Immunological Studies on Cattle Naturally Infected with FMD Compared with the Vaccinated Cattle in Sharkia Governorate, Egypt

Foot and mouth disease (FMD) is a highly contagious viral disease. Despite annual mass vaccination programs in Egypt, the incidence and economic losses are high. The Samples were collected after clinical examination of animals in 2022 and divided into three groups; Group 1(G1): Fifteen samples were collected from apparently healthy cattle (control group). Twenty-five samples were collected from cattle with suspected symptoms of FMD and divided into 2 groups 15 non-vaccinated Group 2(G2) and 10 vaccinated cattle Group 3(G3). Isolation of FMDV using BHK-21 cells was done. Molecular identification, sequencing, and phylogenetic analysis of new FMDV serotypes A and O circulating variants from G2 and G3 regarding targeting the VP1 gene. Results demonstrated that genotype O was related to East Africa-3 (EA-3) topotype. Identification of FMDV using PCR for field isolates, revealed that genotype A was related to genotype IV of the African topotype. The isolated virus on BHK-21 showed characteristic cytopathic effects (CPE). Hematological analysis showed a significant decrease in most hematological parameters with neutrophilia and lymphopenia. Serum biochemical constituents revealed a significant increase in AST, ALT, urea, creatinine, and cortisol, while a significant decrease in the total protein, albumin, and globulins in (G2). The vaccinated group showed a significant increase in serum AST and cortisol. The highest values of IL10, CRP, Troponin 1, Haptoglobin, and Complement 3 in the infected group (G2). Pathological examination revealed ulcerative dermatitis on the tongues and the coronary bands of the hoof. The heart revealed severe lymphocytic myocarditis with depletion of lymph nodes and spleen. While vaccinated (G3) revealed a significant decrease in RBC count and Hb concentration also lymphocytosis, neutropenia, and leukocytosis were founded. while the lowest values of IL10, and Haptoglobin were reported in the vaccinated one (G3). The recorded level of IgG showed a severe decrease in G2 and a high increase in G3. The pathological lesions recorded in G3 were milder than in G2. It could be concluded that the isolates in this study from both groups non-vaccinated and vaccinated were matching each other while not matching with the local vaccinal strain. The vaccinated cattle in 2022 had mild to moderate immunity against the disease

Studies on exogenous elicitors promotion of sulforaphane content in broccoli sprouts and its effect on the MDA-MB-231 breast cancer cell line

Broccoli (Brassica oleracea var. italica) belongs to the Brassicaceae family and is one of the most important cruciferous vegetables. It has gained popularity due to its high glucosinolate concentrations that have positive potential in cancer treatment. In this study, the effects of two elicitors; methyl jasmonate (MeJA) and salicylic acid (SA), on the production of sulforaphane from broccoli 7-days old seedlings, its antiapoptotic activity and its gene expression have been studied. Real-time PCR was used to quantify myrosinase (MY) gene expression associated with sulforaphane production. The antiapoptotic activity of sulforaphane treatments was evaluated and tested using MDA-MB-231 breast cancer cell line. The highest amount of sulforaphane was produced at 80 μM SA and 40 μM MeJA after 24 h of elicitation. Increased production of sulforaphane was found to be associated with over-expression of myrosinase gene. The sulforaphane extract obtained from broccoli seedlings treated with MeJA exerted a higher inhibitory effect on the MDA-MB-231 breast cancer cell line than sulforaphane extract obtained from broccoli seedlings treated with SA. The inhibitory effect increased by using purified sulforaphane. Studies on apoptosis gene transcription showed that all sulforaphane treatments down-regulated the B-cell lymphoma 2 (Bcl-2) gene (antiapoptotic) transcription while up-regulating the pro-apoptotic Bcl2 Associated X, Apoptosis Regulator (Bax) gene, Caspase-3, Caspase-8 and Caspase-9. It may be concluded that increased sulforaphane production could increase its antiapoptotic activity as indicated by induction of more apoptosis in MDA-MB-231 breast cancer cells

Functional Assembly of <i>Caenorhabditis elegans</i> Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity

Among seven homologs of cytochrome b561 in a model organism C. elegans, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in Pichia pastoris cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis–Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc

Functional Assembly of Caenorhabditis elegans Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity

Among seven homologs of cytochrome b561 in a model organism C. elegans, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in Pichia pastoris cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis&ndash;Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc

Identification of RNA aptamer which specifically interacts with PtdIns(3)P

The phosphinositide Ptdlns(3)P plays an important role in autophagy; however, the detailed mechanism of its activity remains unclear. Here, we used a Systematic Evolution of Ligands by EXponential enrichment (SELEX) screening approach to identify an RNA aptamer of 40 nucleotides that specifically recognizes and binds to intracellular lysosomal Ptdlns(3)P. Binding occurs in a magnesium concentration- and pH-dependent manner, and consequently inhibits autophagy as determined by LC3II/I conversion, p62 degradation, formation of LC3 puncta, and lysosomal accumulation of Phafin2. These effects in turn inhibited lysosomal acidification, and the subsequent hydrolytic activity of cathepsin D following induction of autophagy. Given the essential role of Ptdlns(3)P as a key targeting molecule for autophagy induction, identification of this novel Ptdlns(3)P RNA aptamer provides new opportunities for investigating the biological functions and mechanisms of phosphoinositides. (C) 2019 The Authors. Published by Elsevier Inc

Functional characterization of lysosomal interaction of Akt with VRK2

Serine-threonine kinase Akt (also known as PKB, protein kinase B), a core intracellular mediator of cell survival, is involved in various human cancers and has been suggested to play an important role in the regulation of autophagy in mammalian cells. Nonetheless, the physiological function of Akt in the lysosomes is currently unknown. We have reported previously that PtdIns (3)P-dependent lysosomal accumulation of the Akt-Phafin2 complex is a critical step for autophagy induction. Here, to characterize the molecular function of activated Akt in the lysosomes in the process of autophagy, we searched for the molecules that interact with the Akt complex at the lysosomes after induction of autophagy. By time-of-flight-mass spectrometry (TOF/MS) analysis, kinases of the VRK family, a unique serine-threonine family of kinases in the human kinome, were identified. VRK2 interacts with Akt1 and Akt2, but not with Akt3; the C terminus of Akt and the N terminus of VRK2 facilitate the interaction of Akt and VRK2 in mammalian cells. The kinase-dead form of VRK2A (KD VRK2A) failed to interact with Akt in coimmunoprecipitation assays. Bimolecular fluorescence complementation (BiFC) experiments showed that, in the lysosomes, Akt interacted with VRK2A but not with VRK2B or KD VRK2A. Immunofluorescent assays revealed that VRK2 and phosphorylated Akt accumulated in the lysosomes after autophagy induction. WT VRK2A, but not KD VRK2A or VRK2B, facilitated accumulation of phosphorylated Akt in the lysosomes. Downregulation of VRK2 abrogated the lysosomal accumulation of phosphorylated Akt and impaired nuclear localization of TFEB; these events coincided to inhibition of autophagy induction. The VRK2-Akt complex is required for control of lysosomal size, acidification, bacterial degradation, and for viral replication. Moreover, lysosomal VRK2-Akt controls cellular proliferation and mitochondria) outer-membrane stabilization. Given the roles of autophagy in the pathogenesis of human cancer, the current study provides a novel insight into the oncogenic activity of VRK2-Akt complexes in the lysosomes via modulation of autophagy