Search results for: TUNEL assay

Authors: Shanakr Bhattarai, V. S. Tiwari, Barak Akabayov

Abstract:

The plummeting number of infections and death is due to the development of drug-resistant bacteria. In addition, the number of approved antibiotic drugs by the Food and Drug Administration (FDA) is insufficient. Therefore, developing new drugs and finding novel targets for central metabolic pathways in bacteria is urgently needed. One of the promising targets is DNA replication machinery which consists of many essential proteins and enzymes. DnaG primase is an essential enzyme and a central part of the DNA replication machinery. DnaG primase synthesizes short RNA primers that initiate the Okazaki fragments by the lagging strand DNA polymerase. Therefore, it is reasonable to assume that inhibition of primase activity will stall DNA replication and prevent bacterial proliferation. We did the expression and purification of eight different bacterial DnaGs (Mycobacterium tuberculosis(Mtb), Bacillus anthracis (Ba), Mycobacterium smegmatis (Msmeg), Francisella tularencis (Ft), Vibrio cholerae (Vc) and Yersinia pestis (Yp), Staphylococcus aureus(Saureus), Escherichia coli(Ecoli)) followed by the radioactive activity assay. After obtaining the pure and active protein DnaG, we synthesized the inhibitors for them. The inhibitors were divided into five different groups, each containing five molecules, and the cocktail inhibition assay was performed against each DnaGs. The groups of molecules inhibiting the DnaGs were further tested with individual molecules belonging to inhibiting groups. Each molecule showing inhibition was titrated against the corresponding DnaGs to find IC50. We got a molecule(VS167) that acted as broad inhibitors, inhibiting all eight DnaGs. Molecules VS180 and VS186 inhibited seven DnaGs (except Saureus). Similarly, two molecules(VS 173, VS176) inhibited five DnaGs (Mtb, Ba, Ft, Yp, Ecoli). VS261 inhibited four DnaGs (Mtb, Ba, Ft, Vc). MS50 inhibited Ba and Vc DnaGs. And some of the inhibitors inhibited only one DnaGs. Thus we found the broad and specific inhibitors for different bacterial DnaGs, and their Structure-activity analysis(SAR) was done. Further, We tried to explain the similarities among the enzyme DnaGs from different bacteria based on their inhibition pattern.

Keywords: DNA replication, DnaG, okazaki fragments, antibiotic drugs

Procedia PDF Downloads 67

Authors: Julie Connelly, Tanvi Toprani, Xin Xie, Dhinoth Kumar Bangarusamy, Kris C. Gunsalus

Abstract:

The overuse of antibiotics worldwide has contributed to the development of multi-drug resistant (MDR) pathogenic bacterial strains. There is an increasing urgency to discover antibiotics to combat MDR pathogens. The microbiome of the Arabian Gulf is a largely unexplored and potentially rich source of novel bioactive compounds. Microbes that inhabit the Abu Dhabi coastal regions adapt to extreme environments with high salinity, hot temperatures, large temperature fluctuations, and acute exposure to solar energy. The microbes native to this region may produce unique metabolites with therapeutic potential as antibiotics and antifungals. We have isolated 200 pure bacterial strains from mangrove sediments, cyanobacterial mats, and coral reefs of the Abu Dhabi region. In this project, we aim to screen the marine bacterial strains to identify antibiotics, in particular undocumented compounds that show activity against existing antibiotic-resistant strains. We have acquired the ESKAPE pathogen panel, which consists of six antibiotic-resistant gram-positive and gram-negative bacterial pathogens that collectively cause most clinical infections. Our initial efforts of the primary screen using colony-picking co-culture assay have identified several candidate marine strains producing potential antibiotic compounds. We will next apply different assays, including disk-diffusion and broth turbidity growth assay, to confirm the results. This will be followed by bioactivity-guided purification and characterization of target compounds from the scaled-up volume of candidate strains, including SPE fraction, HPLC fraction, LC-MS, and NMR. For antimicrobial compounds with unknown structures, our final goal is to investigate their mode of action by identifying the molecular target.

Keywords: marine bacteria, natural products, drug discovery, ESKAPE panel

Procedia PDF Downloads 47

Authors: Sutapa Som Chaudhury, Chitrangada Das Mukhopadhyay

Abstract:

Alzheimer’s disease is a well-known form of dementia since its discovery in 1906. Current Food and Drug Administration approved medications e.g. cholinesterase inhibitors, memantine offer modest symptomatic relief but do not play any role in disease modification or recovery. In last three decades many small molecules, chaperons, synthetic peptides, partial β-secretase enzyme blocker have been tested for the development of a drug against Alzheimer though did not pass the 3rd clinical phase trials. Here in this study, we designed a PEGylated, aminogenic, tripeptidic polymer with two different molecular weights based on the aggregation prone amino acid sequence 17-20 in amyloid beta (Aβ) 1-42. Being conjugated with poly-ethylene glycol (PEG) which self-assembles into hydrophilic nanoparticles, these PEGylated tripeptides constitute a very good drug delivery system crossing the blood brain barrier while the peptide remains protected from proteolytic degradation and non-specific protein interactions. Moreover, being completely aminogenic they would not raise any side effects. These peptide inhibitors were evaluated for their effectiveness against Aβ42 fibrillation at an early stage of oligomer to fibril formation as well as preformed fibril clearance via Thioflavin T (ThT) assay, dynamic light scattering analyses, atomic force microscopy and scanning electron microscopy. The inhibitors were proved to be safe at a higher concentration of 20µM by the reduction assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye. Moreover, SHSY5Y neuroblastoma cells have shown a greater survivability when treated with the inhibitors following Aβ42 fibril and oligomer treatment as compared with the control Aβ42 fibril and/or oligomer treated neuroblastoma cells. These make the peptidic inhibitors a promising compound in the aspect of the discovery of alternative medication for Alzheimer’s disease.

Keywords: Alzheimer’s disease, alternative medication, amyloid beta, PEGylated peptide

Procedia PDF Downloads 183

Authors: Abdul Matin, Muazzama Akhtar, Shahid Nisar, Saddaf Mazzar, Umer Rashid

Abstract:

Antibiotic resistance is an increasing concern worldwide now a day. Neisseria gonorrhea and Staphylococcus aureus are known to cause major human sexually transmitted and respiratory diseases respectively. Nanotechnology is an emerging discipline and its application in various fields especially in medical sciences is gigantic. In the present study, we synthesized multi-walled carbon nanotubes (MWNTs) using acid oxidation method and solubilized MWNTs were with length predominantly >500 nm and diameters ranging from 40 to 50 nm. The locally synthesized MWNTs were used against gram positive and negative bacteria to determine their impact on bacterial growth. Clinical isolates of Neisseria gonorrhea (isolate: 4C-11) and Staphylococcus aureus (isolate: 38541) were obtained from local hospital and normally cultured in LB broth at 37°C. Both clinical strains can be obtained on request from University of Gujarat. Spectophometric assay was performed to determine the impact of MWNTs on bacterial growth in vitro. To determine the effect of MWTNs on test organisms, various concentration of MWNTs were used and recorded observation on various time intervals to understand the growth inhibition pattern. Our results demonstrated that MWNTs exhibited toxic effects to Staphylococcus aureus while showed very limited growth inhibition to Neisseria gonorrhea, which suggests the resistant potential of Neisseria against nanoparticles. Our results clearly demonstrate the gradual decrease in bacterial numbers with passage of time when compared with control. Maximum bacterial inhibition was observed at maximum concentration (50 µg/ml). Our future work will include further characterization and mode of action of our locally synthesized MWNTs. In conclusion, we investigated and reported for the first time the inhibitory potential of locally synthesized MWNTs on local clinical isolates of Staphylococcus aureus and Neisseria gonorrhea.

Keywords: antibacterial activity, multi walled carbon nanotubes, Neisseria gonorrhea, spectrophotometer assay, Staphylococcus aureus

Procedia PDF Downloads 286

Authors: Maria Lucia G. Lourenco, Viviane Y. Hibaru, Keylla H. N. P. Pereira, Fabiana F. Souza, Joao C. P. Ferreira, Simone B. Chiacchio, Luiz H. A. Machado

Abstract:

Isosporiasis is an affliction caused by coccidial protozoa belonging to genera Isospora spp. or Cystoisospora spp., which may parasitize the small and large intestines of dogs, of which neonates and young animals present higher risk of infection. This study aims at reporting a case of isosporiasis in neonate Pitbull dogs, as well as the diagnosis and treatment. Seven Pitbull puppies were admitted to the São Paulo State University (UNESP) Veterinary Hospital, Botucatu, São Paulo, Brazil, with history of yellowish diarrhea without mucus or blood for the past two days. The animals were five days old. The history of the mother, a primiparous two-year-old, revealed that she was properly vaccinated, not de-wormed and did not present diarrhea. The clinical examination revealed that the neonates weighted between 308 and 360 grams, and presented normal reflexes, moderate dehydration, body temperatures between 36.8 and 37.2 ºC, blood sugar between 103 and 124 mg/dL and normal appetite. A full blood count and a parasitology assay were performed to aid in the diagnosis. The full blood count detected eosinophilia, without any other relevant alterations. The parasitology assay (Willis-Molly & Faust) revealed the presence of Cystoisospora spp. The treatment was instituted with heated fluid therapy with Ringer’s Lactate (4 mL/100 g, subcutaneous) and antibiotic therapy with sulfamethoxazole-trimethoprim (15 mg/kg, orally) every 12 hours for ten days. The mother and other dogs that came in contact with the newborns were also treated. The environment was disinfected for 10 minutes with 1.6% quaternary ammonium. After 10 days, the newborns presented normal clinical signs and no alterations in the full blood count. Isosporiasis is an affliction with high mortality rates in litters that should be diagnosed and treated as soon as possible to increase the survival rates in these patients.

Keywords: Cystoisospora spp., neonatal infection, puppies, diarrhea,

Procedia PDF Downloads 102

Authors: Liang-Jen Wang, Sung-Chou Li, Yuan-Ming Yeh, Sheng-Yu Lee, Ho-Chang Kuo, Chia-Yu Yang

Abstract:

Background: Dysbiosis in the gut microbial community might be involved in the pathophysiology of attention deficit/hyperactivity disorder (ADHD). The fungal component of the gut microbiome, namely the mycobiota, is a hyperdiverse group of multicellular eukaryotes that can influence host intestinal permeability. This study therefore aimed to investigate the impact of fungal mycobiome dysbiosis and intestinal permeability on ADHD. Methods: Faecal samples were collected from 35 children with ADHD and from 35 healthy controls. Total DNA was extracted from the faecal samples, and the internal transcribed spacer (ITS) regions were sequenced using high-throughput next-generation sequencing (NGS). The fungal taxonomic classification was analysed using bioinformatics tools, and the differentially expressed fungal species between the ADHD and healthy control groups were identified. An in vitro permeability assay (Caco-2 cell layer) was used to evaluate the biological effects of fungal dysbiosis on intestinal epithelial barrier function. Results: The β-diversity (the species diversity between two communities), but not α-diversity (the species diversity within a community), reflected the differences in fungal community composition between ADHD and control groups. At the phylum level, the ADHD group displayed a significantly higher abundance of Ascomycota and significantly lower abundance of Basidiomycota than the healthy control group. At the genus level, the abundance of Candida (especially Candida albicans) was significantly increased in ADHD patients compared to the healthy controls. In addition, the in vitro cell assay revealed that C. albicans secretions significantly enhanced the permeability of Caco-2 cells. Conclusions: The current study is the first to explore altered gut mycobiome dysbiosis using the NGS platform in ADHD. The findings from this study indicated that dysbiosis of the fungal mycobiome and intestinal permeability might be associated with susceptibility to ADHD.

Keywords: ADHD, fungus, gut–brain axis, biomarker, child psychiatry

Procedia PDF Downloads 77

Authors: Christian Fercher, Jiaul Islam, Simon R. Corrie

Abstract:

Fluorescence-based immunodiagnostics are an emerging field in biosensor development and exhibit several advantages over traditional detection methods. While various affinity biosensors have been developed to generate a fluorescence signal upon sensing varying concentrations of analytes, reagentless, reversible, and continuous monitoring of complex biological samples remains challenging. Here, we aimed to genetically engineer biosensors based on single-chain antibody fragments (scFv) that are site-specifically labeled with environmentally sensitive fluorescent unnatural amino acids (UAA). A rational design approach resulted in quantifiable analyte-dependent changes in peak fluorescence emission wavelength and enabled antigen detection in vitro. Incorporation of a polarity indicator within the topological neighborhood of the antigen-binding interface generated a titratable wavelength blueshift with nanomolar detection limits. In order to ensure continuous analyte monitoring, scFv candidates with fast binding and dissociation kinetics were selected from a genetic library employing a high-throughput phage display and affinity screening approach. Initial rankings were further refined towards rapid dissociation kinetics using bio-layer interferometry (BLI) and surface plasmon resonance (SPR). The most promising candidates were expressed, purified to homogeneity, and tested for their potential to detect biomarkers in a continuous microfluidic-based assay. Variations of dissociation kinetics within an order of magnitude were achieved without compromising the specificity of the antibody fragments. This approach is generally applicable to numerous antibody/antigen combinations and currently awaits integration in a wide range of assay platforms for one-step protein quantification.

Keywords: antibody engineering, biosensor, phage display, unnatural amino acids

Procedia PDF Downloads 116

Authors: Raudone Lina, Raudonis Raimondas, Gaivelyte Kristina, Pukalskas Audrius, Janulis Valdimaras, Viskelis Pranas

Abstract:

Sorbus L. species are widely distributed in the Northern hemisphere and have been used for medicinal purposes in various traditional medicine systems and as food ingredients. Various Sorbus L. raw materials, fruits, leaves, inflorescences, barks, possess diuretic, anti-inflammatory, hypoglycemic, anti-diarrheal and vasoprotective activities. Phenolics, to whom main pharmacological activities are attributed, are compounds of interest due to their notable antioxidant activity. The aim of this study was to determine the antioxidant profiles of fruits and leaves of selected Sorbus L. species (S. anglica, S. aria f. latifolia, S. arranensis, S. aucuparia, S. austriaca, S. caucasica, S. commixta, S. discolor, S. gracilis, S. hostii, S. semi-incisa, S. tianschanica) and to identify the phenolic compounds with potent contribution to antioxidant activity. Twenty two constituents were identified in Sorbus L. species using ultra high performance liquid chromatography coupled to quadruple and time-of-flight mass spectrometers (UPLC–QTOF–MS). Reducing activity of individual constituents was determined using high performance liquid chromatography (HPLC) coupled to post-column FRAP assay. Signicantly greatest trolox equivalent values corresponding up to 45% of contribution to antioxidant activity were assessed for neochlorogenic and chlorogenic acids, which were determined as markers of antioxidant activity in samples of leaves and fruits. Characteristic patterns of antioxidant profiles obtained using HPLC post-column FRAP assay significantly depend on specific Sorbus L. species and raw materials and are suitable for equivalency research of Sorbus L. fruits and leaves. Selecting species and target plant organs with richest phenolic composition and strongly expressed antioxidant power is the first step in further research of standardized extracts.

Keywords: FRAP, antioxidant, phenolic, Sorbus L., chlorogenic acid, neochlorogenic acid

Procedia PDF Downloads 419

Authors: M. R. Vijayakumar, Sanjay Kumar Singh, Lakshmi, Hithesh Dewangan, Sanjay Singh

Abstract:

Trans resveratrol (RES) is a natural molecule proved for cancer preventive and therapeutic activities devoid of any potential side effects. However, the therapeutic application of RES in disease management is limited because of its rapid elimination from blood circulation thereby low biological half life in mammals. Therefore, the main objective of this study is to enhance the circulation as well as therapeutic efficacy using PEGylated liposomes. D-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS) is applied as steric surface decorating agent to prepare RES liposomes by thin film hydration method. The prepared nanoparticles were evaluated by various state of the art techniques such as dynamic light scattering (DLS) technique for particle size and zeta potential, TEM for shape, differential scanning calorimetry (DSC) for interaction analysis and XRD for crystalline changes of drug. Encapsulation efficiency and invitro drug release were determined by dialysis bag method. Cancer cell viability studies were performed by MTT assay, respectively. Pharmacokinetic studies were performed in sprague dawley rats. The prepared liposomes were found to be spherical in shape. Particle size and zeta potential of prepared formulations varied from 64.5±3.16 to 262.3±7.45 nm and -2.1 to 1.76 mV, respectively. DSC study revealed absence of potential interaction. XRD study revealed presence of amorphous form in liposomes. Entrapment efficiency was found to be 87.45±2.14 % and the drug release was found to be controlled up to 24 hours. Minimized MEC in MTT assay and tremendous enhancement in circulation time of RES PEGylated liposomes than its pristine form revealed that the stearic stabilized PEGylated liposomes can be an alternative tool to commercialize this molecule for chemopreventive and therapeutic applications in cancer.

Keywords: trans resveratrol, cancer nanotechnology, long circulating liposomes, bioavailability enhancement, liposomes for cancer therapy, PEGylated liposomes

Procedia PDF Downloads 558

Authors: D. P. Houhoula, J. Papaparaskevas, S. Konteles, A. Dargenta, A. Farka, C. Spyrou, M. Ziaka, S. Koussisis, E. Charvalos

Abstract:

Objectives: Nanotechnology is providing revolutionary opportunities for the rapid and simple diagnosis of many infectious diseases. Staphylococcus aureus, Listeria monocytogenes and Salmonella enteritis are important human pathogens. Diagnostic assays for bacterial culture and identification are time consuming and laborious. There is an urgent need to develop rapid, sensitive, and inexpensive diagnostic tests. In this study, a gold nanoprobe strategy developed and relies on the colorimetric differentiation of specific DNA sequences based approach on differential aggregation profiles in the presence or absence of specific target hybridization. Method: Gold nanoparticles (AuNPs) were purchased from Nanopartz. They were conjugated with thiolated oligonucleotides specific for the femA gene for the identification of members of Staphylococcus aureus, the mecA gene for the differentiation of Staphylococcus aureus and MRSA Staphylococcus aureus, hly gene encoding the pore-forming cytolysin listeriolysin for the identification of Listeria monocytogenes and the invA sequence for the identification of Salmonella enteritis. DNA isolation from Staphylococcus aureus Listeria monocytogenes and Salmonella enteritis cultures was performed using the commercial kit Nucleospin Tissue (Macherey Nagel). Specifically 20μl of DNA was diluted in 10mMPBS (pH5). After the denaturation of 10min, 20μl of AuNPs was added followed by the annealing step at 58oC. The presence of a complementary target prevents aggregation with the addition of acid and the solution remains pink, whereas in the opposite event it turns to purple. The color could be detected visually and it was confirmed with an absorption spectrum. Results: Specifically, 0.123 μg/μl DNA of St. aureus, L.monocytogenes and Salmonella enteritis was serially diluted from 1:10 to 1:100. Blanks containing PBS buffer instead of DNA were used. The application of the proposed method on isolated bacteria produced positive results with all the species of St. aureus and L. monocytogenes and Salmonella enteritis using the femA, mecA, hly and invA genes respectively. The minimum detection limit of the assay was defined at 0.2 ng/μL of DNA. Below of 0.2 ng/μL of bacterial DNA the solution turned purple after addition of HCl, defining the minimum detection limit of the assay. None of the blank samples was positive. The specificity was 100%. The application of the proposed method produced exactly the same results every time (n = 4) the evaluation was repeated (100% repeatability) using the femA, hly and invA genes. Using the gene mecA for the differentiation of Staphylococcus aureus and MRSA Staphylococcus aureus the method had a repeatability 50%. Conclusion: The proposed method could be used as a highly specific and sensitive screening tool for the detection and differentiation of Staphylococcus aureus Listeria monocytogenes and Salmonella enteritis. The use AuNPs for the colorimetric detection of DNA targets represents an inexpensive and easy-to-perform alternative to common molecular assays. The technology described here, may develop into a platform that could accommodate detection of many bacterial species.

Keywords: gold nanoparticles, pathogens, nanotechnology, bacteria

Procedia PDF Downloads 315

Authors: S. Reshma Reghu, Shiburaj Sugathan, T. G. Nandu, K. B. Ramesh Kumar, Mathew Dan

Abstract:

Bacterial diseases are considered to be one of the most prevalent health hazards in the developing world and many bacteria are becoming resistant to existing antibiotics making the treatment ineffective. Thus, it is necessary to find novel targets and develop new antibacterial drugs with a novel mechanism of action. The process of bacterial cell division is a novel and attractive target for new antibacterial drug discovery. FtsZ, a homolog of eukaryotic tubulin, is the major protein of the bacterial cell division machinery and is considered as an important antibacterial drug target. Zingiberaceae, the Ginger family consists of aromatic herbs with creeping rhizomes. Many of these plants have antimicrobial properties.This study aimed to determine the anti-bacterial activity of selected Zingiberaceous plants by targeting bacterial cell division protein, FtsZ. Essential oils and methanol extracts of Amomum ghaticum, Alpinia galanga, Kaempferia galanga, K. rotunda, and Zingiber officinale were tested to find its antibacterial efficiency using disc diffusion method against authentic bacterial strains obtained from MTCC (India). Essential oil isolated from A.galanga and Z.officinale were further assayed for FtsZ inhibition assay following non-radioactive malachite green-phosphomolybdate assay using E. coli FtsZ protein obtained from Cytoskelton Inc., USA. Z.officinale essential oil possess FtsZ inhibitory property. A molecular docking study was conducted with the known bioactive compounds of Z. officinale as ligands with the E. coli FtsZ protein homology model. Some of the major constituents of this plant like catechin, epicatechin, and gingerol possess agreeable docking scores. The results of this study revealed that several chemical constituents in Ginger plants can be utilised as potential source of antibacterial activity and it can warrant further investigation through drug discovery studies.

Keywords: antibacterial, FtsZ, zingiberaceae, docking

Procedia PDF Downloads 447

Authors: Samar Saadeldin Abdelmotalab Omer, Ikram Mohammed Eltayeb Elsiddig, Amna Beshir Medani Ahmed, Saad Mohammed Hussein Ayoub

Abstract:

Many herbal medicinal products are considered as potential hypocholesterolemic agents with encouraging safety profiles, however, only a limited amount of clinical research exists to support their efficacy. The present study was designed to compare the antihypercholesterolaemic and antioxidant activities of the crude ethanolic extracts of Citrus reticulata peel, Zingiber officinale rhizome, and Sesamum indicum seeds. Forty-five rats were used throughout the experiment, which were divided into nine groups, five rats in each as follows; normal control group (normal rats fed with standard normal diet), rats fed hypercholesterolemic diet consisting of 1% cholesterol and 10% saturated animal fat, which were further divided into eight groups; hypercholesterolemic control group (rats only fed hypercholesterolemic diet), groups 3,4,5,6,7, and 8 were given Citrus reticulata, Zingiber officinale, and Sesamum indicum ethanolic extracts at doses of (250mg/kg and 500mg/kg, respectively) orally; and group 9 rats were given atorvastatin (0.18mg/kg) orally as a reference antihypercholesterolaemic drug. Blood samples were obtained four weeks following treatment from the retro-orbital venous plexus after fasting overnight from all groups and the lipid profile (serum total cholesterol (TC), high-density-lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), and triglycerides levels) was measured and the risk ratio (TC/HDL-C) was assessed. The antioxidant activity of the three plant extracts was determined using DPPH free-radical assay. Results of in vivo and in vitro antihypercholesterolaemic and antioxidant assay, respectively, revealed that the three extracts possess comparable antioxidant and anti-hypercholesterolaemic activities.

Keywords: anti hypercholesterolemic effects, antioxidant activity, HDL, LDL, TC, TGs, citrus reticulata, sesamum indicum, zingiber officinale

Procedia PDF Downloads 433

Authors: Shama Ranjan Barua, Tofazzal M. Rakib, Mohammad Alamgir Hossain, Tania Ferdushy, Sharmin Chowdhury

Abstract:

Rotavirus is one of the main etiologies of neonatal diarrhea in bovine calves that causes significant economic loss in Bangladesh. The present study was carried out to investigate the pathology of neonatal enteritis in calves due to bovine rotavirus infection in south-eastern part of Bangladesh. Rotavirus was identified by using ELISA, RT-PCR (Reverse Transcription Polymerase Chain Reaction), real-time RT-PCR. We examined 12 dead calves with history of diarrhea during necropsy. Among 12 dead calves, in gross examination, 6 were found with pathological changes in intestine, 5 calves had congestion of small intestine and rest one had no distinct pathological changes. Intestinal contents and/or faecal samples of all dead calves were collected and examined to confirm the presence of bovine rotavirus A using Enzyme linked immunosorbant assay (ELISA), RT-PCR and real-time RT-PCR. Out 12 samples, 5 (42%) samples revealed presence of bovine rotavirus A in three diagnostic tests. The histopathological changes were found almost exclusively limited in the small intestine. The lesions of rotaviral enteritis ranged from slight to moderate shortening (atrophy) of villi in the jejunum and ileum with necrotic crypts. The villi were blunt and covered by immature epithelial cells. Infected cells, stained with Haematoxylin and Eosin staining method, showed characteristic syncytia and eosinophilc intracytoplasmic inclusion body. The presence of intracytoplasmic inclusion bodies in enterocytes is the indication of viral etiology. The presence of rotavirus in the affected tissues and/or lesions was confirmed by three different immunological and molecular tests. The findings of histopathological changes will be helpful in future diagnosis of rotaviral infection in dead calves.

Keywords: calves, diarrhea, pathology, rotavirus

Procedia PDF Downloads 224

Authors: Soledad Carinelli, Iñigo Fernández, José Luis González-Mora, Pedro A. Salazar-Carballo

Abstract:

Mastitis is the most relevant inflammatory disease in cattle, affecting the animal health and causing important economic losses on dairy farms. This disease takes place in the mammary gland or udder when some opportunistic microorganisms, such as Staphylococcus aureus, Streptococcus agalactiae, Corynebacterium bovis, etc., invade the teat canal. According to the severity of the inflammation, mastitis can be classified as sub-clinical, clinical and chronic. Standard methods for mastitis detection include counts of somatic cells, cell culture, electrical conductivity of the milk, and California test (evaluation of “gel-like” matrix consistency after cell lysed with detergents). However, these assays present some limitations for accurate detection of subclinical mastitis. Currently, haptoglobin, an acute phase protein, has been proposed as novel and effective biomarker for mastitis detection. In this work, an electrochemical biosensor based on polydopamine-modified magnetic nanoparticles (MNPs@pDA) for haptoglobin detection is reported. Thus, MNPs@pDA has been synthesized by our group and functionalized with hemoglobin due to its high affinity to haptoglobin protein. The protein was labeled with specific antibodies modified with alkaline phosphatase enzyme for its electrochemical detection using an electroactive substrate (1-naphthyl phosphate) by differential pulse voltammetry. After the optimization of assay parameters, the haptoglobin determination was evaluated in milk. The strategy presented in this work shows a wide range of detection, achieving a limit of detection of 43 ng/mL. The accuracy of the strategy was determined by recovery assays, being of 84 and 94.5% for two Hp levels around the cut off value. Milk real samples were tested and the prediction capacity of the electrochemical biosensor was compared with a Haptoglobin commercial ELISA kit. The performance of the assay has demonstrated this strategy is an excellent and real alternative as screen method for sub-clinical bovine mastitis detection.

Keywords: bovine mastitis, haptoglobin, electrochemistry, magnetic nanoparticles, polydopamine

Procedia PDF Downloads 132

Authors: Jun Hyung Lee, Robin B. Guevarra, Jin Ho Cho, Bo-Ra Kim, Jiwon Shin, Doo Wan Kim, Young Hwa Kim, Minho Song, Hyeun Bum Kim

Abstract:

Salmonella is one of the most important swine pathogens, causing acute or chronic digestive diseases, such as enteritis. The acute form of enteritis is common in young pigs of 2-4 months of age. Salmonellosis in swine causes a huge economic burden to swine industry by reducing production. Therefore, it is necessary that swine industries should strive to decrease Salmonellosis in pigs in order to reduce economic losses. Thus, we tested three types of natural plant extracts(PEs) to evaluate antibacterial effects against Salmonella enterica Typhimurium isolated from the piglet with Salmonellosis. Three PEs including turmeric oleoresin (containing curcumin 79 to 85%), capsicum oleoresin (containing capsaicin 40%-40.1%), and garlic essential oil (100% natural garlic) were tested using the direct contact agar diffusion test, minimum inhibitory concentration test, growth curve assay, and heat stability test. The tests were conducted with PEs at each concentration of 2.5%, 5%, and 10%. For the heat stability test, PEs with 10% concentration were incubated at each 4, 20, 40, 60, 80, and 100 °C for 1 hour; then the direct contact agar diffusion test was used. For the positive and negative controls, 0.5N HCl and 1XPBS were used. All the experiments were duplicated. In the direct contact agar diffusion test, garlic essential oil with 2.5%, 5%, and 10% concentration showed inhibit zones of 1.5cm, 2.7cm, and 2.8cm diameters compared to that of 3.5cm diameter for 0.5N HCl. The minimum inhibited concentration of garlic essential oil was 2.5%. Growth curve assay showed that the garlic essential oil was able to inhibit Salmonella growth significantly after 4hours. The garlic essential oil retained the ability to inhibit Salmonella growth after heat treatment at each temperature. However, turmeric and capsicum oleoresins were not able to significantly inhibit Salmonella growth by all the tests. Even though further in-vivo tests will be needed to verify effects of garlic essential oil for the Salmonellosis prevention for piglets, our results showed that the garlic essential oil could be used as a potential natural agent to prevent Salmonellosis in swine.

Keywords: garlic essential oil, pig, salmonellosis, Salmonella enterica

Procedia PDF Downloads 143

Authors: Jun Hyung Lee, Robin B. Guevarra, Jin Ho Cho, Bo-Ra Kim, Jiwon Shin, Doo Wan Kim, Young Hwa Kim, Minho Song, Hyeun Bum Kim

Abstract:

Colibacillosis is one of the major health problems in young piglets ultimately resulting in their death, and it is common especially in young piglets. For the swine industry, colibacillosis is one of the important economic burdens. Therefore, it is necessary for the swine industries to prevent Colibacillosis in piglets in order to reduce economic losses. Thus, we tested three types of natural plant extracts (PEs) to evaluate antibacterial effects against Shiga toxin-producing Escherichia coli (STEC) isolated from the piglet. Three PEs including turmeric oleoresin (containing curcumin 79 to 85%), capsicum oleoresin (containing capsaicin 40%-40.1%), and garlic essential oil (100% natural garlic) were tested using the direct contact agar diffusion test, minimum inhibitory concentration test, growth curve assay, and heat stability test. The tests were conducted with PEs at each concentration of 2.5%, 5%, and 10%. For the heat stability test, PEs with 10% concentration were incubated at each 4, 20, 40, 60, 80, and 100 °C for 1 hour, then the direct contact agar diffusion test was used. For the positive and negative controls, 0.5N HCl and 1XPBS were used. All the experiments were duplicated. In the direct contact agar diffusion test, garlic essential oil with 2.5%, 5%, and 10% concentration showed inhibit zones of 1.1cm, 3.0cm, and 3.6 cm in diameters compared to that of 3.5cm diameter for 0.5N HCl. The minimum inhibited concentration of garlic essential oil was 2.5%. Growth curve assay showed that the garlic essential oil was able to inhibit STEC growth significantly after 4 hours. The garlic essential oil retained the ability to inhibit STEC growth after heat treatment at each temperature. However, turmeric and capsicum oleoresins were not able to significantly inhibit STEC growth by all the tests. Even though further tests using the piglets will be required to evaluate effects of garlic essential oil for the Colibacillosis prevention for piglets, our results showed that the garlic essential oil could be used as a potential natural agent to prevent Colibacillosis in swine.

Keywords: garlic essential oil, pig, Colibacillosis, Escherichia coli

Procedia PDF Downloads 230

Authors: Minhal Abidi, Moinuddin

Abstract:

Diabetes mellitus (DM) induced metabolic abnormalities causes oxidative stress which leads to the pathogenesis of complications associated with diabetes like retinopathy, nephropathy periodontitis etc. Combination of glycation and oxidation 'glycoxidation' occurs when oxidative reactions affect the early state of glycation products. Low density lipoprotein (LDL) is prone to glycoxidative attack by sugars and methylglyoxal (MGO) being a strong glycating agent may have severe impact on its structure and consequent role in diabetes. Pro-inflammatory cytokines like IL1β and TNFα produced by the action of gram negative bacteria in periodontits (PD) can in turn lead to insulin resistance. This work discusses modifications to LDL as a result of glycoxidation. The changes in the protein molecule have been characterized by various physicochemical techniques and the immunogenicity of the modified molecules was also evaluated as they presented neo-epitopes. Binding of antibodies present in diabetes patients to the native and glycated LDL has been evaluated. Role of modified epitopes in the generation of antibodies in diabetes and periodontitis has been discussed. The structural perturbations induced in LDL were analyzed by UV–Vis, fluorescence, circular dichroism and FTIR spectroscopy, molecular docking studies, thermal denaturation studies, Thioflavin T assay, isothermal titration calorimetry, comet assay. MALDI-TOF, ketoamine moieties, carbonyl content and HMF content were also quantitated in native and glycated LDL. IL1β and TNFα levels were also measured in the type 2 DM and PD patients. We report increased carbonyl content, ketoamine moieties and HMF content in glycated LDL as compared to native analogue. The results substantiate that in hyperglycemic state MGO modification of LDL causes structural perturbations making the protein antigenic which could obstruct normal physiological functions and might contribute in the development of secondary complications in diabetic patients like periodontitis.

Keywords: advanced glycation end products, diabetes mellitus, glycation, glycoxidation, low density lipoprotein, periodontitis

Procedia PDF Downloads 168

Authors: Elisangela Sobreira, Denise Teixeira

Abstract:

Visceral leishmaniasis is a public health problem in Brazil, it is the main reservoir dog. In the period 2012-2016 78 diagnoses were performed in dogs suspected. Blood samples were collected from the cephalic vein obtaining serum used for the indirect immunofluorescence test and enzyme-linked immunosorbent assay, while it collected a drop of blood for the rapid chromatographic immunoassay. Obtained in 32 dogs positive. The test is important for the control of this disease and is used routinely in the Zoonoses Control Center.

Keywords: Brazil, dogs, Leismaniasis, Zoonoses center

Procedia PDF Downloads 224

Authors: Chetna Dhand, Venkatesh Mayandi, Silvia Marrero Diaz, Roger W. Beuerman, Seeram Ramakrishna, Rajamani Lakshminarayanan

Abstract:

Sturdy Insect Cuticle Sclerotization, Incredible Substrate independent Mussel’s bioadhesion, Tanning of Leather are some of catechol(amine)s mediated natural processes. Chemical contemplation spots toward a mechanism instigated with the formation of the quinone moieties from the respective catechol(amine)s, via oxidation, followed by the nucleophilic addition of the amino acids/proteins/peptides to this quinone leads to the development of highly strong, cross-linked and water-resistant proteinacious structures. Inspired with this remarkable catechol(amine)s chemistry towards amino acids/proteins/peptides, we attempted to design highly stable and water-resistant antifungal wound dressing mats with exceptional durability using collagen (protein), dopamine (catecholamine) and antifungal drugs (Amphotericin B and Caspofungin) as the key materials. Electrospinning technique has been used to fabricate desired nanofibrous mat including Collagen (COLL), COLL/Dopamine (COLL/DP) and calcium incorporated COLL/DP (COLL-DP-Ca2+). The prepared protein-based scaffolds have been studied for their microscopic investigations (SEM, TEM, and AFM), structural analysis (FT-IR), mechanical properties, water wettability characteristics and aqueous stability. Biocompatibility of these scaffolds has been analyzed for dermal fibroblast cells using MTS assay, Cell TrackerTM Green CMFDA and confocal imaging. Being the winner sample, COLL-DP-Ca2+ scaffold has been selected for incorporating two antifungal drugs namely Caspofungin (Peptide based) and Amphotericin B (Non-Peptide based). Antifungal efficiency of the designed mats has been evaluated for eight diverse fungal strains employing different microbial assays including disc diffusion, cell-viability assay, time kill kinetics etc. To confirm the durability of these mats, in term of their antifungal activity, drug leaching studies has been performed and monitored using disc diffusion assay each day. Ex-vivo fungal infection model has also been developed and utilized to validate the antifungal efficacy of the designed wound dressings. Results clearly reveal dopamine mediated crosslinking within COLL-antifungal scaffolds that leads to the generation of highly stable, mechanical tough, biocompatible wound dressings having the zone of inhabitation of ≥ 2 cm for almost all the investigated fungal strains. Leaching studies and Ex-vivo model has confirmed the durability of these wound dressing for more than 3 weeks and certified their suitability for commercialization. A model has also been proposed to enlighten the chemical mechanism involved for the development of these antifungal wound dressings with exceptional robustness.

Keywords: catecholamine chemistry, electrospinning technique, antifungals, wound dressings, collagen

Procedia PDF Downloads 344

Authors: Hallal Nouria, Kharoubi Omar

Abstract:

Wormwood (Artemisia absinthium L.) is a medicinal and aromatic bitter herb, which has been used as a medicine from ancient times. It has traditionally been used as anthelmintic, choleretic, antiseptic, balsamic, depurative, digestive, diuretic, emmenagogue and in treating leukemia and sclerosis. The species was cited to be used externally as cataplasm of crushed leaves for snake and scorpion bites or decoction for wounds and sores applied locally as antiseptic and antifungal. Wormwood extract have high contents of total phenolic compounds and total flavonoids indicating that these compounds contribute to antiradical and antioxidative activity. Most of the degenerative diseases are caused by free radicals. Antioxidants are the agents responsible for scavenging free radicals. The aim of present study was to evaluate the phytochemical and in vitro antioxidant properties of Wormwood extract. DPPH assay and reducing power assay were the method adopted to study antioxidant potentials of extracts. Standard methods were used to screen preliminary phytochemistry and quantitative analysis of tannin, phenolics and flavanoids. Aqueous and alcoholic extracts were showed good antioxidant effect with IC50 ranges from 62 μg/ml for aqueous and 116μg/ml for alcoholic extracts. Phenolic compounds, tannins and flavonoids were the major phytochemicals present in both the extracts. Percentage of inhibition increased with the increased concentration of extracts. The aqueous and alcoholic extract yielded 20, 15& 3, 59 mg/g gallic acid equivalent phenolic content 2, 78 & 1,83 mg/g quercetin equivalent flavonoid and 2, 34 & 6, 40 g tannic acid equivalent tannins respectively. The aqueous and methanol extracts of the aerial parts showed a positive correlation between the total phenolic content and the antioxidant activity measured in the plant samples. The present study provides evidence that both extracts of Artemisia absinthium is a potential source of natural antioxidant.

Keywords: pharmaceutical industries, medicinal and aromatic plant, antioxidants, phenolic compounds, Artemisia absinthium

Procedia PDF Downloads 401

Authors: Abdul Matin, Muhammad Ajmal, Uzma Yunus, Noaman-ul Haq, Hafiz M. Shohaib, Ambreen G. Muazzam

Abstract:

Carbon nanoparticles (CNPs) have unique properties that are useful for the diagnosis and treatment of cancer due to their precise properties like small size (ideal for delivery within the body) stability in solvent and tunable surface chemistry for targeted delivery. Here, highly fluorescent, monodispersed and water-soluble CNPs were synthesized directly from a suitable carbohydrate source (glucose and sucrose) by one-step acid assisted ultrasonic treatment at 35 KHz for 4 hours. This method is green, simple, rapid and economical and can be used for large scale production and applications. The average particle sizes of CNPs are less than 10nm and they emit bright and colorful green-blue fluorescence under the irradiation of UV-light at 365nm. The CNPs were characterized by scanning electron microscopy, fluorescent spectrophotometry, Fourier transform infrared spectrophotometry, ultraviolet-visible spectrophotometry and TGA analysis. Fluorescent CNPs were used as fluorescent probe and nano-carriers for anticancer drug. Functionalized CNPs (with ethylene diamine) were attached with anticancer drug-Methotrexate. In vitro bioactivity and biocompatibility of CNPs-drug conjugates was evaluated by LDH assay and Sulforhodamine B assay using human lung carcinoma cell lines (H157). Our results reveled that CNPs showed biocompatibility and CNPs-anticancer drug conjugates have shown potent cytotoxic effects and high antitumor activities in lung cancer cell lines. CNPs are proved to be excellent substitute for conventional drug delivery cargo systems and anticancer therapeutics in vitro. Our future studies will be more focused on using the same nanoparticles in vivo model system.

Keywords: carbon nanoparticles, carbon nanoparticles-methotrexate conjugates, human lung carcinoma cell lines, lactate dehydrogenase, methotrexate

Procedia PDF Downloads 272

Authors: Eman Abdullah Morsi, Hend Okasha, Heba Abdel Hady, Mortada El-Sayed, Mohamed Abbas Shemis

Abstract:

Context: Linum usitatissimum (Linn), known as Flaxseed, is one of the most important medicinal plants traditionally used for various health as nutritional purposes. Objective: Estimation of total phenolic and flavonoid contents as well as evaluate the antioxidant using α, α-diphenyl-β-picrylhydrazyl (DPPH), 2-2'azinobis (3-ethylbenzthiazoline-6-sulphonic acid (ABTS) and total antioxidant capacity (TAC) assay and investigation of anti-inflammatory by Bovine serum albumin (BSA) and anticancer activities of hepatocellular carcinoma cell line (HepG2) and breast cancer cell line (MCF7) have been applied on hexane, ethyl acetate, n-butanol and methanol extracts and also, fractions of methonal extract (hexane, ethyl acetate and n-butanol). Materials and Methods: Phenolic and flavonoid contents were detected using spectrophotometric and colorimetric assays. Antioxidant and anti-inflammatory activities were estimated in-vitro. Anticancer activity of extracts and fractions of methanolic extract were tested on (HepG2) and (MCF7). Results: Methanolic extract and its ethyl acetate fraction contain higher contents of total phenols and flavonoids. In addition, methanolic extract had higher antioxidant activity. Butanolic and ethyl acetate fractions yielded higher percent of inhibition of protein denaturation. Meanwhile, ethyl acetate fraction and methanolic extract had anticancer activity against HepG2 and MCF7 (IC50=60 ± 0.24 and 29.4 ± 0.12µg.mL⁻¹) and (IC50=94.7 ± 0.21 and 227 ± 0.48µg.mL⁻¹), respectively. In Gas chromatography-mass spectrometry (GC-MS) analysis, methanolic extract has 32 compounds, whereas; ethyl acetate and butanol fractions contain 40 and 36 compounds, respectively. Conclusion: Flaxseed contains totally different biologically active compounds that have been found to possess good variable activities, which can protect human body against several diseases.

Keywords: phenolic content, flavonoid content, HepG2, MCF7, hemolysis-assay, flaxseed

Procedia PDF Downloads 98

Authors: Isaac Martin, Abigail Lark, Sandra Morales, Eric W. Alton, Jane C. Davies

Abstract:

Objectives: The cystic fibrosis (CF) lung is a unique microbiological niche, wherein harmful bacteria persist for many years despite antibiotic therapy. Pseudomonas aeruginosa (Pa), the major culprit leading to lung decline and increased mortality, thrives in the lungs of patients with CF due to several factors that have been linked with poor antibiotic performance. Our group is investigating alternative therapies including bacteriophage cocktails with which we have previously demonstrated efficacy against planktonic organisms. In this study, we explored the effects of a 4-phage cocktail on Pa grown in two different conditions, intended to mirror the CF lung: a) alongside standard antibiotic treatment in pre-formed biofilms (structures formed by Pa-secreted exopolysaccharides which provide both physical and cell division barriers to antimicrobials and host defenses and b) in an acidic environment postulated to be present in the CF airway due both to the primary defect in bicarbonate secretion and secondary effects of inflammation. Methods: 16 Pa strains from CF patients at the Royal Brompton Hospital were selected based on sensitivity to a) ceftazidime/ tobramycin and b) the phage cocktail in a conventional plaque assay. To assess efficacy of phage in biofilms, 96 well plates with Pa (5x10⁷ CFU/ ml) were incubated in static conditions, allowing adherent bacterial colonies to form for 24 hr. Ceftazidime and tobramycin (both at 2 × MIC) were added, +/- bacteriophage (4x10⁸ PFU/mL) for a further 24 hr. Cell viability and biomass were estimated using fluorescent resazurin and crystal violet assays, respectively. To evaluate the effect of pH, strains were grown planktonically in shaking 96 well plates at pH 6.0, 6.6, 7.0 and 7.5 with tobramycin or phage, at varying concentrations. Cell viability was quantified by fluorescent resazurin assay. Results: For the biofilm assay, treatment groups were compared with untreated controls and expressed as percent reduction in cell viability and biomass. Addition of the 4-phage cocktail resulted in a 1.3-fold reduction in cell viability and 1.7-fold reduction in biomass (p < 0.001) when compared to standard antibiotic treatment alone. Notably, there was a 50 ± 15% reduction in cell viability and 60 ± 12% reduction in biomass (95% CI) for the 4 biofilms demonstrating the most resistance to antibiotic treatment. 83% of strains tested (n=6) showed decreased bacterial killing by tobramycin at acidic pHs (p < 0.01). However, 25% of strains (n=12) showed improved phage killing at acidic pHs (p < 0.05), with none showing the pattern of reduced efficacy at acidic pH demonstrated by tobramycin. Conclusion: The 4-phage anti-Pa cocktail tested against Pa performs well in pre-formed biofilms and in acidic environments; two conditions intended to mimic the CF lung. To our knowledge, these are the first data looking at the effects of subtle pH changes on phage-mediated bacterial killing in the context of Pa infection. These findings contribute to a growing body of evidence supporting the use of nebulised lytic bacteriophage as a treatment in the context of lung infection.

Keywords: biofilm, cystic fibrosis, pH, Pseudomonas aeruginosa, lytic bacteriophage

Procedia PDF Downloads 152

Authors: Vera Lukasova, Eva Filova, Jana Dankova, Vera Sovkova, Matej Daniel, Michala Rampichova

Abstract:

Proper bone implants should promote fast adhesion of cells, stimulate cell differentiation and support the formation of bone tissue. Nowadays titanium is used as a biocompatible material capable of bone tissue integration. This study was focused on comparison of bioactive properties of two titanium alloys - beta titanium alloy Ti36Nb6Ta and standard medical titanium alloy Ti6A14V. The advantage of beta titanium alloy Ti36Nb6Ta is mainly that this material does not contain adverse elements like vanadium or aluminium. Titanium alloys were sterilized in ethanol, placed into 48 well plates and seeded with porcine mesenchymal stem cells. Cells were cultivated for 14 days in standard growth cultivation media with osteogenic supplements. Cell metabolic activity was quantified using MTS assay (Promega). Cell adhesion on day 1 and cell proliferation on further days were verified immunohistochemically using beta-actin monoclonal antibody and secondary antibody conjugated with AlexaFluor®488. Differentiation of cells was evaluated using alkaline phosphatase assay. Additionally, gene expression of collagen I was measured by qRT-PCR. Porcine mesenchymal stem cells adhered and spread well on beta titanium alloy Ti36Nb6Ta on day 1. During the 14 days’ time period the cells were spread confluently on the surface of the beta titanium alloy Ti36Nb6Ta. The metabolic activity of cells increased during the whole cultivation period. In comparison to standard medical titanium alloy Ti6A14V, we did not observe any differences. Moreover, the expression of collagen I gene revealed no statistical differences between both titanium alloys. Therefore, a beta titanium alloy Ti36Nb6Ta promotes cell adhesion, metabolic activity, proliferation and collagen I expression equally to standard medical titanium alloy Ti6A14V. Thus, beta titanium is a suitable material that provides sufficient biocompatible properties. This project was supported by the Czech Science Foundation: grant No. 16-14758S.

Keywords: beta titanium alloy, biocompatibility, differentiation, mesenchymal stem cells

Procedia PDF Downloads 461

Authors: Elena I. Oprita, Oana Craciunescu, Rodica Tatia, Teodora Ciucan, Reka Barabas, Orsolya Raduly, Anca Oancea

Abstract:

Healing of osteochondral defects requires repair of the damaged articular cartilage, the underlying subchondral bone and the interface between these tissues (the functional calcified layer). For this purpose, developing a single monophasic scaffold that can regenerate two specific lineages (cartilage and bone) becomes a challenge. The aim of this work was to develop variants of biodegradable cross-linked composite hydrogel based on natural polypeptides (gelatin), polysaccharides components (chondroitin-4-sulphate and hyaluronic acid), in a ratio of 2:0.08:0.02 (w/w/w) and mixed with Si-hydroxyapatite (Si-Hap), in two ratios of 1:1 and 2:1 (w/w). Si-Hap was synthesized and characterized as a better alternative to conventional Hap. Subsequently, both composite hydrogel variants were cross-linked with (N, N-(3-dimethylaminopropyl)-N-ethyl carbodiimide (EDC) and enriched with a small bioactive molecule (icariin). The small molecule icariin (Ica) (C33H40O15) is the main active constituent (flavonoid) of Herba epimedium used in traditional Chinese medicine to cure bone- and cartilage-related disorders. Ica enhances osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), facilitates matrix calcification and increases the specific extracellular matrix (ECM) components synthesis by chondrocytes. Afterward, the composite hydrogels were characterized for their physicochemical properties in terms of the enzymatic biodegradation in the presence of type I collagenase and trypsin, the swelling capacity and the degree of crosslinking (TNBS assay). The cumulative release of Ica and real-time concentration were quantified at predetermined periods of time, according to the standard curve of standard Ica, after hydrogels incubation in saline buffer at physiological parameters. The obtained cross-linked composite hydrogels enriched with small-molecule Ica were also characterized for morphology by scanning electron microscopy (SEM). Their cytocompatibility was evaluated according to EN ISO 10993-5:2009 standard for medical device testing. Thus, analyses regarding cell viability (Live/Dead assay), cell proliferation (Neutral Red assay) and cell adhesion to composite hydrogels (SEM) were performed using NCTC clone L929 cell line. The final results showed that both cross-linked composite hydrogel variants enriched with Ica presented optimal physicochemical, structural and biological properties to be used as a natural scaffold able to repair osteochondral defects. The data did not reveal any toxicity of composite hydrogels in NCTC stabilized cell lines within the tested range of concentrations. Moreover, cells were capable of spreading and proliferating on both composite hydrogel surfaces. In conclusion, the designed biodegradable cross-linked composites enriched with Si and Ica are recommended for further testing as natural temporary scaffolds, which can allow cell migration and synthesis of new extracellular matrix within osteochondral defects.

Keywords: composites, gelatin, osteochondral defect, small molecule

Procedia PDF Downloads 149

Authors: Anna Höfer, Johannes Herrmann, Patrick Meybohm, Christopher Lotz

Abstract:

Mitochondria are pivotal for energy supply and regulation of cellular functions. Deficiencies of mitochondrial metabolism have been implicated in diverse stressful conditions including infections. Platelets are key mediators for thrombo-inflammation during development and resolution of acute respiratory distress syndrome (ARDS). Previous data point to an exhausted platelet phenotype in critically-ill patients with coronavirus 19 disease (COVID-19) impacting the course of disease. The objective of this work was to characterize platelet mitochondrial metabolism in patients suffering from COVID-19 ARDSA longitudinal analysis of platelet mitochondrial metabolism in 24 patients with COVID-19 induced ARDS compared to 35 healthy controls (ctrl) was performed. Blood samples were analyzed at two time points (t1=day 1; t2=day 5-7 after study inclusion). The activity of mitochondrial citrate synthase was photometrically measured. The impact of oxidative stress on mitochondrial permeability was assessed by a photometric calcium-induced swelling assay and the activity of superoxide dismutase (SOD) by a SOD assay kit. The amount of protein carbonylation and the activity of mitochondria complexes I-IV were photometrically determined. Levels of interleukins (IL)-1α, IL-1β and tumor necrosis factor (TNF-) α were measured by a Multiplex assay kit. Median age was 54 years, 63 % were male and BMI was 29.8 kg/m2. SOFA (12; IQR: 10-15) and APACHE II (27; IQR: 24-30) indicated critical illness. Median Murray Score was 3.4 (IQR: 2.8-3.4), 21/24 (88%) required mechanical ventilation and V-V ECMO support in 14/24 (58%). Platelet counts in ARDS did not change during ICU stay (t1: 212 vs. t2: 209 x109/L). However, mean platelet volume (MPV) significantly increased (t1: 10.6 vs. t2: 11.9 fL; p<0.0001). Citrate synthase activity showed no significant differences between ctrl and ARDS patients. Calcium induced swelling was more pronounced in patients at t1 compared to t2 and to ctrl (50µM; t1: 0.006 vs. ctrl: 0.016 ΔOD; p=0.001). The amount of protein carbonylation as marker for irreversible proteomic modification constantly increased during ICU stay and compared to ctrl., without reaching significance. In parallel, superoxid dismutase activity gradually declined during ICU treatment vs. ctrl (t2: - 29 vs. ctrl.: - 17 %; p=0.0464). Complex I analysis revealed significantly stronger activity in ARDS vs. ctrl. (t1: 0.633 vs. ctrl.: 0.415 ΔOD; p=0.0086). There were no significant differences in complex II, III or IV activity in platelets from ARDS patients compared to ctrl. IL-18 constantly increased during the observation period without reaching significance. IL-1α and TNF-α did not differ from ctrl. However, IL-1β levels were significantly elevated in ARDS (t1: 16.8; t2: 16.6 vs. ctrl.: 12.4 pg/mL; p1=0.0335, p2=0.0032). This study reveals new insights in platelet mitochondrial metabolism during COVID-19 caused ARDS. it data point towards enhanced platelet activity with a pronounced turnover rate. We found increased activity of mitochondria complex I and evidence for enhanced oxidative stress. In parallel, protective mechanisms against oxidative stress were narrowed with elevated levels of IL-1β likely causing a pro-apoptotic environment. These mechanisms may contribute to platelet exhaustion in ARDS.

Keywords: acute respiratory distress syndrome (ARDS), coronavirus 19 disease (COVID-19), oxidative stress, platelet mitochondrial metabolism

Procedia PDF Downloads 14

Authors: Wenjie Wu, Peng Cheng, Zehua Zhang, Fei Luo, Feng Wu, Min Zhong, Jianzhong Xu

Abstract:

Background: There has been limited research into the therapeutic efficacy of rapid diagnosis of spinal tuberculosis complicated with pulmonary tuberculosis. We attempted to discover whether the utilization of a DNA microarray assay to detect multidrug-resistant spinal tuberculosis complicated with pulmonary tuberculosis can improve clinical outcomes. Methods: A prospective study was conducted from February 2006 to September 2015. One hundred and forty-three consecutive culture–confirmed, clinically and imaging diagnosed MDR-TB patients with spinal tuberculosis complicated by pulmonary tuberculosis were enrolled into the study. The initial time to treatment for MDR-TB, the method of infection control, radiological indicators of spinal tubercular infectious foci, culture conversion, and adverse drug reactions were compared with the standard culture methods. Results: Of the total of 143 MDR-TB patients, 68 (47.6%) were diagnosed by conventional culture methods and 75 (52.4%) following the implementation of detection using the DNA microarray. Patients in the microarray group began rational use of the second-line drugs schedule more speedily than sufferers in the culture group (17.3 vs. 74.1 days). Among patients were admitted to a general tuberculosis ward, those from the microarray group spent less time in the ward than those from the culture group (7.8 vs. 49.2 days). In those patients with six months follow-up (n=134), patients in the microarray group had a higher rate of sputum negativity conversion at six months (89% vs. 73%). In the microarray group, the rate of drug adverse reactions was significantly lower (22.2% vs. 67.7%). At the same time, they had a more obvious reduction of the area with spinal tuberculous lesions in radiological examinations (77% vs. 108%). Conclusions: The application of the CapitalBio™ DNA Microarray assay caused noteworthy clinical advances including an earlier time to begin MDR-TB treatment, increased sputum culture conversion, improved infection control measures and better radiographical results

Keywords: tuberculosis, multidrug-resistant, tuberculous spondylitis, DNA microarray, clinical outcomes

Procedia PDF Downloads 259

Authors: Neslihan Taskale Karatug, Mustafa Akcelik

Abstract:

Some literature data show that misL protein play a role on host immune response formed against Salmonella Typhimurium. The aim of the present study is to learn the role of the protein in S. Typhimurium pathogenicity. To describe certain functions of the protein, primarily recombinant misL protein was produced and purified. PCR was performed using a primer set targeted to passenger domain of the misL gene on S. Typhimurium LT2 genome. Amplicon and pet28a vector were enzymatically cleaved with EcoRI and NheI. The digested DNA materials were purified with High Pure PCR Product Purification Kit. The ligation reaction was achieved with the pure products. After preparation of competent Escherichia coli Dh5α, ligation mix was transformed into the cell by electroporation. To confirm the existence of insert gene, recombinant plasmid DNA of Dh5α was isolated with high pure plasmid DNA kit. Proved the correctness of recombinant plasmid was electroporated to BL21. The cell was induced by IPTG. After induction, the presence of recombinant protein was checked by SDS-PAGE. The recombinant misL protein was purified using HisPur Ni-NTA spin colon. The pure protein was shown by SDS-PAGE and western blot immünoassay. The concentration of the protein was measured BCA Protein Assay kit. In the wake of ligation with digested products (2 kb misL and 5.4 kb pet28a) visualised on gel size of the band was about 7.4 kb and was named as pNT01. The pNT01 recombinant plasmid was transformed into Dh5α and colonies were chosen in selective medium. Plasmid DNA isolation from them was carried out. PCR was achieved on the pNT01 to check misL and 2 kb band was observed on the agarose gel. After electroporation of the plasmid and induction of the cell, 68 kDa misL protein was seen. Subsequent to the purification of the protein, only a band was observed on SDS-PAGE. Association of the pure protein with anti-his antibody was verified by the western blot assay. The concentration of the pure misL protein was determined as 345 μg/mL. Production of polyclonal antibody will be achieved by using the obtained pure recombinant misL protein as next step. The role of the protein will come out on the immune system together some assays.

Keywords: cloning, Escherichia coli, recombinant protein purification, Salmonella Typhimurium

Procedia PDF Downloads 360

Authors: Zakaria Baka, Halima Alem

Abstract:

Cancer is a major worldwide health problem that has caused around ten million deaths in 2020. In addition, efforts to develop new anti-cancer drugs still face a high failure rate. This is partly due to the lack of preclinical models that recapitulate in-vivo drug responses. Indeed conventional cell culture approach (known as 2D cell culture) is far from reproducing the complex, dynamic and three-dimensional environment of tumors. To set up more in-vivo-like cancer models, 3D bioprinting seems to be a promising technology due to its ability to achieve 3D scaffolds containing different cell types with controlled distribution and precise architecture. Moreover, the introduction of microfluidic technology makes it possible to simulate in-vivo dynamic conditions through the so-called “cancer-on-chip” platforms. Whereas several cancer types have been modeled through the cancer-on-chip approach, such as lung cancer and breast cancer, only a few works describing ovarian cancer models have been described. The aim of this work is to combine 3D bioprinting and microfluidic technics with setting up a 3D dynamic model of ovarian cancer. In the first phase, alginate-gelatin hydrogel containing SKOV3 cells was used to achieve tumor-like structures through an extrusion-based bioprinter. The desired form of the tumor-like mass was first designed on 3D CAD software. The hydrogel composition was then optimized for ensuring good and reproducible printability. Cell viability in the bioprinted structures was assessed using Live/Dead assay and WST1 assay. In the second phase, these bioprinted structures will be included in a microfluidic device that allows simultaneous testing of different drug concentrations. This microfluidic dispositive was first designed through computational fluid dynamics (CFD) simulations for fixing its precise dimensions. It was then be manufactured through a molding method based on a 3D printed template. To confirm the results of CFD simulations, doxorubicin (DOX) solutions were perfused through the dispositive and DOX concentration in each culture chamber was determined. Once completely characterized, this model will be used to assess the efficacy of anti-cancer nanoparticles developed in the Jean Lamour institute.

Keywords: 3D bioprinting, ovarian cancer, cancer-on-chip models, microfluidic techniques

Procedia PDF Downloads 166

Authors: Salman Akrokayan

Abstract:

Synthetic cDNA (ScDNA) of granulocyte colony-stimulating factor (G-CSF) was constructed using a DNA synthesizer with the aim to increase its expression level. 5' end of the ScDNA of G-CSF coding region was modified by decreasing the GC content without altering the predicted amino acids sequence. The identity of the resulting protein from ScDNA was confirmed by the highly specific enzyme-linked immunosorbent assay. In conclusion, a synthetic G-CSF cDNA in combination with the recombinant DNA protocol offers a rapid and reliable strategy for synthesizing the target protein. However, the commercial utilization of this methodology requires rigorous validation and quality control.

Keywords: synthetic cDNA, recombinant G-CSF, cloning, gene expression

Procedia PDF Downloads 249