Search results for: cell penetrating peptide

Authors: Zeynep Busra Velioglu, Deniz Pulat, Beril Demirbakan, Burak Ozcan, Ece Bayrak, Cevat Erisken

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Bone tissue has the ability to perform a wide array of functions including providing posture, load-bearing capacity, protection for the internal organs, initiating hematopoiesis, and maintaining the homeostasis of key electrolytes via calcium/phosphate ion storage. The most common cause for bone defects is extensive trauma and subsequent infection. Bone tissue has the self-healing capability without a scar tissue formation for the majority of the injuries. However, some may result with delayed union or fracture non-union. Such cases include reconstruction of large bone defects or cases of compromised regenerative process as a result of avascular necrosis and osteoporosis. Several surgical methods exist to treat bone defects, including Ilizarov method, Masquelete technique, growth factor stimulation, and bone replacement. Unfortunately, these are technically demanding and come with noteworthy disadvantages such as lengthy treatment duration, adverse effects on the patient’s psychology, repeated surgical procedures, and often long hospitalization times. These limitations associated with surgical techniques make bone substitutes an attractive alternative. Here, it was hypothesized that a 3D printed scaffold will mimic trabecular bone in terms of biomechanical properties and that such scaffolds will support cell attachment and survival. To test this hypothesis, this study aimed at fabricating poly(lactic acid), PLA, structures using 3D printing technology for trabecular bone defects, characterizing the scaffolds and comparing with bovine trabecular bone. Capacity of scaffolds on human bone marrow stem cell (hBMSC) attachment and survival was also evaluated. Cubes with a volume of 1 cm³ having pore sizes of 0.50, 1.00 and 1.25 mm were printed. The scaffolds/grafts were characterized in terms of porosity, contact angle, compressive mechanical properties as well cell response. Porosities of the 3D printed scaffolds were calculated based on apparent densities. For contact angles, 50 µl distilled water was dropped over the surface of scaffolds, and contact angles were measured using ‘Image J’ software. Mechanical characterization under compression was performed on scaffolds and native trabecular bone (bovine, 15 months) specimens using a universal testing machine at a rate of 0.5mm/min. hBMSCs were seeded onto the 3D printed scaffolds. After 3 days of incubation with fully supplemented Dulbecco’s modified Eagle’s medium, the cells were fixed using 2% formaldehyde and glutaraldehyde mixture. The specimens were then imaged under scanning electron microscopy. Cell proliferation was determined by using EZQuant dsDNA Quantitation kit. Fluorescence was measured using microplate reader Spectramax M2 at the excitation and emission wavelengths of 485nm and 535nm, respectively. Findings suggested that porosity of scaffolds with pore dimensions of 0.5mm, 1.0mm and 1.25mm were not affected by pore size, while contact angle and compressive modulus decreased with increasing pore size. Biomechanical characterization of trabecular bone yielded higher modulus values as compared to scaffolds with all pore sizes studied. Cells attached and survived in all surfaces, demonstrating higher proliferation on scaffolds with 1.25mm pores as compared with those of 1mm. Collectively, given lower mechanical properties of scaffolds as compared to native bone, and biocompatibility of the scaffolds, the 3D printed PLA scaffolds of this study appear as candidate substitutes for bone repair and regeneration.

Keywords: 3D printing, biomechanics, bone repair, stem cell

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Authors: Serdal Pamuk, Irem Cay

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Our model is based on the theory of reinforced random walks coupled with Michealis-Menten mechanisms which view endothelial cell receptors as the catalysts for transforming both tumor and macrophage derived tumor angiogenesis factor (TAF) into proteolytic enzyme which in turn degrade the basal lamina. The model consists of two main parts. First part has seven differential equations (DE’s) in one space dimension over the capillary, whereas the second part has the same number of DE’s in two space dimensions in the extra cellular matrix (ECM). We connect these two parts via some boundary conditions to move the cells into the ECM in order to initiate capillary formation. But, when does this movement begin? To address this question we estimate the thresholds that activate the transport equations in the capillary. We do this by using steady-state analysis of TAF equation under some assumptions. Once these equations are activated endothelial, pericyte and macrophage cells begin to move into the ECM for the initiation of angiogenesis. We do believe that our results play an important role for the mechanisms of cell migration which are crucial for tumor angiogenesis. Furthermore, we estimate the long time tendency of these three cells, and find that they tend to the transition probability functions as time evolves. We provide our numerical solutions which are in good agreement with our theoretical results.

Keywords: angiogenesis, capillary formation, mathematical analysis, steady-state, transition probability function

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Authors: Ji Jiazheng, Wang Jingrao, Jin Xin, Zhang Hong

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Diabetes mellitus is a metabolic disease characterized by high blood sugar. A long-standing hyperglycemic state can lead to various tissue damage. Diabetic retinopathy is the most common and widely studied ocular complication and has become the leading cause of blindness in my country. At the same time, diabetes has profound clinically relevant effects on the cornea, leading to keratopathy and vision-threatening. The cornea is an avascular tissue and is sensitive to hyperglycemia, Keratopathy caused by diabetes is usually chronic, they are called diabetic keratopathy or diabetic neurotrophic keratopathy, leading to several diabetic corneal complications including delayed epithelial wound healing, recurrent erosions, neuropathy, loss of sensitivity. Corneal stem cell dysfunction in diabetic patients as an important influencing factor of diabetic keratopathy. The consequences of this condition are often underestimated. The limbus is located between the cornea and the sclera tissue. The limbal stroma consists of a series of radial elevations with fibrovascular centers known as palisades of Vogt (POV). Previous studies have shown that palisades of Vogt (POV), as the main site of limbal stem cells, plays an important role in the homeostasis of the corneal epithelium. Therefore, POV plays a vital role in the healing of corneal epithelial surgery and postoperative evaluation. IVCM can observe the condition of the corneal epithelium at the cellular level. It has profound significance and guidance for the evaluation of limbal and limbal stem cells. We have previously observed structural changes in POV in HSK and HZO patients on IVCM. At present, there have been reports involving limbal stem cell dysfunction in diabetic patients, but the specific pathogenesis is still unclear. However, there are no studies on POV morphological changes in patients with DM. Therefore, we performed statistics and compared the correlation between POV morphological changes and corneal epithelial basal cell density, corneal nerves, and length of disease in DM patients and normal humans using IVCM studies. At the same time, fundoscopy was used to observe the correlation between the thickness of RNFL and the thickness of GCC and POV in diabetic patients. And to observe the correlation between SVD, DVD and POV for research.

Keywords: confocal microscopy, fundus, limbal stem cells, diabetes

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Authors: Elvin P. Chizenga, Heidi Abrahamse

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Cancer cell resistance to therapy is the main cause of treatment failures and the poor prognosis of cancer convalescence. The progression of cervical cancer to other parts of the genitourinary system and the reported recurrence rates are overwhelming. Current treatments, including surgery, chemo and radiation have been inefficient in eradicating the tumor cells. These treatments are also associated with poor prognosis and reduced quality of life, including fertility loss. This has inspired the need for the development of new treatment modalities to eradicate cervical cancer successfully. Photodynamic Therapy (PDT) is a modern treatment modality that induces cell death by photochemical interactions of light and a photosensitizer, which in the presence of molecular oxygen, yields a set of chemical reactions that generate Reactive Oxygen Species (ROS) and other free radical species causing cell damage. Enhancing PDT using modified drug delivery can increase the concentration of the photosensitizer in the tumor cells, and this has the potential to maximize its therapeutic efficacy. In cervical cancer, all infected cells constitutively express genes of the E6 and E7 HPV viral oncoproteins, resulting in high concentrations of E6 and E7 in the cytoplasm. This provides an opportunity for active targeting of cervical cancer cells using immune-mediated drug delivery to maximize therapeutic efficacy. The use of nanoparticles in PDT has also proven effective in enhancing therapeutic efficacy. Gold nanoparticles (AuNps) in particular, are explored for their use in biomedicine due to their biocompatibility, low toxicity, and enhancement of drug uptake by tumor cells. In this present study, a biomolecule comprising of AuNPs, anti-E6 monoclonal antibodies, and Aluminium Phthalocyanine photosensitizer was synthesized for use in targeted PDT of cervical cancer. The AuNp-Anti-E6-Sulfonated Aluminium Phthalocyanine mix (AlPcSmix) photosensitizing biomolecule was synthesized by coupling AuNps and anti-E6 monoclonal antibodies to the AlPcSmix via Polyethylene Glycol (PEG) chemical links. The final product was characterized using Transmission Electron Microscope (TEM), Zeta Potential, Uv-Vis Spectrophotometry, Fourier Transform Infrared Spectroscopy (FTIR), and X-ray diffraction (XRD), to confirm its chemical structure and functionality. To observe its therapeutic role in treating cervical cancer, cervical cancer cells, HeLa cells were seeded in 3.4 cm² diameter culture dishes at a concentration of 5x10⁵ cells/ml, in vitro. The cells were treated with varying concentrations of the photosensitizing biomolecule and irradiated using a 673.2 nm wavelength of laser light. Post irradiation cellular responses were performed to observe changes in morphology, viability, proliferation, cytotoxicity, and cell death pathways induced. Dose-Dependent response of the cells to treatment was demonstrated as significant morphologic changes, increased cytotoxicity, and decreased cell viability and proliferation This study presented a synthetic biomolecule for targeted PDT of cervical cancer. The study suggested that PDT using this AuNp- Anti-E6- AlPcSmix photosensitizing biomolecule is a very effective treatment method for the eradication of cervical cancer cells, in vitro. Further studies in vivo need to be conducted to support the use of this biomolecule in treating cervical cancer in clinical settings.

Keywords: anti-E6 monoclonal antibody, cervical cancer, gold nanoparticles, photodynamic therapy

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Authors: Susan Hall, Gary D. Grant, Catherine McDermott, Devinder Arora

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Introduction /Aim: The kynurenine pathway is thought to play an important role in the pathophysiology of numerous neurodegenerative diseases including depression, Alzheimer’s disease, and Parkinson’s disease. Numerous neuroactive compounds, including the neurotoxic 3-hydroxyanthranilic acid, 3-hydroxykynurenine and quinolinic acid and the neuroprotective kynurenic acid and picolinic acid, are produced through the metabolism of kynurenine and are thought to be the causative agents responsible for neurodegeneration. The toxicity of 3-hydroxykynurenine, 3-hydroxyanthranilic acid and quinolinic acid has been widely evaluated and demonstrated in primary cell cultures but to date only 3-hydroxykynurenine and 3-hydroxyanthranilic acid have been shown to cause toxicity in immortal tumour cells. The aim of this study was to evaluate the toxicity of kynurenine metabolites, both individually and in combination, on SH-SY5Y neuroblastoma cells after 24 and 72 h exposure in order to explore a cost-effective model to study their neurotoxic effects and potential protective agents. Methods: SH-SY5Y neuroblastoma cells were exposed to various concentrations of the neuroactive kynurenine metabolites, both individually and in combination, for 24 and 72 h, and viability was subsequently evaluated using the Resazurin (Alamar blue) proliferation assay. Furthermore, the effects of these compounds, alone and in combination, on specific death pathways including apoptosis, necrosis and free radical production was evaluated using various assays. Results: Consistent with literature, toxicity was shown with short-term 24-hour treatments at 1000 μM concentrations for both 3-hydroxykynurenine and 3-hydroxyanthranilic acid. Combinations of kynurenine metabolites showed modest toxicity towards SH-SY5Y neuroblastoma cells in a concentration-dependent manner. Specific cell death pathways, including apoptosis, necrosis and free radical production were shown to be increased after both 24 and 72 h exposure of SH-SY5Y neuroblastoma cells to 3-hydroxykynurenine and 3-hydroxyanthranilic acid and various combinations of neurotoxic kynurenine metabolites. Conclusion: It is well documented that neurotoxic kynurenine metabolites show toxicity towards primary human neurons in the nanomolar to low micromolar concentration range. Results show that the concentrations required to show significant cell death are in the range of 1000 µM for 3-hydroxykynurenine and 3-hydroxyanthranilic acid and toxicity of quinolinic acid towards SH-SY5Y was unable to be shown. This differs significantly from toxicities observed in primary human neurons. Combinations of the neurotoxic metabolites were shown to have modest toxicity towards these cells with increased toxicity and activation of cell death pathways observed after 72 h exposure. This study suggests that the 24 h model is unsuitable for use in neurotoxicity studies, however, the 72 h model better represents the observations of the studies using primary human neurons and may provide some benefit in providing a cost-effective model to assess possible protective agents against kynurenine metabolite toxicities.

Keywords: kynurenine metabolites, neurotoxicity, quinolinic acid, SH-SY5Y neuroblastoma

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Authors: N. Slepickova Kasalkova, P. Slepicka, L. Bacakova, V. Svorcik

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Tissue engineering includes combination of materials and techniques used for the improvement, repair or replacement of the tissue. Scaffolds, permanent or temporally material, are used as support for the creation of the "new cell structures". For this important component (scaffold), a variety of materials can be used. The advantage of some polymeric materials is their cytocompatibility and possibility of biodegradation. Poly(L-lactic acid) (PLLA) is a biodegradable,  semi-crystalline thermoplastic polymer. PLLA can be fully degraded into H₂O and CO₂. In this experiment, the effect of the surface modification of biodegradable polymer (performed by plasma treatment) on the various cell types was studied. The surface parameters and changes of the physicochemical properties of modified PLLA substrates were studied by different methods. Surface wettability was determined by goniometry, surface morphology and roughness study were performed with atomic force microscopy and chemical composition was determined using photoelectron spectroscopy. The physicochemical properties were studied in relation to cytocompatibility of human osteoblast (MG 63 cells), rat vascular smooth muscle cells (VSMC), and human stem cells (ASC) of the adipose tissue in vitro. A fluorescence microscopy was chosen to study and compare cell-material interaction. Important parameters of the cytocompatibility like adhesion, proliferation, viability, shape, spreading of the cells were evaluated. It was found that the modification leads to the change of the surface wettability depending on the time of modification. Short time of exposition (10-120 s) can reduce the wettability of the aged samples, exposition longer than 150 s causes to increase of contact angle of the aged PLLA. The surface morphology is significantly influenced by duration of modification, too. The plasma treatment involves the formation of the crystallites, whose number increases with increasing time of modification. On the basis of physicochemical properties evaluation, the cells were cultivated on the selected samples. Cell-material interactions are strongly affected by material chemical structure and surface morphology. It was proved that the plasma treatment of PLLA has a positive effect on the adhesion, spreading, homogeneity of distribution and viability of all cultivated cells. This effect was even more apparent for the VSMCs and ASCs which homogeneously covered almost the whole surface of the substrate after 7 days of cultivation. The viability of these cells was high (more than 98% for VSMCs, 89-96% for ASCs). This experiment is one part of the basic research, which aims to easily create scaffolds for tissue engineering with subsequent use of stem cells and their subsequent "reorientation" towards the bone cells or smooth muscle cells.

Keywords: poly(L-lactic acid), plasma treatment, surface characterization, cytocompatibility, human osteoblast, rat vascular smooth muscle cells, human stem cells

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Authors: Yufan Mu

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High salinity process water (HSPW) is often applied in copper ore flotation to alleviate freshwater shortage; however, it is detrimental to copper flotation as it strongly enhances copper activation of pyrite. In this study, the depression effect of a strong oxidiser, potassium ferrate (𝐾₂𝐹₄), on the flotation of copper-activated pyrite was tested to realise the selective separation of pyrite from copper minerals (e.g., chalcopyrite) in flotation using HSPW. The flotation results show that when (𝐾₂𝐹₄) was added in the flotation cell during conditioning, (𝐾₂𝐹₄) could selectively depress copper-activated pyrite while improving chalcopyrite flotation. The depression mechanism of (𝐾₂𝐹₄) on pyrite was ascribed to the significant increase in the pulp potential (Eₕ), dissolved oxygen (DO) concentration and the amount of ferric oxyhydroxides as a result of ferrate decomposition. In the flotation cell, the high Eh and DO concentration promoted the oxidation of low valency metal species (𝐶⁺𝐹e²⁺) released from mineral surfaces and forged steel grinding media, and the resultant high valency metal oxyhydroxides 𝐶u(𝑂H)₂⁄Fe(OH)₃ together with the ferric oxyhydroxides from ferrate decomposition preferentially precipitated on pyrite surface due to its more cathodic nature compared with chalcopyrite, which increased pyrite surface hydrophilicity and reduced its floatability. This study reveals that (𝐾₂𝐹₄) is a highly efficient depressant for pyrite when separating copper minerals from pyrite in flotation using HSPW if dosed properly.

Keywords: copper flotation, pyrite depression, copper-activated pyrite, potassium ferrate, high salinity process water

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Authors: Shujun Xie, Shirong Zhang, Shenglin Ma

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Background: Chronic inflammation might lead to many malignancies, and inadequate resolution could play a crucial role in tumor invasion, progression, and metastases. A randomised, double-blind, placebo-controlled trial shows that IL-1β inhibition with canakinumab could reduce incident lung cancer and lung cancer mortality in patients with atherosclerosis. The process and secretion of proinflammatory cytokine IL-1β are controlled by the inflammasome. Here we showed the correlation of the innate immune system and afatinib, a tyrosine kinase inhibitor targeting epidermal growth factor receptor (EGFR) in non-small cell lung cancer. Methods: Murine Bone marrow derived macrophages (BMDMs), peritoneal macrophages (PMs) and THP-1 were used to check the effect of afatinib on the activation of NLRP3 inflammasome. The assembly of NLRP3 inflammasome was check by co-immunoprecipitation of NLRP3 and apoptosis-associated speck-like protein containing CARD (ASC), disuccinimidyl suberate (DSS)-cross link of ASC. Lipopolysaccharide (LPS)-induced sepsis and Alum-induced peritonitis were conducted to confirm that afatinib could inhibit the activation of NLRP3 in vivo. Peripheral blood mononuclear cells (PBMCs) from non-small cell lung cancer (NSCLC) patients before or after taking afatinib were used to check that afatinib inhibits inflammation in NSCLC therapy. Results: Our data showed that afatinib could inhibit the secretion of IL-1β in a dose-dependent manner in macrophage. Moreover, afatinib could inhibit the maturation of IL-1β and caspase-1 without affecting the precursors of IL-1β and caspase-1. Next, we found that afatinib could block the assembly of NLRP3 inflammasome and the ASC speck by blocking the interaction of the sensor protein NLRP3 and the adaptor protein ASC. We also found that afatinib was able to alleviate the LPS-induced sepsis in vivo. Conclusion: Our study found that afatinib could inhibit the activation of NLRP3 inflammasome in macrophage, providing new evidence that afatinib could target the innate immune system to control chronic inflammation. These investigations will provide significant experimental evidence in afatinib as therapeutic drug for non-small cell lung cancer or other tumors and NLRP3-related diseases and will explore new targets for afatinib.

Keywords: inflammasome, afatinib, inflammation, tyrosine kinase inhibitor

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Authors: Fadilah Aleanizy

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Colicins are a family of bacterial toxins that kill Escherichia coli and other closely related species. The mode of action of colicins involves binding to an outer membrane receptor and translocation across the cell envelope, leading to cytotoxicity through specific targets. The mechanism of colicin cytotoxicity includes a non-specific endonuclease activity or depolarization of the cytoplasmic membrane by pore-forming activity. For Group A colicins, translocation requires an interaction between the N-terminal domain of the colicin and a series of membrane- bound and periplasmic proteins known as the Tol system (TolB, TolR, TolA, TolQ, and Pal and the active domain must be translocated through the outer membranes. Protein-protein interactions are intrinsic to virtually every cellular process. The transient protein-protein interactions of the colicin include the interaction with much more complicated assemblies during colicin translocation across the cellular membrane to its target. The potassium release assay detects variation in the K+ content of bacterial cells (K+in). This assays is used to measure the effect of pore-forming colicins such as ColA on an indicator organism by measuring the changes of the K+ concentration in the external medium (K+out ) that are caused by cell killing with a K+ selective electrode. One of the goals of this work is to employ a quantifiable in-vivo method to spot which Tol protein are more implicated in the interaction with colicin A as it is translocated to its target.

Keywords: K+ efflux, Colicin A, Tol-proteins, E. coli

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Authors: Victor Pena Ribeiro, Caroline Arruda, Jennyfer Andrea Aldana Mejia, Jairo Kenupp Bastos

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Leishmaniasis is a serious health problem around the world. The treatment of infected individuals with pentavalent antimonial drugs is the main therapeutic strategy. However, they present high toxicity and persistence side effects. Therefore, the discovery of new and safe natural-derived therapeutic agents against leishmaniasis is important. Propolis is a resin of viscous consistency produced by Apis mellifera bees from parts of plants. The main types of Brazilian propolis are green, red, yellow and brown. Thus, the aim of this work was to investigate the chemical composition and leishmanicidal properties of a brown propolis (BP). For this purpose, the hydroalcoholic crude extract of BP was obtained and was fractionated by liquid-liquid chromatography. The chemical profile of the extract and its fractions were obtained by HPLC-UV-DAD. The fractions were submitted to preparative HPLC chromatography for isolation of the major compounds of each fraction. They were analyzed by NMR for structural determination. The volatile compounds were obtained by hydrodistillation and identified by GC/MS. Promastigote forms of Leishmania amazonensis were cultivated in M199 medium and then 2×106 parasites.mL-1 were incubated in 96-well microtiter plates with the samples. The BP was dissolved in dimethyl sulfoxide (DMSO) and diluted into the medium, to give final concentrations of 1.56, 3.12, 6.25, 12.5, 25 and 50 µg.mL⁻¹. The plates were incubated at 25ºC for 24 h, and the lysis percentage was determined by using a Neubauer chamber. The bioassays were performed in triplicate, using a medium with 0.5% DMSO as a negative control and amphotericin B as a positive control. The leishimnicidal effect against promastigote forms was also evaluated at the same concentrations. Cytotoxicity experiments also were performed in 96-well plates against normal (CHO-k1) and tumor cell lines (AGP01 and HeLa) using XTT colorimetric method. Phenolic compounds, flavonoids, and terpenoids were identified in brown propolis. The major compounds were identified as follows: p-coumaric acid (24.6%) for a methanolic fraction, Artepelin-C (29.2%) for ethyl acetate fraction and the compounds of hexane fraction are in the process of structural elucidation. The major volatile compounds identified were β-caryophyllene (10.9%), germacrene D (9.7%), nerolidol (10.8%) and spathulenol (8.5%). The propolis did not show cytotoxicity against normal cell lines (CHO) with IC₅₀ > 100 μg.mL⁻¹, whereas the IC₅₀ < 10 μg.mL⁻¹ showed a potential against the AGP01 cell line, propolis did not demonstrate cytotoxicity against HeLa cell lines IC₅₀ > 100 μg.mL⁻¹. In the determination of the leishmanicidal activity, the highest (50 μg.mL⁻¹) and lowest (1.56 μg.mL⁻¹) concentrations of the crude extract caused the lysis of 76% and 45% of promastigote forms of L. amazonensis, respectively. To the amastigote form, the highest (50 μg.mL⁻¹) and lowest (1.56 μg.mL⁻¹) concentrations caused the mortality of 89% and 75% of L. amazonensis, respectively. The IC₅₀ was 2.8 μg.mL⁻¹ to amastigote form and 3.9 μg.mL⁻¹ to promastigote form, showing a promising activity against Leishmania amazonensis.

Keywords: amastigote, brown propolis, cytotoxicity, promastigote

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Authors: Tie-Qing Zhang, Seunghun Jung, Young-Bae Kim

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This research is devoted to developing a membrane reactor to flexibly meet the hydrogen demand of onboard fuel cells, which is an important part of green energy development. Among many renewable chemical products, dimethyl ether (DME) has the advantages of low reaction temperature (400 °C in this study), high hydrogen atom content, low toxicity, and easy preparation. Autothermal reforming, on the other hand, has a high hydrogen recovery rate and exhibits thermal neutrality during the reaction process, so the additional heat source in the hydrogen production process can be omitted. Therefore, the DME auto thermal reforming process was adopted in this study. To control the temperature of the reaction catalyst bed and hydrogen production rate, a Model Predictive Control (MPC) scheme was designed. Taking the above two variables as the control objectives, stable operation of the reformer can be achieved by controlling the flow rates of DME, steam, and high-purity air in real-time. To prevent catalyst poisoning in the fuel cell, the hydrogen needs to be purified to reduce the carbon monoxide content to below 50 ppm. Therefore, a Pd-Ag hydrogen semi-permeable membrane with a thickness of 3-5 μm was inserted into the auto thermal reactor, and the permeation efficiency of hydrogen was improved by steam purging on the permeation side. Finally, hydrogen with a purity of 99.99 was obtained.

Keywords: hydrogen production, auto thermal reforming, membrane, fuel cell

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Authors: Emma R. Arakelova, Stepan G. Grigoryan, Flora G. Arsenyan, Nelli S. Babayan, Ruzanna M. Grigoryan, Natalia K. Sarkisyan

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Novel nanosize zinc oxide composites of doxorubicin obtained by deposition of 180 nm thick zinc oxide film on the drug surface using DC-magnetron sputtering of a zinc target in the form of gels (PEO+Dox+ZnO and Starch+NaCMC+Dox+ZnO) were studied for drug delivery applications. The cancer specificity was revealed both in in vitro and in vivo models. The cytotoxicity of the test compounds was analyzed against human cancer (HeLa) and normal (MRC5) cell lines using MTT colorimetric cell viability assay. IC50 values were determined and compared to reveal the cancer specificity of the test samples. The mechanistic study of the most active compound was investigated using Flow cytometry analyzing of the DNA content after PI (propidium iodide) staining. Data were analyzed with Tree Star FlowJo software using cell cycle analysis Dean-Jett-Fox module. The in vivo anticancer activity estimation experiments were carried out on mice with inoculated ascitic Ehrlich’s carcinoma at intraperitoneal introduction of doxorubicin and its zinc oxide compositions. It was shown that the nanosize zinc oxide film deposition on the drug surface leads to the selective anticancer activity of composites at the cellular level with the range of selectivity index (SI) from 4 (Starch+NaCMC+Dox+ZnO) to 200 (PEO(gel)+Dox+ZnO) which is higher than that of free Dox (SI = 56). The significant increase in vivo antitumor activity (by a factor of 2-2.5) and decrease of general toxicity of zinc oxide compositions of doxorubicin in the form of the above mentioned gels compared to free doxorubicin were shown on the model of inoculated Ehrlich's ascitic carcinoma. Mechanistic studies of anticancer activity revealed the cytostatic effect based on the high level of DNA biosynthesis inhibition at considerable low concentrations of zinc oxide compositions of doxorubicin. The results of studies in vitro and in vivo behavior of PEO+Dox+ZnO and Starch+NaCMC+Dox+ZnO composites confirm the high potential of the nanosize zinc oxide composites as a vector delivery system for future application in cancer chemotherapy.

Keywords: anticancer activity, cancer specificity, doxorubicin, zinc oxide

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Authors: Adeel Zulifqar, Hong Hu

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Auxetic fabrics are a special kind of textile materials which possess negative Poisson’s ratio. Opposite to most of the conventional fabrics, auxetic fabrics get bigger in the transversal direction when stretched or get smaller when compressed. Auxetic fabrics are superior to conventional fabrics because of their counterintuitive properties, such as enhanced porosity under the extension, excellent formability to a curved surface and high energy absorption ability. Up till today, auxetic fabrics have been produced based on two approaches. The first approach involves using auxetic fibre or yarn and weaving technology to fabricate auxetic fabrics. The other method to fabricate the auxetic fabrics is by using non-auxetic yarns. This method has gained extraordinary curiosity of researcher in recent years. This method is based on realizing auxetic geometries into the fabric structure. In the woven fabric structure auxetic geometries can be realized by creating a differential shrinkage phenomenon into the fabric structural unit cell. This phenomenon can be created by using loose and tight weave combinations within the unit cell of interlacement pattern along with elastic and non-elastic yarns. Upon relaxation, the unit cell of interlacement pattern acquires a non-uniform shrinkage profile due to different shrinkage properties of loose and tight weaves in designed pattern, and the auxetic geometry is realized. The development of uni-stretch auxetic woven fabrics and bi-stretch auxetic woven fabrics by using this method has already been reported. This study reports the development of another kind of bi-stretch auxetic woven fabric. The fabric is first designed by transforming the auxetic geometry into interlacement pattern and then fabricated, using the available conventional weaving technology and non-auxetic elastic and non-elastic yarns. The tensile tests confirmed that the developed bi-stretch auxetic woven fabrics exhibit negative Poisson’s ratio over a wide range of tensile strain. Therefore, it can be concluded that the auxetic geometry can be realized into the woven fabric structure by creating the phenomenon of differential shrinkage and bi-stretch woven fabrics made of non-auxetic yarns having auxetic behavior and stretchability are possible can be obtained. Acknowledgement: This work was supported by the Research Grants Council of Hong Kong Special Administrative Region Government (grant number 15205514).

Keywords: auxetic, differential shrinkage, negative Poisson's ratio, weaving, stretchable

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Authors: M. Hossain, H. P. Zhu, A. B. Yu

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This paper presents a numerical investigation of regime transition of flow of ellipsoidal particles and a comparison with that of spherical particle assembly. Particle assemblies constituting spherical and ellipsoidal particle of 2.5:1 aspect ratio are examined at separate instances in similar flow conditions in a shear cell model that is numerically developed based on the discrete element method. Correlations among elastically scaled stress, kinetically scaled stress, coordination number and volume fraction are investigated, and show important similarities and differences for the spherical and ellipsoidal particle assemblies. In particular, volume fractions at points of regime transition are identified for both types of particles. It is found that compared with spherical particle assembly, ellipsoidal particle assembly has higher volume fraction for the quasistatic to intermediate regime transition and lower volume fraction for the intermediate to inertial regime transition. Finally, the relationship between coordination number and volume fraction shows strikingly distinct features for the two cases, suggesting that different from spherical particles, the effect of the shear rate on the coordination number is not significant for ellipsoidal particles. This work provides a glimpse of currently running work on one of the most attractive scopes of research in this field and has a wide prospect in understanding rheology of more complex shaped particles in light of the strong basis of simpler spherical particle rheology.

Keywords: DEM, granular rheology, non-spherical particles, regime transition

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Authors: T. H. Huang, C. L. Fern, Y. K. Tseng

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The design and reliability of solar photovoltaic modules are crucial to the development of solar energy, and efforts are still being made to extend the life of photovoltaic modules to improve their efficiency because natural aging is time-consuming and does not provide manufacturers and investors with timely information, accelerated aging is currently the best way to estimate the life of photovoltaic modules. Bifacial solar cells not only absorb light from the front side but also absorb light reflected from the ground on the back side, surpassing the performance of single-sided solar cells. Due to the asymmetry of the two sides of the light, in addition to the difference in photovoltaic conversion efficiency, there will also be differences in heat distribution, which will affect the electrical properties and material structure of the bifacial solar cell itself. In this study, there are two types of experimental samples: packaged and unpackaged and then irradiated with UVC light sources and halogen lamps for accelerated aging, as well as a control group without aging. After two weeks of accelerated aging, the bifacial solar cells were visual observation, and infrared thermal images were taken; then, the samples were subjected to IV measurement, and samples were taken for SEM, Raman, and XRD analyses in order to identify the defects that lead to failure and chemical changes, as well as to analyze the reasons for the degradation of their characteristics. From the results of the analysis, it is found that aging will cause carbonization of the polymer material on the surface of bifacial solar cells, and the crystal structure will be affected.

Keywords: bifacial solar cell, accelerated aging, temperature, characterization, electrical measurement

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Authors: Nandhakumar Elumalai, Purushothaman Ayyakannu, Sachidanandam T. Panchanatham

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Background: Understanding the basic mechanisms and factors underlying the tumor growth and invasion has gained attention in recent times. The processes of angiogenesis and apoptosis are known to play a vital role in various stages of cancer. The vascular endothelial growth factor (VEGF) is well established as one of the key regulators of tumor angiogenesis while MMPs are known for their exclusive ability to degrade ECM. Objective: The present study was designed to evaluate the pro apoptotic and anti angiogenic activity of the herbal formulation Shemamruthaa. The anticancer activity of Shemamruthaa was tested in breast cancer cell line (MCF-7). Results of MTT, trypan blue and flow cytometric analysis of apoptotis suggested that Shemamruthaa can induce cytotoxicity in cancer cells, in a concentration- and time dependent manner and induce apoptosis. With these results, we further evaluated the antiangiogenic and pro-apoptotic activities of Shemamruthaa in DMBA induced mammary carcinoma in Sprague Dawley rats. Flavono tumour was induced in 8-week-old Sprague-Dawley rats by gastric intubation of 25 mg DMBA in 1ml olive oil. After 90 days of induction period, the rats were orally administered with Shemamruthaa (400 mg/kg body wt) for 45 days. Treatment with the drug SM significantly modulated the expression of p53, MMP-2, MMP-3, MMP-9 and VEGF by means of its anti angiogenic and protease inhibiting activity. Conclusion: Based on these results, it might be concluded that the formulation, Shemamruthaa, constituted of dried flowers of Hibiscus rosa-sinensis, fruits of Emblica officinalis, and honey has been found to exhibit pronounced antiproliferative and apoptotic effects. This enhanced anticancer effect of Shemamruthaa might be attributed to the synergistic action of polyphenols such as flavonoids, tannins, alkaloids, glycosides, saponins, steroids, terpenoids, vitamin C, niacin, pyrogallol, hydroxymethylfurfural, trilinolein, and other compounds present in the formulation. Collectively, these results demonstrate that Shemamruthaa holds potential to be developed as a potent chemotherapeutic agent against mammary carcinoma.

Keywords: Shemamruthaa, flavonoids, MCF-7 cell line, mammary cancer

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Authors: S. D. Abdul, F. B. J. Sawa, D. Z. Andrawus, G. Dan'ilu

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Fresh leaves of twelve accessions of S. indicum were studied to examine their stomatal features, trichomes, epidermal cell shapes and anticlinal cell-wall patterns which may be used for the delimitation of the varieties. The twelve accessions of S. indicum studied have amphistomatic leaves, i.e. having stomata on both surfaces. Four types of stomatal complex types were observed namely, diacytic, anisocytic, tetracytic and anomocytic. Anisocytic type was the most common occurring on both surfaces of all the varieties and occurred 100% in varieties lale-duk, ex-sudan and ex-gombe 6. One-way ANOVA revealed that there was no significant difference between the stomatal densities of ex-gombe 6, ex-sudan, adawa-wula, adawa-ting, ex-gombe 4 and ex-gombe 2 . Accession adawa-ting (improved) has the smallest stomatal size (26.39µm) with highest stomatal density (79.08mm2) while variety adawa-wula possessed the largest stomatal size (74.31µm) with lowest stomatal density (29.60mm2), the exception was found in variety adawa-ting whose stomatal size is larger (64.03µm) but with higher stomatal density (71.54mm2). Wavy, curve or undulate anticlinal wall patterns with irregular and or isodiametric epidermal cell shapes were observed. These accessions were found to exhibit high degree of heterogeneity in their trichome features. Ten types of trichomes were observed: unicellular, glandular peltate, capitate glandular, long unbranched uniseriate, short unbranched uniseriate, scale, multicellular, multiseriate capitate glandular, branched uniseriate and stallate trichomes. The most frequent trichome type is short-unbranched uniseriate, followed by long-unbranched uniseriate (72.73% and 72.5%) respectively. The least frequent was multiseriate capitate glandular (11.5%). The high variation in trichome types and density coupled with the stomatal complex types suggest that these varieties of S. indicum probably have the capacity to conserve water. Furthermore, the leaf micromorphological features varied from one accession to another, hence, are found to be good diagnostic and additional tool in identification as well as nomenclature of the accessions of S. indicum.

Keywords: Sesamum indicum, stomata, trichomes, epidermal cells, taxonomy

Procedia PDF Downloads 274

Authors: Sajeli A. Begum, Ameer Basha, Kirti Hira, Rukaiyya Khan

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Cyclic dipeptides are simple diketopiperazine derivatives being investigated by several scientists for their biological effects which include anticancer, antimicrobial, haematological, anticonvulsant, immunomodulatory effect, etc. They are potentially active microbial metabolites having been synthesized too, for developing into drug candidates. Cultures of Pseudomonas species have earlier been reported to produce cyclic dipeptides, helping in quorum sensing signals and bacterial–host colonization phenomena during infections, causing cell anti-proliferation and immunosuppression. Fluorescing Pseudomonas species have been identified to secrete lipid derivatives, peptides, pyrroles, phenazines, indoles, aminoacids, pterines, pseudomonic acids and some antibiotics. In the present work, results of investigation on the cyclic dipeptide metabolites secreted by the culture broth of Pseudomonas species as potent pro-inflammatory cytokine inhibitors are discussed. The bacterial strain was isolated from the rhizospheric soil of groundnut crop and identified as Pseudomonas aeruginosa by 16S rDNA sequence (GenBank Accession No. KT625586). Culture broth of this strain was prepared by inoculating into King’s B broth and incubating at 30 ºC for 7 days. The ethyl acetate extract of culture broth was prepared and lyophilized to get a dry residue (EEPA). Lipopolysaccharide (LPS)-induced ELISA assay proved the inhibition of tumor necrosis factor-alpha (TNF-α) secretion in culture supernatant of RAW 264.7 cells by EEPA (IC50 38.8 μg/mL). The effect of oral administration of EEPA on plasma TNF-α level in rats was tested by ELISA kit. The LPS mediated plasma TNF-α level was reduced to 45% with 125 mg/kg dose of EEPA. Isolation of the chemical constituents of EEPA through column chromatography yielded ten cyclic dipeptides, which were characterized using nuclear magnetic resonance and mass spectroscopic techniques. These cyclic dipeptides are biosynthesized in microorganisms by multifunctional assembly of non-ribosomal peptide synthases and cyclic dipeptide synthase. Cyclo (Gly-L-Pro) was found to be more potentially (IC50 value 4.5 μg/mL) inhibiting TNF-α production followed by cyclo (trans-4-hydroxy-L-Pro-L-Phe) (IC50 value 14.2 μg/mL) and the effect was equal to that of standard immunosuppressant drug, prednisolone. Further, the effect was analyzed by determining mRNA expression of TNF-α in LPS-stimulated RAW 264.7 macrophages using quantitative real-time reverse transcription polymerase chain reaction. EEPA and isolated cyclic dipeptides demonstrated diminution of TNF-α mRNA expression levels in a dose-dependent manner under the tested conditions. Also, they were found to control the expression of other pro-inflammatory cytokines like IL-1β and IL-6, when tested through their mRNA expression levels in LPS-stimulated RAW 264.7 macrophages under LPS-stimulated conditions. In addition, significant inhibition effect was found on Nitric oxide production. Further all the compounds exhibited weak toxicity to LPS-induced RAW 264.7 cells. Thus the outcome of the study disclosed the effectiveness of EEPA and the isolated cyclic dipeptides in down-regulating key cytokines involved in pathophysiology of autoimmune diseases.In another study led by the investigators, microbial cyclic dipeptides were found to exhibit excellent antimicrobial effect against Fusarium moniliforme which is an important causative agent of Sorghum grain mold disease. Thus, cyclic dipeptides are emerging small molecular drug candidates for various autoimmune diseases.

Keywords: cyclic dipeptides, cytokines, Fusarium moniliforme, Pseudomonas, TNF-alpha

Procedia PDF Downloads 211

Authors: Emanuela Chiarella, Annamaria Aloisio, Nisticò Clelia, Maria Mesuraca

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ZNF521 is a stem cell-associated transcription co-factor, that plays a crucial role in the homeostatic regulation of the stem cell compartment in the hematopoietic, osteo-adipogenic, and neural system. In normal hematopoiesis, primary human CD34+ hematopoietic stem cells display typically a high expression of ZNF521, while its mRNA levels rapidly decrease when these progenitors progress towards erythroid, granulocytic, or B-lymphoid differentiation. However, most acute myeloid leukemias (AMLs) and leukemia-initiating cells keep high ZNF521 expression. In particular, AMLs are often characterized by chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene, which MLL gene includes a variety of fusion oncogenes arisen from genes normally required during hematopoietic development; once they are fused, they promote epigenetic and transcription factor dysregulation. The chromosomal translocation t(9;11)(p21-22;q23), fusing the MLL gene with AF9 gene, results in a monocytic immune phenotype with an aggressive course, frequent relapses, and a short survival time. To better understand the dysfunctional transcriptional networks related to genetic aberrations, AML gene expression profile datasets were queried for ZNF521 expression and its correlations with specific gene rearrangements and mutations. The results showed that ZNF521 mRNA levels are associated with specific genetic aberrations: the highest expression levels were observed in AMLs involving t(11q23) MLL rearrangements in two distinct datasets (MILE and den Boer); elevated ZNF521 mRNA expression levels were also revealed in AMLs with t(7;12) or with internal rearrangements of chromosome 16. On the contrary, relatively low ZNF521 expression levels seemed to be associated with the t(8;21) translocation, that in turn is correlated with the AML1-ETO fusion gene or the t(15;17) translocation and in AMLs with FLT3-ITD, NPM1, or CEBPα double mutations. Invitro, we found that the enforced co-expression of ZNF521 in cord blood-derived CD34+ cells induced a significant proliferative advantage, improving MLL-AF9 effects on the induction of proliferation and the expansion of leukemic progenitor cells. Transcriptome profiling of CD34+ cells transduced with either MLL-AF9, ZNF521, or a combination of the two transgenes highlighted specific sets of up- or down-regulated genes that are involved in the leukemic phenotype, including those encoding transcription factors, epigenetic modulators, and cell cycle regulators as well as those engaged in the transport or uptake of nutrients. These data enhance the functional cooperation between ZNF521 and MA9, resulting in the development, maintenance, and clonal expansion of leukemic cells. Finally, silencing of ZNF521 in MLL-AF9-transformed primary CD34+ cells inhibited their proliferation and led to their extinction, as well as ZNF521 silencing in the MLL-AF9+ THP-1 cell line resulted in an impairment of their growth and clonogenicity. Taken together, our data highlight ZNF521 role in the control of self-renewal and in the immature compartment of malignant hematopoiesis, which, by altering the gene expression landscape, contributes to the development and/or maintenance of AML acting in concert with the MLL-AF9 fusion oncogene.

Keywords: AML, human zinc finger protein 521 (hZNF521), mixed lineage leukemia gene (MLL) AF9 (MLLT3 or LTG9), cord blood-derived hematopoietic stem cells (CB-CD34+)

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Authors: Jeet Chakraborty, Sanjay Dutta

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Antibiotic resistance by bacteria has proved to be a severe threat to mankind in recent times, and this fortifies an urgency to design and develop potent antibacterial small molecules/compounds with nonconventional mechanisms than the conventional ones. DNA carries the genetic signature of any organism, and bacteria maintain their genomic DNA inside the cell in a well-regulated compact form with the help of various nucleoid associated proteins like HU, HNS, etc. These proteins control various fundamental processes like gene expression, replication, etc., inside the cell. Alteration of the native DNA structure of bacteria can lead to severe consequences in cellular processes inside the bacterial cell that ultimately result in the death of the organism. The change in the global DNA structure by small molecules initiates a plethora of cellular responses that have not been very well investigated. Echinomycin and Triostin-A are biologically active Quinoxaline small molecules that typically consist of a quinoxaline chromophore attached with an octadepsipeptide ring. They bind to double-stranded DNA in a sequence-specific way and have high activity against a wide variety of bacteria, mainly against Gram-positive ones. To date, few synthetic quinoxaline scaffolds were synthesized, displaying antibacterial potential against a broad scale of pathogenic bacteria. QNOs (Quinoxaline N-oxides) are known to target DNA and instigate reactive oxygen species (ROS) production in bacteria, thereby exhibiting antibacterial properties. The divergent role of Quinoxaline small molecules in medicinal research qualifies them for the evaluation of their antimicrobial properties as a potential candidate. The previous study from our lab has given new insights on a 6-nitroquinoxaline derivative 1d as an intercalator of DNA, which induces conformational changes in DNA upon binding.7 The binding event observed was dependent on the presence of a crucial benzyl substituent on the quinoxaline moiety. This was associated with a large induced CD (ICD) appearing in a sigmoidal pattern upon the interaction of 1d with dsDNA. The induction of DNA superstructures by 1d at high Drug:DNA ratios was observed that ultimately led to DNA condensation. Eviction of invitro-assembled nucleosome upon treatment with a high dose of 1d was also observed. In this work, monoquinoxaline derivatives of 1d were synthesized by various modifications of the 1d scaffold. The set of synthesized 6-nitroquinoxaline derivatives along with 1d were all subjected to antibacterial evaluation across five different bacteria species. Among the compound set, 3a displayed potent antibacterial activity against Staphylococcus aureus bacteria. 3a was further subjected to various biophysical studies to check whether the DNA structural alteration potential was still intact. The biological response of S. aureus cells upon treatment with 3a was studied using various cell biology processes, which led to the conclusion that 3d can initiate DNA damage in the S. aureus cells. Finally, the potential of 3a in disrupting preformed S.aureus and S.epidermidis biofilms was also studied.

Keywords: DNA structural change, antibacterial, intercalator, DNA superstructures, biofilms

Procedia PDF Downloads 169

Authors: Kiran Shehzadi, Yasmeen Akhtar, Mujahid Ameen, Tabinda Ijaz, Shoukat Siddique

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Nanoparticles, due to their different sizes and morphologies, are employed in various fields such as the medical field, cosmetics, pharmaceutical, textile industry as well as in paints, adhesives, and electronics. Metal nanoparticles exhibit excellent antimicrobial activity, dye degradation and can be used as anti-cancerous drug loading agents. In this study, sZilver nanoparticles (Ag-NPs) were synthesized employing doxycycline (antibiotic) as a reducing and capping agent (biological/green synthesis). Produced Ag-NPS were characterized using UV/VIS spectrophotometry, XRD, SEM, and FTIR. Surface plasmon resonance (SPR) of silver nanoparticles was observed at 411nm with 90nm size with homogenized spherical shape. These particles revealed good inhibition zones for Fungi such as Candida albicans and Candida tropicalis. In this study, toxic properties of Ag-NPs were monitored by allowing them to penetrate in the cell, causing an abrupt increase in oxidative stress, which resulted ultimately in cell death. Histopathological analysis of mice organs was performed by administering definite concentrations of silver nanoparticles orally to mice for 14 days. Toxic properties were determined, and it was revealed that the toxicity of silver nanoparticles mainly depends on the size. Silver nanoparticles of this work presented mild toxicity for different organs (liver, kidney, spleen, heart, and stomach) of mice.

Keywords: metal nanoparticles, green/biological methods, toxicity, Candida albicans, Candida tropicalis

Procedia PDF Downloads 129

Authors: Malgorzata J. Rybak-Smith, Benedicte Thiebaut, Simon Johnson, Peter Bishop, Helen E. Townley

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Gadolinium doped titania (TiO2:Gd) nanoparticles (NPs) can be activated by X-ray radiation to generate Reactive Oxygen Species (ROS), which can be effective in killing cancer cells. As such, treatment with these NPs can be used to enhance the efficacy of conventional radiotherapy. Incorporation of the NPs in to tumour tissue will permit the extension of radiotherapy to currently untreatable tumours deep within the body, and also reduce damage to neighbouring healthy cells. In an attempt to find a fast and scalable method for the synthesis of the TiO2:Gd NPs, the use of Flame Spray Pyrolysis (FSP) was investigated. A series of TiO2 NPs were generated with 1, 2, 5 and 7 mol% gadolinium dopant. Post-synthesis, the TiO2:Gd NPs were silica-coated to improve their biocompatibility. Physico-chemical characterisation was used to determine the size and stability in aqueous suspensions of the NPs. All analysed TiO2:Gd NPs were shown to have relatively high photocatalytic activity. Furthermore, the FSP synthesized silica-coated TiO2:Gd NPs generated enhanced ROS in chemico. Studies on rhabdomyosarcoma (RMS) cell lines (RD & RH30) demonstrated that in the absence of irradiation all TiO2:Gd NPs were inert. However, application of TiO2:Gd NPs to RMS cells, followed by irradiation, showed a significant decrease in cell proliferation. Consequently, our studies showed that the X-ray-activatable TiO2:Gd NPs can be prepared by a high-throughput scalable technique to provide a novel and affordable anticancer therapy.

Keywords: cancer, gadolinium, ROS, titania nanoparticles, X-ray

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Authors: Aida Kalantari, Boyang Ji, Tao Chen, Ivan Mijakovic

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3-hydroxypropanoic acid (3-HP) is one of the most important biomass-derivable platform chemicals that can be converted into a number of industrially important compounds. There have been several attempts at production of 3-HP from renewable sources in cell factories, focusing mainly on Escherichia coli, Klebsiella pneumoniae, and Saccharomyces cerevisiae. Despite the significant progress made in this field, commercially exploitable large-scale production of 3-HP in microbial strains has still not been achieved. In this study, we investigated the potential of Bacillus subtilis to be used as a microbial platform for bioconversion of glycerol into 3-HP. Our recombinant B. subtilis strains overexpress the two-step heterologous pathway containing glycerol dehydratase and aldehyde dehydrogenase from various backgrounds. The recombinant strains harboring the codon-optimized synthetic pathway from K. pneumoniae produced low levels of 3-HP. Since the enzymes in the heterologous pathway are sensitive to oxygen, we had to perform our experiments in micro-aerobic conditions. Under these conditions, the cell produces lactate in order to regenerate NAD+, and we found the lactate production to be in competition with the production of 3-HP. Therefore, based on the in silico predictions, we knocked out the glycerol kinase (glpk), which in combination with growth on glucose, resulted in improving the 3-HP titer to 1 g/L and the removal of lactate. Cultivation of the same strain in an enriched medium improved the 3-HP titer up to 7.6 g/L. Our findings provide the first report of successful introduction of the biosynthetic pathway for conversion of glycerol into 3-HP in B. subtilis.

Keywords: bacillus subtilis, glycerol, 3-hydroxypropanoic acid, metabolic engineering

Procedia PDF Downloads 247

Authors: Arati Inamdar, Siddharth Bhattacharyya, Kester Haye

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Collision tumors are rare entities characterized by neoplasms of two different cell populations with distinct separating boundaries. Such tumors could be benign, malignant, or a combination of both. The exact mechanism of origin for collision tumors is predicted to be tumor heterogeneity or concurrent occurrence of neoplasm in the same organ. We present two cases of plasmacytoma presenting as a collision tumor, one with a tumor of hematological origin and another with a non-hematological origin, namely Chronic Lymphocytic Leukemia and Adenocarcinoma of the colon, respectively. The immunohistochemical stains and flowcytometry analysis performed on the specimens aided incorrect diagnosis. Interestingly, neoplastic cells of plasmacytoma in the first case demonstrated strong cytokeratin along with weak Epithelial Specific Antigen/ Epithelial cell adhesion molecule Monoclonal Antibody (MOC31) positivity, indicating that the tumor may influence the microenvironment of the tumor in the vicinity. Furthermore, the next-generation sequencing studies performed on the specimen with plasmacytoma and chronic lymphocytic lymphoma demonstrated BReast CAncer gene (BRCA2) and Tumor Necrosis Factor Alpha Induced Protein 3 (TNFAIP3) as a disease associated variants suggestive of risk for multiple tumors including collision tumors. Our reports highlight the unique collision tumors involving plasmacytoma, which have never been reported previously, as well as provide necessary insights about the underline genetic aberrations and tumor heterogeneity through sequencing studies and allow clonality assessment for subsequent tumors.

Keywords: BRCA2, collision tumor, chronic lymphocytic leukemia, plasmacytoma

Procedia PDF Downloads 190

Authors: H. Jeries, N. Volkova, C. G. Iglesias, M. Najjar, M. Rosenblat, M. Aviram, T. Hayek

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Background: Synthetic forms of glucocorticoids (e.g., prednisone, prednisolone) are anti-inflammatory drugs which are widely used in clinical practice. The role of glucocorticoids (GCs) in cardiovascular diseases including atherosclerosis is highly controversial, and their impact on macrophage foam cell formation is still unknown. Our aim was to investigate the effects of prednisone or its active metabolite, prednisolone, on macrophage oxidative stress and lipid metabolism using in-vivo, ex-vivo and in-vitro systems. Methods: The in-vivo study included C57BL/6 mice which were intraperitoneally injected with prednisone or prednisolone (5mg/kg) for 4 weeks, followed by lipid metabolism analyses in the mice aorta, and in peritoneal macrophages (MPM). In the ex-vivo study, we analyzed the effect of serum samples obtained from 9 healthy volunteers before or after treatment with oral prednisone (20mg for 5 days), on J774A.1 macrophage atherogenicity. In-vitro studies were conducted using J774A.1 macrophages, human monocyte derived macrophages (HMDM) and fibroblasts. Cells were incubated with increasing concentrations (0-200 ng/ml) of prednisone or prednisolone, followed by determination of cellular oxidative status, triglyceride and cholesterol metabolism. Results: Prednisone or prednisolone treatment resulted in a significant reduction in triglycerides and mainly in cholesterol cellular accumulation in MPM or in J774A.1 macrophages incubated with human serum. Similar resulted were noted in HMDM or in J774A.1 macrophages which were directly incubated with the GCs. These effects were associated with GCs inhibitory effect on triglycerides and cholesterol biosynthesis rates, throughout downregulation of diacylglycerol acyltransferase1 (DGAT1) expression, and of the sterol regulatory element binding protein (SREBP2) and HMGCR expression, respectively. In parallel to prednisone or prednisolone induced reduction in macrophage triglyceride content, paraoxonase 2 (PON2) expression was significantly upregulated. GCs-induced reduction of cellular triglyceride and cholesterol mass was mediated by the GCs receptors on macrophages since the GCs receptor antagonist (RU 486) abolished these effects. In fibroblasts, unlike macrophages, prednisone or prednisolone showed no anti-atherogenic effects. Conclusions: Prednisone or prednisolone are anti-atherogenic since they protected macrophages from lipid accumulation and foam cell formation.

Keywords: atherosclerosis, cholesterol, foam cell, macrophage, prednisone, prednisolone, triglycerides

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Authors: Sweta Pandey, Samridhi Dhyani, Susmita Chaudhuri

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Klebsiella pneumoniae bacteria is a global threat to human health due to an increase in multi-drug resistance among strains. The hypervirulent strains of Klebsiella pneumoniae is a major trouble due to their association with life-threatening infections in a healthy population. One of the major virulence factors of hyper virulent strains of Klebsiella pneumoniae is the T6SS (Type six secretary system) which is majorly involved in microbial antagonism and causes interaction with the host eukaryotic cells during infections. T6SS mediates some of the crucial factors for establishing infection by the bacteria, such as cell adherence, invasion, and subsequent in vivo colonisation. The antibacterial activity and the cell invasion property of the T6SS system is a major requirement for the establishment of K. pneumoniae infections within the gut. The T6SS can be an appropriate target for developing therapeutics. The T6SS consists of an inner tube comprising hexamers of Hcp (Haemolysin -regulated protein) protein, and at the top of this tube sits VgrG (Valine glycine repeat protein G); the tip of the machinery consists of PAAR domain containing proteins which act as a delivery system for bacterial effectors. For this study, immune response to recombinant VgrG protein was generated to establish this protein as a potential immunogen for the development of therapeutic leads. The immunogenicity of the selected protein was determined by predicting the B cell epitopes by the BCEP analysis tool. The gene sequence for multiple domains of VgrG protein (phage_base_V, T6SS_Vgr, DUF2345) was selected and cloned in pMAL vector in E. coli. The construct was subcloned and expressed as a fusion protein of 203 residue protein with mannose binding protein tag (MBP) to enhance solubility and purification of this protein. The purified recombinant VgrG fusion protein was used for mice immunisation. The antiserum showed reactivity with the recombinant VgrG in ELISA and western blot. The immunised mice were challenged with K. pneumoniae bacteria and showed bacterial clearance in immunised mice. The recombinant VgrG protein can further be used for studying downstream signalling of VgrG protein in mice during infection and for therapeutic MAb development to eradicate K. pneumoniae infections.

Keywords: immune response, Klebsiella pneumoniae, multi-drug resistance, recombinant protein expression, T6SS, VgrG

Procedia PDF Downloads 102

Authors: Tauseef Ahmad, Muhammad Ishaq, Mathew Eapen, Ahyoung Park, Sam Karpiniec, Vanni Caruso, Rajaraman Eri

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Fucoidans are complex, fucose-rich sulfated polymers discovered in brown seaweeds. Fucoidans are popular around the world, particularly in the nutraceutical and pharmaceutical industries, due to their promising medicinal properties. Fucoidans have been shown to have a variety of biological activities, including anti-inflammatory effects. They are known to inhibit inflammatory processes through a variety of mechanisms, including enzyme inhibition and selectin blockade. Inflammation is a part of the complicated biological response of living systems to damaging stimuli, and it plays a role in the pathogenesis of a variety of disorders, including arthritis, inflammatory bowel disease, cancer, and allergies. In the current investigation, various fucoidan extracts from Undaria pinnatifida, Fucus vesiculosus, Macrocystis pyrifera, Ascophyllum nodosum, and Laminaria japonica were assessed for inhibition of pro-inflammatory cytokine production (TNF-α, IL-1β, and IL-6) in LPS induced human macrophage cell line (THP-1) and human peripheral blood mononuclear cells (PBMCs). Furthermore, we also sought to catalogue these extracts based on their anti-inflammatory effects in the current in-vitro cell model. Materials and Methods: To assess the cytotoxicity of fucoidan extracts, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5, -diphenyltetrazolium bromide) cell viability assay was performed. Furthermore, a dose-response for fucoidan extracts was performed in LPS induced THP-1 cells and PBMCs after pre-treatment for 24 hours, and levels of TNF-α, IL-1β, and IL-6 cytokines were measured using Enzyme-Linked Immunosorbent Assay (ELISA). Results: The MTT cell viability assay demonstrated that fucoidan extracts exhibited no evidence of cytotoxicity in THP-1 cells or PBMCs after 48 hours of incubation. The results of the sandwich ELISA revealed that all fucoidan extracts suppressed cytokine production in LPS-stimulated PBMCs and human THP-1 cells in a dose-dependent manner. Notably, at lower concentrations, the lower molecular fucoidan (5-30 kDa) extract from Macrocystis pyrifera was a highly efficient inhibitor of pro-inflammatory cytokines. Fucoidan extracts from all species including Undaria pinnatifida, Fucus vesiculosus, Macrocystis pyrifera, Ascophyllum nodosum, and Laminaria japonica exhibited significant anti-inflammatory effects. These findings on several fucoidan extracts provide insight into strategies for improving their efficacy against inflammation-related diseases. Conclusion: In the current research, we have successfully catalogued several fucoidan extracts based on their efficiency in LPS-induced macrophages and PBMCs in downregulating the key pro-inflammatory cytokines (TNF-, IL-1 and IL-6), which are prospective targets in human inflammatory illnesses. Further research would provide more information on the mechanism of action, allowing it to be tested for therapeutic purposes as an anti-inflammatory medication.

Keywords: fucoidan, PBMCs, THP-1, TNF-α, IL-1β, IL-6, inflammation

Procedia PDF Downloads 59

Authors: Aashutosh A. Tadse, Kush Mehta, Hardik Vyas

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Friction Stir Welding is a process in which an FSW tool produces friction heat and thus penetrates through the junction and upon rotation carries out the weld by exchange of material within the 2 metals being welded. It involves holding the workpieces stiff enough to bear the force of the tool moving across the junction to carry out a successful weld. The weld that has flat plates as workpieces, has a quite simpler geometry in terms of fixture holding them. In the case of FSW of pipes, the pipes need to be held firm with the chucks and jaws according to the diameter of the pipes being welded; the FSW tool is then revolved around the pipes to carry out the weld. Machine requires a larger area and it becomes more costly because of such a setup. To carry out the weld on the Milling machine, the newly designed fixture must be set-up on the table of milling machine and must facilitate rotation of pipes by the motor being shafted to one end of the fixture, and the other end automatically rotated because of the rotating jaws held tight enough with the pipes. The set-up has tapered cones as the jaws that would go in the pipes thus holding it with the help of its knurled surface providing the required grip. The process has rotation of pipes with the stationary rotating tool penetrating into the junction. The FSW on pipes in this process requires a very low RPM of pipes to carry out a fine weld and the speed shall change with every combination of material and diameter of pipes, so a variable speed setting motor shall serve the purpose. To withstand the force of the tool, an attachment to the shaft is provided which will be diameter specific that will resist flow of material towards the center during the weld. The welded joint thus carried out will be proper to required standards and specifications. Current industrial requirements state the need of space efficient, cost-friendly and more generalized form of fixtures and set-ups of machines to be put up. The proposed design considers every mentioned factor and thus proves to be positive in the same.

Keywords: force of tool, friction stir welding, milling machine, rotation of pipes, tapered cones

Procedia PDF Downloads 114

Authors: Nourhan Hussein Fanaki, Hoda Mohamed Gamal El-Din Omar, Nihal Kadry Moussa, Eva Adel Edward Farid

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The resistance of staphylococci to various antibiotics has become a major concern for health care professionals. The efficacy of the combinations of selected glycopeptides (vancomycin and teicoplanin) with gentamicin or rifampicin, as well as that of gentamicin/rifampicin combination, was studied against selected pathogenic staphylococcus isolated from Egypt. The molecular distribution of genes conferring resistance to these four antibiotics was detected among tested clinical isolates. Antibiotic combinations were studied using the checkerboard technique and the time-kill assay (in both the stationary and log phases). Induction of resistance to glycopeptides in staphylococci was tried in the absence and presence of diclofenac sodium as inducer. Transmission electron microscopy was used to study the effect of glycopeptides on the ultrastructure of the cell wall of staphylococci. Attempts were made to cure gentamicin resistance plasmids and to study the transfer of these plasmids by conjugation. Trials for the transformation of the successfully isolated gentamicin resistance plasmid to competent cells were carried out. The detection of genes conferring resistance to the tested antibiotics was performed using the polymerase chain reaction. The studied antibiotic combinations proved their efficacy, especially when tested during the log phase. Induction of resistance to glycopeptides in staphylococci was more promising in presence of diclofenac sodium, compared to its absence. Transmission electron microscopy revealed the thickening of bacterial cell wall in staphylococcus clinical isolates due to the presence of tested glycopeptides. Curing of gentamicin resistance plasmids was only successful in 2 out of 9 tested isolates, with a curing rate of 1 percent for each. Both isolates, when used as donors in conjugation experiments, yielded promising conjugation frequencies ranging between 5.4 X 10-2 and 7.48 X 10-2 colony forming unit/donor cells. Plasmid isolation was only successful in one out of the two tested isolates. However, low transformation efficiency (59.7 transformants/microgram plasmid DNA) of such plasmids was obtained. Negative regulators of autolysis, such as arlR, lytR and lrgB, as well as cell-wall associated genes, such as pbp4 and/or pbp2, were detected in staphylococcus isolates with reduced susceptibility to the tested glycopeptides. Concerning rifampicin resistance genes, rpoBstaph was detected in 75 percent of the tested staphylococcus isolates. It could be concluded that in vitro studies emphasized the usefulness of the combination of vancomycin or teicoplanin with gentamicin or rifampicin, as well as that of gentamicin with rifampicin, against staphylococci showing varying resistance patterns. However, further in vivo studies are required to ensure the safety and efficacy of such combinations. Diclofenac sodium can act as an inducer of resistance to glycopeptides in staphylococci. Cell-wall thickness is a major contributor to such resistance among them. Gentamicin resistance in these strains could be chromosomally or plasmid mediated. Multiple mutations in the rpoB gene could mediate staphylococcus resistance to rifampicin.

Keywords: glycopeptides, combinations, induction, diclofenac, transmission electron microscopy, polymerase chain reaction

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Authors: Sundaram Arulmozhiraja, Kanade Shimizu, Yuta Yamamoto, Satoshi Ichikawa, Maenaka Katsumi, Hiroaki Tokiwa

Abstract:

Drug discovery is shifting from small molecule based drugs targeting local active site to middle molecules (MM) targeting large, flat, and groove-shaped binding sites, for example, protein-protein interface because at least half of all targets assumed to be involved in human disease have been classified as “difficult to drug” with traditional small molecules. Hence, MMs such as peptides, natural products, glycans, nucleic acids with various high potent bioactivities become important targets for drug discovery programs in the recent years as they could be used for ‘undruggable” intracellular targets. Cell membrane permeability is one of the key properties of pharmacodynamically active MM drug compounds and so evaluating this property for the potential MMs is crucial. Computational prediction for cell membrane permeability of molecules is very challenging; however, recent advancement in the molecular dynamics simulations help to solve this issue partially. It is expected that MMs with high membrane permeability will enable drug discovery research to expand its borders towards intracellular targets. Further to understand the chemistry behind the permeability of MMs, it is necessary to investigate their conformational changes during the permeation through membrane and for that their interactions with the membrane field should be studied reliably because these interactions involve various non-bonding interactions such as hydrogen bonding, -stacking, charge-transfer, polarization dispersion, and non-classical weak hydrogen bonding. Therefore, parameters-based classical mechanics calculations are hardly sufficient to investigate these interactions rather, quantum mechanical (QM) calculations are essential. Fragment molecular orbital (FMO) method could be used for such purpose as it performs ab initio QM calculations by dividing the system into fragments. The present work is aimed to study the cell permeability of middle molecules using molecular dynamics simulations and FMO-QM calculations. For this purpose, a natural compound syringolin and its analogues were considered in this study. Molecular simulations were performed using NAMD and Gromacs programs with CHARMM force field. FMO calculations were performed using the PAICS program at the correlated Resolution-of-Identity second-order Moller Plesset (RI-MP2) level with the cc-pVDZ basis set. The simulations clearly show that while syringolin could not permeate the membrane, its selected analogues go through the medium in nano second scale. These correlates well with the existing experimental evidences that these syringolin analogues are membrane-permeable compounds. Further analyses indicate that intramolecular -stacking interactions in the syringolin analogues influenced their permeability positively. These intramolecular interactions reduce the polarity of these analogues so that they could permeate the lipophilic cell membrane. Conclusively, the cell membrane permeability of various middle molecules with potent bioactivities is efficiently studied using molecular dynamics simulations. Insight of this behavior is thoroughly investigated using FMO-QM calculations. Results obtained in the present study indicate that non-bonding intramolecular interactions such as hydrogen-bonding and -stacking along with the conformational flexibility of MMs are essential for amicable membrane permeation. These results are interesting and are nice example for this theoretical calculation approach that could be used to study the permeability of other middle molecules. This work was supported by Japan Agency for Medical Research and Development (AMED) under Grant Number 18ae0101047.

Keywords: fragment molecular orbital theory, membrane permeability, middle molecules, molecular dynamics simulation

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