Search results for: cellular uptake

Authors: Janneth Gonzalez, Andrés Pinzon Velasco, Maria Angarita

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Astrocytes play an important role in various processes in the brain, including pathological conditions such as neurodegenerative diseases. Recent studies have shown that the increase in saturated fatty acids such as palmitic acid (PA) triggers pro-inflammatorypathways in the brain. The use of synthetic neurosteroids such as tibolone has demonstrated neuro-protective mechanisms. However, broad studies with a systemic point of view on the neurodegenerative role of PA and the neuro-protective mechanisms of tibolone are lacking. In this study, we performed the integration of multi-omic data (transcriptome and proteome) into a human astrocyte genomic scale metabolic model to study the astrocytic response during palmitate treatment. We evaluated metabolic fluxes in three scenarios (healthy, induced inflammation by PA, and tibolone treatment under PA inflammation). We also applied a control theory approach to identify those reactions that exert more control in the astrocytic system. Our results suggest that PA generates a modulation of central and secondary metabolism, showing a switch in energy source use through inhibition of folate cycle and fatty acid β‐oxidation and upregulation of ketone bodies formation. We found 25 metabolic switches under PA‐mediated cellular regulation, 9 of which were critical only in the inflammatory scenario but not in the protective tibolone one. Within these reactions, inhibitory, total, and directional coupling profiles were key findings, playing a fundamental role in the (de)regulation of metabolic pathways that may increase neurotoxicity and represent potential treatment targets. Finally, the overall framework of our approach facilitates the understanding of complex metabolic regulation, and it can be used for in silico exploration of the mechanisms of astrocytic cell regulation, directing a more complex future experimental work in neurodegenerative diseases.

Keywords: astrocytes, data integration, palmitic acid, computational model, multi-omics

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Authors: Rutuja Upadhyay, Anurag Mehra

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Solid food foams/cellular foods are colloidal systems which impart structure, texture and mouthfeel to many food products such as bread, cakes, ice-cream, meringues, etc. Their heterogeneous morphology makes the quantification of structure/mechanical relationships complex. The porous structure of solid food foams is highly influenced by the processing conditions, ingredient composition, and their interactions. Sensory perceptions of food foams are dependent on bubble size, shape, orientation, quantity and distribution and determines the texture of foamed foods. The state and structure of the solid matrix control the deformation behavior of the food, such as elasticity/plasticity or fracture, which in turn has an effect on the force-deformation curves. The obvious step in obtaining the relationship between the mechanical properties and the porous structure is to quantify them simultaneously. Here, we attempt to research food foams such as bread dough, baked bread and steamed rice cakes to determine the link between ingredients and the corresponding effect of each of them on the rheology, microstructure, bubble size and texture of the final product. Dynamic rheometry (SAOS), confocal laser scanning microscopy, flatbed scanning, image analysis and texture profile analysis (TPA) has been used to characterize the foods studied. In all the above systems, there was a common observation that when the mean bubble diameter is smaller, the product becomes harder as evidenced by the increase in storage and loss modulus (G′, G″), whereas when the mean bubble diameter is large the product is softer with decrease in moduli values (G′, G″). Also, the bubble size distribution affects texture of foods. It was found that bread doughs with hydrocolloids (xanthan gum, alginate) aid a more uniform bubble size distribution. Bread baking experiments were done to study the rheological changes and mechanisms involved in the structural transition of dough to crumb. Steamed rice cakes with xanthan gum (XG) addition at 0.1% concentration resulted in lower hardness with a narrower pore size distribution and larger mean pore diameter. Thus, control of bubble size could be an important parameter defining final food texture.

Keywords: food foams, rheology, microstructure, texture

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Authors: Agatha Bebbington, Terry Tetley, Kathryn Hadler

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Background: Astronauts will set foot on the Moon later this decade, and are at high risk of lunar dust inhalation. Freshly-fractured lunar dust produces reactive oxygen species in solution, which are known to cause cellular damage and inflammation. Cytotoxicity and inflammatory mediator release was measured in pulmonary alveolar epithelial cells (cells that line the gas-exchange zone of the lung) exposed to a lunar dust simulant, LMS-1. It was hypothesised that freshly-fractured LMS-1 would result in increased cytotoxicity and inflammatory mediator release, owing to the angular morphology and high reactivity of fractured particles. Methods: A human alveolar epithelial type 1-like cell line (TT1) and a human macrophage-like cell line (THP-1) were exposed to 0-200μg/ml of unground, aged-ground, and freshly-ground LMS-1 (screened at <22μm). Cell viability, cytotoxicity, and inflammatory mediator release (IL-6, IL-8) were assessed using MMT, LDH, and ELISA assays, respectively. LMS-1 particles were characterised for their size, surface area, and morphology before and after grinding. Results: Exposure to LMS-1 particles did not result in overt cytotoxicity in either TT1 epithelial cells or THP-1 macrophage-like cells. A dose-dependent increase in IL-8 release was observed in TT1 cells, whereas THP-1 cell exposure, even at low particle concentrations, resulted in increased IL-8 release. Both cytotoxic and pro-inflammatory responses were most marked and significantly greater in TT1 and THP-1 cells exposed to freshly-fractured LMS-1. Discussion: LMS-1 is a novel lunar dust simulant; this is the first study to determine its toxicological effects on respiratory cells in vitro. An increased inflammatory response in TT1 and THP-1 cells exposed to ground LMS-1 suggests that low particle size, increased surface area, and angularity likely contribute to toxicity. Conclusions: Evenlow levels of exposure to LMS-1 could result in alveolar inflammation. This may have pathological consequences for astronauts exposed to lunar dust on future long-duration missions. Future research should test the effect of low-dose, intermittent lunar dust exposure on the respiratory system.

Keywords: lunar dust, LMS-1, lunar dust simulant, long-duration space travel, lunar dust toxicity

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Authors: Han-Qin Zheng, Yi-Fan Chiang-Hsieh, Chia-Hung Chien, Wen-Chi Chang

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As the most important non-vascular plants, algae have many research applications, including high species diversity, biofuel sources, adsorption of heavy metals and, following processing, health supplements. With the increasing availability of next-generation sequencing (NGS) data for algae genomes and transcriptomes, an integrated resource for retrieving gene expression data and metabolic pathway is essential for functional analysis and systems biology in algae. However, gene expression profiles and biological pathways are displayed separately in current resources, and making it impossible to search current databases directly to identify the cellular response mechanisms. Therefore, this work develops a novel AlgaePath database to retrieve gene expression profiles efficiently under various conditions in numerous metabolic pathways. AlgaePath, a web-based database, integrates gene information, biological pathways, and next-generation sequencing (NGS) datasets in Chlamydomonasreinhardtii and Neodesmus sp. UTEX 2219-4. Users can identify gene expression profiles and pathway information by using five query pages (i.e. Gene Search, Pathway Search, Differentially Expressed Genes (DEGs) Search, Gene Group Analysis, and Co-Expression Analysis). The gene expression data of 45 and 4 samples can be obtained directly on pathway maps in C. reinhardtii and Neodesmus sp. UTEX 2219-4, respectively. Genes that are differentially expressed between two conditions can be identified in Folds Search. Furthermore, the Gene Group Analysis of AlgaePath includes pathway enrichment analysis, and can easily compare the gene expression profiles of functionally related genes in a map. Finally, Co-Expression Analysis provides co-expressed transcripts of a target gene. The analysis results provide a valuable reference for designing further experiments and elucidating critical mechanisms from high-throughput data. More than an effective interface to clarify the transcript response mechanisms in different metabolic pathways under various conditions, AlgaePath is also a data mining system to identify critical mechanisms based on high-throughput sequencing.

Keywords: next-generation sequencing (NGS), algae, transcriptome, metabolic pathway, co-expression

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Authors: Natalia Moreno-Castellanos, Amaia Rodriguez, Gema Frühbeck

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Introduction: In adipocytes, the cytoskeleton undergoes important expression and distribution in adipocytes rearrangements during adipogenesis and in obesity. Indeed, a role for these proteins in the regulation of adipocyte differentiation and response to insulin has been demonstrated. Recently, septins have been considered as new components of the cytoskeletal network that interact with other cytoskeletal elements (actin and tubulin) profoundly modifying their dynamics. However, these proteins have not been characterized as yet in adipose tissue. In this work, were examined the cellular, molecular and functional features of a member of this family, septin 11 (SEPT11), in adipocytes and evaluated the impact of obesity on the expression of this protein in human adipose tissue. Methods: Adipose gene and protein expression levels of SEPT11 were analysed in human samples. SEPT11 distribution was evaluated by immunocytochemistry, electronic microscopy, and subcellular fractionation techniques. GST-pull down, immunoprecipitation and a Yeast-Two Hybrid (Y2H) screening were used to identify the SEPT11 interactome. Gene silencing was employed to assess the role of SEPT11 in the regulation of insulin signaling and lipid metabolism in adipocytes. Results: SEPT11 is expressed in human adipocytes, and its levels increased in both omental and subcutaneous adipose tissue in obesity, with SEPT11 mRNA content positively correlating with parameters of insulin resistance in subcutaneous fat. In non-stimulated adipocytes, SEPT11 immunoreactivity showed a ring-like distribution at the cell surface and associated to caveolae. Biochemical analyses showed that SEPT11 interacted with the main component of caveolae, caveolin-1 (CAV1) as well as with the fatty acid-binding protein, FABP5. Notably, the three proteins redistributed and co-localized at the surface of lipid droplets upon exposure of adipocytes to oleate. In this line, SEPT11 silencing in 3T3-L1 adipocytes impaired insulin signaling and decreased insulin-induced lipogenesis. Conclusions: Those findings demonstrate that SEPT11 is a novel component of the adipocyte cytoskeleton that plays an important role in the regulation of lipid traffic, metabolism and can thus represent a potential biomarker of insulin resistance in obesity in adipocytes through its interaction with both CAV1 and FABP5.

Keywords: caveolae, lipid metabolism, obesity, septins

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Authors: Violina Angelova, Krasimir Ivanov, Stefan Krustev, Dimitar Dimitrov

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Availability of a metal is characterised by its quantity transgressing from soil into different extractants or by its content in plants. In literature, the terms 'available forms of compounds' and 'mobile' are often considered as equivalents of the term 'accessible' to plants. Rapid and a sufficiently reliable method for defining the accessible for plants forms turns out to be their extraction through different extractants, imitating the functioning of the root system. As a criterion for the pertinence of the extractant to this purpose usually serves the significant statistic correlation between the extracted quantities of the element from soil and its content in plants. The aim of this work was to evaluate the effectiveness of various extractions (DTPA-TEA, AB-DTPA, Mehlich 3, 0.01 M CaCl₂, 1M NH₄NO₃) for the determination of bioavailability of heavy metals in industrially polluted soils from the metallurgical activity near Plovdiv and Kardjali, Bulgaria. Quantity measurements for contents of heavy metals were performed with ICP-OES. The results showed that extraction capacity was as follows: Mehlich 3>ABDTPA>DTPA-TEA>CaCl₂>NaNO₃. The content of the mobile form of heavy metals depends on the nature of metal ion, the nature of extractant and pH. The obtained results show that CaCl₂ extracts a greater quantity of mobile forms of heavy metals than NH₄NO₃. DTPA-TEA and AB-DTPA are capable of extracting from the soil not only the heavy metals participating in the exchange processes but also the heavy metals bound in carbonates and organic complexes, as well as bound and occluded in oxide and secondary clay minerals. AB-DTPA extracts a bit more heavy metals than DTPA-TEA. The darker color of the solutions obtained with AB-DTPA indicates that considerable quantities organic matter are being destructed. A comparison of the mobile forms of heavy metals extracted from clean and highly polluted soils has revealed that in the polluted soils the greater portion of heavy metals exists in a mobile form. High correlation coefficients are obtained between the metals extracted with different extractants and their total content in soil (r=0.9). A positive correlation between the pH, soil organic matter and the extracted quantities of heavy metals has been found. The results of correlation analysis revealed that the heavy metals extracted by DTPA-TEA, AB-DTPA, Mehlich 3, CaCl₂ and NaNO₃ correlated significantly with plant uptake. Significant correlation was found between DTPA-TEA, AB-DTPA, and CaCl₂ with heavy metals concentration in plants. Application of extracting methods contains chelating agents would be recommended in the future research onthe availabilityof heavy metals in polluted soils.

Keywords: availability, chemical extractants, heavy metals, mobile forms

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Authors: Maria Lopez-Ibarra, Ana Velazquez-Wong, Lucelli Yañez-Gutierrez, Maria Araujo-Solis, Fabio Salamanca-Gomez, Alfonso Mendez-Tenorio, Haydeé Rosas-Vargas

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Congenital heart diseases (CHD) are the most common congenital abnormalities. These conditions can occur as both an element of distinct chromosomal malformation syndromes or as non-syndromic forms. Their etiology is not fully understood. Genetic variants such copy number variants have been associated with CHD. The aim of our study was to analyze these genomic variants in peripheral blood from Mexican children diagnosed with non-syndromic CHD. We included 16 children with atrial and ventricular septal defects and 5 healthy subjects without heart malformations as controls. To exclude the most common heart disease-associated syndrome alteration, we performed a fluorescence in situ hybridization test to identify the 22q11.2, responsible for congenital heart abnormalities associated with Di-George Syndrome. Then, a microarray based comparative genomic hybridization was used to identify global copy number variants. The identification of copy number variants resulted from the comparison and analysis between our results and data from main genetic variation databases. We identified copy number variants gain in three chromosomes regions from pediatric patients, 4q13.2 (31.25%), 9q34.3 (25%) and 20q13.33 (50%), where several genes associated with cellular, biosynthetic, and metabolic processes are located, UGT2B15, UGT2B17, SNAPC4, SDCCAG3, PMPCA, INPP6E, C9orf163, NOTCH1, C20orf166, and SLCO4A1. In addition, after a hierarchical cluster analysis based on the fluorescence intensity ratios from the comparative genomic hybridization, two congenital heart disease groups were generated corresponding to children with atrial or ventricular septal defects. Further analysis with a larger sample size is needed to corroborate these copy number variants as possible biomarkers to differentiate between heart abnormalities. Interestingly, the 20q13.33 gain was present in 50% of children with these CHD which could suggest that alterations in both coding and non-coding elements within this chromosomal region may play an important role in distinct heart conditions.

Keywords: aCGH, bioinformatics, congenital heart diseases, copy number variants, fluorescence in situ hybridization

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Authors: Somayeh Fani, Mahmood Ameen Abdulla

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Costus speciosus is an important Malaysian medicinal plant commonly used traditionally in the treatment of many aliments. The present investigation is designed to elucidate preventive effects of ethanolic extracts of C. speciosus rhizome against absolute ethanol-induced gastric mucosal injury in Sprague-Dawley rats. Five groups of rats were orally pre-treated with vehicle, carboxymethylcellulose (CMC) as normal control group (Group 1), ethanol as ulcer control group (Group 2), omeprazole 20 mg/kg (reference group) (Group 3), and 250 and 500 mg/kg of C. speciosus extract (experimental groups) (Group 4 and 5), respectively. An hour later, CMC was given orally to Group 1 rats and absolute ethanol was given orally to Group 2-5 rats to generate gastric mucosal injury. After an additional hour, the rats were sacrificed. Grossly, ulcer control group exhibited severe of gastric mucosal hemorrhagic injury and increased in ulcer area, whereas groups pre-treated with omeprazole or plant’s rhizomes exhibited the significant reduction of gastric mucosal injury. Significant increase in the pH and mucous of gastric content was observed in rats re-treated with C. speciosus rhizome. Histology, ulcer control rats, demonstrated remarkable disruption of gastric mucosa, increased in edema and inflammatory cells infiltration of submucosal layer compared to rats pre-treated with rhizomes extract. Periodic acid Schiff staining for glycoprotein, rats pre-fed with C. speciosus C. displayed remarkably intense uptake of magenta color by glandular gastric mucosa compared with ulcer control rats. Immunostaining of gastric epithelium, rats pre-treatment with rhizome extract provide evidence of up-regulation of HSP70 and down-regulation of Bax proteins compared to ulcer control animals. Gastric tissue homogenate, C. speciosus significantly increased the activity of superoxide dismutase (SOD), and catalase (CAT), increased the level of non-protein sulfhydryl (NP-SH) and decreased the level of lipid peroxidation after ethanol administration. Acute toxicity test did not show any signs of toxicity. The mechanisms implicated the gasrtoprotective property of C. speciosus depend upon the antisecretory activity, increased in gastric mucus glycoprotein, up-regulation of HSP70 protein and down-regulation of Bax proteins, reduction in the lipid peroxidation and increase in the level of NP-SH and antioxidant enzymes activity in gastic homogenate.

Keywords: antioxidant, Costus speciosus, gastric ulcer, histology, omeprazole

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Authors: Mateusz Hermyt, Pawel Kaczmarek, Weronika Rupik

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The egg tooth is a crucial structure during hatching of lizards and snakes. In contrast to birds, turtles, crocodiles, and monotremes, egg tooth of squamate reptiles is a true tooth sharing common features of structure and development with all the other teeth of vertebrates. The egg tooth; however, due to its function, exhibits structural differences in relation to regular teeth. External morphology seems to be important in the context of phylogenetic relationships within Squamata but up to date, there is scarce information concerning structure and development of the egg tooth at the submicroscopical level. In presented studies detailed analysis of the egg tooth development in grass snake has been performed with the usage of light (including fluorescent), transmission and scanning electron microscopy. Grass snake embryo’s heads have been used in our studies. Grass snake is common snake species occurring in most of Europe including Poland. The grass snake is characterized by the presence of single unpaired egg tooth (as in most squamates) in contrast to geckos and dibamids possessing paired egg teeth. Studies show changes occurring on the external morphology, tissue and cellular levels of differentiating egg tooth. The egg tooth during its development changes its curvature. Initially, faces directly downward and in the course of its differentiation, it gradually changes to rostro-ventral orientation. Additionally, it forms conical dentinal protrusions on the sides. Histological analysis showed that egg tooth development occurs in similar steps in relation to regular teeth. It undergoes initiation, bud, cap and bell morphological stages. Analyses focused on describing morphological changes in hard tissues (mainly dentin and predentin) of egg tooth and in cells which enamel organ consists of. It included: outer enamel epithelium, stratum intermedium, inner enamel epithelium, odontoblasts, and cells of dental pulp. All specimens used in the study were captured according to the Polish regulations concerning the protection of wild species. Permission was granted by the Local Ethics Commission in Katowice (41/2010; 87/2015) and the Regional Directorate for Environmental Protection in Katowice (WPN.6401.257.2015.DC).

Keywords: hatching, organogenesis, reptile, Squamata

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Authors: Dugeree Otgongerel, Kyong Jin Cho, Yu-Han Kim, Sangmee Ahn Jo

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Excessive activation of glutamate receptor is thought to be involved in retinal ganglion cell (RGC) death after ischemia- reperfusion damage. Glutamate carboxypeptidase II (GCPII) is an enzyme responsible for the synthesis of glutamate. Several studies showed that inhibition of GCPII prevents or reduces cellular damage in brain diseases. Thus, in this study, we examined the expression of GCPII in rat retina and the role of GCPII in acute high IOP ischemia-reperfusion damage of eye by using a GCPII inhibitor, 2-(phosphonomethyl) pentanedioic acid (2-PMPA). Animal model of ischemia-reperfusion was induced by raising the intraocular pressure for 60 min and followed by reperfusion for 3 days. Rats were randomly divided into four groups: either intra-vitreous injection of 2-PMPA (11 or 110 ng per eye) or PBS after ischemia-reperfusion, 2-PMPA treatment without ischemia-reperfusion and sham-operated normal control. GCPII immunoreactivity in normal rat retina was detected weakly in retinal nerve fiber layer (RNFL) and retinal ganglionic cell layer (RGL) and also inner plexiform layer (IPL) and outer plexiform layer (OPL) strongly where are co-stained with an anti-GFAP antibody, suggesting that GCPII is expressed mostly in Muller and astrocytes. Immunostaining with anti-BRN antibody showed that ischemia- reperfusion caused RGC death (31.5 %) and decreased retinal thickness in all layers of damaged retina, but the treatment of 2-PMPA twice at 0 and 48 hour after reperfusion blocked these retinal damages. GCPII level in RNFL layer was enhanced after ischemia-reperfusion but was blocked by PMPA treatment. This result was confirmed by western blot analysis showing that the level of GCPII protein after ischemia- reperfusion increased by 2.2- fold compared to control, but this increase was blocked almost completely by 110 ng PMPA treatment. Interestingly, GFAP immunoreactivity in the retina after ischemia- reperfusion followed by treatment with PMPA showed similar pattern to GCPII, increase after ischemia-reperfusion but reduction to the normal level by PMPA treatment. Our data demonstrate that increase of GCPII protein level after ischemia-reperfusion injury is likely to cause glial activation and/or retinal cell death which are mediated by glutamate, and GCPII inhibitors may be useful in treatment of retinal disorders in which glutamate excitotoxicity is pathogenic.

Keywords: glutamate carboxypepptidase II, glutamate excitotoxicity, ischemia-reperfusion, retinal ganglion cell

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Authors: Abdulkarem Qasem Dammag, Basema Qasim Dammag, Jian Dai

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Change in Land use and Land cover (LU/LC) has a profound impact on the area's natural, economic, and ecological development, and the search for drivers of land cover change is one of the fundamental issues of LU/LC change. The study aimed to assess the temporal and Spatio-temporal dynamics of LU/LC in the past and to predict the future using Landsat images by exploring the characteristics of different LU/LC types. Spatio-temporal patterns of LU/LC change in Ibb Governorate, Yemen, were analyzed based on RS and GIS from 1990, 2005, and 2020. A socioeconomic survey and key informant interviews were used to assess potential drivers of LU/LC. The results showed that from 1990 to 2020, the total area of vegetation land decreased by 5.3%, while the area of barren land, grassland, built-up area, and waterbody increased by 2.7%, 1.6%, 1.04%, and 0.06%, respectively. Based on socio-economic surveys and key informant interviews, natural factors had a significant and long-term impact on land change. In contrast, site construction and socio-economic factors were the main driving forces affecting land change in a short time scale. The analysis results have been linked to the CA-Markov Land Use simulation and forecasting model for the years 2035 and 2050. The simulation results revealed from the period 2020 to 2050, the trend of dynamic changes in land use, where the total area of barren land decreased by 7.0% and grassland by 0.2%, while the vegetation land, built-up area, and waterbody increased by 4.6%, 2.6%, and 0.1 %, respectively. Overall, these findings provide LULC's past and future trends and identify drivers, which can play an important role in sustainable land use planning and management by balancing and coordinating urban growth and land use and can also be used at the regional level in different levels to provide as a reference. In addition, the results provide scientific guidance to government departments and local decision-makers in future land-use planning through dynamic monitoring of LU/LC change.

Keywords: LU/LC change, CA-Markov model, driving forces, change detection, LU/LC change simulation

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Authors: Y. Landkocz, C. Lepers, P.J. Martin, B. Fougère, F. Roy Saint-Georges. A. Verdin, F. Cazier, F. Ledoux, D. Courcot, F. Sichel, P. Gosset, P. Shirali, S. Billet

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In 2013, the International Agency for Research on Cancer (IARC) classified air pollution and fine particles as carcinogenic to humans. Causal relationships exist between elevated ambient levels of airborne particles and increase of mortality and morbidity including pulmonary diseases, like lung cancer. However, due to a double complexity of both physicochemical Particulate Matter (PM) properties and tumor mechanistic processes, mechanisms of action remain not fully elucidated. Furthermore, because of several common properties between air pollution PM and tobacco smoke, like the same route of exposure and chemical composition, potential mechanisms of synergy could exist. Therefore, smoking could be an aggravating factor of the particles toxicity. In order to identify some mechanisms of action of particles according to their size, two samples of PM were collected: PM0.03 2.5 and PM0.33 2.5 in the urban-industrial area of Dunkerque. The overall cytotoxicity of the fine particles was determined on human bronchial cells (BEAS-2B). Toxicological study focused then on the metabolic activation of the organic compounds coated onto PM and some genetic and epigenetic changes induced on a co-culture model of BEAS-2B and alveolar macrophages isolated from bronchoalveolar lavages performed in smokers and non-smokers. The results showed (i) the contribution of the ultrafine fraction of atmospheric particles to genotoxic (eg. DNA double-strand breaks) and epigenetic mechanisms (eg. promoter methylation) involved in tumor processes, and (ii) the influence of smoking on the cellular response. Three main conclusions can be discussed. First, our results showed the ability of the particles to induce deleterious effects potentially involved in the stages of initiation and promotion of carcinogenesis. The second conclusion is that smoking affects the nature of the induced genotoxic effects. Finally, the in vitro developed cell model, using bronchial epithelial cells and alveolar macrophages can take into account quite realistically, some of the existing cell interactions existing in the lung.

Keywords: air pollution, fine and ultrafine particles, genotoxic and epigenetic alterations, smoking

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Authors: Brooke Sweeney, David White, Felix Amparano, Nick A. Clark, Amy R. Beck, Mathew Lindquist, Lora Edwards, Julie Vandal, Jennifer Lisondra, Katie Cox, Renee Arensberg, Allen Cummins, Jazmine Cedeno, Jason D. Fraser, Kelsey Dean, Helena H. Laroche, Cristina Fernandez

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Background: National organizations call for standardized pre-surgical requirements and education to optimize postoperative outcomes. Since 2017 our surgery program has used defined protocols and educational curricula pre- and post-surgery. In response to patient outcomes, our educational content was refined to include quizzes to assess patient knowledge and surgical preparedness. We aim to optimize adolescent pre-bariatric surgery preparedness by improving overall aggregate pre-surgical assessment performance from 68% to 80% within 12 months. Methods: A multidisciplinary improvement team was developed within the weight management clinic (WMC) of our tertiary care, free-standing children’s hospital. A manual has been utilized since 2017, with limitations in consistent delivery and patient uptake of information. The curriculum has been improved to include quizzes administered during WMC visits prior to bariatric surgery. The initial outcome measure is the pre-surgical quiz score of adolescents preparing for bariatric surgery. Process measure was the number of questions answered correctly to test the questions. Baseline performance was determined by a patient assessment survey of pre-surgical preparedness at patient visits. Plan-Do-Study-Act cycles (PDSA) included: 1) creation and implementation of a refined curriculum, 2) development of 5 new quizzes based upon learning objectives, and 3) improving provider-lead teaching and quiz administration within clinic workflow. Run charts assessed impact over time. Results: A total of 346 quiz questions were administered to 34 adolescents. The outcome measure improved from a baseline mean of 68% to 86% following PDSA 2 cycles, and it was sustained. Conclusion/Implication: Patient/family comprehension of surgical preparedness improved with standardized education via team member-led teaching and assessment using quizzes during pre-surgical clinic visits. The next steps include launching redesigned teaching materials with modules correlated to quizzes and assessment of comprehension and outcomes post-surgically.

Keywords: bariatric surgery, adolescent, clinic, pre-bariatric training

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Authors: Ludmila A. Gavriliuc, Jerry McLarty, Heather E. Kleiner, J. Michael Mathis

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Abstract -- Background: The citrus coumarine Auraptene (Aur) is an effective chemopreventive agent, as manifested in many models of diseases and cancer. Nuclear factor erythroid 2-related factor (Nrf2) is an important regulator of genes induced by oxidative stress, such as glutathione S-transferases, heme oxygenase-1, and peroxiredoxin 1, by activating the antioxidant response element (ARE). Genetic and biochemical evidence has demonstrated that glutathione (GSH) and glutathione-dependent enzymes, glutathione reductase (GR), glutathione peroxidases (GPs), glutathione S-transferases (GSTs) are responsible for the control of intracellular reduction-oxidation status and participate in cellular adaptation to oxidative stress. The effect of Aur on the activity of GR, GPs (Se-GP and Se-iGP), and content of GSH in the liver, kidney, and spleen is insufficiently explored. Aim: Our goal was the examination of the Aur influence on the redox-system of GSH in Nrf2 wild type and Nrf2 knockout mice via activation of Nrf2 and ARE. Methods: Twenty female mice, 10 Nrf2 wild-type (WT) and 10 Nrf2 (-/-) knockout (KO), were bred and genotyped for our study. The activity of GR, Se-GP, Se-iGP, GST, G6PD, CytP450 reductase, catalase (Cat), and content of GSH were analyzed in the liver, kidney, and spleen using Spectrophotometry methods. The results of the specific activity of enzymes and the amount of GSH were analyzed with ANOVA and Spearman statistical methods. Results: Aur (200 mg/kg) treatment induced hepatic GST, GR, Se-GP activity and inhibited their activity in the spleen of mice, most likely via activation of the ARE through Nrf2. Activation in kidney Se-GP and G6PD by Aur is also controlled, apparently through Nrf2. Results of the non-parametric Spearman correlation analysis indicated the strong positive correlation between GR and G6PD only in the liver in WT control mice (r=+0.972; p < 0.005) and in the kidney KO control mice (r=+0.958; p < 0.005). The observed low content of GSH in the liver of KO mice indicated an increase in its participation in the neutralization of toxic substances with the absence of induction of GSH-dependent enzymes, such as GST, GR, Se-GP, and Se-iGP. Activation of CytP450 in kidney and spleen and Cat in the liver in KO mice probably revealed another regulatory mechanism for these enzymes. Conclusion: Thereby, obtained results testify that Aur can modulate the activity of genes and antioxidant enzymatic redox-system of GSH, responsible for the control of intracellular reduction-oxidation status.

Keywords: auraptene, glutathione, GST, Nrf2

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Authors: Saeed Bagherifam, Trevor Brown, Chris Fellows, Ravi Naidu

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Over the last century, rapid urbanization, industrial emissions, and mining activities have resulted in widespread contamination of the environment by heavy metal(loid)s. Arsenic (As) is a toxic metalloid belonging to group 15 of the periodic table, which occurs naturally at low concentrations in soils and the earth’s crust, although concentrations can be significantly elevated in natural systems as a result of dispersion from anthropogenic sources, e.g., mining activities. Bioavailability is the fraction of a contaminant in soils that is available for uptake by plants, food chains, and humans and therefore presents the greatest risk to terrestrial ecosystems. Numerous attempts have been made to establish in situ and ex-situ technologies of remedial action for remediation of arsenic-contaminated soils. In situ stabilization techniques are based on deactivation or chemical immobilization of metalloid(s) in soil by means of soil amendments, which consequently reduce the bioavailability (for biota) and bioaccessibility (for humans) of metalloids due to the formation of low-solubility products or precipitates. This study investigated the effectiveness of two different types of synthetic manganese and iron oxides (birnessite and goethite) for stabilization of As in a soil spiked with 1000 mg kg⁻¹ of As and treated with 10% dosages of soil amendments. Birnessite was made using HCl and KMnO₄, and goethite was synthesized by the dropwise addition of KOH into Fe(NO₃) solution. The resulting contaminated soils were subjected to a series of chemical extraction studies including sequential extraction (BCR method), single-step extraction with distilled (DI) water, 2M HNO₃ and simplified bioaccessibility extraction tests (SBET) for estimation of bioaccessible fractions of As in two different soil fractions ( < 250 µm and < 2 mm). Concentrations of As in samples were measured using inductively coupled plasma mass spectrometry (ICP-MS). The results showed that soil with birnessite reduced bioaccessibility of As by up to 92% in both soil fractions. Furthermore, the results of single-step extractions revealed that the application of both birnessite and Goethite reduced DI water and HNO₃ extractable amounts of arsenic by 75, 75, 91, and 57%, respectively. Moreover, the results of the sequential extraction studies showed that both birnessite and goethite dramatically reduced the exchangeable fraction of As in soils. However, the amounts of recalcitrant fractions were higher in birnessite, and Goethite amended soils. The results revealed that the application of both birnessite and goethite significantly reduced bioavailability and the exchangeable fraction of As in contaminated soils, and therefore birnessite and Goethite amendments might be considered as promising adsorbents for stabilization and remediation of As contaminated soils.

Keywords: arsenic, bioavailability, in situ stabilisation, metalloid(s) contaminated soils

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Authors: Foong Kiew Ooi, Aidi Naim Mohamad Samsani, Chee Keong Chen, Mohamed Saat Ismail

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Introduction: Prolonged exercise in a hot and humid environment can result in glycogen depletion and associated with loss of body fluid. Carbohydrate contained in sports beverages is beneficial for improving sports performance and preventing dehydration. Carbohydrate contained in honey is believed can be served as an alternative form of carbohydrate for enhancing sports performance. Objective: To investigate the effectiveness of honey drink compared to sports drink as a recovery aid for running performance and physiological parameters in the heat. Method: Ten male recreational athletes (age: 22.2 ± 2.0 years, VO2max: 51.5 ± 3.7 ml.kg-1.min-1) participated in this randomized cross-over study. On each trial, participants were required to run for 1 hour in the glycogen depletion phase (Run-1), followed by a rehydration phase for 2 hours and subsequently a 20 minutes time trial performance (Run-2). During Run-1, subjects were required to run on the treadmill in the heat (31°C) with 70% relative humidity at 70 % of their VO2max. During rehydration phase, participants drank either honey drink or sports drink, or plain water with amount equivalent to 150% of body weight loss in dispersed interval (60 %, 50 % and 40 %) at 0 min, 30 min and 60 min respectively. Subsequently, time trial was performed by the participants in 20 minutes and the longest distance covered was recorded. Physiological parameters were analysed using two-way ANOVA with repeated measure and time trial performance was analysed using one-way ANOVA. Results: Result showed that Acacia honey elicited a better time trial performance with significantly longer distance compared to water trial (P<0.05). However, there was no significant difference between Acacia honey and sport drink trials (P > 0.05). Acacia honey and sports drink trials elicited 249 m (8.24 %) and 211 m (6.79 %) longer in distance compared to the water trial respectively. For physiological parameters, plasma glucose, plasma insulin and plasma free fatty acids in Acacia honey and sports drink trials were significantly higher compared to the water trial respectively during rehydration phase and time trial running performance phase. There were no significant differences in body weight changes, oxygen uptake, hematocrit, plasma volume changes and plasma cortisol in all the trials. Conclusion: Acacia honey elicited greatest beneficial effects on sports performance among the drinks, thus it has potential to be used for rehydration in athletes who train and compete in hot environment.

Keywords: honey drink, rehydration, sports performance, plasma glucose, plasma insulin, plasma cortisol

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Authors: Xiaojie Cheng, Ulrike Frank, Feng Zhao, Karin Pritsch

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Ragweed (Ambrosia artemisiifolia) is an invasive weed that has become an increasing global problem. In addition to affecting land use and crop yields, ragweed has a strong impact on human health as it produces highly allergenic pollen. Global warming will result in an earlier and longer pollen season enhanced pollen production and an increase in pollen allergenicity with a negative effect on atopic patients. The aims of this study were to investigate the effects of increasing temperature, the future climate scenario in the Munich area, southern Germany, predicted on the basis of RCP8.5 until the end of 2050s, or/and NO₂, a major air pollutant, 1) on the vegetative and reproductive characteristics of ragweed plants, 2) on the total allergenicity of ragweed pollen, 3) on the total pollen proteomic patterns. Ragweed plants were cultivated for the whole plant vegetation period under controlled conditions either under ambient climate conditions or 4°C higher temperatures with or without additional NO₂. Higher temperature resulted in bigger plant sizes, longer male inflorescences, and longer pollen seasons. The total allergenic potential of the pollen was accessed by dot blot using serum from ragweed pollen sensitized patients. The comparative immunoblot analysis revealed that the in vivo fumigation of ragweed plants with elevated NO₂-concentrations significantly increased the allergenic potential of the pollen, and in combination with increased temperature, the allergenic potential was even higher. On the other hand, label-free protein quantification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed. The results showed that more proteins were significantly up- and down-regulated under higher temperatures with/without elevated NO₂ conditions. Most of the highly expressed proteins were participating intensively in the metabolic process, the cellular process, and the stress defense process. These findings suggest that rising temperature and elevated NO₂ are important environmental factors for higher abiotic stress activities, catalytic activities, and thus higher allergenic potential observed in pollen proteins.

Keywords: climate change, NO₂, pollen proteome, ragweed, temperature

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Authors: Rasha Al-Saedi, Anthony Meijer

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The progress of new porous materials has increased rapidly over the past decade for use in applications such as catalysis, gas storage and removal of environmentally unfriendly species due to their high surface area and high thermal stability. In this work, a theoretical study of the zirconium-based metal organic framework (MOFs) were examined in order to determine their potential for gas adsorption of various guest molecules: CO2, N2, CH4 and H2. The zirconium cluster consists of an inner Zr6O4(OH)4 core in which the triangular faces of the Zr6- octahedron are alternatively capped by O and OH groups which bound to nine formate groups and three benzoate groups linkers. General formula is [Zr(μ-O)4(μ-OH)4(HCOO)9((phyO2C)3X))] where X= CH2OH, CH2NH2, CH2CONH2, n(NH2); (n = 1-3). Three types of adsorption sites on the Zr metal center have been studied, named according to capped chemical groups as the ‘−O site’; the H of (μ-OH) site removed and added to (μ-O) site, ‘–OH site’; (μ-OH) site removed, the ‘void site’ where H2O molecule removed; (μ-OH) from one site and H from other (μ-OH) site, in addition to no defect versions. A series of investigations have been performed aiming to address this important issue. First, density functional theory DFT-B3LYP method with 6-311G(d,p) basis set was employed using Gaussian 09 package in order to evaluate the gas adsorption performance of missing-linker defects in zirconium cluster. Next, study the gas adsorption behaviour on different functionalised zirconium clusters. Those functional groups as mentioned above include: amines, alcohol, amide, in comparison with non-substitution clusters. Then, dispersion-corrected density functional theory (DFT-D) calculations were performed to further understand the enhanced gas binding on zirconium clusters. Finally, study the water effect on CO2 and N2 adsorption. The small functionalized Zr clusters were found to result in good CO2 adsorption over N2, CH4, and H2 due to the quadrupole moment of CO2 while N2, CH4 and H2 weakly polar or non-polar. The adsorption efficiency was determined using the dispersion method where the adsorption binding improved as most of the interactions, for example, van der Waals interactions are missing with the conventional DFT method. The calculated gas binding strengths on the no defect site are higher than those on the −O site, −OH site and the void site, this difference is especially notable for CO2. It has been stated that the enhanced affinity of CO2 of no defect versions is most likely due to the electrostatic interactions between the negatively charged O of CO2 and the positively charged H of (μ-OH) metal site. The uptake of the gas molecule does not enhance in presence of water as the latter binds to Zr clusters more strongly than gas species which attributed to the competition on adsorption sites.

Keywords: density functional theory, gas adsorption, metal- organic frameworks, molecular simulation, porous materials, theoretical chemistry

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Authors: Kamila Dus-Szachniewicz, Artur Bednarkiewicz, Katarzyna Gdesz-Birula, Slawomir Drobczynski

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In modern oncology hyperthermia (HT) is defined as a controlled tumor heating. HT treatment temperatures range between 40–48 °C and can selectively damage heat-sensitive cancer cells or limit their further growth, usually with minimal injury to healthy tissues. Despite many advantages, conventional whole-body and regional hyperthermia have clinically relevant side effects, including cardiac and vascular disorders. Additionally, the lack of accessibility of deep-seated tumor sites and impaired targeting micrometastases renders HT less effective. It is believed that above disadvantages can significantly overcome by the application of biofunctionalized microparticles, which can specifically target tumor sites and become activated by an external stimulus to provide a sufficient cellular response. In our research, the unique optical tweezers system have enabled capturing the silica microparticles, primary cells and tumor spheroids in highly controllable and reproducible environment to study the impact of localized heat stimulation on normal and pathological cell and within multicellular tumor spheroid. High throughput spheroid model was introduced to better mimic the response to HT treatment on tumors in vivo. Additionally, application of local heating of tumor spheroids was performed in strictly controlled conditions resembling tumor microenvironment (temperature, pH, hypoxia, etc.), in response to localized and nonhomogeneous hyperthermia in the extracellular matrix, which promotes tumor progression and metastatic spread. The lack of precise control over these well- defined parameters in basic research leads to discrepancies in the response of tumor cells to the new treatment strategy in preclinical animal testing. The developed approach enables also sorting out subclasses of cells, which exhibit partial or total resistance to therapy, in order to understand fundamental aspects of the resistance shown by given tumor cells in response to given therapy mode and conditions. This work was funded by the National Science Centre (NCN, Poland) under grant no. UMO-2017/27/B/ST7/01255.

Keywords: cancer spheroids, hyperthermia, microparticles, optical tweezers

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Authors: Mauricio A. Sarabia-Vallejos, Carmen M. Gonzalez-Henriquez, Adolfo Del Campo-Garcia, Aitzibier L. Cortajarena, Juan Rodriguez-Hernandez

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In this study, it was explored the formation and the morphological differences between wrinkled hydrogel patterns obtained via generation of surface instabilities. The slight variations in the polymerization conditions produce important changes in the material composition and pattern structuration. The compounds were synthesized using three main components, i.e. an amphiphilic monomer, hydroxyethyl methacrylate (HEMA), a hydrophobic monomer, trifluoroethyl methacrylate (TFMA), and a hydrophilic crosslinking agent, poly(ethylene glycol) diacrylate (PEGDA). The first part of this study was related to the formation of wrinkled surfaces using only HEMA and PEGDA and varying the amount of water added in the reaction. The second part of this study involves the gradual insertion of TFMA into the hydrophilic reaction mixture. Interestingly, the manipulation of the chemical composition of this hydrogel affects both surface morphology and physicochemical characteristics of the patterns, inducing transitions from one particular type of structure (wrinkles or ripples) to different ones (creases, folds, and crumples). Contact angle measurements show that the insertion of TFMA produces a slight decrease in surface wettability of the samples, remaining however highly hydrophilic (contact angle below 45°). More interestingly, by using confocal Raman spectroscopy, important information about the wrinkle formation mechanism is obtained. The procedure involving two consecutive thermal and photopolymerization steps lead to a “pseudo” two-layer system. Thus, upon photopolymerization, the surface is crosslinked to a higher extent than the bulk and water evaporation drives the formation of wrinkled surfaces. Finally, cellular, and bacterial proliferation studies were performed to the samples, showing that the amount of TFMA included in each sample slightly affects the proliferation of both (bacteria and cells), but in the case of bacteria, the morphology of the sample also plays an important role, importantly reducing the bacterial proliferation.

Keywords: antibiofouling properties, hydrophobic/hydrophilic balance, morphologic characterization, wrinkled hydrogel patterns

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Authors: Kumari Rekha, Mathur K Dhananjay, Maheshwari Deepanshu, Nautiyal Nidhi, Shubham Smriti, Laal Deepika, Sinha Swati, Kumar Anupam, Biswas Subhrajit, Shiv Kumar Sarin

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Background: Mesenchymal stem cells are multipotent stem cells derived from mesoderm and are used for therapeutic purposes because of their self-renewal, homing capacity, Immunomodulatory capability, low immunogenicity and mitochondrial transfer signaling. MSCs have the ability to regulate the mechanism of both innate as well as adaptive immune responses through the modulation of cellular response and the secretion of inflammatory mediators. Different sources of MSC are UC MSC, BM MSC, Dental Pulp, and Adipose MSC. The most frequent source used is umbilical cord tissue due to its being easily available and free of limitations of collection procedures from respective hospitals. The immunosuppressive role of MSCs is particularly interesting for clinical use since it confers resistance to rejection by the host immune response. Methodology: In this study, T helper cells (TH4), Cytotoxic T cells (CD-8), immunoregulatory cells (CD25 +FOXP3+) are compared from isolated MSC from three different methods, UC Dissociation Kit (Miltenyi), Explant Culture and Collagenase Type-IV. To check the immunomodulatory property, these MSCs were seeded with PBMC(Coculture) in CD3 coated 24 well plates. Cd28 antibody was added in coculture for six days. The coculture was analyzed in FACS Verse flow cytometry. Results: From flow cytometry analysis of coculture, it found that All over T helper cells (CD4+) number p<0.0264 increases in (All Enzymes) MSC rather than explant MSC(p>0.0895) as compared to Collagenase(p>0.7889) in a coculture of Activated T cell and Mesenchymal Stem Cell. Similar T reg cells (CD25+, FOXP3+) expression p<0.0234increases in All Enzymes), decreases in Explant and Collagenase. Experiments have shown that MSCs can also directly prevent the cytotoxic activity of CD8 lymphocytes mainly by blocking their proliferation rather than by inhibiting the cytotoxic effect. And promoting the t-reg cells, which helps in the mediation of immune response in various diseases. Conclusion: MSC suppress Cytotoxic CD8 T cell and Enhance immunoregulatory T reg (CD4+, CD25+, FOXP3+) Cell expression. Thus, MSC maintains a proper balance(ratio) between CD4 T cells and Cytotoxic CD8 T cells.

Keywords: MSC, disease, T cell, T regulatory

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Authors: Epameinondas Xanthakis, Erik Kaunisto, Alain Le-Bail, Lilia Ahrné

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Fruits and vegetables are characterized as perishable food matrices due to their short shelf life as several deterioration mechanisms are being involved. Prior to the common preservation methods like freezing or canning, fruits and vegetables are being blanched in order to inactivate deteriorative enzymes. Both conventional blanching pretreatments and conventional freezing methods hide drawbacks behind their beneficial impacts on the preservation of those matrices. Conventional blanching methods may require longer processing times, leaching of minerals and nutrients due to the contact with the warm water which in turn leads to effluent production with large BOD. An important issue of freezing technologies is the size of the formed ice crystals which is also critical for the final quality of the frozen food as it can cause irreversible damage to the cellular structure and subsequently to degrade the texture and the colour of the product. Herein, the developed microwave blanching methodology and the results regarding quality aspects and enzyme inactivation will be presented. Moreover, heat transfer phenomena, mass balance, temperature distribution, and enzyme inactivation (such as Pectin Methyl Esterase and Ascorbic Acid Oxidase) of our microwave blanching approach will be evaluated based on measurements and computer modelling. The present work is part of the COLDμWAVE project which aims to the development of an innovative environmentally sustainable process for blanching and freezing of fruits and vegetables with improved textural and nutritional quality. In this context, COLDµWAVE will develop tailored equipment for MW blanching of vegetables that has very high energy efficiency and no water consumption. Furthermore, the next steps of this project regarding the development of innovative pathways in MW assisted freezing to improve the quality of frozen vegetables, by exploring in depth previous results acquired by the authors, will be presented. The application of MW assisted freezing process on fruits and vegetables it is expected to lead to improved quality characteristics compared to the conventional freezing. Acknowledgments: COLDμWAVE has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grand agreement No 660067.

Keywords: blanching, freezing, fruits, microwave blanching, microwave

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Authors: Andleeb Zahra, Itrat Rubab, Sumaira Malik, Amina Khan, Muhammad Jawad Khan, M. Qaiser Fatmi

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Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide. Recent studies have highlighted the role of miRNA in disease pathology, indicating its potential use in an early diagnostic tool. miRNAs are small, double stranded, non-coding RNAs that regulate gene expression by deregulating mRNAs. miRNAs play important roles in modifying various cellular processes such as cell growth, differentiation, apoptosis, and immune response. Dis-regulated expression of miRNAs is known to affect the cell growth, and this may function as tumor suppressors or oncogenes in various cancers. Objectives: The main objectives of this study were to characterize the extracellular miRNAs involved in oral cancer (OC) to assist early detection of cancer as well as to propose a list of genes that can potentially be used as biomarkers of OC. We used gene expression data by microarrays already available in literature. Materials and Methods: In the first step, a total of 318 miRNAs involved in oral carcinoma were shortlisted followed by the prediction of their target genes. Simultaneously, the differentially expressed genes (DEGs) of oral carcinoma from all experiments were identified. The common genes between lists of DEGs of OC based on experimentally proven data and target genes of each miRNA were identified. These common genes are the targets of specific miRNA, which is involved in OC. Finally, a list of genes was generated which may be used as biomarker of OC. Results and Conclusion: In results, we included some of pathways in cancer to show the change in gene expression under the control of specific miRNA. Ingenuity pathway analysis (IPA) provided a list of major biomarkers like CDH2, CDK7 and functional enrichment analysis identified the role of miRNA in major pathways like cell adhesion molecules pathway affected by cancer. We observed that at least 25 genes are regulated by maximum number of miRNAs, and thereby, they can be used as biomarkers of OC. To better understand the role of miRNA with respect to their target genes further experiments are required, and our study provides a platform to better understand the miRNA-OC relationship at genomics level.

Keywords: biomarkers, gene expression, miRNA, oral carcinoma

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Authors: M. Gowri, E. K. Girija, V. Ganesh

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Background and Significance: Among the dental infections, inflammation and infection of the root canal are common among all age groups. Currently, the management of root canal infections involves cleaning the canal with powerful irrigants followed by intracanal medicament application. Though these treatments have been in vogue for a long time, root canal failures do occur. Treatment for root canal infections is limited due to the anatomical complexity in terms of small micrometer volumes and poor penetration of drugs. Thus, infections of the root canal seem to be a challenge that demands development of new agents that can eradicate C. albicans. Methodology: In the present study, we synthesized and screened silver-curcumin nanoparticle against Candida albicans. Detailed molecular studies were carried out with silver-curcumin nanoparticle on C. albicans pathogenicity. Morphological cell damage and antibiofilm activity of silver-curcumin nanoparticle on C. albicans was studied using scanning electron microscopy (SEM). Biochemical evidence for membrane damage was studied using flow cytometry. Further, the antifungal activity of silver-curcumin nanoparticle was evaluated in an ex vivo dentinal tubule infection model. Results: Screening data showed that silver-curcumin nanoparticle was active against C. albicans. Silver-curcumin nanoparticle exerted time kill effect and post antifungal effect. When used in combination with fluconazole or nystatin, silver-curcumin nanoparticle revealed a minimum inhibitory concentration (MIC) decrease for both drugs used. In-depth molecular studies with silver-curcumin nanoparticle on C. albicans showed that silver-curcumin nanoparticle inhibited yeast to hyphae (Y-H) conversion. Further, SEM images of C. albicans showed that silver-curcumin nanoparticle caused membrane damage and inhibited biofilm formation. Biochemical evidence for membrane damage was confirmed by increased propidium iodide (PI) uptake in flow cytometry. Further, the antifungal activity of silver-curcumin nanoparticle was evaluated in an ex vivo dentinal tubule infection model, which mimics human tooth root canal infection. Confocal laser scanning microscopy studies showed eradication of C. albicans and reduction in colony forming unit (CFU) after 24 h treatment in the infected tooth samples in this model. Conclusion: The results of this study can pave the way for developing new antifungal agents with well deciphered mechanisms of action and can be a promising antifungal agent or medicament against root canal infection.

Keywords: C. albicans, ex vivo dentine model, inhibition of biofilm formation, root canal infection, yeast to hyphae conversion inhibition

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Authors: Johanna Perdomo, Riki Lamont, Edmund Pickering, Naomi C. Paxton, Maria A. Woodruff

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Background: Tissue Engineering (TE) has combined advanced materials, such as biomaterials, to create affordable scaffolds and dynamic systems to generate stimulation of seeded cells on these scaffolds, improving and maintaining the cellular growth process in a cell culture. However, Few TE skin products have been clinically translated, and more research is required to produce highly biomimetic skin substitutes that mimic the native elasticity of skin in a controlled manner. Therefore, this work will be focused on the fabrication of a novel mechanical system to enhance the TE treatment approaches for the reparation of damaged tissue skin. Aims: To archive this, a soft robotic device will be created to emulate different deformation of skin stress. The design of this soft robot will allow the attachment of scaffolds, which will then be mechanically actuated. This will provide a novel and highly adaptable platform for dynamic cell culture. Methods: Novel, low-cost soft robot is fabricated via 3D printed moulds and silicone. A low cost, electro-mechanical device was constructed to actuate the soft robot through the controlled combination of positive and negative air pressure to control the different state of movements. Mechanical tests were conducted to assess the performance and calibration of each electronic component. Similarly, pressure-displacement test was performed on scaffolds, which were attached to the soft robot, applying various mechanical loading regimes. Lastly, digital image correlation test was performed to obtain strain distributions over the soft robot’s surface. Results: The control system can control and stabilise positive pressure changes for long hours. Similarly, pressure-displacement test demonstrated that scaffolds with 5µm of diameter and wavy geometry can displace at 100%, applying a maximum pressure of 1.5 PSI. Lastly, during the inflation state, the displacement of silicone was measured using DIC method, and this showed a parameter of 4.78 mm and strain of 0.0652. Discussion And Conclusion: The developed soft robot system provides a novel and low-cost platform for the dynamic actuation of tissue scaffolds with a target towards dynamic cell culture.

Keywords: soft robot, tissue engineering, mechanical stimulation, dynamic cell culture, bioreactor

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Authors: Jeroen Rotteveel, Zeger De Groot

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The small satellite community is more and more aware of the need to start operating responsibly and sustainably in order to secure the use of outer space in the long run. On the technical side, many debris mitigation techniques have been investigated and demonstrated on board small satellites, showing that technically, a lot of things can be done to curb the growth of space debris and operate more responsible. However, in the absence of strict laws and constraints, one cannot help but wonder what the incentive is to incur significant costs (paying for debris mitigation systems and the launch mass of these systems) and to lose performance onboard resource limited small satellites (mass, volume, power)? Many small satellite developers are operating under tight budgets, either from their sponsors (in case of academic and research projects) or from their investors (in case of startups). As long as it is not mandatory to act more responsibly, we might need to consider the implementation of incentives to stimulate developers to accommodate deorbiting modules, etc. ISISPACE joined the NetZeroSpace initiative in 2021 with the aim to play its role in secure the use of low earth orbit for the next decades by facilitating more sustainable use of space. The company is in a good position as both a satellite builder, a rideshare launch provider, and a technology development company. ISISPACE operates under one of the stricter space laws in the world in terms of maximum orbital lifetime and has been active in various debris mitigation and debris removal in-orbit demonstration missions in the past 10 years. ISISPACE proposes to introduce together with launch partners and regulators an incentive scheme for CubeSat developers to baseline debris mitigation systems on board their CubeSats in such a way that is does not impose too many additional costs to the project. Much like incentives to switch to electric cars or install solar panels on your house, such an incentive can help to increase market uptake of behavior or solutions prior to legislation or bans of certain practices. This can be achieved by: Introducing an extended launch volume in CubeSat deployers to accommodate debris mitigation systems without compromising available payload space for the payload of the main mission Not charging the fee for the launch mass for the additional debris mitigation module Whenever possible, find ways to further co-fund the purchase price, or otherwise reduce the cost of flying debris mitigation modules onboard the CubeSats. The paper will outline the framework of such an incentive scheme and provides ISISPACE’s way forward to make this happen in the near future.

Keywords: netZerospace, cubesats, debris mitigation, small satellite community

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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: Yunjia Yang, Peng Li, Gordon Xu, Timothy Mahony, Bing Zhang, Neena Mitter, Karishma Mody

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Sheep flystrike is one of the most economically important diseases affecting the Australian sheep and wool industry (>356M/annually). Currently, control of Lucillia cuprina relies almost exclusively on chemicals controls, and the parasite has developed resistance to nearly all control chemicals used in the past. It is, therefore, critical to develop an alternative solution for the sustainable control and management of flystrike. RNA interference (RNAi) technologies have been successfully explored in multiple animal industries for developing parasites controls. This research project aims to develop a RNAi based biological control for sheep blowfly. Double-stranded RNA (dsRNA) has already proven successful against viruses, fungi, and insects. However, the environmental instability of dsRNA is a major bottleneck for successful RNAi. Bentonite polymer (BenPol) technology can overcome this problem, as it can be tuned for the controlled release of dsRNA in the gut challenging pH environment of the blowfly larvae, prolonging its exposure time to and uptake by target cells. To investigate the potential of BenPol technology for dsRNA delivery, four different BenPol carriers were tested for their dsRNA loading capabilities, and three of them were found to be capable of affording dsRNA stability under multiple temperatures (4°C, 22°C, 40°C, 55°C) in sheep serum. Based on stability results, dsRNA from potential targeted genes was loaded onto BenPol carriers and tested in larvae feeding assays, three genes resulting in knockdowns. Meanwhile, a primary blowfly embryo cell line (BFEC) derived from L. cuprina embryos was successfully established, aim for an effective insect cell model for testing RNAi efficacy for preliminary assessments and screening. The results of this study establish that the dsRNA is stable when loaded on BenPol particles, unlike naked dsRNA rapidly degraded in sheep serum. The stable nanoparticle delivery system offered by BenPol technology can protect and increase the inherent stability of dsRNA molecules at higher temperatures in a complex biological fluid like serum, providing promise for its future use in enhancing animal protection.

Keywords: lucilia cuprina, primary cell line establishment, RNA interference, insect cell transfection

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Authors: Reshu Saxena, R. K. Tripathi

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HIV-1 transmission and spread involves significant host-virus interaction. Potential targets for prevention of HIV-1 lies at the site of mucosal barriers. Thus a better understanding of how HIV-1 infects target cells at such sites and lead their invasion is required, with prime focus on the host determinants regulating HIV-1 spread. HIV-1 Nef is important for viral infectivity and pathogenicity. It promotes HIV-1 replication, facilitating immune evasion by interacting with various host factors and altering cellular pathways via multiple protein-protein interactions. In this study nef was sequenced from HIV-1 patients, and showed specific mutations revealing sequence variability in nef. To explore the difference in Nef functionality based on sequence variability we have studied the effects of HIV-1 Nef in human SupT1 T cell line and (THP-1) monocyte-macrophage cell lines through proteomics approach. 2D-Gel Electrophoresis in control and Nef-transfected SupT1 cells demonstrated several differentially expressed proteins with significant modulation of alpha-enolase. Through further studies, effects of Nef on alpha-enolase regulation were found to be cell lineage-specific, being stimulatory in macrophages/monocytes, inhibitory in T cells and without effect in HEK-293 cells. Cell migration and invasion studies were employed to determine biological function affected by Nef mediated regulation of alpha-enolase. Cell invasion was enhanced in THP-1 cells but was inhibited in SupT1 cells by wildtype nef. In addition, the modulation of enolase and cell invasion remained unaffected by a unique nef variant. These results indicated that regulation of alpha-enolase expression and invasive property of host cells by Nef is sequence specific, suggesting involvement of a particular motif of Nef. To precisely determine this site, we designed a heptapeptide including the suggested alpha-enolase regulating sequence of nef and a nef mutant with deletion of this site. Macrophages/monocytes being the major cells affected by HIV-1 at mucosal barriers, were particularly investigated by the nef mutant and peptide. Both the nef mutant and heptapeptide led to inhibition of enhanced enolase expression and increased invasiveness in THP-1 cells. Together, these findings suggest a possible mechanism of host invasion by HIV-1 through Nef mediated regulation of alpha-enolase and identifies a potential therapeutic target for HIV-1 entry at mucosal barriers.

Keywords: HIV-1 Nef, nef variants, host-virus interaction, tissue invasion

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Authors: Bonnie Clough, Tamara Ownsworth, Vladimir Estivill-Castro, Matt Stainer, Rene Hexel, Andrew Bulmer, Wendy Moyle, Allison Waters, David Neumann, Jayke Bennett

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The success of global vaccination programs is reliant on the uptake of vaccines to achieve herd immunity. Yet, many individuals do not obtain vaccines or venipuncture procedures when needed. Whilst health education may be effective for those individuals who are hesitant due to safety or efficacy concerns, for many of these individuals, the primary concern relates to blood or injection fear or phobia (BII). BII is highly prevalent and associated with a range of negative health impacts, both at individual and population levels. Exposure therapy is an efficacious treatment for specific phobias, including BII, but has high patient dropout and low implementation by therapists. Whilst virtual reality approaches exposure therapy may be more acceptable, they have similarly low rates of implementation by therapists and are often difficult to tailor to an individual client’s needs. It was proposed that a piloted robot may be able to adequately facilitate fear induction and be an acceptable approach to exposure therapy. The current study examined fear induction responses, acceptability, and feasibility of a piloted robot for BII exposure. A Nao humanoid robot was programmed to connect with a virtual reality head-mounted display, enabling live streaming and exploration of real environments from a distance. Thirty adult participants with BII fear were randomly assigned to robot-pilot or virtual reality exposure conditions in a laboratory-based fear exposure task. All participants also completed a computer-based two-dimensional exposure task, with an order of conditions counterbalanced across participants. Measures included fear (heart rate variability, galvanic skin response, stress indices, and subjective units of distress), engagement with a feared stimulus (eye gaze: time to first fixation and a total number of fixations), acceptability, and perceived treatment credibility. Preliminary results indicate that fear responses can be adequately induced via a robot-piloted platform. Further results will be discussed, as will implications for the treatment of BII phobia and other fears. It is anticipated that piloted robots may provide a useful platform for facilitating exposure therapy, being more acceptable than in-vivo exposure and more flexible than virtual reality exposure.

Keywords: anxiety, digital mental health, exposure therapy, phobia, robot, virtual reality

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