Chimerism of avian IgY-scFv and truncated IgG-Fc: A novel strategy in cross-species antibody generation and enhancement.

Chicken single-chain fragment variable (IgY-scFv) is a functional fragment and an emerging development in genetically engineered antibodies with a wide range of biomedical applications. However, scFvs have considerably shorter serum half-life due to the absence of antibody Fc region compared with the full-length antibody, and usually requires continuous intravenous administration for efficacy. A promising approach to overcome this limitation is to fuse scFv with immunoglobulin G (IgG) Fc region, for better recognition and mediation by the neonatal Fc receptor (FcRn) in the host. In this study, engineered mammalian ΔFc domains (CH2, CH3, and intact Fc region) were fused with anti-canine parvovirus-like particles avian IgY-scFv to produce chimeric antibodies and expressed in the HEK293 cell expression system. The obtained scFv-CH2, scFv-CH3, and scFv-Fc can bind with antigen specifically and dose-dependently. Surface plasmon resonance investigation confirmed that scFv-CH2, scFv-CH3, and scFv-Fc had different degrees of binding to FcRn, with scFv-Fc showing the highest affinity. scFv-Fc had a significantly longer half-life in mice compared with the unfused scFv. The identified ΔFcs are promising for the development of engineered Fc-based therapeutic antibodies and proteins with longer half-lives. The avian IgY-scFv-mammalian IgG Fc region opens up new avenues for antibody engineering, and it is a novel strategy to enhance the rapid development and screening of functional antibodies in veterinary and human medicine.

Transgenic expression of Hyp-1 gene from Hypericum perforatum L. alters expression of defense-related genes and modulates recalcitrance to Agrobacterium tumefaciens.

MAIN CONCLUSION: Phenolic oxidative coupling protein (Hyp-1) isolated from Hypericum perforatum L. was characterized as a defense gene involved in H. perforatum recalcitrance to Agrobacterium tumefaciens-mediated transformation Hypericum perforatum L. is a reservoir of high-value secondary metabolites of increasing interest to researchers and to the pharmaceutical industry. However, improving their production via genetic manipulation is a challenging task, as H. perforatum is recalcitrant to Agrobacterium tumefaciens-mediated transformation. Here, phenolic oxidative coupling protein (Hyp-1), a pathogenesis-related (PR) class 10 family gene, was selected from a subtractive cDNA library from A. tumefaciens-treated H. perforatum suspension cells. The role of Hyp-1 in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum and Lactuca sativa overexpressing Hyp-1, and in Catharanthus roseus silenced for its homologous Hyp-1 gene, CrIPR. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in Hyp-1 transgenic plants. However, silencing of CrIPR induced CrPR-5 expression and decreased expression efficiency of Agrobacterium. The expression of core genes involved in several defense pathways was also analyzed in Hyp-1 transgenic tobacco plants. Overexpression of Hyp-1 led to an ample down-regulation of key genes involved in auxin signaling, microRNA-based gene silencing, detoxification of reactive oxygen species, phenylpropanoid pathway and PRs. Moreover, Hyp-1 was detected in the nucleus, plasma membrane and the cytoplasm of epidermal cells by confocal microscopy. Overall, our findings suggest Hyp-1 modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.

Curcumin Encapsulated into Methoxy Poly(Ethylene Glycol) Poly(ε-Caprolactone) Nanoparticles Increases Cellular Uptake and Neuroprotective Effect in Glioma Cells.

Curcumin is a natural polyphenolic compound isolated from turmeric (Curcuma longa) with well-demonstrated neuroprotective and anticancer activities. Although curcumin is safe even at high doses in humans, it exhibits poor bioavailability, mainly due to poor absorption, fast metabolism, and rapid systemic elimination. To overcome these issues, several approaches, such as nanoparticle-mediated targeted delivery, have been undertaken with different degrees of success. The present study was conducted to compare the neuroprotective effect of curcumin encapsulated in poly(ε-caprolactone) and methoxy poly(ethylene glycol) poly(ε-caprolactone) nanoparticles in U251 glioblastoma cells. Prepared nanoparticles were physically characterized by laser doppler anemometry, transmission electron microscopy, and X-ray diffraction. The results from laser doppler anemometry confirmed that the size of poly(ε-caprolactone) and poly(ethylene glycol) poly(ε-caprolactone) nanoparticles ranged between 200-240 nm for poly(ε-caprolactone) nanoparticles and 30-70 nm for poly(ethylene glycol) poly(ε-caprolactone) nanoparticles, and transmission electron microscopy images revealed their spherical shape. Treatment of U251 glioma cells and zebrafish embryos with poly(ε-caprolactone) and poly(ethylene glycol) poly(ε-caprolactone) nanoparticles loaded with curcumin revealed efficient cellular uptake. The cellular uptake of poly(ethylene glycol) poly(ε-caprolactone) nanoparticles was higher in comparison to poly(ε-caprolactone) nanoparticles. Moreover, poly(ethylene glycol) poly(ε-caprolactone) di-block copolymer-loaded curcumin nanoparticles were able to protect the glioma cells against tBHP induced-oxidative damage better than free curcumin. Together, our results show that curcumin-loaded poly(ethylene glycol) poly(ε-caprolactone) di-block copolymer nanoparticles possess significantly stronger neuroprotective effect in U251 human glioma cells compared to free curcumin and curcumin-loaded poly(ε-caprolactone) nanoparticles.

Baccharis dracunculifolia DC Consumption Improves Nociceptive and Depressive-like Behavior in Rats with Experimental Osteoarthritis.

Osteoarthritis (OA) persistently activates nociceptors, leading to chronic pain, which is often accompanied by the comorbid development of emotional impairments (anxiety and depression), an effect associated with microgliosis. Baccharis dracunculifolia DC (Asteraceae), a Brazilian edible plant, is an important source of active compounds with anti-inflammatory abilities. Thus, we evaluated its ability to reverse OA-induced nociceptive and emotional-like impairments in osteoarthritic ovariectomized female rats using the kaolin/carrageenan (K/C) model. Four weeks after OA induction, mechanical hyperalgesia was confirmed, and the treatment started. Control animals (SHAMs) were treated with phosphate-buffered saline (PBS), while arthritic animals (ARTHs) either received PBS or B. dracunculifolia 50 mg/kg (Bd50) and 100 mg/kg (Bd100), via gavage, daily for five weeks. At the end of the treatment, anxiety-like behavior was assessed using the Open Field Test (OFT), anhedonia was assessed using the Sucrose Preference Test (SPT), and learned helplessness was assessed using the Forced Swimming Test (FST). After occision, microglia were stained with IBA-1 and quantified in brain sections of target areas (prefrontal cortex, amygdala, and periaqueductal grey matter). Treatment with B. dracunculifolia extract reversed OA-induced mechanical hyperalgesia and partly improved depressive-like behavior in OA animals' concomitant to a decrease in the number of M1 microglia. Our findings suggest that B. dracunculifolia extracts can potentially be used in the food industry and for the development of nutraceuticals and functional foods.

Copaiba oil minimizes inflammation and promotes parenchyma re-epithelization in acute allergic asthma model induced by ovalbumin in BALB/c mice.

Introduction: Asthma is a condition of airflow limitation, common throughout the world, with high mortality rates, especially as it still faces some obstacles in its management. As it constitutes a public health challenge, this study aimed to investigate the effect of copaiba oil (e.g., Copaifera langsdorffii), as a treatment resource, at doses of 50 and 100 mg/kg on certain mediators of acute lung inflammation (IL-33, GATA3, FOXP3, STAT3, and TBET) and early mechanisms of lung remodeling (degradation of elastic fiber tissues, collagen deposition, and goblet cell hyperplasia). Methods: Using an ovalbumin-induced acute allergic asthma model in BALB/c mice, we analyzed the inflammatory mediators through immunohistochemistry and the mechanisms of lung remodeling through histopathology, employing orcein, Masson's trichrome, and periodic acid-Schiff staining. Results: Copaiba oil treatment (CO) reduced IL-33 and increased FOXP3 by stimulating the FOXP3/GATA3 and FOXP3/STAT3 pathways. Additionally, it upregulated TBET, suggesting an additional role in controlling GATA3 activity. In the respiratory epithelium, CO decreased the fragmentation of elastic fibers while increasing the deposition of collagen fibers, favoring epithelial restructuring. Simultaneously, CO reduced goblet cell hyperplasia. Discussion: Although additional research is warranted, the demonstrated anti-inflammatory and re-epithelializing action makes CO a viable option in exploring new treatments for acute allergic asthma.

Genista tridentata Phytochemical Characterization and Biological Activities: A Systematic Review.

Genista tridentata (L.) Willk., known as "prickled broom", is a Leguminosae (Fabaceae) species native to the Iberian Peninsula, Morocco, Algeria, and Tunisia. It is used in folk medicine as an anti-inflammatory, for gastrointestinal and respiratory disorders, rheumatism, and headaches, to lower blood pressure, against hypercholesterolemia and hyperglycemia. This study aimed to systematically review the literature on the bioactivities and phytochemical profile of Genista tridentata to understand its pharmacological potential. For this, four electronic databases (PubMed, GoogleScholar, Repositórios Cientificos de Acesso Aberto de Portugal (RCCAP), and ScienceDirect) were searched from inception up to 31 December 2022. From a total of 264 potentially eligible studies considered for screening, 34 papers were considered eligible for this systematic review. The sampling included 71 extracts, collected mainly in Portugal. Genista tridentata extracts present a high level of flavonoids and phenolic compounds. The flowers and aerial parts of the plant were the most studied, and aqueous extracts were the most used. The results predict a high potential for the application of Genista tridentata as a new source of natural antioxidants and preservatives for the food industry with subsequent health benefits, such as the production of nutraceuticals. Moreover, the results indicate that the plant can be collected at all seasons of the year, which represents a benefit for the industry.

Crocetin and Its Glycoside Crocin, Two Bioactive Constituents From Crocus sativus L. (Saffron), Differentially Inhibit Angiogenesis by Inhibiting Endothelial Cytoskeleton Organization and Cell Migration Through VEGFR2/SRC/FAK and VEGFR2/MEK/ERK Signaling Pathways.

Crocetin and crocin are two important carotenoids isolated from saffron (Crocus sativus L.), which have been used as natural biomedicines with beneficial effects for improving the suboptimal health status associated with abnormal angiogenesis. However, the anti-angiogenic effects and underlying mechanisms of the effects of crocetin and crocin have not been investigated and compared. The anti-angiogenic effects of crocetin and crocin were tested on human umbilical vein endothelial cells (HUVECs) in vitro, and in zebrafish in vivo. In vivo, crocetin (20 µM) and crocin (50 and 100 µM) significantly inhibited subintestinal vein vessels formation, and a conversion process between them existed in zebrafish, resulting in a difference in their effective concentrations. In the HUVEC model, crocetin (10, 20 and 40 µM) and crocin (100, 200 and 400 µM) inhibited cell migration and tube formation, and inhibited the phosphorylation of VEGFR2 and its downstream pathway molecules. In silico analysis further showed that crocetin had a higher ability to bind with VEGFR2 than crocin. These results suggested that crocetin was more effective than crocin in inhibiting angiogenesis through regulation of the VEGF/VEGFR2 signaling pathway. These compounds, especially crocetin, are potential candidate natural biomedicines for the management of diseases associated with abnormal blood vessel growth, such as age-related macular degeneration.

Overexpression of polygalacturonase-inhibiting protein (PGIP) gene from Hypericum perforatum alters expression of multiple defense-related genes and modulates recalcitrance to Agrobacterium tumefaciens in tobacco.

Hypericum perforatum L is a remarkable source of high-value secondary metabolites with increasing applications in pharmaceutical industry. However, improvement in the production of secondary metabolites through genetic engineering is a demanding task, as H. perforatum is not amenable to Agrobacterium tumefaciens-mediated transformation. In this study, we identified a Polygalacturonase-inhibiting protein (PGIP) gene from a subtractive cDNA library of A. tumefaciens-treated H. perforatum suspension cells. The role of HpPGIP in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum overexpressing HpPGIP alone or fused at the N-terminus to Phenolic oxidative coupling protein (Hyp-1), a gene that positively modulates resistance to A. tumefaciens. Furthermore, virus-induced gene silencing was employed to knock down the expression of the PGIP homologous in N. benthamiana. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in both HpPGIP and Hyp-1-PGIP transgenic plants, as assessed by GUS staining assays. However, silencing of PGIP in N. benthamiana increased the resistance to A. tumefaciens rather than susceptibility, which correlated with induction of pathogenesis-related proteins (PRs). The expression of core genes involved in several defense pathways was also analyzed in transgenic tobacco plants. Overexpression of HpPGIP led to up-regulation of key genes involved in hormone signaling, microRNA-based gene silencing, homeostasis of reactive oxygen species, and the phenylpropanoid pathway. Overexpression of Hyp-1-PGIP seemed to enhance the effect of PGIP on the expression of most genes analyzed. Moreover, HpPGIP was detected in the cytoplasm, nucleus and the plasma membrane or cell wall by confocal microscopy. Overall, our findings suggest HpPGIP modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.