Search results for: hematopoietic stem cell transplant

Authors: Jayprakash Yadav, Nivedita Patra

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Polyhydroxybutyrate (PHB) is an interesting material in the field of medical science, pharmaceutical industries, and tissue engineering because of its properties such as biodegradability, biocompatibility, hydrophobicity, and elasticity. PHB is naturally accumulated by several microbes in their cytoplasm during the metabolic process as energy reserve material. PHB can be extracted from cell biomass using halogenated hydrocarbons, chemicals, and enzymes. In this study, a cheaper and non-toxic solvent, acetone, was used for the extraction process. The different parameters like acetone percentage, and solvent pH, process temperature, and incubation periods were optimized using the Response Surface Methodology (RSM). RSM was performed and the determination coefficient (R2) value was found to be 0.8833 from the quadratic regression model with no significant lack of fit. The designed RSM model results indicated that the fitness of the response variable was significant (P-value < 0.0006) and satisfactory to denote the relationship between the responses in terms of PHB recovery and purity with respect to the values of independent variables. Optimum conditions for the maximum PHB recovery and purity were found to be solvent pH 7, extraction temperature - 43 °C, incubation time - 70 minutes, and percentage acetone – 30 % from this study. The maximum predicted PHB recovery was found to be 0.845 g/g biomass dry cell weight and the purity was found to be 97.23 % using the optimized conditions.

Keywords: acetone, PHB, RSM, halogenated hydrocarbons, extraction, bacillus subtilis.

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Authors: Trenten Theis, Trevor Daubert, Kennedy Kluthe, Austin Nuxoll

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Staphylococcus aureus is a gram-positive bacterium responsible for an estimated 23,000 deaths in the United States and 25,000 deaths in the European Union annually. Recurring S. aureus bacteremia is associated with biofilm-mediated infections and can occur in 5 - 20% of cases, even with the use of antibiotics. Despite these infections being caused by drug-susceptible pathogens, they are surprisingly difficult to eradicate. One potential explanation for this is the presence of persister cells—a dormant type of cell that shows a high tolerance to antibiotic treatment. Recent studies have shown a connection between low intracellular ATP and persister cell formation. Specifically, this decrease in ATP, and therefore increase in persister cell formation, is due to an interrupted tricarboxylic acid (TCA) cycle. However, S. aureus persister cells’ role in pathogenesis remains unclear. Initial studies have shown that a fumC (TCA cycle gene) knockout survives challenge from aspects of the innate immune system better than wild-type S. aureus. Specifically, challenges from two antimicrobial peptides--LL-37 and hBD-3—show a log increase in survival of the fumC::N∑ strain compared to wild type S. aureus after 18 hours. Furthermore, preliminary studies show that the fumC knockout has a log more survival within a macrophage. These data lead us to hypothesize that the fumC knockout is better suited to other aspects of the innate immune system compared to wild-type S. aureus. To further investigate the mechanism for increased survival of fumC::N∑ within a macrophage, we tested bacterial growth in the presence of reactive oxygen species (ROS), reactive nitrogen species (RNS), and a low pH. Preliminary results suggest that the fumC knockout has increased growth compared to wild-type S. aureus in the presence of all three antimicrobial factors; however, no difference was observed in any single factor alone. To investigate survival within a host, a nine-day biofilm-associated catheter infection was performed on 6–8-week-old male and female C57Bl/6 mice. Although both sexes struggled to clear the infection, female mice were trending toward more frequently clearing the HG003 wild-type infection compared to the fumC::N∑ infection. One possible reason for the inability to reduce the bacterial burden is that biofilms are largely composed of persister cells. To test this hypothesis further, flow cytometry in conjunction with a persister cell marker was used to measure persister cells within a biofilm. Cap5A (a known persister cell marker) expression was found to be increased in a maturing biofilm, with the lowest levels of expression seen in immature biofilms and the highest expression exhibited by the 48-hour biofilm. Additionally, bacterial cells in a biofilm state closely resemble persister cells and exhibit reduced membrane potential compared to cells in planktonic culture, further suggesting biofilms are largely made up of persister cells. These data may provide an explanation as to why infections caused by antibiotic-susceptible strains remain difficult to treat.

Keywords: antibiotic tolerance, Staphylococcus aureus, host-pathogen interactions, microbial pathogenesis

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Authors: M. Areekijseree, W. Pongsawat, M. Pumipaiboon, C. Thepsithar, S. Sengsai, T. Chuen-Im

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Morphological interaction of porcine cumulus-oocyte complexes (pCOCs) was investigated on in vitro condition using electron microscope (SEM and TEM). The totals of 1,923 oocytes were round in shape, surrounded by zona pellucida with layer of cumulus cells ranging between 59.29-202.14 µm in size. They were classified into intact-, multi-, partial cumulus cell layer oocyte, and completely denuded oocyte, at the percentage composition of 22.80% 32.70%, 18.60%, and 25.90 % respectively. The pCOCs classified as intact- and multi cumulus cell layer oocytes were further culturing at 37°C with 5% CO2, 95% air atmosphere and high humidity for 44 h in M199 with Earle’s salts supplemented with 10% HTFCS, 2.2 mg/mL NaHCO3, 1 M Hepes, 0.25 mM pyruvate, 15 µg/mL porcine follicle-stimulating hormone, 1 µg/mL LH, 1µg/mL estradiol with ethanol, and 50 µg/mL gentamycin sulfate. On electron microscope study, cumulus cells were found to stick their processes to secrete substance from the sac-shape end into zona pellucida of the oocyte and also communicated with the neighboring cells through their microvilli on the beginning of incubation period. It is believed that the cumulus cells communicate with the oocyte by inserting the microvilli through this gap and embedded in the oocyte cytoplasm before secreting substance, through the sac-shape end of the microvilli, to inhibit primary oocyte development at the prophase I. Morphological changes of the complexes were observed after culturing for 24-44 h. One hundred percentages of the cumulus layers were expanded and cumulus cells were peeling off from the oocyte surface. In addition, the round-shape cumulus cells transformed themselves into either an elongate shape or a columnar shape, and no communication between cumulus neighboring cells. After 44 h of incubation time, diameter of oocytes surrounded by cumulus cells was larger than 0 h incubation. The effect of hormones in culture medium is exerted by their receptors present in porcine oocyte. It is likely that all morphological changes of the complexes after hormone treatment were to allow maturation of the oocyte. This study demonstrated that the association of hormones in M199 could promote porcine follicle activation in 44 h in vitro condition. This culture system should be useful for studying the regulation of early follicular growth and development, especially because these follicles represent a large source of oocytes that could be used in vitro for cell technology.

Keywords: cumulus cells, electron microscopy, in vitro, porcine oocyte

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Authors: Karol Rodriguez-Peña, Martha L. Macias-Rubalcava, Leticia Rocha-Zavaleta, Sergio Sanchez

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A bioassay-guided study for anti-cancer compounds from endophytes of the Mexican medicinal plant Amphipteryygium adstringens resulted in the isolation of a streptomycete capable of producing a group of compounds with high cytotoxic activity. Microorganisms from surface sterilized samples of various sections of the plant were isolated and all the actinomycetes found were evaluated for their potential to produce compounds with cytotoxic activity against cancer cell lines MCF7 (breast cancer) and HeLa (cervical cancer) as well as the non-tumoural cell line HaCaT (keratinocyte). The most active microorganism was picked for further evaluation. The identification of the microorganism was carried out by 16S rDNA gene sequencing, finding the closest proximity to Streptomyces scabrisporus, but with the additional characteristic that the strain isolated in this study was capable of producing colorful compounds never described for this species. Crude extracts of dichloromethane and ethyl acetate showed IC50 values of 0.29 and 0.96 μg/mL for MCF7, 0.51 and 1.98 μg/mL for HeLa and 0.96 and 2.7 μg/mL for HaCaT. Scaling the fermentation to 10 L in a bioreactor generated 1 g of total crude extract, which was fractionated by silica gel open column to yield 14 fractions. Nine of the fractions showed cytotoxic activity. Fraction 4 was chosen for subsequent purification because of its high activity against cancerous cell lines, lower activity against keratinocytes. HPLC-UV-MS/ESI was used for the evaluation of this fraction, finding at least 10 different compounds with high values of m/z (≈588). Purification of the compounds was carried out by preparative thin-layer chromatography. The prevalent compound was Steffimycin B, a molecule known for its antibiotic and cytotoxic activities and also for its low solubility in aqueous solutions. Along with steffimycin B, another five compounds belonging to the steffimycin family were isolated and at this moment their structures are being elucidated, some of which display better solubility in water: an attractive property for the pharmaceutical industry. As a conclusion to this study, the isolation of endophytes resulted in the discovery of a strain capable of producing compounds with high cytotoxic activity that need to be studied for their possible utilization.

Keywords: amphipterygium adstringens, cytotoxicity, streptomyces scabrisporus, steffimycin

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Authors: Ujjval J. Solanki, Prof.(Dr.) P.J. Gundaliya, Prof.M.D. Barasara

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The application of Falling Weight Deflectometer is to evaluate structural performance of the flexible pavement. The exercise of back calculation is required to know the modulus of elasticity of existing in-service pavement. The process of back calculation needs in-depth field experience for the input of range of modulus of elasticity of bituminous, granular and subgrade layer, and its required number of trial to find such matching moduli with the observed FWD deflection on the field. The study carried out at Barnala-Mansa State Highway Punjab-India using FWD before and after overlay; the deflections obtained at 0 on the load cell, 300, 600, 900,1200, 1500 and 1800 mm interval from the load cell these seven deflection results used to calculate Surface Curvature Index (SCI), Base damage Index (BDI), Base curvature index (BCI). This SCI, BCI and BDI indices are useful to predict the structural performance of in-service pavement and also useful to identify homogeneous section for condition assessment. The SCI, BCI and BDI range are determined for before and after overlay the range of SCI 520 to 51 BDI 294 to 63 BCI 83 to 0.27 for old pavement and SCI 272 to 23 BDI 228 to 28, BCI 25.85 to 4.60 for new pavement. It also shows good correlation with back calculated modulus of elasticity of all the three layer.

Keywords: back calculation, base damage index, base curvature index, FWD (Falling Weight Deflectometer), surface curvature index

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Authors: Fhatuwani Herman Nndwambi, P. W. Mashela

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Salinity is a major constraint limiting crop productivity. It has been predicted that by the year 2050, more than 50% of the arable land will be affected by salinity. Two similar salinity experiments were conducted in two seasons under greenhouse condition. Six levels of salinity plus control (viz; control, 2, 4, 8, 16, 32 and 64 % NaCl and CaCl2 at 3:1 ratio) were applied in a form of irrigation water in a single factor experiment arranged in a complete block design with 20 replications. Plant growth and yield were negatively affected by salinity treatments especially at the high levels of salinity. For example, our results suggest that the 32 and 64% of NaCl and CaCl2 treatment were too much for the plant to withstand as determined by reduced dry shoot mass, stem diameter and plant height in both seasons. On the other hand, stomatal conductance and chlorophyll content increased with an increased level of salinity.

Keywords: growth, salinity, season, yield

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Authors: Bhavini Patel, Aslihan Ugun-Klusek, Ellen Billet

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The two main contributing factors to familial and idiopathic form of Parkinson’s disease (PD) are oxidative stress and altered proteolysis. Monoamine oxidase-A (MAO-A) plays a significant role in redox homeostasis by producing reactive oxygen species (ROS) via deamination of for example, dopamine. The ROS generated induces chemical modification of proteins resulting in altered biological function. The ubiquitin-proteasome system, which consists of three different types or proteolytic activity, namely “chymotrypsin-like” activity (CLA), “trypsin-like” activity (TLA) and “post acidic-like” activity (PLA), is responsible for the degradation of ubiquitinated proteins. Defects in UPS are known to be strongly correlated to PD. Herein, the effect of ROS generated by MAO-A on proteasome activity and the effects of proteasome inhibition on MAO-A protein levels in WT, mock and MAO-A overexpressed (MAO-A+) SHSY5Y neuroblastoma cell lines were investigated. The data in this study report increased proteolytic activity when MAO-A protein levels are significantly increased, in particular CLA and PLA. Additionally, 20S proteasome inhibition induced a decrease in MAO-A levels in WT and mock cells in comparison to MAO-A+ cells in which 20S proteasome inhibition induced increased MAO-A levels to be further increased at 48 hours of inhibition. This study supports the fact that MAO-A could be a potential pharmaceutical target for neuronal protection as data suggests that endogenous MAO-A levels may be essential for modulating cell death and survival.

Keywords: monoamine oxidase, neurodegeneration, Parkinson's disease, proteasome

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Authors: Ferreira RC, Simons HZ, Thompson WS, Cutler AJ, Dopico XC, Smyth DJ, Mashar M, Schuilenburg H, Walker NM, Dunger DB, Wallace C, Todd JA, Wicker LS, Pekalski ML

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Type 1 diabetes is caused by autoimmune destruction of insulin-secreting beta cells in the pancreas. T cells are known to play an important role in this immune-mediated destruction; however, there is no general consensus regarding alterations in cytokine production or T cell subsets in peripheral blood of patients with type 1 diabetes. Using polychromatic flow cytometry of peripheral blood mononuclear cells (PBMCs), we assessed production of the proinflammatory cytokines IL-21, IFN-γ and IL-17 by memory CD4 T effector (Teff) cells in 69 patients with type 1 diabetes and 61 healthy donors. We found a 21.9% (95% CI 5.8, 40.2; p = 3.9 × 10(-3)) higher frequency of IL-21(+) CD45RA(-) memory CD4(+) Teffs in patients with type 1 diabetes (geometric mean 5.92% [95% CI 5.44, 6.44]) compared with healthy donors (geometric mean 4.88% [95% CI 4.33, 5.50]). In a separate cohort of 30 patients with type 1 diabetes and 32 healthy donors, we assessed the frequency of circulating T follicular helper (Tfh) cells in whole blood. Consistent with the increased production of IL-21, we also found a 14.9% increase in circulating Tfh cells in the patients with type 1 diabetes (95% CI 2.9, 26.9; p = 0.016). Analysis of IL-21 production by PBMCs from a subset of 46 of the 62 donors immunophenotyped for Tfh showed that frequency of Tfh cells was associated with the frequency of IL-21+ cells (r2 = 0.174, p = 0.004). These results indicate that increased IL-21 production is likely to be an aetiological factor in the pathogenesis of type 1 diabetes that could be considered as a potential therapeutic target.

Keywords: T follicular helper cell, IL-21, IL-17, type 1 diabetes

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Authors: Ashit Syngle, Inderjit Verma, Pawan Krishan

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Aim: Endothelial progenitor cells (EPCs) are unique populations which have reparative potential in overcoming the endothelial damage and reducing cardiovascular risk. Patients with ankylosing spondylitis (AS) have increased risk of cardiovascular morbidity and mortality. The aim of this study was to investigate the endothelial progenitor cell population in AS patients and its potential relationships with disease variables. Methods: Endothelial progenitor cells were measured in peripheral blood samples from 20 AS and 20 healthy controls by flow cytometry on the basis of CD34 and CD133 expression. Disease activity was evaluated by using Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). Functional ability was monitored by using Bath Ankylosing Spondylitis Functional Index (BASFI). Results: EPCs were depleted in AS patients as compared to the healthy controls (CD34+/CD133+: 0.027 ± 0.010 % vs. 0.044 ± 0.011 %, p<0.001). EPCs depletion were significantly associated with disease duration (r=-0.52, p=0.01) and BASDAI (r=-0.45, p=0.04). Conclusion: This is the first study to demonstrate endothelial progenitor cells depletion in AS patients. EPCs depletion inversely correlates with disease duration and disease activity, suggesting the pivotal role of inflammation in depletion of EPCs. EPC would possibly also serve as a therapeutic target for preventing cardiovascular disease in AS.

Keywords: ankylosing spondylitis, endothelial progenitor cells, inflammation, vascular damage

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Authors: Michael P. Mannino, Gerald W. Hart

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The majority of glucose metabolism proceeds through glycolytic pathways such as glycolysis or pentose phosphate pathway, however, about 5% is shunted through the hexosamine biosynthetic pathway, producing uridine diphosphate N-acetyl glucosamine (UDP-GlcNAc). This precursor can then be incorporated into complex oligosaccharides decorating the cell surface or remain as an intracellular post-translational-modification (PTM) of serine/threonine residues (O-GlcNAcylation, OGN), which has been identified on over 4,000 cytosolic or nuclear proteins. Intracellular OGN has major implications on cellularprocesses, typically by modulating protein localization, protein-protein interactions, protein degradation, and gene expression. Additionally, OGN is known to have an extensive cross-talk with phosphorylation, be in a competitive or cooperative manner. Unlike other PTMs there are only two cycling enzymes that are capable of adding or removing the GlcNAc moiety, O-linked N-aceytl glucosamine Transferase (OGT) and O-linked N-acetyl glucoamidase (OGA), respectively. The activity of OGT has been shown to be sensitive to cellular UDP-GlcNAc levels, even changing substrate affinity. Owing to this and that the concentration of UDP-GlcNAc is related to the metabolisms of glucose, amino acid, fatty acid, and nucleotides, O-GlcNAc is often referred to as a nutrient sensing rheostat. Indeed OGN is known to regulate several signaling pathways as a result of nutrient levels, such as insulin signaling. Dysregulation of OGN is associated with several disease states such as cancer, diabetes, and neurodegeneration. Improvements in glycomics over the past 10-15 years has significantly increased the OGT substrate pool, suggesting O-GlcNAc’s involvement in a wide variety of signaling pathways. However, O-GlcNAc’s role at the receptor level has only been identified in a case-by-case basis of known pathways. Examining the OGN of the plasma membrane (PM) may better focus our understanding of O-GlcNAc-effected signaling pathways. In this current study, PM fractions were isolated from several cell types via ultracentrifugation, followed by purification and MS/MS analysis in several cell lines. This process was repeated with or without OGT/OGA inhibitors or with increased/decreased glucose levels in media to ascertain the importance of OGN. Various pathways are followed up on in more detailed studies employing methods to localize OGN at the PM specifically.

Keywords: GlcNAc, nutrient sensitive, post-translational-modification, receptor

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Authors: Sirus Mohammadi, Rohala Moghimi

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The main advantages of microgrids are high energy efficiency through the application of Combined Heat and Power (CHP), high quality and reliability of the delivered electric energy and environmental and economic advantages. This study presents an energy management system (EMS) to optimize the operation of the microgrid (MG). In this paper an Adaptive Modified Firefly Algorithm (AMFA) is presented for optimal operation of a typical MG with renewable energy sources (RESs) accompanied by a back-up Micro-Turbine/Fuel Cell/Battery hybrid power source to level the power mismatch or to store the energy surplus when it’s needed. The problem is formulated as a nonlinear constraint problem to minimize the total operating cost. The management of Energy storage system (ESS), economic load dispatch and operation optimization of distributed generation (DG) are simplified into a single-object optimization problem in the EMS. The proposed algorithm is tested on a typical grid-connected MG including WT/PV/Micro Turbine/Fuel Cell and Energy Storage Devices (ESDs) then its superior performance is compared with those from other evolutionary algorithms such as Genetic Algorithm (GA), Particle Swarm Optimization (PSO), Fuzzy Self Adaptive PSO (FSAPSO), Chaotic Particle PSO (CPSO), Adaptive Modified PSO (AMPSO), and Firefly Algorithm (FA).

Keywords: microgrid, operation management, optimization, firefly algorithm (AMFA)

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Authors: Obsi Terfasa, Bhanupriya Das, Mithilish Passawan

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The large-scale application of microbial fuel cell (MFC) technology is significantly hindered by the high cost of the commonly used proton exchange membrane, Nafion. This has led to the recent development of ceramic membranes using various clay minerals. This study evaluates the characteristics and potential use of a new ceramic membrane made from potter’s clay © mixed with different proportions (0, 5, 10 wt%) of fly ash (FA), labeled as CFA0, CFA5, CFA10, for cost-effective and sustainable MFC use. Among these, the CFA10 membrane demonstrated superior quality with a fine pore size distribution (average 0.41 μm), which supports higher water uptake and reduced oxygen diffusion. Its oxygen mass transfer coefficient was 4.13 ± 0.13 × 10⁻⁴ cm/s, about 40% lower than the control. X-ray diffraction analysis revealed that the CFA membrane is rich in quartz, which enhances proton conductance and water retention. Electrochemical kinetics studies, including cyclic voltammetry and electrochemical impedance spectroscopy (EIS), also confirmed the effectiveness of the CFA10 membrane in MFC, showing a peak current output of 15.35 mA and low ohmic resistance (78.2 Ω). The novel CFA10 ceramic membrane, incorporating coal fly ash, a waste material, shows promise for high MFC performance at a significantly reduced cost (96%), making it suitable for sustainable scaling up of the technology.

Keywords: ceramic membrane, Coulombic efficiency, electro-chemical kinetics, fly ash, proton conductivity, microbial fuel cell

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Authors: Carmela Nardelli, Laura Iaffaldano, Valentina Capobianco, Antonietta Tafuto, Maddalena Ferrigno, Angela Capone, Giuseppe Maria Maruotti, Maddalena Raia, Rosa Di Noto, Luigi Del Vecchio, Pasquale Martinelli, Lucio Pastore, Lucia Sacchetti

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Maternal obesity and nutrient excess in utero increase the risk of future metabolic diseases in the adult life. The mechanisms underlying this process are probably based on genetic, epigenetic alterations and changes in foetal nutrient supply. In mammals, the placenta is the main interface between foetus and mother, it regulates intrauterine development, modulates adaptive responses to sub optimal in uterus conditions and it is also an important source of human amniotic mesenchymal stem cells (hA-MSCs). We previously highlighted a specific microRNA (miRNA) profiling in amnion from obese (Ob) pregnant women, here we compared the miRNA expression profile of hA-MSCs isolated from (Ob) and control (Co) women, aimed to search for any alterations in metabolic pathways that could predispose the new-born to the obese phenotype. Methods: We isolated, at delivery, hA-MSCs from amnion of 16 Ob- and 7 Co-women with pre-pregnancy body mass index (mean/SEM) 40.3/1.8 and 22.4/1.0 kg/m2, respectively. hA-MSCs were phenotyped by flow cytometry. Globally, 384 miRNAs were evaluated by the TaqMan Array Human MicroRNA Panel v 1.0 (Applied Biosystems). By the TargetScan program we selected the target genes of the miRNAs differently expressed in Ob- vs Co-hA-MSCs; further, by KEGG database, we selected the statistical significant biological pathways. Results: The immunophenotype characterization confirmed the mesenchymal origin of the isolated hA-MSCs. A large percentage of the tested miRNAs, about 61.4% (232/378), was expressed in hA-MSCs, whereas 38.6% (146/378) was not. Most of the expressed miRNAs (89.2%, 207/232) did not differ between Ob- and Co-hA-MSCs and were not further investigated. Conversely, 4.8% of miRNAs (11/232) was higher and 6.0% (14/232) was lower in Ob- vs Co-hA-MSCs. Interestingly, 7/232 miRNAs were obesity-specific, being expressed only in hA-MSCs isolated from obese women. Bioinformatics showed that these miRNAs significantly regulated (P<0.001) genes belonging to several metabolic pathways, i.e. MAPK signalling, actin cytoskeleton, focal adhesion, axon guidance, insulin signaling, etc. Conclusions: Our preliminary data highlight an altered miRNA profile in Ob- vs Co-hA-MSCs and suggest that an epigenetic miRNA-based mechanism of gene regulation could affect pathways involved in placental growth and function, thereby potentially increasing the newborn’s risk of metabolic diseases in the adult life.

Keywords: hA-MSCs, obesity, miRNA, biosystem

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Authors: Okyu Kwon, Dongho Kang, Byungsik Kim, Seungkwon Jung

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We propose a technique to measure the impact of loss of traffic function in a particular area to surrounding areas. The proposed method is applied to the city of Seoul, which is the capital of South Korea, with a population of about ten million. Based on the actual road network in Seoul, we construct an abstract road network between 1kmx1km grid cells. The link weight of the abstract road network is re-adjusted considering traffic volume measured at several survey points. On the modified abstract road network, we evaluate the traffic vulnerability by calculating a network measure of betweenness centrality (BC) for every single grid cells. This study analyzes traffic impacts caused by road dysfunction due to heavy rainfall in urban areas. We could see the change of the BC value in all other grid cells by calculating the BC value once again when the specific grid cell lost its traffic function, that is, when the node disappeared on the grid-based road network. The results show that it is appropriate to use the sum of the BC variation of other cells as the influence index of each lattice cell on traffic. This research was supported by a grant (2017-MOIS31-004) from Fundamental Technology Development Program for Extreme Disaster Response funded by Korean Ministry of Interior and Safety (MOIS).

Keywords: vulnerability, road network, beweenness centrality, heavy rainfall, road impact

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Authors: Jannis Kountouras, Nikolaos Kapetanakis, Stergios A. Polyzos, Apostolis Papaeftymiou, Panagiotis Katsinelos, Ioannis Venizelos, Christina Nikolaidou, Christos Zavos, Iordanis Romiopoulos, Elena Tsiaousi, Evangelos Kazakos, Michael Doulberis

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Background & Aims: Helicobacter pylori infection (Hp-I) has been recognized as a substantial risk agent involved in gastrointestinal (GI) tract oncogenesis by stimulating cancer stem cells (CSCs), oncogenes, immune surveillance processes, and triggering GI microbiota dysbiosis. We aimed to investigate the possible involvement of active Hp-I in the sequence: chronic inflammation–adenoma–colorectal cancer (CRC) development. Methods: Four pillars were investigated: (i) endoscopic and conventional histological examinations of patients with CRC, colorectal adenomas (CRA) versus controls to detect the presence of active Hp-I; (ii) immunohistochemical determination of the presence of Hp; expression of CD44, an indicator of CSCs and/or bone marrow-derived stem cells (BMDSCs); expressions of oncogene Ki67 and anti-apoptotic Bcl-2 protein; (iii) expression of CD45, indicator of immune surveillance locally (assessing mainly T and B lymphocytes locally); and (iv) correlation of the studied parameters with the presence or absence of Hp-I. Results: Among 50 patients with CRC, 25 with CRA, and 10 controls, a significantly higher presence of Hp-I in the CRA (68%) and CRC group (84%) were found compared with controls (30%). The presence of Hp-I with accompanying immunohistochemical expression of CD44 in biopsy specimens was revealed in a high proportion of patients with CRA associated with moderate/severe dysplasia (88%) and CRC patients with moderate/severe degree of malignancy (91%). Comparable results were also obtained for Ki67, Bcl-2, and CD45 immunohistochemical expressions. Concluding Remarks: Hp-I seems to be involved in the sequence: CRA – dysplasia – CRC, similarly to the upper GI tract oncogenesis, by several pathways such as the following: Beyond Hp-I associated insulin resistance, the major underlying mechanism responsible for the metabolic syndrome (MetS) that increase the risk of colorectal neoplasms, as implied by other Hp-I related MetS pathologies, such as non-alcoholic fatty liver disease and upper GI cancer, the disturbance of the normal GI microbiota (i.e., dysbiosis) and the formation of an irritative biofilm could contribute to a perpetual inflammatory upper GIT and colon mucosal damage, stimulating CSCs or recruiting BMDSCs and affecting oncogenes and immune surveillance processes. Further large-scale relative studies with a pathophysiological perspective are necessary to demonstrate in-depth this relationship.

Keywords: Helicobacter pylori, colorectal cancer, colorectal adenomas, gastrointestinal oncogenesis

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Authors: Khobaib Khobaib, Alexander Mikkelsen, Zbigniew Rozynek

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Electric fields have proven useful for inducing droplet deformation and to structure particles adsorbed at droplet interfaces. In this experimental research, direct current electric fields were applied to deform particle-covered droplets made out of silicone oil and immersed in castor oil. The viscosity of the drop and surrounding fluid were changed by external heating. We designed an experimental system in such a way that electric field-induced electrohydrodynamic (EHD) flows were asymmetric and only present on one side of the drop, i.e., the droplet adjoined a washer and adhered to one of the electrodes constituting the sample cell. The study investigated the influence of viscosity on the steady-state deformation magnitude of particle-laden droplets, droplet compression, and relaxation, as well as particle arrangements at drop interfaces. Initially, before the application of an electric field, we changed the viscosity of the fluids by heating the sample cell at different temperatures. The viscosity of the fluids was varied by changing the temperature of the fluids from 25 to 50°C. Under the application of a uniform electric field of strength 290 Vmm⁻¹, electric stress was induced at the drop interface, yielding drop deformation. In our study, we found that by lowering the fluid viscosity, the velocity of the EHD flows was increased, which also increases the deformation of the drop.

Keywords: drop deformation and relaxation, electric field, electrohydrodynamic flow, particle assembly, viscosity

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Authors: Ádám Vass, István Bakos, Irina Borbáth, Zoltán Pászti, István Sajó, András Tompos

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Important requirements for the anode side electrocatalysts of polymer electrolyte membrane (PEM) fuel cells are CO-tolerance, stability and corrosion resistance. Carbon is still the most common material for electrocatalyst supports due to its low cost, high electrical conductivity and high surface area, which can ensure good dispersion of the Pt. However, carbon becomes degraded at higher potentials and it causes problem during application. Therefore it is important to explore alternative materials with improved stability. Molybdenum-oxide can improve the CO-tolerance of the Pt/C catalysts, but it is prone to leach in acidic electrolyte. The Mo was stabilized by isovalent substitution of molybdenum into the rutile phase titanium-dioxide lattice, achieved by a modified multistep sol-gel synthesis method optimized for preparation of Ti0.7Mo.3O2-C composite. High degree of Mo incorporation into the rutile lattice was developed. The conductivity and corrosion resistance across the anticipated potential/pH window was ensured by mixed oxide – activated carbon composite. Platinum loading was carried out using NaBH4 and ethylene glycol; platinum content was 40 wt%. The electrocatalyst was characterized by both material investigating methods (i.e. XRD, TEM, EDS, XPS techniques) and electrochemical methods (cyclic-voltammetry, COads stripping voltammetry, hydrogen oxidation reaction on rotating disc electrode). The electrochemical activity of the sample was compared to commercial 40 wt% Pt/C (Quintech) and PtRu/C (Quintech, Pt= 20 wt%, Ru= 10 wt%) references. Enhanced CO tolerance of the electrocatalyst prepared using the Ti0.7Mo.3O2-C composite material was evidenced by the appearance of a CO-oxidation related 'pre-peak' and by the pronounced shift of the maximum of the main CO oxidation peak towards less positive potential compared to Pt/C. Fuel cell polarization measurements were also carried out using Bio-Logic and Paxitech FCT-150S test device. All details on the design, preparation, characterization and testing by both electrochemical measurements and fuel cell test device of electrocatalyst supported on Ti0.7Mo.3O2-C composite material will be presented and discussed.

Keywords: anode electrocatalyst, composite material, CO-tolerance, TiMoOx

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Authors: Nkechi V. Enwuru, Bullum Nkeki, Elizabeth A. Adekoya, Olumide A. Adebesin, Rebecca F. Peters, Victoria A. Aikhomu, Mendie E. U.

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Background: Probiotics are live microbial feed supplement beneficial for host. Probiotics and their postbiotic products have been used to prevent or treat various health conditions. However, the products cell viability is often low due to harsh conditions subjected during processing, handling, storage, and gastrointestinal transit. These strongly influence probiotics’ benefits; thus, viability is essential for probiotics to produce health benefits for the host. Microencapsulation is a promising technique with considerable effects on probiotic survival. The study is aimed to formulate a microencapsulated probiotic and evaluate its viability, antimicrobial efficacy, and cytotoxic activity of its postbiotic on the MCF-7 breast cancer cell line. Method: Human and animal raw milk were sampled for lactic acid bacteria. The isolated bacteria were identified using conventional and VITEK 2 systems. The identified lactic acid bacterium was encapsulated using spray-dried and extrusion methods. The free, encapsulated, and chitosan-coated encapsulated probiotics were tested for viability in simulated-gastric intestinal (SGI) fluid and different storage conditions at refrigerated (4oC) and room (25oC) temperatures. The disintegration time and weight uniformity of the spray-dried hard gelatin capsules were tested. The antimicrobial property of free and encapsulated probiotics was tested against enteric pathogenic isolates from antiretroviral therapy (ART) treated HIV-positive patients. The postbiotic of the free cells was extracted, and its cytotoxic effect on the MCF-7 breast cancer cell line was tested through an MTT assay. Result: The Lactobacillus plantarum was isolated from animal raw milk. Zero-size hard gelatin L. plantarum capsules with granules within a size range of 0.71–1.00 mm diameter was formulated. The disintegration time ranges from 2.14±0.045 to 2.91±0.293 minutes, while the average weight is 502.1mg. Simulated gastric solution significantly affected viability of both free and microcapsules. However, the encapsulated cells were more protected and viable due to impermeability in the microcapsules. Furthermore, the viability of free cells stored at 4oC and 25oC were less than 4 log CFU/g and 6 log CFU/g respectively after 12 weeks. However, the microcapsules stored at 4oC achieved the highest viability among the free and microcapsules stored at 25oC and the free cells stored at 4oC. Encapsulated cells were released in the simulated gastric fluid, viable and effective against the enteric pathogens tested. However, chitosan-coated calcium alginate encapsulated probiotics significantly inhibited Shigella flexneri, Candida albicans, and Escherichia coli. The Postbiotic Metabolites (PM) of L. plantarum produced a cytotoxic effect on the MCF-7 breast cancer cell line. The postbiotic showed significant cytotoxic activity similar to 5FU, a standard antineoplastic agent. The inhibition concentration of 50% growth (IC50) of postbiotic metabolite K3 is low and consistent with the IC50 of the positive control (Cisplatin). Conclusions: Lactobacillus plantarum postbiotic exhibited a cytotoxic effect on the MCF-7 breast cancer cell line and could be used as combined adjuvant therapy in breast cancer management. The microencapsulation technique protects the probiotics, improving their viability and delivery to the gastrointestinal tract. Chitosan enhances antibacterial efficacy; thus, chitosan-coated microencapsulated L. plantarum probiotics could be more effective and used as a combined therapy in HIV management of opportunistic enteric infection.

Keywords: probiotics, encapsulation, gastrointestinal conditions, antimicrobial effect, postbiotic, cytotoxicity effect

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Authors: Yanni Chang, Dezhi Dai, Albert Y. Tong

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Piecewise linear interface calculation (PLIC) schemes are widely used in the volume-of-fluid (VOF) method to capture interfaces in numerical simulations of multiphase flows. Dynamic overset meshes can be especially useful in applications involving component motions and complex geometric shapes. In the present study, the VOF value of an acceptor cell is evaluated in a geometric way that transfers the fraction field between the meshes precisely with reconstructed interfaces from the corresponding donor elements. The acceptor cell value is evaluated by using a weighted average of its donors for most of the overset interpolation schemes for continuous flow variables. The weighting factors are obtained by different algebraic methods. Unlike the continuous flow variables, the VOF equation is a step function near the interfaces, which ranges from zero to unity rapidly. A geometric interpolation scheme of the VOF field in overset meshes for the PLIC-VOF method has been proposed in the paper. It has been tested successfully in quadrilateral/hexahedral overset meshes by employing several VOF advection tests with imposed solenoidal velocity fields. The proposed algorithm has been shown to yield higher accuracy in mass conservation and interface reconstruction compared with three other algebraic ones.

Keywords: interpolation scheme, multiphase flows, overset meshes, PLIC-VOF method

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Authors: Mehdi Ghanavati

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The Effects of salinity level and duration on carbon isotope discrimination (Δ) of Matricaria chamomilla and Matricaria aurea were evaluated. Four ecotypes of M. chamomilla and four ecotypes of M. aurea were grown at different NaCl concentrations (control, 6, 12 and 18 dS/m) in sand culture condition. Carbon isotope discrimination (Δ) varied significantly (p<0.001) among ecotypes. The amount of carbon isotope discrimination (Δ) increased in first salinity level (6 dS/m), but in other levels (12 and 18 dS/m) it did not increase. Stages of salinity treatments (two stages: first from seedling stage until the end of the experiment and second stage of stress exertion began at stem elongation and seedlings emergence from rosette stage to harvest) had not a significant difference. Study of two spices of chamomile showed the M. aurea had a higher amount of carbon isotope discrimination (Δ) (22.9%) than M. chamomilla (22.48%).

Keywords: salinity, carbon isotope discrimination, Matricaria chamomilla, Matricaria aurea

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Authors: F. Kashanian, M. M. Masoudi, A. Akbari, A. Shamloo, M. R. Zand, S. S. Salehi

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Nano-sized materials present new opportunities in biology and medicine and they are used as biomedical tools for investigation, separation of molecules and cells. To achieve more effective cancer therapy, it is essential to select cancer cells exactly. This research suggests that using the antibody-functionalized nontoxic Arginine-doped magnetic nanoparticles (A-MNPs), has been prosperous in detection, capture, and magnetic separation of circulating tumor cells (CTCs) in tumor tissue. In this study, A-MNPs were synthesized via a simple precipitation reaction and directly immobilized Ep-CAM EBA-1 antibodies over superparamagnetic A-MNPs for Mucin BCA-225 in breast cancer cell. The samples were characterized by vibrating sample magnetometer (VSM), FT-IR spectroscopy, Tunneling Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). These antibody-functionalized nontoxic A-MNPs were used to capture breast cancer cell. Through employing a strong permanent magnet, the magnetic separation was achieved within a few seconds. Antibody-Conjugated nontoxic Arginine-doped Fe₃O₄ nanoparticles have the potential for the future study to capture CTCs which are released from tumor tissue and for drug delivery, and these results demonstrate that the antibody-conjugated A-MNPs can be used in magnetic hyperthermia techniques for cancer treatment.

Keywords: tumor tissue, antibody, magnetic nanoparticle, CTCs capturing

Procedia PDF Downloads 358

Authors: Luksanaporn Krungkraipetch

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Pterygium is a frequent ocular surface lesion that begins in the limbal conjunctiva within the palpebral fissure and spreads to the cornea. The lesion is more common in the nasal limbus than in the temporal, and it has a wing-like aspect. Indications for surgery, in decreasing order of significance, are growth over the corneal center, decreased vision due to corneal deformation, documented growth, sensations of discomfort, and esthetic concerns. The aim of this study is twofold: first, to determine the frequency of pterygium recurrence after surgery at the mentioned hospital, and second, to identify the factors that influence the recurrence of pterygium. The research design is a retrospective examination of 164 patient samples in an eastern Thai university hospital (Code 13766). Data analysis is descriptive statistics analysis, i.e., basic data details about pterygium surgery and the risk of recurrent pterygium, and for factor analysis, the inferential statistics chi-square and ANOVA are utilized. Twenty-four of the 164 patients who underwent surgery exhibited recurrent pterygium. Consequently, the incidence of recurrent pterygium after surgery was 14.6%. There were an equal number of men and women present. The participants' ages ranged from 41 to 60 years (62, 8 percent). According to the findings, the majority of patients were female (60.4%), over the age of 60 (51.2%), did not live near the beach (83.5%), did not have an underlying disease (92.1%), and 95.7% did not have any other eye problems. Gender (X² = 1.26, p = .289), age (X² = 5.86, p = .119), an address near the sea (X² = 3.30, p = .081)), underlying disease (X² = 0.54, p = .694), and eye disease (X² = 0.00, p = 1.00) had no effect on pterygium recurrence. Recurrences occurred in 79.1% of all surgical procedures and 11.6% of all patients using the bare sclera technique. The recurrence rate for conjunctival autografts was 20.9% for all procedures and 3.0% for all participants. Mitomycin-C and amniotic membrane transplant techniques had no recurrence following surgery. Comparing the surgeries done on people with recurrent pterygium did not show anything important (F = 1.13, p = 0.339). In conclusion, the prevalence of pterygium recurrence following pterygium, 14.6%, does not differ from earlier research. Underlying disease, other eye conditions, and surgical procedures such as pterygium recurrence are unaffected by pterygium surgery.

Keywords: pterygium, recurrence pterygium, pterygium surgery, excision pterygium

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Authors: Lynn Lehmann, Dinorah Leyva, Ana Lazic, Stefan Duhr, Philipp Baaske

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Characterization of biomolecular interactions, such as protein-protein, protein-nucleic acid or protein-small molecule, provides critical insights into cellular processes and is essential for the development of drug diagnostics and therapeutics. Here we present a novel, label-free, and tether-free technology to analyze picomolar to millimolar affinities of biomolecular interactions by Microscale Thermophoresis (MST). The entropy of the hydration shell surrounding molecules determines thermophoretic movement. MST exploits this principle by measuring interactions using optically generated temperature gradients. MST detects changes in the size, charge and hydration shell of molecules and measures biomolecule interactions under close-to-native conditions: immobilization-free and in bioliquids of choice, including cell lysates and blood serum. Thus, MST measures interactions under close-to-native conditions, and without laborious sample purification. We demonstrate how MST determines the picomolar affinities of antibody::antigen interactions, and protein::protein interactions measured from directly from cell lysates. MST assays are highly adaptable to fit to the diverse requirements of different and complex biomolecules. NanoTemper´s unique technology is ideal for studies requiring flexibility and sensitivity at the experimental scale, making MST suitable for basic research investigations and pharmaceutical applications.

Keywords: biochemistry, biophysics, molecular interactions, quantitative techniques

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Authors: Grazia Disciglio, Francesco Lops, Annalisa Tarantino, Emanuele Tarantino

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The parasitic weed specie Phelipanche ramosa (L) Pomel is one of the major constraints in tomato crop in Apulia region (southern Italy). The experimental was considered to investigate the effect of six organic compounds (Olive miller wastewater, Allil isothiocyanate®, Alfa plus K®, Radicon®, Rizosum Max®, Kendal Nem®) on the naturally infested field of tomato growing season in 2016. The randomized block design with 3 replicates was adopted. Tomato seedling were transplant on 19 May 2016. During the growing cycle of the tomato at 74, 81, 93 and 103 days after transplantation (DAT), the number of parasitic shoots (branched plants) that had emerged in each plot was determined. At harvesting on 13 September 2016 the major quanti-qualitative yield parameters were determined, including marketable yield, mean weight, dry matter, soluble solids, fruit colour, pH and titratable acidity. The treatments provided the results show that none of treatments provided complete control against P. ramosa. However, among the products tested Olive miller wastewater, Alfa plus K®, Rizosum Max® and Kendal Nem® products applied to the soil show the number of emerged shoots significantly lower than Radicon® and especially than the Allil isothiocyanate® treatment and the untreated control. Regarding the effect of different treatments on the tomato productive parameters, the marketable yield resulted significantly higher in the same mentioned treatments which gave the lower P. ramosa infestation. No significative differences for the other fruit characteristics were observed.

Keywords: processing tomato crop, Phelipanche ramosa, olive-mill wastewater, organic fertilizers

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Authors: E. Nakamachi, S. Tanaka, K. Yamamoto, Y. Morita

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In this study, we developed a three-dimensional (3D) direct current electric field (DCEF) stimulation bio-reactor for axonal outgrowth enhancement to generate the neural network of the central nervous system (CNS). By using our newly developed 3D DCEF stimulation bio-reactor, we cultured the rat pheochromocytoma cells (PC12) and investigated the effects on the axonal extension enhancement and network generation. Firstly, we designed and fabricated a 3D bio-reactor, which can load DCEF stimulation on PC12 cells embedded in the collagen gel as extracellular environment. The connection between the electrolyte and the medium using salt bridges for DCEF stimulation was introduced to avoid the cell death by the toxicity of metal ion. The distance between the salt bridges was adopted as the design variable to optimize a structure for uniform DCEF stimulation, where the finite element (FE) analyses results were used. Uniform DCEF strength and electric flux vector direction in the PC12 cells embedded in collagen gel were examined through measurements of the fabricated 3D bio-reactor chamber. Measurement results of DCEF strength in the bio-reactor showed a good agreement with FE results. In addition, the perfusion system was attached to maintain pH 7.2 ~ 7.6 of the medium because pH change was caused by DCEF stimulation loading. Secondly, we disseminated PC12 cells in collagen gel and carried out 3D culture. Finally, we measured the morphology of PC12 cell bodies and neurites by the multiphoton excitation fluorescence microscope (MPM). The effectiveness of DCEF stimulation to enhance the axonal outgrowth and the neural network generation was investigated. We confirmed that both an increase of mean axonal length and axogenesis rate of PC12, which have been exposed 5 mV/mm for 6 hours a day for 4 days in the bioreactor. We found following conclusions in our study. 1) Design and fabrication of DCEF stimulation bio-reactor capable of 3D culture nerve cell were completed. A uniform electric field strength of average value of 17 mV/mm within the 1.2% error range was confirmed by using FE analyses, after the structure determination through the optimization process. In addition, we attached a perfusion system capable of suppressing the pH change of the culture solution due to DCEF stimulation loading. 2) Evaluation of DCEF stimulation effects on PC12 cell activity was executed. The 3D culture of PC 12 was carried out adopting the embedding culture method using collagen gel as a scaffold for four days under the condition of 5.0 mV/mm and 10mV/mm. There was a significant effect on the enhancement of axonal extension, as 11.3% increase in an average length, and the increase of axogenesis rate. On the other hand, no effects on the orientation of axon against the DCEF flux direction was observed. Further, the network generation was enhanced to connect longer distance between the target neighbor cells by DCEF stimulation.

Keywords: PC12, DCEF stimulation, 3D bio-reactor, axonal extension, neural network generation

Procedia PDF Downloads 183

Authors: Chang-Hui Shen, Paulina Konarzewska

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Cell homeostasis is regulated by vacuolar activity and it has been shown that lipid composition of the vacuole plays an important role in vacuolar function. The major phosphoinositide species present in the vacuolar membrane include phosphatidylinositol 3,5-biphosphate (PI(3,5)P₂) which is generated from PI(3)P controlled by Fab1p. Deletion of FAB1 gene reduce the synthesis of PI(3,5)P₂ and thus result in enlarged or fragmented vacuoles, with neutral vacuolar pH due to reduced vacuolar H⁺-ATPase activity. These mutants also exhibited poor growth at high extracellular pH and in the presence of CaCl₂. Conversely, VPS34 regulates the synthesis of PI(3)P from phosphatidylinositol (PI), and the lack of Vps34p results in the reduction of vacuolar activity. Although the cellular observations are clear, it is still unknown about the molecular mechanism between the phospholipid biosynthesis pathway and vacuolar activity. Since both VPS34 and FAB1 are important in vacuolar activity, we hypothesize that the molecular mechanism of vacuolar function might be regulated by the transcriptional regulators of phospholipid biosynthesis. In this study, we study the role of the major phospholipid biosynthesis transcription factor, INO2, in the regulation of vacuolar activity. We first performed qRT-PCR to examine the effect of Ino2p on the expression of VPS34 and FAB1. Our results showed that VPS34 was upregulated in the presence of inositol for both WT and ino2Δ cells. However, FAB1 was only upregulated significantly in ino2Δ cells. This indicated that Ino2p might be the negative regulator for FAB1 expression. Next, growth sensitivity experiment showed that WT, vma3Δ, and ino2Δ grew well in growth medium buffered to pH 5.5 containing 10 mM CaCl₂. As cells were switched to growth medium buffered to pH 7 containing CaCl₂ WT, ino2Δ and opi1Δ showed growth reduction, whereas vma3Δ was completely nonviable. As the concentration of CaCl₂ was increased to 60 mM, ino2Δ cells showed moderate growth reduction compared to WT. This result suggests that ino2Δ cells have better vacuolar activity. Microscopic analysis and vacuolar acidification were employed to further elucidate the importance of INO2 in vacuolar homeostasis. Analysis of vacuolar morphology indicated that WT and vma3Δ cells displayed vacuoles that occupied a small area of the cell when grown in media buffered to pH 5.5. Whereas, ino2Δ displayed fragmented vacuoles. On the other hand, all strains grown in media buffered to pH 7, exhibited enlarged vacuoles that occupied most of the cell’s surface. This indicated that the presence of INO2 may play negative effect in vacuolar morphology when cells are grown in media buffered to pH 5.5. Furthermore, vacuolar acidification assay showed that only vma3Δ cells displayed notably less acidic vacuoles as cells were grown in media buffered to pH 5.5 and pH 7. Whereas, ino2Δ cells displayed more acidic pH compared to WT at pH7. Taken together, our results demonstrated the molecular mechanism of the vacuolar activity regulated by the phospholipid biosynthesis transcription factors Ino2p. Ino2p negatively regulates vacuolar activity through the expression of FAB1.

Keywords: vacuole, phospholipid, homeostasis, Ino2p, FAB1

Procedia PDF Downloads 125

Authors: Ayda Youssef, Tarek Rafaat, Iman zaky

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Purpose: Langerhans’ cell histiocytosis (LCH) is not uncommon pathology that implies aberrant proliferation of a specific dendritic (Langerhans) cell. These atypical but mature cells of monoclonal origin can infiltrate many sites of the body and may occur as localized lesions or as widespread systemic disease. Liver is one of the uncommon sites of affection. The twofold objective of this study is to illustrate the radiological presentation of this disease, and to compare these results with previously reported series. Methods and Materials: Between 2007 and 2012, 150 patients with biopsy-proven LCH were treated in our hospital, a paediatric cancer tertiary care center. A retrospective review of radiographic images and reports was performed. There were 33 patients with liver affection are stratified. All patients underwent imaging studies, mostly US and CT. A chart review was performed to obtain demographic, clinical and radiological data. They were analyzed and compared to other published series. Results: Retrospective assessment of 150 patients with LCH was performed, among them 33 patients were identified who had liver involvement. All these patients developed multisystemic disease; They were 12 females and 21 males with (n= 32), seven of them had marked hepatomegaly. Diffuse hypodense liver parenchyma was encountered in five cases, the periportal location has a certain predilection in cases of focal affection where three cases has a hypodense periportal soft tissue sheets, one of them associated with dilated biliary radicals, only one case has multiple focal lesions unrelated to portal tracts. On follow up of the patients, two cases show abnormal morphology of liver with bossy outline. Conclusion: LCH is a not infrequent disease. A high-index suspicion should be raised in the context of diagnosis of liver affection. A biopsy is recommended in the presence of radiological suspicion. Chemotherapy is the preferred therapeutic modality. Liver histiocytosis are not disease specific features but should be interpreted in conjunction with the clinical history and the results of biopsy. Clinical Relevance/Application: Radiologist should be aware of different patterns of hepatobiliary histiocytosis, Thus early diagnosis and proper management of patient can be conducted.

Keywords: langerhans’ cell histiocytosis, liver, medical and health sciences, radiology

Procedia PDF Downloads 275

Authors: Anna Cameron, Chunxia Zhao, Haofei Wang, Yun Liu, Guang Ze Yang

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Since the first publication in 1775, cancer research has built a comprehensive understanding of how cellular components of the tumor niche promote disease development. However, only within the last decade has research begun to establish the impact of non-cellular components of the niche, particularly the extracellular matrix (ECM). The ECM, a three-dimensional scaffold that sustains the tumor microenvironment, plays a crucial role in disease progression. Cancer cells actively deregulate and remodel the ECM to establish a tumor-promoting environment. Recent work has highlighted the need to further our understanding of the complexity of this cancer-ECM relationship. In vitro models use hydrogels to mimic the ECM, as hydrogel matrices offer biological compatibility and stability needed for long term cell culture. However, natural hydrogels are being used in these models verbatim, without tuning their biophysical characteristics to achieve pathophysiological relevance, thus limiting their broad use within cancer research. The biophysical attributes of these gels dictate cancer cell proliferation, invasion, metastasis, and therapeutic response. Evaluating the three most widely used natural hydrogels, Matrigel, collagen, and agarose gel, the permeability, stiffness, and pore-size of each gel were measured and compared to the in vivo environment. The pore size of all three gels fell between 0.5-6 µm, which coincides with the 0.1-5 µm in vivo pore size found in the literature. However, the stiffness for hydrogels able to support cell culture ranged between 0.05 and 0.3 kPa, which falls outside the range of 0.3-20,000 kPa reported in the literature for an in vivo ECM. Permeability was ~100x greater than in vivo measurements, due in large part to the lack of cellular components which impede permeation. Though, these measurements prove important when assessing therapeutic particle delivery, as the ECM permeability decreased with increasing particle size, with 100 nm particles exhibiting a fifth of the permeability of 10 nm particles. This work explores ways of adjusting the biophysical characteristics of hydrogels by changing protein concentration and the trade-off, which occurs due to the interdependence of these factors. The global aim of this work is to produce a more pathophysiologically relevant model for each tumor type.

Keywords: cancer, extracellular matrix, hydrogel, microfluidic

Procedia PDF Downloads 89

Authors: Drago Bratkovic, Curtis Gravance, David Ketteridge, Ravi Krishnan, Michael Imperiale

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The mucopolysaccharidoses (MPSs) are a group of lysosomal storage diseases that have a common defect in the catabolism of glycosaminoglycans (GAGs). MPS I is the most common of the MPS diseases. Manifestations of MPS I include coarsening of facial features, corneal clouding, developmental delay, short stature, skeletal manifestations, hearing loss, cardiac valve disease, hepatosplenomegaly, and umbilical and inguinal hernias. Treatments for MPS I restore or activate the missing or deficient enzyme in the case of enzyme replacement therapy (ERT) and haematopoietic stem cell transplantation (HSCT). Pentosan polysulfate sodium (PPS) is a potential treatment to improve bone and joint manifestations of MPS I. The mechanisms of action of PPS that are relevant to the treatment of MPS I are the ability to: (i) Reduce systemic and accumulated GAG, (ii) Reduce inflammatory effects via the inhibition of NF-kB, resulting in the reduction in pro-inflammatory mediators. (iii) Reduce the expression of the pain mediator nerve growth factor in osteocytes from degenerating joints. (iv) Inhibit the cartilage degrading enzymes related to joint dysfunction in MPS I. PPS is being evaluated as an adjunctive therapy to ERT and/or HSCT in an open-label, single-centre, phase 2 study. Patients are ≥ 5 years of age with a diagnosis of MPS I and previously received HSCT and/or ERT. Three white, female, patients with MPS I-Hurler, ages 14, 15, and 19 years, and one, white male patient aged 15 years are enrolled. All were diagnosed at ≤2 years of age. All patients received HSCT ≤ 6 months after diagnosis. Two of the patients were treated with ERT prior to HSCT, and 1 patient received ERT commencing 3 months prior to HSCT. Two patients received 0.75mg/kg and 2 patients received 1.5mg/kg of PPS. PPS was well tolerated at doses of 0.75 and 1.5 mg/kg to 47 weeks of continuous dosing. Of the 19 adverse events (AEs), 2 were related to PPS. One AE was moderate (pre-syncope) and 1 was mild (injection site bruising), experienced in the same patient. All AEs were reported as mild or moderate. There have been no SAEs. One subject experienced a COVID-19 infection and PPS was interrupted. The MPS I signature GAG fragments, sulfated disaccharide and UA-HNAc S, tended to decrease in 3 patients from baseline through Week 25. Week 25 GAG data are pending for the 4th patient. Overall, most biomarkers (inflammatory, cartilage degeneration, and bone turnover) evaluated in the 3 patients with 25-week assessments have indicated either no change or a reduction in levels compared to baseline. In 3 patients, there was a trend toward improvement in the 2MWT from baseline to Week 48 with > 100% increase in 1 patient (01-201). In the 3 patients that had Week 48 assessments, patients and proxies reported improvement in PGIC, including “worthwhile difference” (n=1), or “made all the difference” (n=2).

Keywords: MPS I, pentosan polysulfate sodium, clinical study, 2MWT, QoL

Procedia PDF Downloads 109

Authors: Shanna Swart, Caryn Fenner, Athanasios Kotsiopoulos, Susan Harrison

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Linear alkanes are produced as by-products from the increasing use of gas-to-liquid fuel technologies for synthetic fuel production and offer great potential for value addition. Their current use as low-value fuels and solvents do not maximize this potential. Therefore, attention has been drawn towards direct activation of these aliphatic alkanes to more useful products such as alcohols, aldehydes, carboxylic acids and derivatives. Cytochrome P450 monooxygenases (P450s) can be used for activation of these aliphatic alkanes using whole-cells or cell-free systems. Some limitations of whole-cell systems include reduced mass transfer, stability and possible side reactions. Since the P450 systems are little studied as cell-free systems, they form the focus of this study. Challenges of a cell-free system include co-factor regeneration, substrate availability and enzyme stability. Enzyme immobilization offers a positive outlook on this dilemma, as it may enhance stability of the enzyme. In the present study, 2 different P450s (CYP153A6 and CYP102A1) as well as the relevant accessory enzymes required for electron transfer (ferredoxin and ferredoxin reductase) and co-factor regeneration (glucose dehydrogenase) have been expressed in E. coli and purified by metal affinity chromatography. Glucose dehydrogenase (GDH), was used as a model enzyme to assess the potential of various enzyme immobilization strategies including; surface attachment on MagReSyn® microspheres with various functionalities and on electrospun nanofibers, using self-assembly based methods forming Cross Linked Enzymes (CLE), Cross Linked Enzyme Aggregates (CLEAs) and spherezymes as well as in a sol gel. The nanofibers were synthesized by electrospinning, which required the building of an electrospinning machine. The nanofiber morphology has been analyzed by SEM and binding will be further verified by FT-IR. Covalent attachment based methods showed limitations where only ferredoxin reductase and GDH retained activity after immobilization which were largely attributed to insufficient electron transfer and inactivation caused by the crosslinkers (60% and 90% relative activity loss for the free enzyme when using 0.5% glutaraldehyde and glutaraldehyde/ethylenediamine (1:1 v/v), respectively). So far, initial experiments with GDH have shown the most potential when immobilized via their His-tag onto the surface of MagReSyn® microspheres functionalized with Ni-NTA. It was found that Crude GDH could be simultaneously purified and immobilized with sufficient activity retention. Immobilized pure and crude GDH could be recycled 9 and 10 times, respectively, with approximately 10% activity remaining. The immobilized GDH was also more stable than the free enzyme after storage for 14 days at 4˚C. This immobilization strategy will also be applied to the P450s and optimized with regards to enzyme loading and immobilization time, as well as characterized and compared with the free enzymes. It is anticipated that the proposed immobilization set-up will offer enhanced enzyme stability (as well as reusability and easy recovery), minimal mass transfer limitation, with continuous co-factor regeneration and minimal enzyme leaching. All of which provide a positive outlook on this robust multi-enzyme system for efficient activation of linear alkanes as well as the potential for immobilization of various multiple enzymes, including multimeric enzymes for different bio-catalytic applications beyond alkane activation.

Keywords: alkane activation, cytochrome P450 monooxygenase, enzyme catalysis, enzyme immobilization

Procedia PDF Downloads 222