In Vitro Cytotoxicity of a New Nano Root Canal Sealer on Human Gingival Fibroblasts

Introduction: The aim of this in vitro study was to evaluate the cytotoxicity of a new nano zinc-oxide eugenol (NZOE) sealer on human gingival fibroblasts (HGFs) compared with Pulpdent (micro-sized ZOE sealer) and AH-26 (resin-based sealer). Methods and Materials: The Pulpdent, AH-26, and NZOE sealers were prepared and exposed to cell culture media immediately after setting, and 24 h and one week after setting. Then, the primary cultured HGFs were incubated for 24 h with different dilutions (1:1 to 1:32) of each sealer extract. Cell viability was evaluated by methyl thiazolyl diphenyl tetrazolium bromide (MTT) assay. The results were compared using two-way analysis of variance followed by Tukey’s post hoc test. The level of significance was set at 0.05. Results: All sealer extracts, up to 32 times dilutions, showed cytotoxicity when exposed to HGF immediately after setting. The extracts obtained 24 h or one week after setting showed lower cytotoxicity than extracts obtained immediately after setting. At all setting times, NZOE showed lower cytotoxicity than Pulpdent and AH-26. While one-week extracts of NZOE had no significant effect on the viability of HGF at dilutions 1:4 to 1:32, both Pulpdent and AH-26 decreased the cell viability at dilutions of 1:4 and 1:8. Conclusion: NZOE exhibited lower cytotoxicity compared to Pulpdent and AH-26 on HGF and has the potential to be considered as a new root canal filling material.Keywords: Cytotoxicity; Human Gingival Fibroblast; MTT assay; Nano; Seale

Production and Purification of a Novel Anti-TNF-α Single Chain Fragment Variable Antibody

Purpose: TNF-α is an inflammatory cytokine with a key role in initiation of inflammatory responses. Anti-TNF-α antibodies are being used in clinic for the purpose of diagnosis and treatment due to their high specificity. The objective of the current study was to express and purify an anti-TNF-α scFv antibody identified by phage display technology. Methods: The DNA coding sequence of the identified scFv was cloned into pET28a vector and the corresponding protein was expressed as 6×His tagged using E.coli BL21 (DE3) pLysS expression system followed by affinity purification on Ni-Sepharose affinity column. Results: The J44 scFv antibody was cloned into the expression vector and successfully expressed and purified. The purity of the scFv fraction was confirmed using SDS-PAGE analysis. Western blotting technique was used to detect expression of 6×His tagged protein. Conclusion: In the current study an anti-TNF-α scFv antibody was successfully expressed in bacterial expression system and purified on affinity column. The purified protein can be used in different in vitro and in vivo experiments in order to elucidate its functionality

Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain

Introduction: The bacterial enzyme has gained more attention in therapeutic application because of the higher substrate specificity and longer half-life. L-asparaginase is an important enzyme with known antineoplastic effect against acute lymphoblastic leukemia (ALL). Methods: Novel L-asparaginase genes were identified from a locally isolated halo-thermotolerant Bacillus strain and the recombinant enzymes were overexpressed in modified E. coli strains, OrigamiTM B and BL21. In addition, the biochemical properties of the purified enzymes were characterized, and the enzyme activity was evaluated at different temperatures, pH, and substrate concentrations. Results: The concentration of pure soluble enzyme obtained from Origami strain was ~30 mg/L of bacterial culture, which indicates the significant improvement compared to L-asparaginase produced by E. coli BL21 strain. The catalytic activity assay on the identified L-asparaginases (ansA1 and ansA3 genes) from Bacillus sp. SL-1 demonstrated that only ansA1 gene codes an active and stable homologue (ASPase A1) with high substrate affinity toward L-asparagine. The Kcat and Km values for the purified ASPase A1 enzyme were 23.96s-1 and 10.66 µM, respectively. In addition, the recombinant ASPase A1 enzyme from Bacillus sp. SL-1 possessed higher specificity to L-asparagine than L-glutamine. The ASPase A1 enzyme was highly thermostable and resistant to the wide range of pH 4.5–10. Conclusion: The biochemical properties of the novel ASPase A1 derived from Bacillus sp. SL-l indicated a great potential for the identified enzyme in pharmaceutical and industrial applications

Anti-PL-7 (anti-threonyl-tRNA synthetase) antisynthetase syndrome: clinical manifestations in a series of patients from a European multicenter study (EUMYONET) and review of the literature

K

Effects on muscle tissue remodeling and lipid metabolism in muscle tissue from adult patients with polymyositis or dermatomyositis treated with immunosuppressive agents.

BACKGROUND: Polymyositis (PM) and dermatomyositis (DM) are autoimmune muscle diseases, conventionally treated with high doses of glucocorticoids in combination with immunosuppressive drugs. Treatment is often dissatisfying, with persisting muscle impairment. We aimed to investigate molecular mechanisms that might contribute to the persisting muscle impairment despite immunosuppressive treatment in adult patients with PM or DM using gene expression profiling of repeated muscle biopsies. METHODS: Paired skeletal muscle biopsies from six newly diagnosed adult patients with DM or PM taken before and after conventional immunosuppressive treatment were examined by gene expression microarray analysis. Selected genes that displayed changes in expression were analyzed by Western blot. Muscle biopsy sections were evaluated for inflammation, T lymphocytes (CD3), macrophages (CD68), major histocompatibility complex (MHC) class I expression and fiber type composition. RESULTS: After treatment, genes related to immune response and inflammation, including inflammasome pathways and interferon, were downregulated. This was confirmed at the protein level for AIM-2 and caspase-1 in the inflammasome pathway. Changes in genes involved in muscle tissue remodeling suggested a negative effect on muscle regeneration and growth. Gene markers for fast type II fibers were upregulated and fiber composition was switched towards type II fibers in response to treatment. The expression of genes involved in lipid metabolism was altered, suggesting a potential lipotoxic effect on muscles of the immunosuppressive treatment. CONCLUSION: The anti-inflammatory effect of immunosuppressive treatment was combined with negative effects on genes involved in muscle tissue remodeling and lipid metabolism, suggesting a negative effect on recovery of muscle performance which may contribute to persisting muscle impairment in adult patients with DM and PM

Expression, purification and DNA-binding properties of zinc finger domains of DOF proteins from Arabidopsis thaliana

Introduction: DOF proteins are a family of plant-specific transcription factors with a conserved zinc finger (ZF) DNA-binding domain. Although several studies have demonstrated their specific DNA binding, quantitative affinity data is not available for the binding of DOF domains to their binding sites. Methods: ZF domains of DOF2.1, DOF3.4, and DOF5.8 from Arabidopsis thaliana were expressed and purified. Their DNA binding affinities were assessed using gel retardation assays and microscale thermophoresis with two different oligonucleotide probes containing one and two copies of recognition sequence AAAG. Results: DOF zinc finger domains (DOF-ZFs) were shown to form independently folded structures. Assessments using microscale thermophoresis demonstrated that DOF-ZFs interact more tightly (~ 100 fold) with double-motif probe than the single-motif probe. The overall Kd values for the DOF3.4-ZF and DOF5.8-ZF to the double-motif probe were ~2.3±1 and 2.5±1 µM, respectively. Conclusion: Studied DOF-ZF domains formed stable complexes with the double-motif probe. Although DOF3.4-ZF and DOF5.8-ZF do not dimerize with an appreciable affinity in the absence of DNA (judging from size-exclusion and multiangle laser light scattering data), it is possible that these ZFs form protein-protein contacts when bound to this oligonucleotide, consistent with previous reports that DOF proteins can homo- and hetero-dimerize

Studies of immunopathogenic mechanisms and treatment of chronic, inflammatory myopathies, myositis

p>Polymyosit (PM), dermatomyositis (DM) and sporadic inclusion body myositis (sIBM) are chronic inflammatory myopathies which are characterized by muscle weakness, fatigue and extra-muscular involvement. Mononuclear inflammatory cells are typically found in muscle tissue. Treatment is based on glucocorticoids together with other immunosuppressive agents. Despite favorable response most patients with PM and DM develop sustained muscle weakness and some patients do not respond at all, including sIBM. The explanation for this is unknown. Aims: The overall aim was to achieve increased understanding of disease mechanisms in myositis by evaluating therapeutic effects on muscle performance and variables in muscle tissue of pharmacological (high dose IVIg treatment and TNF-blockade, infliximab) and of non-pharmacological treatment, endurance and resistance training. Patients and methods: Clinical outcome measures (disease activity and muscle performance), laboratory tests and muscle biopsies were performed before and after the interventions. Muscle biopsies were investigated for signs of inflammation, fibrosis and muscle fiber characteristics. Patients with chronic, refractory myositis were treated with high dose IVIg for 12 weeks (study I, n= 13) or with infliximab for 16 weeks (study II, n= 12). In studies III and IV patients with chronic, stable and low active inflammatory DM or PM were applied to endurance training for 12 weeks (study III, n = 8) or resistance training for 7 weeks (study IV, n= 8). Results: After high dose IVIg treatment muscle performance improved in 3 of 13 patients. In two patients there were signs of decreased inflammation in muscle biopsies, but there was no correlation between improved muscle performance and changes of inflammatory markers in muscle tissue. There was still a pronounced expression of inflammatory markers in muscle tissue after treatment. After infliximab treatment 3 patients had decreased, 2 had increased and 5 had unchanged disease activity. Four stopped prematurely due to adverse events. Muscle performance did not improve in any patient. Signs of active inflammation by magnetic resonance imaging were apparent before treatment in two completers and after treatment in five cases. There were minor changes in inflammatory markers in muscle biopsies, but they were still present after treatment with infliximab in all patients. Before training a low proportion of oxidative, type I fibers (mean 32±10%) and an increased proportion of intermediate type IIC fibers (3± 3%) was apparent in muscle biopsies compared to in healthy individuals. After endurance training type I fibers had increased significantly (42±13%) and type IIC fibers were reduced to 1±1%. Improved muscle performance correlated to an increased proportion of type I fibers and increased type II fiber area. After resistance training improved muscle performance was associated with downregulation of several genes signalling for inflammation and fibrosis. Gene transcripts involved in metabolic regulation were also modified. Conclusion: Treatment with high dose IVIg or infliximab had limited clinical effects and there was no consistent decrease of inflammatory molecules expressed in muscle tissue. With infliximab treatment some patients even worsened, arguing against TNF as a key molecule in the underlying disease mechanism in chronic, treatment-resistant PM, DM and sIBM. In contrast, endurance as well as resistance training had positive effects on muscle performance and this was associated with changes in muscle characteristics and molecular expression in muscle, supporting the notion that non-immune mechanisms may have a role in causing muscle weakness

Applied case studies and solutions in molecular docking-based drug design/ Siavoush Dastmalchi, Tabriz University of Medical Sciences, Iran, Maryam Hamzeh-Mivehroud, Tabriz University of Medical Sciences, Iran, and Babak Sokouti, Tabriz University of Medical Sciences, Iran, editors.

Includes bibliographical references and index."This book is a pivotal reference source for the latest scholarly research on the progress of pharmaceutical design and computational approaches in the field of molecular docking, highlighting innovative research perspectives and real-world applications"--Role of molecular docking in computer-aided drug design and development / Rahul Agarwal, Ashutosh Singh, Subhabrata Sen -- Application of docking methodologies in QSAR-based studies / Omar Deeb [and 3 others] -- Molecular docking challenges and limitations / Jahan B. Ghasemi, Azizeh Abdolmaleki, Fereshteh Shiri -- Application of molecular docking in studies on the binding mechanism of three enzymes with natural products / Hua-jin Zeng, Ran Yang, Ling-bo Qu -- Molecular docking of biologically active substances to double helical nucleic acids: problems and solutions / Kateryna V. Miroshnychenko, Anna V. Shestopalova -- Molecular-docking-based drug design and discovery: rational drug design for the subtype selective GPCR ligands / Soo-Kyung Kim, William A. Goddard III -- Molecular modelling, dynamics, and docking of membrane proteins: still a challenge / Nanda Kumar Yellapu -- In silico perspective into interactions and mutations in human TLR4 and Ebola glycoprotein / Sujay Ray, Arundhati Banerjee -- Molecular-docking-based anti-allergic drug design / Anamika Basu, Piyali Basak, Anasua Sarkar -- Protein-protein interactions (PPIs) as an alternative to targeting the ATP binding site of kinase: in silico approach to Identify PPI Inhibitors / Sailu Sarvagalla, Mohane Selvaraj Coumar -- Applications of molecular docking: its impact and importance outside the purview of drug discovery / Josephine Anthony [and 4 others].1 online resource (xvii, 367 pages)

Methods and algorithms for molecular docking-based drug design and discovery/ Siavoush Dastmalchi, Maryam Hamzeh-Mivehroud, and Babak Sokouti, editors.

Includes bibliographical references and index."This book investigates the evolution of pharmaceutical design and computational approaches in the field of molecular docking, highlighting theoretical backgrounds and emergent research in the area of computer-assisted drug design"--Provided by publisher.Molecular docking at a glance / Maryam Hamzeh-Mivehroud, Babak Sokouti, Siavoush Dastmalchi -- Methods for docking and drug designing / Ahmad Abu Turab Naqvi, Md. Imtaiyaz Hassan -- Scoring functions in docking experiments / Pravin Ambure, Kunal Roy -- The comparison of docking search algorithms and scoring functions: an overview and case studies / Marjana Novic [and 4 others] -- Protein ligand interaction fingerprints / Ali HajiEbrahimi [and 3 others] -- Different types of molecular docking based on variations of interacting molecules: variations of molecular docking / Amit Das, Simanti Bhattacharya -- Protein-protein docking: are we there yet? / Horia Jalily Hasani, Khaled H. Barakat -- Protein-ligand docking methodologies and its application in drug discovery / Sanchaita Rajkhowa, Ramesh C. Deka -- Scoring functions of protein-ligand interactions / Zhiqiang Yan, Jin Wang -- Molecular docking technique to understand enzyme-ligand interactions / Kailas Dashrath Sonawane, Maruti Jayram Dhanavade -- Recent advancements in docking methodologies / Vijay Kumar Srivastav, Vineet Singh, Meena Tiwari -- Docking methodologies and recent advances / Ashwani Kumar, Ruchika Goyal, Sandeep Jain -- Current trends in docking methodologies / Shubhandra Tripathi [and 3 others] -- Protein structure prediction using homology modeling: methods and tools / Akanksha Gupta, Pallavi Mohanty, Sonika Bhatnagar -- Online molecular docking resources / Adriana Isvoran.1 online resource (PDFs (456 pages))