific for the human receptor, rapidly endocytosed by cells and shared a common core structure. A minimized variant was found to compete with the natural ligand, transferrin, for receptor binding and cell uptake, but performed ~twofold better than it in competition experiments. Using this molecule, we generated aptamer-targeted siRNA-laden liposomes. Aptamer targeting enhanced both uptake and target gene knockdown in cells grown in culture when compared to nonmodified or nontargeted liposomes. The aptamer should prove useful as a surrogate for transferrin in many applications including cell imaging and targeted drug delivery.