scholarly journals Trichinella spiralis: Knockdown of gamma interferon inducible lysosomal thiol reductase (GILT) results in the reduction of worm burden

2021 ◽  
Vol 15 (11) ◽  
pp. e0009958
Author(s):  
Hong Fei ◽  
Muhammad Ali-ul-Husnain Naqvi ◽  
Sana Zahra Naqvi ◽  
Lixin Xu ◽  
Xiaokai Song ◽  
...  
Keyword(s):  
Mhc Class Ii ◽  
Trichinella Spiralis ◽  
Reductase Activity ◽  
3D Structure ◽  
Gamma Interferon ◽  
Pcr Analysis ◽  
Multiple Sequence ◽  
Small Interference Rna ◽  
Thiol Reductase

Trichinella spiralis is mammalian skeletal muscles parasite which may cause trichinellosis in animals and humans. Gamma interferon inducible lysosomal thiol reductase (GILT) is a widespread superfamily which plays key role in processing and presentation of MHC class II restricted antigen by catalyzing disulfide bond reduction. There are no reports about GILT in T. spiralis. In present study, GILT from T. spiralis (Tsp-GILT) was cloned, analyzed by multiple-sequence alignment, and predicted by 3D structure model. Recombinant Tsp-GILT (about 46 kDa) was efficiently expressed in Escherichia coli and thiol reductase activity suggested that in acidic environment the addition of a reducing agent is needed. Soaking method was used to knockdown expression of Tsp-GILT using small interference RNA (siRNA). Immunofluorescence assay confirmed the transformation of siRNA into muscle larva (ML) and new born larva (NBL). Quantitative real time-PCR (QRT-PCR) analysis revealed that transcription level of Tsp-GILT mRNA can be up-regulated by stimulation of mouse IFN-γ and down-regulated by siRNA2 in vitro. NBLs soaked with siRNA2 showed 32.3% reduction in the generation of MLs. MLs soaked with siRNA2 showed 26.2% reduction in the next generation of MLs, but no significant effect was observed on adult worms or NBLs. These findings concluded that GILT may play important roles in the development of T. spiralis parasite.

scholarly journals Desulfovibrio gigas Flavodiiron Protein Affords Protection against Nitrosative Stress In Vivo

2006 ◽  
Vol 188 (8) ◽  
pp. 2745-2751 ◽  
Author(s):  
Rute Rodrigues ◽  
João B. Vicente ◽  
Rute Félix ◽  
Solange Oliveira ◽  
Miguel Teixeira ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Nitrosative Stress ◽  
Reductase Activity ◽  
Anaerobic Growth ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Desulfovibrio Gigas ◽  
Reverse Transcription Pcr

ABSTRACT Desulfovibrio gigas flavodiiron protein (FDP), rubredoxin:oxygen oxidoreductase (ROO), was proposed to be the terminal oxidase of a soluble electron transfer chain coupling NADH oxidation to oxygen reduction. However, several members from the FDP family, to which ROO belongs, revealed nitric oxide (NO) reductase activity. Therefore, the protection afforded by ROO against the cytotoxic effects of NO was here investigated. The NO and oxygen reductase activities of recombinant ROO in vitro were tested by amperometric methods, and the enzyme was shown to effectively reduce NO and O2. Functional complementation studies of an Escherichia coli mutant strain lacking the ROO homologue flavorubredoxin, an NO reductase, showed that ROO restores the anaerobic growth phenotype of cultures exposed to otherwise-toxic levels of exogenous NO. Additional studies in vivo using a D. gigas roo-deleted strain confirmed an increased sensitivity to NO of the mutant strain in comparison to the wild type. This effect is more pronounced when using the nitrosating agent S-nitrosoglutathione (GSNO), which effectively impairs the growth of the D. gigas Δroo strain. roo is constitutively expressed in D. gigas under all conditions tested. However, real-time reverse transcription-PCR analysis revealed a twofold induction of mRNA levels upon exposure to GSNO, suggesting regulation at the transcription level by NO. The newly proposed role of D. gigas ROO as an NO reductase combined with the O2 reductase activity reveals a versatility which appears to afford protection to D. gigas at the onset of both oxidative and nitrosative stresses.

The mode of action of some benzimidazole drugs on Trichinella spiralis

Parasitology ◽  
1987 ◽  
Vol 95 (1) ◽  
pp. 61-70 ◽  
Author(s):  
A. Criado Fornelio ◽  
F. Rodriguez Caabeiro ◽  
A. Jimenez Gonzalez
Keyword(s):  
Trichinella Spiralis ◽  
Reductase Activity ◽  
Oral Treatment ◽  
Mitochondrial Fraction ◽  
Significant Inhibition ◽  
Fumarate Reductase ◽  
Glucose Levels ◽  
Free Glucose

SUMMARYIn an attempt to find possible targets for benzimidazole action in muscle-stage larvae of Trichinella spiralis, the effects of mebendazole and thiabendazole were tested in vivo by oral treatment of infested mice and in vitro by including these anthelmintics in an adequate maintenance medium containing decapsulated larvae. The effects of the anthelmintics on succinate dehydrogenase and fumarate reductase activities, measured in the mitochondrial fraction obtained from the in vivo- or in vitro-treated larvae showed that only thiabendazole causes significant inhibition of fumarate reductase activity. On the other hand, measurements of free glucose, glycogen reserves and soluble protein in the treated larvae indicate that in vivo, mebendazole and thiabendazole clearly diminish free glucose levels, although in vitro only mebendazole produces the same diminution. Both the glycogen and protein contents of the larvae remained unchanged after treatment in vivo or in vitro. The importance of these findings with regard to a possible site of action for mebendazole and thiabendazole is discussed.

scholarly journals Effects of Peptide-Induced Immune Tolerance on Murine Lupus

2021 ◽  
Vol 12 ◽  
Author(s):  
Ram P. Singh ◽  
Bevra H. Hahn ◽  
David S. Bischoff
Keyword(s):  
T Cells ◽  
B Cells ◽  
Mhc Class Ii ◽  
Molecular Mechanisms ◽  
Cytotoxic T Lymphocyte ◽  
Cell Types ◽  
T Cell Subsets ◽  
Pcr Analysis

The regulation of autoimmunity and the molecular mechanisms by which different immune cells, including T cells, polymorphonuclear leukocytes (PMN-granulocytes), and B cells suppress autoimmune diseases is complex. We have shown previously that BWF1 lupus mice are protected from autoimmunity after i.v. injection or oral administration of tolerogenic doses of pCons, an artificial synthetic peptide based on sequences containing MHC class I and MHC class II determinants in the VH region of a J558-encoded BWF1 anti-DNA Ab. Several T cell subsets can transfer this tolerance. In this study, we determined the potential roles of granulocytes, B cells and regulatory T cells altered by pCons treatment in the BWF1 (NZB/NZW) mouse model of lupus. Immunophenotyping studies indicated that pCons treatment of BWF1 mice significantly increased CD4+FoxP3+ T cells, reduced the percent of B cells expressing CD19+CD5+ but increased the percent of CD19+CD1d+ regulatory B cells and increased the ability of the whole B cell population to suppress IgG anti-DNA production in vitro. pCons treatment significantly decreased the expression of CTLA-4 (cytotoxic T-lymphocyte-associated protein-4) in CD8+ T cells. In addition, peptide administration modified granulocytes so they became suppressive. We co-cultured sorted naïve B cells from mice making anti-DNA Ab (supported by addition of sorted naive CD4+ and CD8+ T cells from young auto-antibody-negative BWF1 mice) with sorted B cells or granulocytes from tolerized mice. Both tolerized granulocytes and tolerized B cells significantly suppressed the production of anti-DNA in vitro. In granulocytes from tolerized mice compared to saline-treated littermate controls, real-time PCR analysis indicated that expression of interferon-induced TNFAIP2 increased more than 2-fold while Ptdss2 and GATA1 mRNA were up-regulated more than 10-fold. In contrast, expression of these genes was significantly down-regulated in tolerized B cells. Further, another IFN-induced protein, Bcl2, was reduced in tolerized B cells as determined by Western blot analyses. In contrast, expression of FoxP3 was significantly increased in tolerized B cells. Together, these data suggest that B cells and granulocytes are altered toward suppressive functions by in vivo tolerization of BWF1 mice with pCons and it is possible these cell types participate in the clinical benefits seen in vivo.

scholarly journals Identification of vaccine targets & design of vaccine against SARS-CoV-2 coronavirus using computational and deep learning-based approaches.

2020 ◽  
Author(s):  
Bilal Ahmed Abbasi ◽  
Devansh Saraf ◽  
Trapti Sharma ◽  
Robin Sinha ◽  
Shachee Singh ◽  
...  
Keyword(s):  
B Cell ◽  
Mhc Class Ii ◽  
Structure Prediction ◽  
3D Structure ◽  
In Vivo Studies ◽  
T Cell Epitopes ◽  
Vaccine Candidates ◽  
Potential Vaccine ◽  
K 12

An unusual pneumonia infection, named COVID-19, was reported on December 2019 in China. It was reported to be caused by a novel coronavirus which has infected approximately 8.7 million people worldwide with a death toll of 463000 till date. This study is focused on finding potential vaccine candidates and designing an in-silico subunit multi-epitope vaccine candidates using a unique computational pipeline, integrating reverse vaccinology and molecular docking methods. A protein named SARS-CoV-spike [S] protein of SARS-CoV-2 having GenBank ID- QHD43416.1 was shortlisted, as a potential vaccine candidate and was examined for the presence of B-cell and T-cell epitopes. We also investigated antigenicity and interaction with distinct polymorphic alleles of the epitopes. High ranking epitopes/peptides such as DLCFTNVY (B cell class), KIADYNKL (MHC Class-I) and VKNKCVNFN (MHC class-II) were shortlisted for subsequent analysis. Digestion analysis verified the safety and stability of the shortlisted peptides. Docking study reported a strong binding of proposed peptides with HLA-A*02 and HLA-B7 alleles. We used standard methods to construct vaccine model and this construct was evaluated further for its antigenicity, physicochemical properties, 2D and 3D structure prediction and validation. Finally, the vaccine construct was reverse transcribed and adapted for E. coli strain K 12 prior to the insertion within the pET-28-a (+) vector for determining translational and microbial expression. Also, six multi-epitope subunit vaccines were constructed using different strategies containing immunogenic epitopes, appropriate adjuvants and linker sequences. We propose that our vaccine constructs can be used for downstream investigations using in-vitro and in-vivo studies to design effective and safe vaccine against COVID19.

T-cell Epitope-based Vaccine Design for Nipah Virus by Reverse Vaccinology Approach

2020 ◽  
Vol 23 (8) ◽  
pp. 788-796
Author(s):  
Praveen K.P. Krishnamoorthy ◽  
Sekar Subasree ◽  
Udhayachandran Arthi ◽  
Mohammad Mobashir ◽  
Chirag Gowda ◽  
...  
Keyword(s):  
T Cell ◽  
Mhc Class Ii ◽  
Vaccine Development ◽  
Nipah Virus ◽  
3D Structure ◽  
Cell Epitope ◽  
Class Ii ◽  
Class I ◽  
Reverse Vaccinology ◽  
Stable Complex

Aim and Objective: Nipah virus (NiV) is a zoonotic virus of the paramyxovirus family that sporadically breaks out from livestock and spreads in humans through breathing resulting in an indication of encephalitis syndrome. In the current study, T cell epitopes with the NiV W protein antigens were predicted. Materials and Methods: Modelling of unavailable 3D structure of W protein followed by docking studies of respective Human MHC - class I and MHC - class II alleles predicted was carried out for the highest binding rates. In the computational analysis, epitopes were assessed for immunogenicity, conservation, and toxicity analysis. T – cell-based vaccine development against NiV was screened for eight epitopes of Indian - Asian origin. Results: Two epitopes, SPVIAEHYY and LVNDGLNII, have been screened and selected for further docking study based on toxicity and conservancy analyses. These epitopes showed a significant score of -1.19 kcal/mol and 0.15 kcal/mol with HLA- B*35:03 and HLA- DRB1 * 07:03, respectively by using allele - Class I and Class II from AutoDock. These two peptides predicted by the reverse vaccinology approach are likely to induce immune response mediated by T – cells. Conclusion: Simulation using GROMACS has revealed that LVNDGLNII epitope forms a more stable complex with HLA molecule and will be useful in developing the epitope-based Nipah virus vaccine.

Healing effects of curcumin nanoparticles in deep tissue injury mouse model.

2020 ◽  
Vol 18 ◽  
Author(s):  
Zirui Zhang ◽  
Shangcong Han ◽  
Panpan Liu ◽  
Xu Yang ◽  
Jing Han ◽  
...  
Keyword(s):  
Wound Healing ◽  
Mrna Expression ◽  
Tissue Injury ◽  
Inflammatory Cells ◽  
Pcr Analysis ◽  
Protective Effects ◽  
Deep Tissue ◽  
Deep Tissue Injury

Background: Chronic inflammation and lack of angiogenesis are the important pathological mechanisms in deep tissue injury (DTI). Curcumin is a well-known anti-inflammatory and antioxidant agent. However, curcumin is unstable under acidic and alkaline conditions, and can be rapidly metabolized and excreted in the bile, which shortens its bioactivity and efficacy. Objective: This study aimed to prepare curcumin-loaded poly (lactic-co-glycolic acid) nanoparticles (CPNPs) and to elucidate the protective effects and underlying mechanisms of wound healing in DTI models. Methods: CPNPs were evaluated for particle size, biocompatibility, in vitro drug release and their effect on in vivo wound healing. Results : The results of in vivo wound closure analysis revealed that CPNP treatments significantly improved wound contraction rates (p<0.01) at a faster rate than other three treatment groups. H&E staining revealed that CPNP treatments resulted in complete epithelialization and thick granulation tissue formation, whereas control groups resulted in a lack of compact epithelialization and persistence of inflammatory cells within the wound sites. Quantitative real-time PCR analysis showed that treatment with CPNPs suppressed IL-6 and TNF-α mRNA expression, and up-regulated TGF-β, VEGF-A and IL-10 mRNA expression. Western blot analysis showed up-regulated protein expression of TGF-β, VEGF-A and phosphorylatedSTAT3. Conclusion: Our results showed that CPNPs enhanced wound healing in DTI models, through modulation of the JAK2/STAT3 signalling pathway and subsequent upregulation of pro-healing factors.

scholarly journals Targeting Toll-Like Receptor 2: Polarization of Porcine Macrophages by a Mycoplasma-Derived Pam2cys Lipopeptide

Vaccines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 692
Author(s):  
Giulia Franzoni ◽  
Antonio Anfossi ◽  
Chiara Grazia De Ciucis ◽  
Samanta Mecocci ◽  
Tania Carta ◽  
...  
Keyword(s):  
Mhc Class Ii ◽  
Macrophage Polarization ◽  
Surface Protein ◽  
Antimicrobial Activities ◽  
Toll Like Receptor ◽  
Activation Markers ◽  
Class I Mhc ◽  
Toll Like Receptor 2

Toll-like receptor 2 (TLR2) ligands are attracting increasing attention as prophylactic and immunotherapeutic agents against pathogens and tumors. We previously observed that a synthetic diacylated lipopeptide based on a surface protein of Mycoplasma agalactiae (Mag-Pam2Cys) strongly activated innate immune cells, including porcine monocyte-derived macrophages (moMΦ). In this study, we utilized confocal microscopy, flow cytometry, multiplex cytokine ELISA, and RT-qPCR to conduct a comprehensive analysis of the effects of scalar doses of Mag-Pam2Cys on porcine moMΦ. We observed enhanced expression of activation markers (MHC class I, MHC class II DR, CD25), increased phagocytotic activity, and release of IL-12 and proinflammatory cytokines. Mag-Pam2Cys also upregulated the gene expression of several IFN-α subtypes, p65, NOS2, and molecules with antimicrobial activities (CD14, beta defensin 1). Overall, our data showed that Mag-Pam2Cys polarized porcine macrophages towards a proinflammatory antimicrobial phenotype. However, Mag-Pam2Cys downregulated the expression of IFN-α3, six TLRs (TLR3, -4, -5, -7, -8, -9), and did not interfere with macrophage polarization induced by the immunosuppressive IL-10, suggesting that the inflammatory activity evoked by Mag-Pam2Cys could be regulated to avoid potentially harmful consequences. We hope that our in vitro results will lay the foundation for the further evaluation of this diacylated lipopeptide as an immunopotentiator in vivo.

scholarly journals Analysis of Sequence Divergence in Mammalian ABCGs Predicts a Structural Network of Residues That Underlies Functional Divergence

2021 ◽  
Vol 22 (6) ◽  
pp. 3012
Author(s):  
James I. Mitchell-White ◽  
Thomas Stockner ◽  
Nicholas Holliday ◽  
Stephen J. Briddon ◽  
Ian D. Kerr
Keyword(s):  
Substrate Specificity ◽  
Functional Divergence ◽  
3D Structure ◽  
Sequence Divergence ◽  
Conformational Flexibility ◽  
Substrate Selectivity ◽  
Multiple Sequence ◽  
Atp Binding Cassette Transporters ◽  
Structural Network ◽  
Wide Substrate Specificity

The five members of the mammalian G subfamily of ATP-binding cassette transporters differ greatly in their substrate specificity. Four members of the subfamily are important in lipid transport and the wide substrate specificity of one of the members, ABCG2, is of significance due to its role in multidrug resistance. To explore the origin of substrate selectivity in members 1, 2, 4, 5 and 8 of this subfamily, we have analysed the differences in conservation between members in a multiple sequence alignment of ABCG sequences from mammals. Mapping sets of residues with similar patterns of conservation onto the resolved 3D structure of ABCG2 reveals possible explanations for differences in function, via a connected network of residues from the cytoplasmic to transmembrane domains. In ABCG2, this network of residues may confer extra conformational flexibility, enabling it to transport a wider array of substrates.

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