Characterization and Functional Test of Canine Probiotics.
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ABSTRACT:
SUBMITTER: Jang HJ
PROVIDER: S-EPMC7982664 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In this study, we provide the first comprehensive annotation of canine interferon-? (CaIFN-?, type III IFN). Phylogenetic analysis based on genomic sequences indicated that CaIFN-? is located in the same branch with Swine IFN-?1 (SwIFN-?), Bat IFN-?1 (BaIFN-?), and human IFN-?1 (HuIFN-?1). CaIFN-? was cloned, expressed in Escherichia coli, and purified to further investigate the biological activity in vitro. The recombinant CaIFN-? (rCaIFN-?) displayed potent antiviral activity on both homologous and heterologous animal cells in terms of inhibiting the replication of the New Jersey serotype of vesicular stomatitis virus (VSV), canine parvovirus, and influenza virus A/WSN/33 (H1N1), respectively. In addition, we also found that rCaIFN-? exhibits a significant antiproliferative response against A72 canine tumor cells and MDCK cells in a dose-dependent manner. Furthermore, CaIFN-? activated the JAK-STAT signaling pathway. To evaluate the expression of CaIFN-? induced by virus and the expression of IFN-stimulated genes (ISGs) induced by rCaIFN-? in the MDCK cells, we measured the relative mRNA level of CaIFN-? and ISGs (ISG15, Mx1, and 2'5'-OAS) by quantitative real-time PCR and found that the mRNA level of CaIFN-? and the ISGs significantly increased after treating the MDCK cells with viruses and rCaIFN-? protein, respectively. Finally, to evaluate the binding activity of rCaIFN-? to its receptor, we expressed the extracellular domain of the canine IFN-? receptor 1 (CaIFN-?R1-EC) and determined the binding activity via ELISA. Our results demonstrated that rCaIFN-? bound tightly to recombinant CaIFN-?R1-EC (rCaIFN-?R1-EC).
| S-EPMC4217014 | biostudies-literature
Project description:In this study, we provide the first comprehensive annotation of the entire family of canine interferons (IFNs). Canine IFN-? (IFNE), IFN-? (IFNK), and IFN-? (IFNL) were discovered for the first time. Ten functional and 2 truncated IFN-? (IFNA) pseudogenes were found in the genome, which also enriched the existing knowledge about canine IFNA. The canine type I IFN genes are clustered on chromosome 11, and their relative arrangements are illustrated. To further investigate the biological activity of canine IFNE, it was expressed and purified in Escherichia coli. Recombinant canine IFNE (rCaIFN-?) displayed potent antiviral activity on both homologous and heterologous animal cells in vitro, indicating that rCaIFN-? has more broad cross-species activity than recombinant canine IFNA (rCaIFN-?). The antiviral activities of rCaIFN-? and rCaIFN-?7 against different viruses on MDCK cells were also evaluated. The antiviral activities of recombinant canine IFNK and IFNL were demonstrated using a VSV-MDCK virus-target cell system. rCaIFN-? exhibited a significant anti-proliferative response against A72 canine tumor cells and MDCK canine epithelial cells in a dose-dependent manner. rCaIFN-?7 was approximately 16-fold more potent than rCaIFN-? in promoting natural killer cell cytotoxicity activity. Further, rCaIFN-? can activate the JAK-STAT signaling pathway.
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Project description:The manufacturing processes of commercial probiotic strains may be affected in different ways in the attempt to optimize yield, costs, functionality, or stability, influencing gene expression, protein patterns, or metabolic output. Aim of this work was to compare different samples of a high concentration (450 billion bacteria) multispecies (8 strains) formulation produced at two different manufacturing sites, USA (US) and Italy (IT), by applying functional proteomics analyses. Several protein-profile differences were detected between IT- and US-made products, with Lactobacillus paracasei, Streptococcus thermophilus, and Bifidobacteria being the main affected probiotics/microorganisms. Integration of omics platforms with in vivo analysis emerged as a powerful tool to assess manufacturing procedures.
2020-01-22 | PXD017086 | Pride
Project description:Functional dyspepsia (FD) is a common disorder in everyday clinical practice identified nowadays as a multi-factorial, difficult to treat condition with a significant burden on patients' quality of life (QoL) and healthcare systems worldwide. Despite its high prevalence in the general population, the precise etiology of the disorder remains elusive, with its pathophysiological spectrum evolving over time, including variable potential mechanisms, i.e., impaired gastric accommodation, gastric motor disorders, hypersensitivity to gastric distention, disorders of the brain-gut axis, as well as less evident ones, i.e., altered duodenal microbiota composition and genetic susceptibility. In light of these implications, a definitive, universal treatment that could be beneficial for all FD patients is not available yet. Recently, probiotics have been suggested to be an effective therapeutic option that could alleviate gastrointestinal symptoms in patients with Irritable Bowel Syndrome (IBS), potentially due to anti-inflammatory properties or by modulating the complex bidirectional interactions between gastrointestinal microbiota and host crosstalk; however, their impact on the multiple aspects of FD remains ambiguous. In this review, we aim to summarize all currently available evidence for the efficacy of probiotics as a novel therapeutic approach for FD.
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Project description:Bacillus subtilis is currently used as an oral probiotic. We examined two commercial B. subtilis probiotic preparations, Enterogermina and Biosubtyl. Surprisingly, physiological and genetic characterization of the bacteria contained in each of these preparations has shown that neither contains B. subtilis.
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Project description:IntroductionProbiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. From this definition, accurate enumeration of probiotic products is a necessity. Nonetheless, this definition does not specify the methods for assessing such viability. Colony forming units is the de facto gold standard for enumerating viable in probiotic products. The notion of microbial viability has been anchored in the concept of cultivability, which refers to a cell's capacity to replicate and form colonies on agar media. However, there is a growing consensus that the term "viability" should not be exclusively tied to the ability to cultivate cells. For example, bacterial cells can exist in a Viable But Non-Culturable (VBNC) state, characterized by the maintenance of characteristics such as membrane integrity, enzymatic activity, pH gradients, and elevated levels of rRNA, despite losing the ability to form colonies.MethodsHerein we present the results of a collaborative inter-laboratory ring test for cytometric bacterial quantification. Specifically, membrane integrity fluorescence flow cytometry (FFC) method and the newer impedance flow cytometry (IFC) method have been used. Both methods interrogate single cells in solution for the presence of intact membranes. FFC exploits fluorochromes that reflect the presence or absence of an intact membrane. IFC probes membrane integrity in a label-free approach by detecting membrane-induced hindrances to the propagation of electricity.ResultsA performance ring-test and comparison design on the FFC method showed that the method is robust against the exchange of equipment, procedures, materials, and operators. After initial method optimization with assessments of rehydration medium, wake-up duration, and phase shift gating on the individual strains, the IFC method showed good agreement with the FFC results. Specifically, we tested 6 distinct species of probiotic bacteria (3 Lactobacillus and 3 Bifidobacterium strains) finding good agreement between FFC and IFC results in terms of total and live cells.DiscussionTogether, these results demonstrate that flow cytometry is a reliable, precise, and user-friendly culture-independent method for bacterial enumeration.
| S-EPMC10667725 | biostudies-literature
Project description:The heme-regulated inhibitor (HRI) negatively regulates protein synthesis by phosphorylating eukaryotic initiation factor-2alpha (eIF2alpha) thereby inhibiting protein translation. The importance of HRI in regulating hemoglobin synthesis in erythroid cells makes it an attractive molecular target in need of further characterization. In this work, we have cloned and expressed the canine form of the HRI kinase. The canine nucleotide sequence has 86%, 82%, and 81% identity to the human, mouse, and rat HRI, respectively. It was noted that an isoleucine residue in the ATP binding site of human, rat, and mouse HRI is replaced by a valine in the canine kinase. The expression of canine HRI protein by in vitro translation using wheat germ lysate or in Sf9 cells using a baculovirus expression system was increased by the addition of hemin. Following purification, the canine protein was found to be 72 kD and showed kinase activity determined by its ability to phosphorylate a synthetic peptide substrate. Quercetin, a kinase inhibitor known to inhibit mouse and human HRI, inhibits canine HRI in a concentration-dependent manner. Additionally, quercetin is able to increase de novo protein synthesis in canine reticulocytes. We conclude that the canine is a suitable model species for studying the role of HRI in erythropoiesis.
| S-EPMC2778829 | biostudies-literature
Project description:BackgroundThere is an urgent need to identify effective therapy to treat coronavirus diseases 2019 (COVID-19). Supplement consumption is becoming popular in this pandemic era. An example of this is probiotic consumption to improve the host's immune system.ObjectiveThis study aimed to prove whether antibodies from people taking probiotics could influence lactate dehydrogenase (LDH), adenosine triphosphate (ATP) values, and cell viability in vitro in peripheral blood mononuclear cells (PBMCs) inoculated with the SARS-CoV-2 spike protein as COVID-19 cells models.MethodsThis was an experimental study with control and intervention groups, totally in 12 groups divided based on antibody levels, probiotic intervention, probiotic non-intervention group, SARS-CoV-2 infection group, and non-SARS-CoV-2 infection group. In vitro assays were carried out on PBMC cell cultures inoculated with S1 SARS-CoV-2 recombinant as a COVID-19 cell model. The COVID-19 cell model was given antibodies divided into three antibody level groups: sRBD levels of <3, 325.76 and 646.18. The cytotoxicity assessment examined increased levels of LDH, cytopathic activity by measuring ATP levels, and cell viability by XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay. Data were analyzed with SPSS 21 for Windows.ResultsThis study showed a significant difference in the LDH value (p < 0.001) between each group. The difference in ATP values between groups was significant (p < 0.001). Meanwhile, the cell viability examination found that there was a tendency of decreased XTT (cell viability in %) when there was an increase of LDH and ATP.ConclusionThe change of LDH values occurred most in the antibody group that did not consume probiotics. The highest cytopathic activity based on the ATP values occurred in the infected cell culture group with antibody levels of 325.76 and consuming probiotics. In addition, LDH and ATP activities provided evidence of a significant influence on cell viability.
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Project description:BACKGROUND:Glucocorticoids, among the most widely utilized drugs in veterinary medicine, are employed to treat a wide variety of diseases; however, their use often induces adverse events in dogs. The efficacy of glucocorticoids usually depends on dosage, although differences in sensitivity to glucocorticoids in individual animals have been reported. Glucocorticoids bind to the cytoplasmic glucocorticoid receptor (GR), which is expressed in almost all cells. These receptors are key factors in determining individual sensitivity to glucocorticoids. This study examined individual differences in glucocorticoid sensitivity in dogs, focusing on reactivity of the GR to prednisolone. RESULTS:We first molecularly cloned the GR gene from a healthy dog. We discovered a mutant GR in a dog suspected to have iatrogenic Cushing syndrome. The mutant GR had extra nucleotides between exons 6 and 7, resulting in a truncated form of GR that was 98 amino acids shorter than the wild-type dog GR. The truncated GR exhibited very low reactivity to prednisolone, irrespective of concentration. CONCLUSIONS:We have identified the truncated form of canine GR in a dog with iatrogenic Cushing syndrome. This truncated form showed the very less sensitivity to glucocorticoid in vitro, unfortunately, we could not elucidate its clinical significance. However, our data is a first report about the function of canine GR, and will facilitate the analysis of canine glucocorticoid sensitivity.
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Project description:BackgroundLysyl oxidase is an extracellular regulatory enzyme with an imperative role in interlinking of collagen and elastin by oxidizing lysine residues. Lysyl oxidase has been implicated in incidence of mammary tumors in bitches. Therefore, it becomes significant to study the structural and functional features of this enzyme for a better understanding of its molecular mechanisms.ResultsThe detailed computational investigation of the canine lysyl oxidase protein was analyzed in silico with respect to its physicochemical properties, secondary and tertiary structure predictions and functional analysis using standard bioinformatic tools. Lysyl oxidase is a flexible protein with an average molecular weight of around 46 kDa, unstable, hydrophilic, and extracellular (secretory) in nature. Twelve cysteine residues and a disulfide bridge were also found. Secondary structure analysis shows that most of the protein has predominant coiled configuration. A putative copper-binding region signature was predicted. The phylogenetic relationship of canine lysyl oxidase with a vast range of mammalian species indicates that the protein was very well conserved throughout the course of evolution. Top 10 interacting proteins were identified using STRING v10.0 analysis, elastin being the closest interacting protein. Functional analysis by InterproScan predicted protein's biological role in oxidation-reduction process.ConclusionUnderstanding the structural and functional properties of the protein will facilitate a better understanding of its mechanism of enzyme action. Further, the predicted 3D model will serve as a cornerstone for further understanding towards the tumorigenesis potential of the protein.
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