Formulation and Characterization of Glucosamine Sulphate Loaded Carbopol Based Hydrogels for the Management of Osteoarthritis

Osteoarthritis is emerging as the most ordinary form of arthritis, affecting 22-39% of the Indian population. A wide range of medications and therapies are available for the treatment of osteoarthritis. With a desire to develop a therapeutically effective dosage form, the present study was carried out to formulate glucosamine sulfate loaded carbopol based hydrogel. Hydrogels H1 to H6 were formulated without permeation enhancers while formulations H7 to H12 were developed with a different class of permeation enhancers such as PEG400, oleic acid, Tween 40, DMSO and PG. Based on viscosity, it was detected that formulation H4 containing polymer 1% was ideal for incorporating drug. Considering H4 as a placebo, H6 was used for further evaluation. Drug content was found to be 99.2±0.64, with in vitro drug release of 15±0.86, 22±1.59, 28±0.72, 35±0.68, 40±0.31, 47±0.83, 58±1.59, and 70±0.9 % at a duration of 1, 2, 3, 4, 5, 6, 7, 8 hours respectively. Skin irritation tests carried out on Wistar rats revealed that skin was intact with no inflammation or erythema detected, compared to untreated site. By diffusion disc method, it was evident that the levels of microbial load were relatively low, and no harmful microorganisms were identified. There were no significant changes in physicochemical properties on stability studies. Due to a simple method of preparation and effective drug delivery, glucosamine sulfate loaded hydrogels could be contemplated as a prominent formulation in the beneficiary treatment of osteoarthritis.

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In vitro and In Silico Approach For Characterization of Antimicrobial Peptide From Probiotics Against Staphylococcus Aureus and Escherichia Coli

10.21203/rs.3.rs-366314/v2 ◽

2021 ◽

Author(s):

Amrutha Bindu ◽

Lakshmi Devi

Keyword(s):

Amino Acid ◽

Antimicrobial Peptide ◽

Lactobacillus Plantarum ◽

Exchange Chromatography ◽

Amino Acid Sequences ◽

Proteinase K ◽

Kinetic Assay ◽

Wide Range

Abstract The focus of present study was to characterize antimicrobial peptide produced by probiotic cultures, Enterococcus durans DB-1aa (MCC4243), Lactobacillus plantarum Cu2-PM7 (MCC4246) and Lactobacillus fermentum Cu3-PM8 (MCC4233) against Staphylococus aureus and E. coli. The growth kinetic assay revealed 24 h of incubation to be optimum for bacteriocin production. The partially purified compound after ion-exchange chromatography was found to be thermoresistant and stable under wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, a-amylase and lipase. The apparent molecular weight of bacteriocin from MCC4243 and MCC4246 was found to be 3.5 KDa. Translated partial amino acid sequence of plnA gene in MCC4246 displayed 48 amino acid sequences showing 100% similarity with plantaricin A of Lactobacillus plantarum (WP_0036419). The sequence revealed 7 β sheets, 6 α sheets, 6 predicted coils and 9 predicted turns. The functions on cytoplasm show 10.82 isoelectric point and 48.6% hydrophobicity. The molecular approach of using Geneious Prime software and protein prediction data base for characterization of bacteriocin is novel and predicts “KSSAYSLQMGATAIKQVKKLFKKWGW” as peptide responsible for antimicrobial activity. The study provides information about broad spectrum bacteriocin in native probiotic culture and paves a way towards its application in functional foods as biopreservative agents.

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Characterization of Andean Blueberry in Bioactive Compounds, Evaluation of Biological Properties and in vitro Bioaccessibility

10.20944/preprints202009.0055.v1 ◽

2020 ◽

Author(s):

Nieves Baenas ◽

Jenny Ruales ◽

Diego A. Moreno ◽

Daniel Alejandro Barrio ◽

Carla M. Stinco ◽

...

Keyword(s):

Antioxidant Capacity ◽

Bioactive Compounds ◽

Biological Activities ◽

In Vitro Digestion ◽

Biological Properties ◽

Wide Range ◽

Zebrafish Embryogenesis

Andean blueberries are wild berries grown and consumed in Ecuador which contain high values of bioactive compounds, mainly anthocyanins, with powerful antioxidant activity. The aim of this study was to evaluate the profile and contents of (poly)phenols and carotenoids in Andean blueberry by HPLC-DAD-MSn and determine a wide range of its biological activities. The antioxidant capacity of this fruit was evaluated in vitro by three different methods and in vivo using the zebrafish animal model, also the toxicity effect was determined by the zebrafish embryogenesis test. Besides, the antimicrobial activity and the capacity of Andean blueberry to produce hemagglutination in blood cells were evaluated. Finally, the bioaccessibility of (poly)phenols and related antioxidant capacity were determined in the different phases of an in vitro digestion. The global results indicated no toxicity of Andean blueberry, weakly bacteriostatic activity, and high contents of anthocyanins and antioxidant capacity, which were partially bioaccesible in vitro (~ 50 % at the final intestinal step), contributing to the knowledge of its health benefits for consumers and its potential use in the food and pharmaceutical industry as functional ingredient.

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Semi-automated high-throughput substrate screening assay for nucleoside kinases

10.33774/chemrxiv-2021-k0w7q ◽

2021 ◽

Author(s):

Katja Hellendahl ◽

Maryke Fehlau ◽

Sebastian Hans ◽

Peter Neubauer ◽

Anke Kurreck

Keyword(s):

High Throughput ◽

Viral Infections ◽

Screening Assay ◽

Liquid Handling ◽

Wide Range ◽

High Throughput Assay ◽

Liquid Handling Robot

Nucleoside kinases (NKs) are key enzymes involved in the in vivo phosphorylation of nucleoside analogues used as drugs to treat cancer or viral infections. Having different specificities, the characterization of NKs is essential for drug design and the production of nucleotide analogues in an in vitro enzymatic process. Therefore, a fast and reliable substrate screening assay for NKs is of great importance. Here, we report the validation of a well-known luciferase-based assay for the detection of NK activity in 96-well plate format. The assay was semi-automated using a liquid handling robot. A good linearity was demonstrated (r² >0.98) in the range of 0 to 500 µM ATP, and it was shown that also alternative phosphate donors like dATP or CTP were accepted by the luciferase. The developed high-throughput assay revealed comparable results to HPLC analysis. The assay was exemplary used for the comparison of the substrate spectra of four nucleoside kinases using 20 (8 natural and 12 modified) substrates. The screening results correlated well with literature data and, additionally, previously unknown substrates were identified for three of the NKs studied. Our results demonstrate that the developed semi-automated high-throughput assay is suitable to identify best performing NKs for a wide range of substrates.

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Characterization of cystathionine synthase as a selectable, liver-specific trait in rat hepatomas

Journal of Cell Science ◽

10.1242/jcs.82.1.309 ◽

1986 ◽

Vol 82 (1) ◽

pp. 309-320

Author(s):

S.J. Goss

Keyword(s):

Cell Growth ◽

Serum Proteins ◽

Selective Medium ◽

Carbamoyl Phosphate ◽

Cycle Enzyme ◽

Wide Range ◽

Specific Trait ◽

Well Differentiated

Cell growth using homocysteine as a source of cysteine-sulphur requires two enzymes, cystathionine synthase (CS) and gamma-cystathionase (CT). The second of these enzymes, CT, is apparently present in most cell lines regardless of their tissues of origin, since most cells can grow in vitro in the absence of cystine if they are provided with cystathionine, the intermediate in the pathway. Likewise, homocysteine will support the growth of many human cells. However, of a wide range of rodent cells, only well-differentiated rat hepatoma cells were found to grow using homocysteine in place of cystine. It is shown that cell growth in homocysteine-medium correlates well with the presence in the cells of detectable levels of CS. Furthermore, in cells able to grow in homocysteine-medium, it is possible to demonstrate the homocysteine-dependent trans-sulphuration of serine to cysteine. Growth in homocysteine-medium is not dependent on the release of preformed cysteine from disulphide complexes with serum proteins. In cell hybrids, and in ‘dedifferentiated’ variants of rat hepatomas, CS, but not CT, is subject to extinction coordinately with well-characterized liver-specific traits. For rodent cells, homocysteine-medium thus acts as a selective medium requiring the expression of a single liver-specific trait, CS. In addition it is shown that, in certain hepatoma variants, CS is regulated co-ordinately with a urea-cycle enzyme (carbamoyl phosphate synthetase I) by glucocorticoids and cyclic-AMP. Cell death through cysteine starvation is briefly considered. The immediate cause of death is apparently an insufficient supply of reduced glutathione. Selenium and vitamin E assist cell growth when the supply of cysteine is limiting.

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Characterization of Hepatitis B Surface Antigen Loaded Polylactic Acid-Based Microneedle and Its Dermal Safety Profile

Pharmaceutics ◽

10.3390/pharmaceutics12060531 ◽

2020 ◽

Vol 12 (6) ◽

pp. 531 ◽

Cited By ~ 3

Author(s):

Young-Guk Na ◽

Minki Kim ◽

Mingu Han ◽

Hyun Wook Huh ◽

Ji-Seok Kim ◽

...

Keyword(s):

Hepatitis B ◽

Surface Antigen ◽

Hepatitis B Surface Antigen ◽

Skin Irritation ◽

Hepatitis B Vaccination ◽

Histological Changes ◽

Release Testing

A surge of interest in microneedle (MN) vaccines as a novel vaccination system has emerged. Before the clinical application of MN vaccine, an assessment of potential biological risks to skin and quality control of MN must be performed. Therefore, the present study aims to evaluate the physicochemical properties of MN and to evaluate the histological changes and inflammatory cell infiltrations after the application of MN with hepatitis B surface antigen (HBsAg). During in vitro and in vivo release testing, HBsAg MN released over 70% of HBsAg at 30 min. During the pyrogen test of HBsAg MN in rabbit, no rabbit showed an individual rise in temperature of 0.5 °C or more. MN with HBsAg produced the moderate immunization in mice. MN application did not alter the thickness of dermal and epidermal layers in mice. In addition, the topical applications of MN and MN for hepatitis B vaccine did not acutely induce the inflammation, allergic reaction, dermal toxicity and skin irritation. Thus, the MN system for the delivery of HBsAg could be the promising technology in the hepatitis B vaccination.

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F.05 Characterization of NBCA glue polymerization for embolization of brain AVM’s

Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques ◽

10.1017/cjn.2016.100 ◽

2016 ◽

Vol 43 (S2) ◽

pp. S19-S20

Author(s):

BH Wang ◽

D Pelz ◽

D Lee ◽

MR Boulton ◽

SP Lownie

Keyword(s):

High Speed ◽

Contact Angles ◽

Polymerization Rate ◽

Physical Interaction ◽

Wide Range ◽

Glue Injection ◽

Glue Embolization

Background: Brain arteriovenous malformations (AVM’s) are abnormal connections between arteries and veins. Endovascular glue embolization with N-butyl cyanoacrylate (NBCA) is an accepted form of treatment, with most complications related to timing of polymerization. Current literature reports a wide range of polymerization times with large discrepancies between in-vivo and in-vitro results. Methods: Polymerization time was measured for mixtures of lipiodol/NBCA of 50/50, 60/40, 70/30. The influence of pH, temperature and presence of biological catalysts on polymerization rate was investigated in-vivo using submerged droplet tests. PVA-C, silicone and endothelium surfaces were compared and contact angles were measured to assess physical interaction with NBCA. High-speed video of glue injection through a microcatheter was captured to characterize coaxial flow. Results: Polymerization rate increases with pH and temperature. A hydrophilic substrate such as PVA-C provides surface properties that are most similar to endothelium. Endothelium provides a catalytic surface that increases the rate of polymerization. Blood products further increase the polymerization rate with RBC’s providing almost instantaneous polymerization of NBCA upon contact. Characterization of coaxial flow shows dripping to jetting transition with significant wall effect. Conclusions: We have successfully deconstructed and characterized the dynamic behavior of NBCA embolization. A refined understanding of NBCA behavior could help reduce embolization-related complications.

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Characterization of Wolbachia Transfection Efficiency by Using Microinjection of Embryonic Cytoplasm and Embryo Homogenate

Applied and Environmental Microbiology ◽

10.1128/aem.71.6.3199-3204.2005 ◽

2005 ◽

Vol 71 (6) ◽

pp. 3199-3204 ◽

Cited By ~ 28

Author(s):

Zhiyong Xi ◽

Stephen L. Dobson

Keyword(s):

Germ Line ◽

Transfection Efficiency ◽

Basic Research ◽

Host Interaction ◽

Wide Range ◽

Insect Populations ◽

Alpha Proteobacteria ◽

Phosphate Buffered Saline

ABSTRACT Wolbachia spp. are intracellular alpha proteobacteria closely related to Rickettsia. The maternally inherited infections occur in a wide range of invertebrates, causing several reproductive abnormalities, including cytoplasmic incompatibility. The artificial transfer of Wolbachia between hosts (transfection) is used both for basic research examining the Wolbachia-host interaction and for applied strategies that use Wolbachia infections to affect harmful insect populations. Commonly employed transfection techniques use embryonic microinjection to transfer Wolbachia-infected embryo cytoplasm or embryo homogenate. Although microinjections of both embryonic cytoplasm and homogenate have been used successfully, their respective transfection efficiencies (rates of establishing stable germ line infections) have not been directly compared. Transfection efficiency may be affected by variation in Wolbachia quantity or quality within the donor embryos and/or the buffer types used in embryo homogenization. Here we have compared Wolbachia bacteria that originate from different embryonic regions for their competencies in establishing stable germ line infections. The following three buffers were compared for their abilities to maintain an appropriate in vitro environment for Wolbachia during homogenization and injection: phosphate-buffered saline, Drosophila Ringer's buffer, and a sucrose-phosphate-glutamate solution (SPG buffer). The results demonstrate that Wolbachia bacteria from both anterior and posterior embryo cytoplasms are competent for establishing infection, although differing survivorships of injected hosts were observed. Buffer comparison shows that embryos homogenized in SPG buffer yielded the highest transfection success. No difference was observed in transfection efficiencies when the posterior cytoplasm transfer and SPG-homogenized embryo techniques were compared. We discuss the results in relation to intra- and interspecific Wolbachia transfection and the future adaptation of the microinjection technique for additional insects.

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Isolation and characterization of an avian isolate of Encephalitozoon hellem

Parasitology ◽

10.1017/s0031182099005995 ◽

2000 ◽

Vol 121 (1) ◽

pp. 9-14 ◽

Cited By ~ 35

Author(s):

K. F. SNOWDEN ◽

K. LOGAN ◽

D. N. PHALEN

Keyword(s):

Small Subunit ◽

Obligate Intracellular ◽

Clinically Normal ◽

Isolation And Characterization ◽

Wide Range ◽

Spore Shedding ◽

Avian Pathogen ◽

Molecular Sequencing

Members of the phylum Microspora are a group of unusual, obligate intracellular eukaryotic parasites that infect a wide range of hosts. However, there are a limited number of microsporidial infections reported in avian hosts, and no parasite species has been defined as an avian pathogen. A microsporidian organism was recovered from the droppings of a clinically normal peach-faced lovebird (Agapornis roseicollis) and established in in vitro culture. Intermittent parasite spore shedding was documented over a 2-month period using calcofluor M2R staining of cloacal swabs. The organism was identified as Encephalitozoon hellem based on protein and antigenic profiles and molecular sequencing of the small subunit and internal transcribed spacer regions of the ribosomal RNA gene.

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Genome-Wide Identification and Functional Characterization of the Trans-Isopentenyl Diphosphate Synthases Gene Family in Cinnamomum camphora

Frontiers in Plant Science ◽

10.3389/fpls.2021.708697 ◽

2021 ◽

Vol 12 ◽

Author(s):

Zerui Yang ◽

Chunzhu Xie ◽

Ting Zhan ◽

Linhuan Li ◽

Shanshan Liu ◽

...

Keyword(s):

Essential Oil ◽

Gene Family ◽

Functional Characterization ◽

Cinnamomum Camphora ◽

Isopentenyl Diphosphate ◽

Geranyl Diphosphate ◽

Wide Range ◽

Camphor Tree

Trans-isopentenyl diphosphate synthases (TIDSs) genes are known to be important determinants for terpene diversity and the accumulation of terpenoids. The essential oil of Cinnamomum camphora, which is rich in monoterpenes, sesquiterpenes, and other aromatic compounds, has a wide range of pharmacological activities and has therefore attracted considerable interest. However, the TIDS gene family, and its relationship to the camphor tree (C. camphora L. Presl.), has not yet been characterized. In this study, we identified 10 TIDS genes in the genome of the C. camphora borneol chemotype that were unevenly distributed on chromosomes. Synteny analysis revealed that the TIDS gene family in this species likely expanded through segmental duplication events. Furthermore, cis-element analyses demonstrated that C. camphora TIDS (CcTIDS) genes can respond to multiple abiotic stresses. Finally, functional characterization of eight putative short-chain TIDS proteins revealed that CcTIDS3 and CcTIDS9 exhibit farnesyl diphosphate synthase (FPPS) activity, while CcTIDS1 and CcTIDS2 encode geranylgeranyl diphosphate synthases (GGPPS). Although, CcTIDS8 and CcTIDS10 were found to be catalytically inactive alone, they were able to bind to each other to form a heterodimeric functional geranyl diphosphate synthase (GPPS) in vitro, and this interaction was confirmed using a yeast two-hybrid assay. Furthermore, transcriptome analysis revealed that the CcTIDS3, CcTIDS8, CcTIDS9, and CcTIDS10 genes were found to be more active in C. camphora roots as compared to stems and leaves, which were verified by quantitative real-time PCR (qRT-PCR). These novel results provide a foundation for further exploration of the role of the TIDS gene family in camphor trees, and also provide a potential mechanism by which the production of camphor tree essential oil could be increased for pharmacological purposes through metabolic engineering.

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Development of an in vitro tissue culture system for hammer coral (Fimbriaphyllia ancora) ovaries

Scientific Reports ◽

10.1038/s41598-021-03810-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yi-Ling Chiu ◽

Ching-Fong Chang ◽

Shinya Shikina

Keyword(s):

Structural Integrity ◽

Culture Method ◽

Culture Conditions ◽

Short Term ◽

Wide Range ◽

Fetal Bovine ◽

Short Term Culture ◽

Mesenterial Filaments

AbstractIn vitro gonad culture systems have proven useful to investigate intrinsic mechanisms of sexual reproduction in animals. Here we describe development of an in vitro culture method for coral ovaries. Mesenterial tissues containing both ovaries and mesenterial filaments were microscopically isolated from the scleractinian coral, Fimbriaphyllia ancora, and culture conditions were optimized. M199 diluted 10× (10% M199, pH 8.1) and supplemented with 25 mM HEPES and the antibiotics, ampicillin, penicillin and streptomycin, supported oocyte survival and maintained the structural integrity of ovaries during short-term culture (~ 6 days). Addition of a commercial antibiotic–antimycotic solution (Anti–Anti) and fetal bovine serum adversely affected ovary maintenance and caused tissue disintegration. Characterization of cultured ovaries showed that there is no difference in cell proliferation of ovarian somatic cells between culture Days 1 and 6. Moreover, the presence of oogonia and expression of a major yolk protein, vitellogenin, were confirmed in ovaries cultured for 6 days. This system will be useful for studying effects of a wide range of substances on coral oogenesis.

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