Search results for: Elisa Valderrama

Authors: Iulianna Taritsa, Kuldeep Neote, Eric Fossel

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Lysosome-induced immunogenic cell death (LIICD) is a powerful mechanism of targeting cancer cells that kills circulating malignant cells and primes the host’s immune cells against future remission. Current immunotherapies for cancer are limited in preventing recurrence – a gap that can be bridged by training the immune system to recognize cancer neoantigens. Lysosomal leakage can be induced therapeutically to traffic antigens from dying cells to dendritic cells, which can later present those tumorigenic antigens to T cells. Previous research has shown that oxidative agents administered in the tumor microenvironment can initiate LIICD. We generated a fusion protein between an oxidative agent known as xanthine oxidase (XO) and a mini-antibody specific for EGFR/HER2-sensitive breast tumor cells. The anti-EGFR single domain antibody fragment is uniquely sourced from llama, which is functional without the presence of a light chain. These llama micro-antibodies have been shown to be better able to penetrate tissues and have improved physicochemical stability as compared to traditional monoclonal antibodies. We demonstrate that the fusion protein created is stable and can induce early markers of immunogenic cell death in an in vitro human breast cancer cell line (SkBr3). Specifically, we measured overall cell death, as well as surface-expressed calreticulin, extracellular ATP release, and HMGB1 production. These markers are consensus indicators of ICD. Flow cytometry, luminescence assays, and ELISA were used respectively to quantify biomarker levels between treated versus untreated cells. We also included a positive control group of SkBr3 cells dosed with doxorubicin (a known inducer of LIICD) and a negative control dosed with cisplatin (a known inducer of cell death, but not of the immunogenic variety). We looked at each marker at various time points after cancer cells were treated with the XO/antibody fusion protein, doxorubicin, and cisplatin. Upregulated biomarkers after treatment with the fusion protein indicate an immunogenic response. We thus show the potential for this fusion protein to induce an anticancer effect paired with an adaptive immune response against EGFR/HER2+ cells. Our research in human cell lines here provides evidence for the success of the same therapeutic method for patients and serves as the gateway to developing a new treatment approach against breast cancer.

Keywords: apoptosis, breast cancer, immunogenic cell death, lysosome

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Authors: I. Cerrillo, A. Carrillo-Vico, M. A. Ortega, B. Escudero-López, N. Álvarez-Sánchez, F. Martín, M. S. Fernández-Pachón

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Melatonin is a bioactive compound involved in multiple biological activities such as glucose tolerance, circadian rhythm regulation, antioxidant defense or immune system action. In elderly subjects the intake of foods and drinks rich in melatonin is very important due to its endogenous level decreases with age. Alcoholic fermentation is a process carried out in fruits, vegetables and legumes to obtain new products with improved bioactive compounds profile in relation to original substrates. Alcoholic fermentation process carried out by Saccharomycetaceae var. Pichia kluyveri induces an important synthesis of melatonin in orange juice. A novel beverage derived of fermented orange juice could be a promising source of this bioactive compound. The aim of the present study was to determine whether the acute intake of fermented orange juice increase the levels of urinary 6-sulfatoxymelatonin in healthy humans. Nine healthy volunteers (7 women and 2 men), aged between 20 and 25 years old and BMI of 21.1  2.4 kg/m2, were recruited. On the study day, participants ingested 500 mL of fermented orange juice. The first urine collection was made before fermented orange juice consumption (basal). The rest of urine collections were made in the following time intervals after fermented orange juice consumption: 0-2, 2-5, 5-10, 10- 15 and 15-24 hours. During the experimental period only the consumption of water was allowed. At lunch time a meal was provided (60 g of white bread, two slices of ham, a slice of cheese, 125 g of sweetened natural yoghurt and water). The subjects repeated the protocol with orange juice following a 2-wk washout period between both types of beverages. The levels of 6-sulfatoxymelatonin (6-SMT) were measured in urine recollected at different time points using the Melatonin-Sulfate Urine ELISA (IBL International GMBH, Hamburg, Germany). Levels of 6-SMT were corrected to those of creatinine for each sample. A significant (p < 0.05) increase in urinary 6-SMT levels was observed between 2-5 hours after fermented orange juice ingestion with respect to basal values (increase of 67,8 %). The consumption of orange juice did not induce any significant change in urinary 6-SMT levels. In addition, urinary 6-SMT levels obtained between 2-5 hours after fermented orange juice ingestion (115,6 ng/mg) were significantly different (p < 0.05) from those of orange juice (42,4 ng/mg). The enhancement of urinary 6-SMT after the ingestion of 500 mL of fermented orange juice in healthy humans compared to orange juice could be an important advantage of this novel product as an excellent source of melatonin. Fermented orange juice could be a new functional food, and its consumption could exert a potentially positive effect on health in both the maintenance of health status and the prevention of chronic diseases.

Keywords: fermented orange juice, functional beverage, healthy human, melatonin

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Authors: Hana Al-Baghaoi, Kumar Shiva Gubbiyappa, Mayuren Candasamy, Kiruthiga Perumal Vijayaraman

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Acne is a chronic inflammatory disease of the sebaceous glands characterized by areas of skin with seborrhea, comedones, papules, pustules, nodules, and possibly scarring. Propionibacterium acnes (P. acnes), plays a key role in the pathogenesis of acne. Their colonization and proliferation trigger the host’s inflammatory response leading to the production of pro-inflammatory cytokines such as interleukin-8 (IL-8) and tumour necrosis factor-α (TNF-α). The usage of honey and natural compounds to treat skin ailments has strong support in the current trend of drug discovery. The present study was carried out evaluate antimicrobial and anti-inflammatory potential of Doani Sidr honey and its fractions against P. acnes and to screen madecassoside alone and in combination with fractions of honey. The broth dilution method was used to assess the antibacterial activity. Also, ultra structural changes in cell morphology were studied before and after exposure to Sidr honey using transmission electron microscopy (TEM). The three non-toxic concentrations of the samples were investigated for suppression of cytokines IL 8 and TNF α by testing the cell supernatants in the co-culture of the human peripheral blood mononuclear cells (hPBMCs) heat killed P. acnes using enzyme immunoassay kits (ELISA). Results obtained was evaluated by statistical analysis using Graph Pad Prism 5 software. The Doani Sidr honey and polysaccharide fractions were able to inhibit the growth of P. acnes with a noteworthy minimum inhibitory concentration (MIC) value of 18% (w/v) and 29% (w/v), respectively. The proximity of MIC and MBC values indicates that Doani Sidr honey had bactericidal effect against P. acnes which is confirmed by TEM analysis. TEM images of P. acnes after treatment with Doani Sidr honey showed completely physical membrane damage and lysis of cells; whereas non honey treated cells (control) did not show any damage. In addition, Doani Sidr honey and its fractions significantly inhibited (> 90%) of secretion of pro-inflammatory cytokines like TNF α and IL 8 by hPBMCs pretreated with heat-killed P. acnes. However, no significant inhibition was detected for madecassoside at its highest concentration tested. Our results suggested that Doani Sidr honey possesses both antimicrobial and anti-inflammatory effects against P. acnes and can possibly be used as therapeutic agents for acne. Furthermore, polysaccharide fraction derived from Doani Sidr honey showed potent inhibitory effect toward P. acnes. Hence, we hypothesize that fraction prepared from Sidr honey might be contributing to the antimicrobial and anti-inflammatory activity. Therefore, this polysaccharide fraction of Doani Sidr honey needs to be further explored and characterized for various phytochemicals which are contributing to antimicrobial and anti-inflammatory properties.

Keywords: Doani sidr honey, Propionibacterium acnes, IL-8, TNF alpha

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Authors: Izuddin Fahmy Abu, Mohamad Haiqal Nizar Mohamad, Muhammad Fattah Fazel, Renu Agarwal, Igor Iezhitsa, Nor Salmah Bakar, Henrik Franzyk, Ian Mellor

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Glaucoma is the global leading cause of irreversible blindness. Currently, the available treatment strategy only involves lowering intraocular pressure (IOP); however, the condition often progresses despite lowered or normal IOP in some patients. N-methyl-D-aspartate receptor (NMDAR) excitotoxicity often occurs in neurodegeneration-related glaucoma; thus it is a relevant target to develop a therapy based on neuroprotection approach. This study investigated the effects of Philanthotoxin-343 (PhTX-343), an NMDAR antagonist, on the neuroprotection of NMDA-induced glaucoma to alleviate visual impairments. Male Sprague-Dawley rats were equally divided: Groups 1 (control) and 2 (glaucoma) were intravitreally injected with phosphate buffer saline (PBS) and NMDA (160nM), respectively, while group 3 was pre-treated with PhTX-343 (160nM) 24 hours prior to NMDA injection. Seven days post-treatments, rats were subjected to visual behavior assessments and subsequently euthanized to harvest their retina and optic nerve tissues for histological analysis and determination of nitrosative stress level using 3-nitrotyrosine ELISA. Visual behavior assessments via open field, object, and color recognition tests demonstrated poor visual performance in glaucoma rats indicated by high exploratory behavior. PhTX-343 pre-treatment appeared to preserve visual abilities as all test results were significantly improved (p < 0.05). H&E staining of the retina showed a marked reduction of ganglion cell layer thickness in the glaucoma group; in contrast, PhTX-343 significantly increased the number by 1.28-folds (p < 0.05). PhTX-343 also increased the number of cell nuclei/100μm2 within inner retina by 1.82-folds compared to the glaucoma group (p < 0.05). Toluidine blue staining of optic nerve tissues showed that PhTX-343 reduced the degeneration changes compared to the glaucoma group which exhibited vacuolation overall sections. PhTX-343 also decreased retinal 3- nitrotyrosine concentration by 1.74-folds compared to the glaucoma group (p < 0.05). All results in PhTX-343 group were comparable to control (p > 0.05). We conclude that PhTX-343 protects against NMDA-induced changes and visual impairments in the rat model by reducing nitrosative stress levels.

Keywords: excitotoxicity, glaucoma, nitrosative stress , NMDA receptor , N-methyl-D-aspartate , philanthotoxin, visual behaviour

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Authors: Hung-Chih Hsu, Pey-Jium Chang, Ching-Hsein Chen, Jer-Liang Andrew Yeh

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Osteoarthritis (OA) is a common disorder in rehabilitation clinic. The main characteristics include joint pain, localized tenderness and enlargement, joint effusion, cartilage destruction, loss of adhesion of perichondrium, synovium hyperplasia. Synoviocytes inflammation might be a cause of local tenderness and effusion. Inflammation cytokines might also play an important role in joint pain, cartilage destruction, decrease adhesion of perichondrium to the bone. Treatments of osteoarthritis include non-steroid anti-inflammation drugs (NSAID), glucosamine supplementation, hyaluronic acid, arthroscopic debridement, and total joint replacement. Total joint replacement is commonly used in patients with severe OA who failed respond to pharmacological treatment. However, some patients received surgery had serious adverse events, including instability of the implants due to insufficient adhesion to the adjacent bony tissue or synovial inflammation. We tried to develop ideal nano-topographic oxidized silicon nanosponges by using with various chemicals to produce thickness difference in nanometers in order to study more about the cell-environment interactions in vitro like the alterations of cell adhesion, morphology, extracellular matrix secretions in the pathogenesis of osteoarthritis. Cytokines studies like growth factor, reactive oxygen species, reactive inflammatory materials (Like nitrous oxide and prostaglandin E2), extracellular matrix (ECM) degradation enzymes, and synthesis of collagen will also be observed and discussed. Extracellular and intracellular expression transforming growth factor beta (TGF-β) will be studied by reverse transcription-polymerase chain reaction (RT-PCR). The degradation of ECM will be observed by the bioactivity ratio of matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase by ELISA (Enzyme-linked immunosorbent assay). When rabbit synoviocytes were cultured on these nano-topographic structures, they demonstrate better cell adhesion rate, decreased expression of MMP-2,9 and PGE2, and increased expression of TGF-β when cultured in nano-topographic oxidized silicon nanosponges than in the planar oxidized silicon ones. These results show cell behavior, cytokine production can be influenced by physical characteristics from different nano-topographic structures. Our study demonstrates the possibility of manipulating cell behavior in these nano-topographic biomaterials.

Keywords: osteoarthritis, synoviocyte, oxidized silicon surfaces, reactive oxygen species

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Authors: Pakize Neslihan Taslı, Alev Cumbul, Gul Merve Yalcın, Fikrettin Sahin

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A key experimental trial in the regeneration of large oral and craniofacial defects is the neogenesis of osseous and ligamentous interfacial structures. Currently, oral regenerative medicine strategies are unpredictable for repair of tooth supporting tissues destroyed as a consequence of trauma, chronic infection or surgical resection. A different approach combining the gel-casting method with Hydroxy Apatite HA-based scaffold and different cell lineages as a hybrid system leads to successively mimic the early stage of tooth development, in vitro. HA is widely accepted as a bioactive material for guided bone and tooth regeneration. In this study, it was reported that, HA porous scaffold preparation, characterization and evaluation of structural and chemical properties. HA is the main factor that exists in tooth and it is in harmony with structural, biological, and mechanical characteristics. Here, this study shows mimicking immature tooth at the late bell stage design and construction of HA scaffolds for cell transplantation of human Adipose Stem Cells (hASCs), human Bone Marrow Stem Cells (hBMSCs) and Gingival Epitelial cells for the formation of human tooth dentin-pulp-enamel complexes in vitro. Scaffold characterization was demonstrated by SEM, FTIR and pore size and density measurements. The biological contraction of dental tissues against each other was demonstrated by mRNA gene expressions, histopatologic observations and protein release profile by ELISA tecnique. The tooth shaped constructs with a pore size ranging from 150 to 300 µm arranged by gathering right amounts of materials provide interconnected macro-porous structure. The newly formed tissue like structures that grow and integrate within the HA designed constructs forming tooth cementum like tissue, pulp and bone structures. These findings are important as they emphasize the potential biological effect of the hybrid scaffold system. In conclusion, this in vitro study clearly demonstrates that designed 3D scaffolds shaped as a immature tooth at the late bell stage were essential to form enamel-dentin-pulp interfaces with an appropriate cell and biodegradable material combination. The biomimetic architecture achieved here is providing a promising platform for dental tissue engineering.

Keywords: tooth regeneration, tissue engineering, adipose stem cells, hydroxyapatite tooth engineering, porous scaffold

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Authors: Sevde Altuntas, Fatih Buyukserin

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Alzheimer’s disease is responsible for irreversible neural damage of brain parts. One of the disease markers is Amyloid-β 1-42 protein that accumulates in the brain in the form plaques. The basic problem for detection of the protein is the low amount of protein that cannot be detected properly in body liquids such as blood, saliva or urine. To solve this problem, tests like ELISA or PCR are proposed which are expensive, require specialized personnel and can contain complex protocols. Therefore, Surface-enhanced Raman Spectroscopy (SERS) a good candidate for detection of Amyloid-β 1-42 protein. Because the spectroscopic technique can potentially allow even single molecule detection from liquid and solid surfaces. Besides SERS signal can be improved by using nanopattern surface and also is specific to molecules. In this context, our study proposes to fabricate diagnostic test models that utilize Au-coated nanopatterned polycarbonate (PC) surfaces modified with Thioflavin - T to detect low concentrations of Amyloid-β 1-42 protein in water and artificial saliva medium by the enhancement of protein SERS signal. The nanopatterned PC surface that was used to enhance SERS signal was fabricated by using Anodic Alumina Membranes (AAM) as a template. It is possible to produce AAMs with different column structures and varying thicknesses depending on voltage and anodization time. After fabrication process, the pore diameter of AAMs can be arranged with dilute acid solution treatment. In this study, two different columns structures were prepared. After a surface modification to decrease their surface energy, AAMs were treated with PC solution. Following the solvent evaporation, nanopatterned PC films with tunable pillared structures were peeled off from the membrane surface. The PC film was then modified with Au and Thioflavin-T for the detection of Amyloid-β 1-42 protein. The protein detection studies were conducted first in water via this biosensor platform. Same measurements were conducted in artificial saliva to detect the presence of Amyloid Amyloid-β 1-42 protein. SEM, SERS and contact angle measurements were carried out for the characterization of different surfaces and further demonstration of the protein attachment. SERS enhancement factor calculations were also completed via experimental results. As a result, our research group fabricated diagnostic test models that utilize Au-coated nanopatterned polycarbonate (PC) surfaces modified with Thioflavin-T to detect low concentrations of Alzheimer’s Amiloid – β protein in water and artificial saliva medium. This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) Grant No: 214Z167.

Keywords: alzheimer, anodic aluminum oxide, nanotopography, surface enhanced Raman spectroscopy

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Authors: Mihai Palade, Gina Cecilia Pistol, Mariana Stancu, Veronica Chedea, Ionelia Taranu

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Nutrition is an important determinant of general health status, with especially focus on prevention and/or attenuation of the inflammatory-associated pathologies. People with chronic kidney disease can experience chronic inflammation that can lead to cardiovascular disease and even an increased rate of death. There are important links between chronic kidney diseases, inflammation and nutritional strategies that may prevent or protect against undesirable inflammation and oxidative stress. The grape by-products either seeds or pomace are rich in polyphenols which may be beneficial in prevention of inflammatory, antioxidant and antimicrobial processes. As a model for studying the impact of grape seeds on renal inflammation and oxidative stress, we used in this study weaned piglets. After a feeding trial of 30 days with a control diet and an experimental diet containing 5% grape seed (GS), kidney samples were collected. In renal tissues were determined the expression and activity of important markers of immune respose and oxidative stress: pro-inflammatory cytokines (TNF-alpha, IL-1 beta, IL-6, IL-8, IFN-gamma), anti-inflammatory cytokines (IL-4, IL-10), anti-oxidant enzymes (catalase CAT, superoxide dismutase SOD, glutathione peroxidise GPx) and important mediators belonging to nuclear receptors (NF-kB1, Nrf-2 and PPAR-gamma). Gene expression was evaluated by qPCR, whereas protein concentration was determined using proteomic techniques (ELISA). The activity of anti-oxidant enzymes was determined using specific kits. Our results showed that GS enriched in polyphenols does not have effect on TNF-alpha, IL-6 and IL-1 beta gene expression and protein concentration in kidney. By contrast, the gene expression and protein level of IL-8 and IFN-gamma were decreased in GS kidney. Anti-inflammatory cytokines IL-4 and IL-10 gene levels were increased in kidneys collected from GS piglets in comparison with controls, with no modification of protein levels between the two groups. The activities of anti-oxidant enzymes CAT and GPx were increased in kidney by GS, whereas SOD activity was unmodified in comparison with control samples. Also, the GS diet was associated with no modulation of mRNAs for nuclear receptors NF-kB1, Nrf-2 and PPAR-gamma gene expressions in kidneys. In conclusion, our results demonstrated that GS enriched in bioactive compounds such polyphenols could modulate inflammation and oxidative stress markers in kidney tissues. Further studies are necessary to elucidate the mechanism of action of GS compounds in case kidney inflammation associated with oxidative stress, and signalling molecules involved in these mechanisms.

Keywords: animal model, kidney inflammation, oxidative stress, grape seed

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Authors: Elisa Esposito, Johannes C. Jansen, Loredana Dellamuzia, Ugo Moretti, Lidietta Giorno

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The reduction of CO₂ emission into the atmosphere as a result of human activity is one of the most important environmental challenges to face in the next decennia. Emission of CO₂, related to the use of fossil fuels, is believed to be one of the main causes of global warming and climate change. In this scenario, the production of biomethane from organic waste, as a renewable energy source, is one of the most promising strategies to reduce fossil fuel consumption and greenhouse gas emission. Unfortunately, biogas upgrading still produces the greenhouse gas CO₂ as a waste product. Therefore, this work presents a case study on biogas upgrading, aimed at the simultaneous purification of methane and CO₂ via different steps, including CO₂/methane separation by polymeric membranes. The original objective of the project was the biogas upgrading to distribution grid quality methane, but the innovative aspect of this case study is the further purification of the captured CO₂, transforming it from a useless by-product to a pure gas with food-grade quality, suitable for commercial application in the food and beverage industry. The study was performed on a pilot plant constructed by Tecno Project Industriale Srl (TPI) Italy. This is a model of one of the largest biogas production and purification plants. The full-scale anaerobic digestion plant (Montello Spa, North Italy), has a digestive capacity of 400.000 ton of biomass/year and can treat 6.250 m3/hour of biogas from FORSU (organic fraction of solid urban waste). The entire upgrading process consists of a number of purifications steps: 1. Dehydration of the raw biogas by condensation. 2. Removal of trace impurities such as H₂S via absorption. 3.Separation of CO₂ and methane via a membrane separation process. 4. Removal of trace impurities from CO₂. The gas separation with polymeric membranes guarantees complete simultaneous removal of microorganisms. The chemical purity of the different process streams was analysed by a certified laboratory and was compared with the guidelines of the European Industrial Gases Association and the International Society of Beverage Technologists (EIGA/ISBT) for CO₂ used in the food industry. The microbiological purity was compared with the limit values defined in the European Collaborative Action. With a purity of 96-99 vol%, the purified methane respects the legal requirements for the household network. At the same time, the CO₂ reaches a purity of > 98.1% before, and 99.9% after the final distillation process. According to the EIGA/ISBT guidelines, the CO₂ proves to be chemically and microbiologically sufficiently pure to be suitable for food-grade applications.

Keywords: biogas, CO₂ separation, CO2 utilization, CO₂ food grade

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Authors: Oluwabori Emmanuel Olukoyejo, Ogra Victor Ogra, Bosede Amodu, Tewogbade Adeoye Adedeji

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Background: Disorders of thyroid function are the second most common endocrine disorders worldwide, with a direct relationship with metabolic bone diseases. These metabolic bone complications are often subtle but manifest as bone pains and an increased risk of fractures. The gold standard for diagnosis, Dual Energy X-ray Absorptiometry (DEXA), is limited in this environment due to unavailability, cumbersomeness and cost. However, bone biomarkers have shown prospects in assessing alterations in bone remodeling, which has not been studied in this environment. Aim: This study evaluates serum levels of bone-specific alkaline phosphatase (bone-specific ALP), osteopontin and osteoprotegerin biomarkers of bone turnover in patients with disorders of thyroid function. Methods: This is a cross-sectional study carried out over a period of one and a half years. Forty patients with thyroid dysfunctions, aged 20 to 50 years, and thirty-eight age and sex-matched healthy euthyroid controls were included in this study. Patients were further stratified into hyperthyroid and hypothyroid groups. Bone-specific ALP, osteopontin, and osteoprotegerin, alongside serum total calcium, ionized calcium and inorganic phosphate, were assayed for all patients and controls. A self-administered questionnaire was used to obtain data on sociodemographic and medical history. Then, 5 ml of blood was collected in a plain bottle and serum was harvested following clotting and centrifugation. Serum samples were assayed for B-ALP, osteopontin, and osteoprotegerin using the ELISA technique. Total calcium and ionized calcium were assayed using an ion-selective electrode, while the inorganic phosphate was assayed with automated photometry. Results: The hyperthyroid and hypothyroid patient groups had significantly increased median serum B-ALP (30.40 and 26.50) ng/ml and significantly lower median OPG (0.80 and 0.80) ng/ml than the controls (10.81 and 1.30) ng/ml respectively, p < 0.05. However, serum osteopontin in the hyperthyroid group was significantly higher and significantly lower in the hypothyroid group when compared with the controls (11.00 and 2.10 vs 3.70) ng/ml, respectively, p < 0.05. Both hyperthyroid and hypothyroid groups had significantly higher mean serum total calcium, ionized calcium and inorganic phosphate than the controls (2.49 ± 0.28, 1.27 ± 0.14 and 1.33 ± 0.33) mmol/l and (2.41 ± 0.04, 1.20 ± 0.04 and 1.15 ± 0.16) mmol/l vs (2.27 ± 0.11, 1.17 ± 0.06 and 1.08 ± 0.16) mmol/l respectively, p < 0.05. Conclusion: Patients with disorders of thyroid function have metabolic imbalances of all the studied bone markers, suggesting a higher bone turnover. The routine bone markers will be an invaluable tool for monitoring bone health in patients with thyroid dysfunctions, while the less readily available markers can be introduced as supplementary tools. Moreover, bone-specific ALP, osteopontin and osteoprotegerin were found to be the strongest independent predictors of metabolic bone markers’ derangements in patients with thyroid dysfunctions.

Keywords: metabolic bone diseases, biomarker, bone turnover, hyperthyroid, hypothyroid, euthyroid

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Authors: Ananya Pal, Neelakshi Sarkar, Debraj Saha, Dipanwita Das, Subhashish Kamal Guha, Bibhuti Saha, Runu Chakravarty

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Presently, tenofovir disoproxil fumurate (TDF) is the most effective anti-viral agent for the treatment of hepatitis B virus (HBV) in individuals co-infected with HIV and HBV as TDF has activity to suppress both wild-type and lamivudine (3TC)-resistant HBV. However, suboptimal response to TDF was reported in HIV-HBV co-infected individuals with prior 3TC therapy from different countries recently. The incidence of 3TC-resistant HBV strains is quite high in HIV-HBV co-infected patients experiencing long-term anti-retroviral therapy (ART) in eastern India. In spite of this risk, most of the patients with long-term 3TC treatment are continued with the same anti-viral agent in this country. Only a few have received TDF in addition to 3TC in the ART regimen since TDF has been available in India for the treatment of HIV-infected patients in 2012. In this preliminary study, we investigated the virologic and biochemical parameters among HIV-HBV co-infected patients who are non-responders to 3TC treatment during the continuation of 3TC or TDF add-on to 3TC in their ART regimen. Fifteen HIV-HBV co-infected patients who experienced long-term 3TC (mean duration months 36.87 ± 24.08 months) were identified with high HBV viremia ( > 20,000 IU/ml) or harbouring 3TC-resistant HBV. These patients receiving ART from School of Tropical Medicine Kolkata, the main ART centre in eastern India were followed-up semi-annually for next three visits. Different virologic parameters including quantification of plasma HBV load by real-time PCR, detection of hepatitis B e antigen (HBeAg) by commercial ELISA and anti-viral resistant mutations by sequencing were studied. During three follow-up among study subjects, 86%, 47%, and 43% had 3TC-mono-therapy (mean treatment-duration 41.54±18.84, 49.67±11.67, 54.17±12.37 months respectively) whereas 14%, 53%, and 57% experienced TDF in addition to 3TC (mean treatment duration 4.5±2.12, 16.56±11.06, and 23±4.07 months respectively). Mean CD4 cell-count in patients receiving 3TC was tended to be lower during third follow-up as compared to the first and the second [520.67±380.30 (1st), 454.8±196.90 (2nd), and 397.5±189.24 (3rd) cells/mm3) and similar trend was seen in patients experiencing TDF in addition to 3TC [334.5±330.218 (1st), 476.5±194.25 (2nd), and 461.17±269.89 (3rd) cells/mm3]. Serum HBV load was increased during successive follow-up of patients with 3TC-mono-therapy. Initiation of TDF lowered serum HBV-load among 3TC-non-responders at the time of second visit ( < 2,000 IU/ml), interestingly during third follow-up, mean HBV viremia increased >1 log IU/ml (mean 3.56±2.84 log IU/ml). Persistence of 3TC-resistant double and triple mutations was also observed in both the treatment regimens. Mean serum alanine aminotransferase remained elevated in these patients during this follow-up study. Persistence of high HBV viraemia and 3TC-resistant mutation in HBV during the continuation of 3TC might lead to major public health threat in India. The inclusion of TDF in the ART regimen of 3TC non-responder HIV-HBV co-infected patients showed adverse treatment response in terms of virologic and biochemical parameters. Therefore, serious attention is necessary for proper management of long-term 3TC experienced HIV-HBV co-infected patients with high HBV viraemia or 3TC-resistant HBV mutants in India.

Keywords: HBV, HIV, TDF, 3TC-resistant

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Authors: Lívia Pinheiro Carvalho, Luciana Di Thommazo-Luporini, Rafael Luís Luporini, José Carlos Bonjorno Junior, Renata Pedrolongo Basso Vanelli, Manoel Carneiro de Oliveira Junior, Rodolfo de Paula Vieira, Renata Trimer, Renata G. Mendes, Mylène Aubertin-Leheudre, Audrey Borghi-Silva

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Rationale: Cardiorespiratory fitness (CRF) and functional capacity in young obese and normal-weight people are associated with metabolic and cardiovascular diseases and mortality. However, it remains unclear whether their metabolically healthy (MH) or at risk (AR) phenotype influences cardiorespiratory fitness in this vulnerable population such as obese adults but also in normal-weight people. HOMA insulin resistance index (HI) and leptin-adiponectin ratio (LA) are strong markers for characterizing those phenotypes that we hypothesized to be associated with physical fitness. We also hypothesized that an easy and feasible exercise test could identify a subpopulation at risk to develop metabolic and related disorders. Methods: Thirty-nine sedentary men and women (20-45y; 18.530 kg.m-2) underwent a clinical evaluation, including the six-minute step test (ST), a well-validated and reliable test for young people. Body composition assessment was done by a tetrapolar bioimpedance in a fasting state and in the folicular phase for women. A maximal cardiopulmonary exercise testing, as well as the ST, evaluated the oxygen uptake at the peak of the test (VO2peak) by an ergospirometer Oxycon Mobile. Lipids, glucose, insulin were analysed and the ELISA method quantified the serum leptin and adiponectin from blood samples. Volunteers were divided in two groups: AR or MH according to a HI cutoff of 1.95, which was previously determined in the literature. T-test for comparison between groups, Pearson´s test to correlate main variables and ROC analysis for discriminating AR from up-and-down cycles in ST (SC) were applied (p<0.05). Results: Higher LA, fat mass (FM) and lower HDL, SC, leg lean mass (LM) and VO2peak were found in AR than in MH. Significant correlations were found between VO2peak and SC (r= 0.80) as well as between LA and FM (r=0.87), VO2peak (r=-0.73), and SC (r=-0.65). Area under de curve showed moderate accuracy (0.75) of SC <173 to discriminate AR phenotype. Conclusion: Our study found that at risk obese and normal-weight subjects showed an unhealthy metabolism as well as a poor CRF and functional daily activity capacity. Additionally, a simple and less costly functional test associated with above-mentioned aspects is able to identify ‘at risk’ subjects for primary intervention with important clinical and health implications.

Keywords: aerobic capacity, exercise, fitness, metabolism, obesity, 6MST

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Authors: Nabeeha Hassan Abdel Jalil, Robert Barigye, Hamda Al Alawi, Afra Al Dhaheri, Fatma Graiban Al Muhairi, Maryam Al Khateri, Nouf Al Alalawi, Susan Olet, Khaja Mohteshamuddin, Ahmad Al Aiyan, Mohamed Elfatih Hamad

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A serological survey was carried out to determine the seroprevalence of Brucella spp, Coxiella burnetii, Chlamydophila abortus, and Toxoplasma gondii in sheep and goat herds in the UAE. A total of 915 blood samples [n= 222, [sheep]; n= 215, [goats]) were collected from livestock farms in the Emirates of Abu Dhabi, Dubai, Sharjah and Ras Al-Khaimah (RAK). An additional 478 samples (n= 244, [sheep]; n= 234, (goats]) were collected from the Al Ain livestock central market and tested by indirect ELISA for pathogen-specific antibodies with the Brucella antibodies being further corroborated by the Rose-Bengal agglutination test. Seropositivity for the four pathogens is variably documented in sheep and goats from the study area. Respectively, the overall livestock farm prevalence rates for Brucella spp, C. burnetii, C. abortus, and T. gondii were 2.7%, 27.9%, 8.1%, and 16.7% for sheep, and 0.0%, 31.6%, 9.3%, and 5.1% for goats. Additionally, the seroprevalence rates Brucella spp, C. burnetii, C. abortus, and T. gondii in samples from the livestock market were 7.4%, 21.7%, 16.4%, and 7.0% for sheep, and 0.9%, 32.5%, 19.2%, and 11.1% for goats respectively. Overall, sheep had 12.59 more chances than goats of testing seropositive for Brucella spp (OR, 12.59 [95% CI 2.96-53.6]) but less likely to be positive for C. burnetii-antibodies (OR, 0.73 [95% CI 0.54-0.97]). Notably, the differences in the seroprevalence rates of C. abortus and T. gondii in sheep and goats were not statistically significant (p > 0.0500). The present data indicate that all the four study pathogens are present in sheep and goat populations in the UAE where coxiellosis is apparently the most seroprevalent followed by chlamydophilosis, toxoplasmosis, and brucellosis. While sheep from the livestock market were more likely than those from farms to be Brucella-seropositive than those, the overall exposure risk of C. burnetii appears to be greater for goats than sheep. As more animals from the livestock market were more likely to be seropositive to Chlamydophila spp, it is possible that under the UAE animal production conditions, at least, coxiellosis and chlamydophilosis are more likely to increase the culling rate of domesticated small ruminants than toxoplasmosis and brucellosis. While anecdotal reports have previously insinuated that brucellosis may be a significant animal health risk in the UAE, the present data suggest C. burnetii, C. abortus and T. gondii to be more significant pathogens of sheep and goats in the country. Despite this possibility, the extent to which these pathogens may nationally be contributing to reproductive failure in sheep and goat herds is not known and needs to be investigated. Potentially, these agents may also carry a potentially zoonotic risk that needs to be investigated in risk groups like farm workers, and slaughter house personnel. An ongoing study is evaluating the seroprevalence of bovine coxiellosis in the Emirate of Abu Dhabi and the data thereof will further elucidate on the broader epidemiological dynamics of the disease in the national herd.

Keywords: Brucella spp, Chlamydophila abortus, goat, sheep, Toxoplasma gondii, UAE

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Authors: Belgheis Ebrahimi, Jun Lu

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In recent years, there has been a growing recognition of the potential of marine-based functional foods and combination therapies in promoting a healthy lifestyle and exploring their effectiveness in preventing or treating diseases. The combination of marine bioactive compounds or extracts offers synergistic or enhancement effects through various mechanisms, including multi-target actions, improved bioavailability, enhanced bioactivity, and mitigation of potential adverse effects. Both the green-lipped mussel (GLM) and fucoidan derived from brown seaweed are rich in bioactivities. These two, mussel and fucoidan, have not been previously formulated together. This study aims to combine GLM oil from Perna canaliculus with low molecular weight fucoidan (LMWF) extracted from Undaria pinnatifida to investigate the unique mixture’s anti-inflammatory and antioxidant properties. The cytotoxicity of individual compounds and combinations was assessed using the MTT assay in (THP-1 and RAW264.7) cell lines. The anti-inflammatory activity of mussel-fucoidan was evaluated by treating LPS-stimulated human monocyte and macrophage (THP1-1) cells. Subsequently, the inflammatory cytokines released into the supernatant of these cell lines were quantified via ELISA. Antioxidant activity was determined by using the free radical scavenging assay (DPPH). DPPH assay demonstrated that the radical scavenging activity of the combinations, particularly at concentrations exceeding 1 mg/ml, showed a significantly higher percentage of inhibition when compared to the individual component. This suggests an enhancement effect when the two compounds are combined, leading to increased antioxidant activity. In terms of immunomodulatory activity, the individual compounds exhibited distinct behaviors. GLM oil displayed a higher ability to suppress the cytokine TNF- compared to LMWF. Interestingly, the LMWF fraction, when used individually, did not demonstrate TNF- suppression. However, when combined with GLM, the TNF- suppression (anti-inflammatory) activity of the combination was better than GLM or LWMF alone. This observation underscores the potential for enhancement interactions between the two components in terms of anti-inflammatory properties. This study revealed that each individual compound, LMWF, and GLM, possesses unique and notable bioactivity. The combination of these two individual compounds results in an enhancement effect, where the bioactivity of each is enhanced, creating a superior combination. This suggests that the combination of LMWF and GLM has the potential to offer a more potent and multifaceted therapeutic effect, particularly in the context of antioxidant and anti-inflammatory activities. These findings hold promise for the development of novel therapeutic interventions or supplements that harness the enhancement effects.

Keywords: combination, enhancement effect, perna canaliculus, undaria pinnatifida

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Authors: Mustafa M. Donma, Orkide Donma

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Obesity is associated with cardiovascular disease risk factors and metabolic syndrome (MetS). In this study, associations between adipokines and adipokine as well as obesity indices were evaluated. Plasma adipokine levels may exhibit variations according to body adipose tissue mass. Besides, upon consideration of obesity as an inflammatory disease, adipokines may play some roles in this process. The ratios of proinflammatory adipokines to adiponectin may act as highly sensitive indicators of body adipokine status. The aim of the study is to present some adipokine indices, which are thought to be helpful for the evaluation of childhood obesity and also to determine the best discriminators in the diagnosis of MetS. 80 prepubertal children (aged between 6-9.5 years) included in the study were divided into three groups; 30 children with normal weight (NW), 25 morbid obese (MO) children and 25 MO children with MetS. Physical examinations were performed. Written informed consent forms were obtained from the parents. The study protocol was approved by Ethics Committee of Namik Kemal University Medical Faculty. Anthropometric measurements, such as weight, height, waist circumference (C), hip C, head C, neck C were recorded. Values for body mass index (BMI), diagnostic obesity notation model assessment Index-II (D2 index) as well as waist-to-hip, head-to-neck ratios were calculated. Adiponectin, resistin, leptin, chemerin, vaspin, progranulin assays were performed by ELISA. Adipokine-to-adiponectin ratios were obtained. SPSS Version 20 was used for the evaluation of data. p values ≤ 0.05 were accepted as statistically significant. Values of BMI and D2 index, waist-to-hip, head-to-neck ratios did not differ between MO and MetS groups (p ≥ 0.05). Except progranulin (p ≤ 0.01), similar patterns were observed for plasma levels of each adipokine. There was not any difference in vaspin as well as resistin levels between NW and MO groups. Significantly increased leptin-to-adiponectin, chemerin-to-adiponectin and vaspin-to-adiponectin values were noted in MO in comparison with those of NW. The most valuable adipokine index was progranulin-to-adiponectin (p ≤ 0.01). This index was strongly correlated with vaspin-to-adiponectin ratio in all groups (p ≤ 0.05). There was no correlation between vaspin-to-adiponectin and chemerin-to--adiponectin in NW group. However, a correlation existed in MO group (r = 0.486; p ≤ 0.05). Much stronger correlation (r = 0.609; p ≤ 0.01) was observed in MetS group between these two adipokine indices. No correlations were detected between vaspin and progranulin as well as vaspin and chemerin levels. Correlation analyses showed a unique profile confined to MetS children. Adiponectin was found to be correlated with waist-to-hip (r = -0.435; p ≤ 0.05) as well as head-to-neck (r = 0.541; p ≤ 0.05) ratios only in MetS children. In this study, it has been investigated if adipokine indices have priority over adipokine levels. In conclusion, vaspin-to-adiponectin, progranulin-to-adiponectin, chemerin-to-adiponectin along with waist-to-hip and head-to-neck ratios were the optimal combinations. Adiponectin, waist-to-hip, head-to-neck, vaspin-to-adiponectin, chemerin-to-adiponectin ratios had appropriate discriminatory capability for MetS children.

Keywords: adipokine indices, metabolic syndrome, obesity indices, pediatric obesity

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Authors: Alireza Barari, Ahmad Abdi

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Regular body trainings cause adaption in various system in body. One of the important effect of body training is its effect on immune system. It seems that cytokines usually release after long period exercises or some exercises which cause skeletal muscular damages. If some of the cytokines which cause responses such as inflammation of cells in skeletal muscles, with manipulating of training program, it can be avoided or limited from those exercises which induct cytokines release. Ginger plant is a kind of medicinal plants which is known as a anti inflammation plant. This plant is as most precedence medicinal plants in medicine science especially in inflammation cure. The aim of the present study was the effect of selected resistance training and consumption of ginger extract on IL-1α and TNFα untrained young women. The population includes young women interested in participating in the study with the average of 30±2 years old from Abbas Abad city among which 32 participants were chosen randomly and divided into 4 four groups, resistance training (R), resistance training and ginger consumption(RG), Ginger consumption(G)and Control group(C). The training groups performed circuit resistance training at the intensity of 65-75% one repeat maximum, 3 days a week for 6 weeks. Besides resistance training, subjects were given either ginseng (5 mg/kg per day) or placebo. Prior to and 48 hours after interventions body composition was measured and blood samples were taken in order to assess serum levels of IL-1α and TNFα. Plasma levels of cytokines were measured with commercially available ELISA Kits.IL-1α kit and TNFα kit were used in this research. To demonstrate the effectiveness of the independent variable and the comparison between groups, t-test and ANOVA were used. To determine differences between the groups, the Scheffe test was used that showed significant changes in any of the variables. we observed that circuit resistance training in R and RG groups can significant decreased in weight and body mass index in untrained females (p<0.05). The results showed a significant decreased in the mean level of IL-1α levels before and after the training period in G group (p=0.046) and RG group (p=0.022). Comparison between groups also showed there was significant difference between groups R-RG and RG-C. Intergroup comparison results showed that the mean levels of TNFα before and after the training in group G (p=0.044) and RG (p=0.037), significantly decreased. Comparison between groups also showed there was significant difference between groups R–RG , R-G ,RG-C and G-C. The research shows that circuit resistance training with reducing overload method results in systemic inflammation had significant effect on IL-1α levels and TNFα. Of course, Ginger can counteract the negative effects of resistance training exercise on immune function and stability of the mast cell membrane. Considerable evidence supported the anti-inflammatory properties of ginger for several constituents, especially gingerols, shogaols, paradols, and zingerones, through decreased cytokine gene TNF α and IL-1Α expression and inhibition of cyclooxygenase 1 and 2. These established biological actions suggest that ingested ginger could block the increase in IL-1α.

Keywords: resistance training, ginger, IL-1α , TNFα

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Authors: M. H. A. D. de Silva, R. P. Hewawasam, M. A. G. Iresha

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Macrosomia is common in infants born to not only women diagnosed with gestational diabetes mellitus but also non-diabetic obese women. Maternal Body Mass Index (BMI) correlates with the incidence of large for gestational age infants. Obesity has reached epidemic levels in modern societies. During the past two decades, obesity in children and adolescents has risen significantly in Asian populations including Sri Lanka. There is increasing evidence to believe that infants who are born large for gestational age are more likely to be obese in childhood and adolescence and are at risk of cardiovascular and metabolic complications later in life. It is also established that Asians have lower skeletal muscle mass, low bone mineral content and excess body fat for a given BMI indicating a genetic predisposition in the occurrence of obesity. The objective of this study is to determine the effect of maternal weight, weight gain during pregnancy, c-peptide and insulin concentrations in the cord blood on the birth of appropriate for and large for gestational age infants in a tertiary care center in Southern Sri Lanka. Umbilical cord blood was collected from 90 newborns (Male 40, Female 50; gestational age 35-42 weeks) after double clamping the umbilical cord before separation of the placenta and the concentration of insulin and C-peptide were measured by ELISA technique. Anthropometric parameters of the newborn such as birth weight, length, ponderal index, occipital frontal, chest, hip and calf circumferences were measured, and characteristics of the mother were collected. The relationship between insulin, C-peptide and anthropometrics were assessed by Spearman correlation. The multiple logistic regression analysis examined influences of maternal weight, weight gain during pregnancy, C-peptide and insulin concentrations in cord blood as covariates on the birth of large for gestational age infants. A significant difference (P<0.001) was observed between the insulin levels of infants born large for gestational age (18.73 ± 0.52 µlU/ml) and appropriate for gestational age (13.08 ± 0.56 µlU/ml). Consistently, A significant decrease in concentration (41.68%, P<0.001) was observed between C-peptide levels of infants born large for gestational age and appropriate for gestational age. Cord blood insulin and C-peptide levels had a significant correlation with birth weight (r=0.35, P<0.05) of the newborn at delivery. Maternal weight and BMI which are indicators of maternal nutrition were proven to be directly correlated with birth weight and length. To our knowledge, this relationship was investigated for the first time in a Sri Lankan setting and was also evident in our results. This study confirmed the fact that insulin and C-peptide play a major role in regulating fetal growth. According to the results obtained in this study, we can suggest that the increased BMI of the mother has a direct influence on increased maternal insulin secretion, which may subsequently affect cord insulin and C-peptide levels and also birth weight of the infant.

Keywords: C-peptide, insulin, large for gestational age, maternal weight

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Authors: Avijit Dey, Antony Gomes, Subir Chandra Dasgupta

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Tea (Camellia sinensis) is the most popular beverages in the world and is ranked second after the water. Tea has been considered as a health promoting beverage since ancient times due to its health-promoting activity. Recently, immunomodulatory, anti-arthritic, antioxidant, anticancer and cardioprotective activity of tea has been established. Very few studies have demonstrated the effect of high dose of black tea on health. The aim of the present study was to evaluate the role of low & high dose of Black Tea Extract (BTE) on the different physiological parameters of mother and pups during prenatal and postnatal developmental period in the experimental rodent. BTE was orally administered in LD (50mg BTE/kg/day) and HD (100mg BTE/kg/day) except control groups of rats (n=6/group) throughout the prenatal (day 0-21) and postnatal (day 21-42) periods. During prenatal period (0, 7th, 14th, 20th days) body weight, urinary calcium, magnesium, urea and creatinine was measured. In postnatal period physical (0, 10th, 21th days) parameters of pups like body weight, cranial length, cranial diameter, neck width, tail length, craniosacral length of pups were analyzed. Liver and lungs from pups and kidney spleen, etc. from mothers were collected on day 42 for histopathological studies. The comparative urine strip and morphology of RBC was also analyzed by SEM from mothers of different groups on day 42. The level of cytokines like IL-1alpha, IL-1beta, IL-6, IL-10, TNF-alpha were analysed by enzyme-linked immunosorbent assay (ELISA) on day 0, day 20 and day 42. The body weight of LD and HD mothers were also significantly (P<0.05) less than control mothers at 20th day of pregnancy and there was also significant changes in urinary calcium, urea, creatinine. The bio morphometric analysis of pups showed significant alteration (P<0.05) in HD groups relative to control. Some histological alterations were also observed in pups and mothers. Comparative urine strip analysis and morphology of RBC showed significant changes in treated groups. LD and HD treated mothers showed an increase in proinflammatory cytokines like IL-1beta, TNF-alpha and decrease in anti-inflammatory cytokine-like IL-10 on day 20 compared to PC mothers. This study clearly indicated that high dose of BTE possesses detrimental effect on pregnant mother and the pup. Further studies are in progress to elucidate the molecular mechanism of actions. This project work has been sponsored by National Tea Research Foundation vide Project Sanction No.: 17 (305)/2013/4423 dated 11th March, 2014. All experimental protocols described in the study were approved by animal ethics committee.

Keywords: black tea extract, pregnancy, prenatal and postnatal development, inflammation

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Authors: Evgeny A. Ermakov, Lyudmila P. Smirnova, Valentina N. Buneva

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Schizophrenia associated with dysregulation of neurotransmitter processes in the central nervous system and disturbances in the humoral immune system resulting in the formation of antibodies (Abs) to the various components of the nervous tissue. Abs to different neuronal receptors and DNA were detected in the blood of patients with schizophrenia. Abs hydrolyzing DNA were detected in pool of polyclonal autoantibodies in autoimmune and infectious diseases, such catalytic Abs were named abzymes. It is believed that DNA-hydrolyzing abzymes are cytotoxic, cause nuclear DNA fragmentation and induce cell death by apoptosis. Abzymes with DNAase activity are interesting because of the mechanism of formation and the possibility of use as diagnostic markers. Therefore, in our work we have set following goals: to determine the level anti-DNA Abs in the serum of patients with schizophrenia and to study DNA-hydrolyzing activity of IgG of patients with schizophrenia. Materials and methods: In our study there were included 41 patients with a verified diagnosis of paranoid or simple schizophrenia and 24 healthy donors. Electrophoretically and immunologically homogeneous IgGs were obtained by sequential affinity chromatography of the serum proteins on protein G-Sepharose and gel filtration. The levels of anti-DNA Abs were determined using ELISA. DNA-hydrolyzing activity was detected as the level of supercoiled pBluescript DNA transition in circular and linear forms, the hydrolysis products were analyzed by agarose electrophoresis followed by ethidium bromide stain. To correspond the registered catalytic activity directly to the antibodies we carried out a number of strict criteria: electrophoretic homogeneity of the antibodies, gel filtration (acid shock analysis) and in situ activity. Statistical analysis was performed in ‘Statistica 9.0’ using the non-parametric Mann-Whitney test. Results: The sera of approximately 30% of schizophrenia patients displayed a higher level of Abs interacting with single-stranded (ssDNA) and double-stranded DNA (dsDNA) compared with healthy donors. The average level of Abs interacting with ssDNA was only 1.1-fold lower than that for interacting with dsDNA. IgG of patient with schizophrenia were shown to possess DNA hydrolyzing activity. Using affinity chromatography, electrophoretic analysis of isolated IgG homogeneity, gel filtration in acid shock conditions and in situ DNAse activity analysis we proved that the observed activity is intrinsic property of studied antibodies. We have shown that the relative DNAase activity of IgG in patients with schizophrenia averaged 55.4±32.5%, IgG of healthy donors showed much lower activity (average of 9.1±6.5%). It should be noted that DNAase activity of IgG in patients with schizophrenia with a negative symptoms was significantly higher (73.3±23.8%), than in patients with positive symptoms (43.3±33.1%). Conclusion: Anti-DNA Abs of patients with schizophrenia not only bind DNA, but quite efficiently hydrolyze the substrate. The data show a correlation with the level of DNase activity and leading symptoms of patients with schizophrenia.

Keywords: anti-DNA antibodies, abzymes, DNA hydrolysis, schizophrenia

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Authors: Laura Gonzalez, Santiago Benavides, Martha Elena Londono, Ana Elisa Casas, Adriana Restrepo-Osorio

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Cotton products are sensitive to microorganisms due to its ability to retain moisture, which might cause change into the coloration, mechanical properties reduction or foul odor generation; consequently, this represents risks to the health of users. Nowadays, have been carried out researches to give antibacterial properties to textiles using different strategies, which included the use of silver nanoparticles (AgNPs). The antibacterial behavior can be affected by laundering process reducing its effectiveness. In the other way, the environmental impact generated for the synthetic antibacterial agents has motivated to seek new and more ecological ways for produce AgNPs. The aims of this work are to determine the antibacterial activity of cotton fabric functionalized with green (G) and commercial (C) AgNPs after twenty washing cycles, also to evaluate morphological and color changes. A plain weave cotton fabric suitable for dyeing and two AgNPs solutions were use. C a commercial product and G produced using an ecological method, both solutions with 0.5 mM concentration were impregnated on cotton fabric without stabilizer, at a liquor to fabric ratio of 1:20 in constant agitation during 30min and then dried at 70 °C by 10 min. After that the samples were subjected to twenty washing cycles using phosphate-free detergent simulated on agitated flask at 150 rpm, then were centrifuged and dried on a tumble. The samples were characterized using Kirby-Bauer test determine antibacterial activity against E. coli y S. aureus microorganisms, the results were registered by photographs establishing the inhibition halo before and after the washing cycles, the tests were conducted in triplicate. Scanning electron microscope (SEM) was used to observe the morphologies of cotton fabric and treated samples. The color changes of cotton fabrics in relation to the untreated samples were obtained by spectrophotometer analysis. The images, reveals the presence of inhibition halo in the samples treated with C and G AgNPs solutions, even after twenty washing cycles, which indicated a good antibacterial activity and washing durability, with a tendency to better results against to S. aureus bacteria. The presence of AgNPs on the surface of cotton fiber and morphological changes were observed through SEM, after and before washing cycles. The own color of the cotton fiber has been significantly altered with both antibacterial solutions. According to the colorimetric results, the samples treated with C lead to yellowing while the samples modified with G to red yellowing Cotton fabrics treated AgNPs C and G from 0.5 mM solutions exhibited excellent antimicrobial activity against E. coli and S. aureus with good laundering durability effects. The surface of the cotton fibers was modified with the presence of AgNPs C and G due to the presence of NPs and its agglomerates. There are significant changes in the natural color of cotton fabric due to deposition of AgNPs C and G which were maintained after laundering process.

Keywords: antibacterial property, cotton fabric, fastness to wash, Kirby-Bauer test, silver nanoparticles

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Authors: Delphine Morales, Florian Lombart, Agathe Truchot, Pauline Maire, Pascale Vigneron, Antoine Galmiche, Catherine Lok, Muriel Vayssade

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Targeted therapy molecules are used as a first-line treatment for metastatic melanoma with B-Raf mutation. Nevertheless, these molecules can cause side effects to patients and are efficient on 50 to 60 % of them. Indeed, melanoma cell sensitivity to targeted therapy molecules is dependent on tumor microenvironment (cell-cell and cell-extracellular matrix interactions). To better unravel factors modulating cell sensitivity to B-Raf inhibitor, we have developed and compared several melanoma models: from metastatic melanoma cells cultured as monolayer (2D) to a co-culture in a 3D dermal equivalent. Cell response was studied in different melanoma cell lines such as SK-MEL-28 (mutant B-Raf (V600E), sensitive to Vemurafenib), SK-MEL-3 (mutant B-Raf (V600E), resistant to Vemurafenib) and a primary culture of dermal human fibroblasts (HDFn). Assays have initially been performed in a monolayer cell culture (2D), then a second time on a 3D dermal equivalent (dermal human fibroblasts embedded in a collagen gel). All cell lines were treated with Vemurafenib (a B-Raf inhibitor) for 48 hours at various concentrations. Cell sensitivity to treatment was assessed under various aspects: Cell proliferation (cell counting, EdU incorporation, MTS assay), MAPK signaling pathway analysis (Western-Blotting), Apoptosis (TUNEL), Cytokine release (IL-6, IL-1α, HGF, TGF-β, TNF-α) upon Vemurafenib treatment (ELISA) and histology for 3D models. In 2D configuration, the inhibitory effect of Vemurafenib on cell proliferation was confirmed on SK-MEL-28 cells (IC50=0.5 µM), and not on the SK-MEL-3 cell line. No apoptotic signal was detected in SK-MEL-28-treated cells, suggesting a cytostatic effect of the Vemurafenib rather than a cytotoxic one. The inhibition of SK-MEL-28 cell proliferation upon treatment was correlated with a strong expression decrease of phosphorylated proteins involved in the MAPK pathway (ERK, MEK, and AKT/PKB). Vemurafenib (from 5 µM to 10 µM) also slowed down HDFn proliferation, whatever cell culture configuration (monolayer or 3D dermal equivalent). SK-MEL-28 cells cultured in the dermal equivalent were still sensitive to high Vemurafenib concentrations. To better characterize all cell population impacts (melanoma cells, dermal fibroblasts) on Vemurafenib efficacy, cytokine release is being studied in 2D and 3D models. We have successfully developed and validated a relevant 3D model, mimicking cutaneous metastatic melanoma and tumor microenvironment. This 3D melanoma model will become more complex by adding a third cell population, keratinocytes, allowing us to characterize the epidermis influence on the melanoma cell sensitivity to Vemurafenib. In the long run, the establishment of more relevant 3D melanoma models with patients’ cells might be useful for personalized therapy development. The authors would like to thank the Picardie region and the European Regional Development Fund (ERDF) 2014/2020 for the funding of this work and Oise committee of "La ligue contre le cancer".

Keywords: 3D human skin model, melanoma, tissue engineering, vemurafenib efficiency

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Authors: Shamim Mushtaq, Moazzam Shahid

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Breast cancer (BC) claims the lives of half a million women every year and is the most common cause of death in the developing world. In 2019, it was estimated that BC alone accounts for 15% of all cancer deaths in younger women (aged < 45 years old) with advanced-stage lung metastasis. According to the World Health Organization & International Union against Cancer, in Asia, a high number of cancer-related deaths will be observed in 2020, whereas the burden will be reduced in Western countries due to awareness about the disease, better health facilities and advanced treatments. In the last 15 years, it has been reported that the incidence of BC has increased by 1.1% among Asian compared to the US population from 2003 to 2012. To date, several BC biological subtypes have been reported so far, which are associated with different treatment responses. The heterogeneity and diversity of BC reflected these different subtypes, including Luminal A (23.7% prevalence) and B (38.8% prevalence) that have pathological estrogen receptor (ER+)-positive tumors, the human epidermal growth factor receptor 2 (HER2) (11.2% prevalence) and triple-negative breast cancer (TNBC) (25% prevalence). According to Shaukat Khanum Memorial Cancer Hospital and Research Centre – Pakistan, ten years of data showed that among 636 BC patients, 30.5% had TNBC who were <40 years of age, which is an extremely alarming situation. Therefore, there is a dire need to explore and develop therapeutic targets for the treatment of early TNBC. Since the last decade, unfortunately, there has been little success in understanding the complexity of TNBC and in discovering new biological therapeutic targets. However, conventional chemotherapy is the only choice of treatment for TNBC patients. Many investigators revealed advances in multi-omics (multiple "omes", e.g., genome, proteome, transcriptome, epigenome, and microbiome) which were later identified as actionable targets and increased prevalence in TNBC patients. However, various drugs have been identified so far which are related to a particular diagnostic and prognostic biomarker. For example, Epidermal growth factor receptor ( EGFR or ErbB-1), HER-2/neu (ErbB-2), HER-3 (ErbB-3), and HER-4 (ErbB-4). Protein Transglin-2 (TAGLN 2 ) and Profilins-1 (Pfn-1 ) are the ubiquitously expressed large family of proteins present in all eukaryotes, enabling actin cytoskeletal reorganization. It is known that the oncogenic transformation of cells is accompanied by alteration in the actin cytoskeleton. There are causal connections between altered expression of actin cytoskeletal regulators and cancer progression. Our case-control study identified TAGLN-2 and Pfn-1 proteins in TNBC blood by mass spectrometry. Both TAGLN-2 and Pfn-1 proteins are differentially expressed in early and advanced stages of TNBS patients, which could be potential predictors or therapeutic targets for TNBC.

Keywords: TNBC, blood biomarkers, mass spectrometry, qPCR, ELISA

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Authors: Daniela Manila Bianchi, Samantha Lupi, Elisa Barcucci, Sandra Fragassi, Clara Tramuta, Lucia Decastelli

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Introduction: The undeclared allergens detection in food products plays a fundamental role in the safety of the allergic consumer. The protection of allergic consumers is guaranteed, in Europe, by Regulation (EU) No 1169/2011 of the European Parliament, which governs the consumer's right to information and identifies 14 food allergens to be mandatorily indicated on food labels: among these, an egg is included. An egg can be present as an ingredient or as contamination in raw and cooked products. The main allergen egg proteins are ovomucoid, ovalbumin, lysozyme, and ovotransferrin. This study presents the results of a survey conducted in Northern Italy aimed at detecting the presence of undeclared egg proteins in food matrices in the latest ten years (2011-2021). Method: In the period January 2011 - October 2021, a total of 1205 different types of food matrices (ready-to-eat, meats, and meat products, bakery and pastry products, baby foods, food supplements, pasta, fish and fish products, preparations for soups and broths) were delivered to Food Control Laboratory of Istituto Zooprofilattico Sperimentale of Piemonte Liguria and Valle d’Aosta to be analyzed as official samples in the frame of Regional Monitoring Plan of Food Safety or in the contest of food poisoning. The laboratory is ISO 17025 accredited, and since 2019, it has represented the National Reference Centre for the detection in foods of substances causing food allergies or intolerances (CreNaRiA). All samples were stored in the laboratory according to food business operator instructions and analyzed within the expiry date for the detection of undeclared egg proteins. Analyses were performed with RIDASCREEN®FAST Ei/Egg (R-Biopharm ® Italia srl) kit: the method was internally validated and accredited with a Limit of Detection (LOD) equal to 2 ppm (mg/Kg). It is a sandwich enzyme immunoassay for the quantitative analysis of whole egg powder in foods. Results: The results obtained through this study showed that egg proteins were found in 2% (n. 28) of food matrices, including meats and meat products (n. 16), fish and fish products (n. 4), bakery and pastry products (n. 4), pasta (n. 2), preparations for soups and broths (n.1) and ready-to-eat (n. 1). In particular, in 2011 egg proteins were detected in 5% of samples, in 2012 in 4%, in 2013, 2016 and 2018 in 2%, in 2014, 2015 and 2019 in 3%. No egg protein traces were detected in 2017, 2020, and 2021. Discussion: Food allergies occur in the Western World in 2% of adults and up to 8% of children. Allergy to eggs is one of the most common food allergies in the pediatrics context. The percentage of positivity obtained from this study is, however, low. The trend over the ten years has been slightly variable, with comparable data.

Keywords: allergens, food, egg proteins, immunoassay

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Authors: Caroline Lavratti, Pedro Dal Lago, Gustavo Reinaldo, Gilson Dorneles, Andreia Bard, Laira Fuhr, Daniela Pochmann, Alessandra Peres, Luciane Wagner, Viviane Elsner

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Approximately 1% of the world's population is affected by schizophrenia (SZ), a chronic and debilitating neurodevelopmental disorder. Among possible factors, reduced levels of Brain-derived neurotrophic factor (BDNF) has been recognized in physiopathogenesis and course of SZ. In this context, peripheral BDNF levels have been used as a biomarker in several clinical studies, since this neurotrophin is able to cross the blood-brain barrier in a bi-directional manner and seems to present a strong correlation with the central nervous system fluid levels. The patients with SZ usually adopts a sedentary lifestyle, which has been partly associated with the increase in obesity incidence rates, metabolic syndrome, type 2 diabetes and coronary heart disease. On the other hand, exercise, a non-invasive and low cost intervention, has been considered an important additional therapeutic option for this population, promoting benefits to physical and mental health. To our knowledge, few studies have been pointed out that the positive effects of exercise in SZ patients are mediated, at least in part, to enhanced levels of BDNF after training. However, these studies are focused on evaluating the effect of single bouts of exercise of chronic interventions, data concerning the short- and long-term exercise outcomes on BDNF are scarce. Therefore, this study aimed to evaluate the effect of a concurrent exercise protocol (CEP) on plasma BDNF levels of SZ patients in different time-points. Material and Methods: This study was approved by the Research Ethics Committee of the Centro Universitário Metodista do IPA (no 1.243.680/2015). The participants (n=15) were subbmited to the CEP during 90 days, 3 times a week for 60 minutes each session. In order to evaluate the short and long-term effects of exercise, blood samples were collected pre, 30, 60 and 90 days after the intervention began. Plasma BDNF levels were determined with the ELISA method, from Sigma-Aldrich commercial kit (catalog number RAB0026) according to manufacturer's instructions. Results: A remarkable increase on plasma BDNF levels at 90 days after training compared to baseline (p=0.006) and 30 days (p=0.007) values were observed. Conclusion: Our data are in agreement with several studies that show significant enhancement on BDNF levels in response to different exercise protocols in SZ individuals. We might suggest that BDNF upregulation after training in SZ patients acts in a dose-dependent manner, being more pronounced in response to chronic exposure. Acknowledgments: This work was supported by Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS)/Brazil.

Keywords: exercise, BDNF, schizophrenia, time-points

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Authors: Elisa Santana-Monagas, Juan L. Núñez, Jaime León, Samuel Falcón, Celia Fernández, Rocío P. Solís

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Teaching practices have been widely studied in relation to students' outcomes, positioning themselves as one of their strongest catalysts and influencing students' emotional experiences. In the higher education context, teachers become even more crucial as many students ground their decisions on which courses to enroll in based on opinions and ratings of teachers from other students. Unfortunately, sometimes universities do not provide the personal, social, and academic stimulation students demand to be actively engaged. To evaluate their teachers, universities often rely on students' evaluations of teaching (SET) collected via Likert scale surveys. Despite its usefulness, such a method has been questioned in terms of validity and reliability. Alternatively, researchers can rely on qualitative answers to open-ended questions. However, the unstructured nature of the answers and a large amount of information obtained requires an overwhelming amount of work. The present work presents an alternative approach to analyse such data: sentiment analysis. To the best of our knowledge, no research before has included results from SA into an explanatory model to test how students' sentiments affect their emotional engagement in class. The sample of the present study included a total of 225 university students (Mean age = 26.16, SD = 7.4, 78.7 % women) from the Educational Sciences faculty of a public university in Spain. Data collection took place during the academic year 2021-2022. Students accessed an online questionnaire using a QR code. They were asked to answer the following open-ended question: "If you had to explain to a peer who doesn't know your teacher how he or she communicates in class, what would you tell them?". Sentiment analysis was performed using Microsoft's pre-trained model. The reliability of the measure was estimated between the tool and one of the researchers who coded all answers independently. The Cohen's kappa and the average pairwise percent agreement were estimated with ReCal2. Cohen's kappa was .68, and the agreement reached was 90.8%, both considered satisfactory. To test the hypothesis relations among SA and students' emotional engagement, a structural equation model (SEM) was estimated. Results demonstrated a good fit of the data: RMSEA = .04, SRMR = .03, TLI = .99, CFI = .99. Specifically, the results showed that student’s sentiment regarding their teachers’ teaching positively predicted their emotional engagement (β == .16 [.02, -.30]). In other words, when students' opinion toward their instructors' teaching practices is positive, it is more likely for students to engage emotionally in the subject. Altogether, the results show a promising future for sentiment analysis techniques in the field of education. They suggest the usefulness of this tool when evaluating relations among teaching practices and student outcomes.

Keywords: sentiment analysis, students' evaluation of teaching, structural-equation modelling, emotional engagement

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Authors: V. T. Gerov, D. I. Gerova, I. D. Micheva, N. F. Nazifova-Tasinova, M. N. Nikolova, M. G. Pasheva, B. T. Galunska

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Multiple myeloma (MM) is the most frequent plasma cell (PC) dyscrasia that involves the skeleton. Myeloma bone disease (MBD) is characterized by osteolytic bone lesions as a result of increased osteoclasts activity not followed by reactive bone formation due to osteoblasts suppression. Skeletal complications cause significant adverse effects on quality of life and lead to increased morbidity and mortality. Last decade studies revealed the implication of different proteins in osteoclast activation and osteoblast inhibition. The aim of the present study was to determine serum levels of periostin, sRANKL and osteopontin and to evaluate their role as bone markers in MBD. Materials and methods. Thirty-two newly diagnosed MM patients (mean age: 62.2 ± 10.7 years) and 33 healthy controls (mean age: 58.9 ± 7.5 years) were enrolled in the study. According to IMWG criteria 28 patients were with symptomatic MM and 4 with monoclonal gammopathy of undetermined significance (MGUS). In respect to their bone involvement all symptomatic patients were divided into two groups (G): 9 patients with 0-3 osteolytic lesions (G1) and 19 patients with >3 osteolytic lesions and/or pathologic fractures (G2). Blood samples were drawn for routine laboratory analysis and for measurement of periostin, sRANKL and osteopontin serum levels by ELISA kits (Shanghai Sunred Biological Technology, China). Descriptive analysis, Mann-Whitney test for assessment the differences between groups and non-parametric correlation analysis were performed using GraphPad Prism v8.01. Results. The median serum levels of periostin, sRANKL and osteopontin of ММ patients were significantly higher compared to controls (554.7pg/ml (IQR=424.0-720.6) vs 396.9pg/ml (IQR=308.6-471.9), p=0.0001; 8.9pg/ml (IQR=7.1-10.5) vs 5.6pg/ml (IQR=5.1-6.4, p<0.0001 and 514.0ng/ml (IQR=469.3-754.0) vs 387.0ng/ml (IQR=335.9-441.9), p<0.0001, respectively). for assessment of differences between groups and non-parametric correlation analysis were performed using GraphPad Prism v8.01. Statistical significance was found for all tested bone markers between symptomatic MM patients and controls: G1 vs controls (p<0.03), G2 vs controls (p<0.0001) for periostin; G1 vs controls (p<0.0001), G2 vs controls (p<0.0001) for sRANKL; G1 vs controls (p=0.002), G2 vs controls (p<0.0001) for osteopontin, as well between symptomatic MM patients and MGUS patients: G1 vs MGUS (p<0.003), G2 vs MGUS (p=0.003) for periostin; G1 vs MGUS (p<0.05), G2 vs MGUS (p<0.001) for sRANKL; G1 vs MGUS (p=0.011), G2 vs MGUS (p=0.0001) for osteopontin. No differences were detected between MGUS and controls and between patients in G1 and G2 groups. Spearman correlation analysis revealed moderate positive correlation between periostin and beta-2-microglobulin (r=0.416, p=0.018), percentage bone marrow myeloma PC (r=0.432, p=0.014), and serum total protein (r=0.427, p=0.015). Osteopontin levels were also positively related to beta-2-microglobulin (r=0.540, p=0.0014), percentage bone marrow myeloma PC (r=0.423, p=0.016), and serum total protein (r=0.413, p=0.019). Serum sRANKL was only related to beta-2-microglobulin levels (r=0.398, p=0.024). Conclusion: In the present study, serum levels of periostin, sRANKL and osteopontin in newly diagnosed MM patients were evaluated. They gradually increased from MGUS to more advanced stages of MM reflecting the severity of bone destruction. These results support the idea that some new protein markers could be used in monitoring the MBD as a most severe complication of MM.

Keywords: myeloma bone disease, periostin, sRANKL, osteopontin

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Authors: Elisa Santana-Monagas, Juan L. Núñez, Jaime León, Samuel Falcón, Celia Fernández, Rocío P. Solís

Abstract:

Students’ emotional experiences have been a widely discussed theme among researchers, proving a central role on students’ outcomes. Yet, up to now, far too little attention has been paid to teaching practices that negatively relate with students’ negative emotions in the higher education. The present work aims to examine the relationship between teachers’ teaching practices (i.e., students’ evaluations of teaching and autonomy support), the students’ feelings of boredom and agentic engagement and motivation in the higher education context. To do so, the present study incorporates one of the most popular tools in natural processing language to address students’ evaluations of teaching: sentiment analysis. Whereas most research has focused on the creation of SA models and assessing students’ satisfaction regarding teachers and courses to the author’s best knowledge, no research before has included results from SA into an explanatory model. A total of 225 university students (Mean age = 26.16, SD = 7.4, 78.7 % women) participated in the study. Students were enrolled in degree and masters’ studies at the faculty of Education of a public university of Spain. Data was collected using an online questionnaire students could access through a QR code they completed during a teaching period where the assessed teacher was not present. To assess students’ sentiments towards their teachers’ teaching, we asked them the following open-ended question: “If you had to explain a peer who doesn't know your teacher how he or she communicates in class, what would you tell them?”. Sentiment analysis was performed with Microsoft's pre-trained model. For this study, we relied on the probability of the students answer belonging to the negative category. To assess the reliability of the measure, inter-rater agreement between this NLP tool and one of the researchers, who independently coded all answers, was examined. The average pairwise percent agreement and the Cohen’s kappa were calculated with ReCal2. The agreement reached was of 90.8% and Cohen’s kappa .68, both considered satisfactory. To test the hypothesis relations a structural equation model (SEM) was estimated. Results showed that the model fit indices displayed a good fit to the data; χ² (134) = 351.129, p < .001, RMSEA = .07, SRMR = .09, TLI = .91, CFI = .92. Specifically, results show that boredom was negatively predicted by autonomy support practices (β = -.47[-.61, -.33]), whereas for the negative sentiment extracted from SET, this relation was positive (β = .23[.16, .30]). In other words, when students’ opinion towards their instructors’ teaching practices was negative, it was more likely for them to feel bored. Regarding the relations among boredom and student outcomes, results showed a negative predictive value of boredom on students’ motivation to study (β = -.46[-.63, -.29]) and agentic engagement (β = -.24[-.33, -.15]). Altogether, results show a promising future for sentiment analysis techniques in the field of education as they proved the usefulness of this tool when evaluating relations among teaching practices and student outcomes.

Keywords: sentiment analysis, boredom, motivation, agentic engagement

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Authors: Ruslin, R. E. Kartasasmita, M. S. Wibowo, S. Ibrahim

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An aphrodisiac is a substance contained in food or drug that can arouse sexual instinct and increase pleasure while working, these substances derived from plants, animals, and minerals. When consuming substances that have aphrodisiac activity and duration can improve the sexual instinct. The natural aphrodisiac effect can be obtained through plants, animals, and minerals. Leonurine compound has aphrodisiac activity, these compounds can be isolated from plants of Leonurus Sp, Sundanese people is known as deundereman, this plant is empirical has aphrodisiac activity and based on the isolation of active compounds from plants known to contain compounds leonurine, so that the compound is expected to have activity aphrodisiac. Leonurine compound can be isolated from plants or synthesized chemically with material dasa siringat acid. Leonurine compound can be obtained commercial and derivatives of these compounds can be synthesized in an effort to increase its activity. This study aims to obtain derivatives leonurine better aphrodisiac activity compared with the parent compound, modified the structure of the compounds in the form leonurin guanidino butyl ester group with butyl amin and bromoetanol. ArgusLab program version 4.0.1 is used to determine the binding energy, hydrogen bonds and amino acids involved in the interaction of the compound PDE5 receptor. The in vivo test leonurine compounds and derivatives as an aphrodisiac ingredients and hormone testosterone levels using 27 male rats Wistar strain and 9 female mice of the same species, ages ranged from 12 weeks rats weighing + 200 g / tail. The test animal is divided into 9 groups according to the type of compounds and the dose given. Each treatment group was orally administered 2 ml per day for 5 days. On the sixth day was observed male rat sexual behavior and taking blood from the heart to measure testosterone levels using ELISA technique. Statistical analysis was performed in this study is the ANOVA test Least Square Differences (LSD) using the program Statistical Product and Service Solutions (SPSS). Aphrodisiac efficacy of the leonurine compound and its derivatives have proven in silico and in vivo test, the in silico testing leonurine derivatives have smaller binding energy derivatives leonurine so that activity better than leonurine compounds. Testing in vivo using rats of wistar strain that better leonurine derivative of this compound shows leonurine that in silico studies in parallel with in vivo tests. Modification of the structure in the form of guanidine butyl ester group with butyl amin and bromoethanol increase compared leonurine compound for aphrodisiac activity, testosterone derivatives of compounds leonurine experienced a significant improvement especial is 1RD compounds especially at doses of 100 and 150 mg/bb. The results showed that the compound leonurine and its compounds contain aphrodisiac activity and increase the amount of testosterone in the blood. The compound test used in this study acts as a steroid precursor resulting in increased testosterone.

Keywords: aphrodisiac dysfunction erectile leonurine 1-RD 2-RD, dysfunction, erectile leonurine, 1-RD 2-RD

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Authors: Faraj M. Elkhatri, Hana Ellafi

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In recent years, there has been a growing recognition of the potential of marine-based functional foods and combination therapies in promoting a healthy lifestyle and exploring their effectiveness in preventing or treating diseases. The combination of marine bioactive compounds or extracts offers synergistic or enhancement effects through various mechanisms, including multi-target actions, improved bioavailability, enhanced bioactivity, and mitigation of potential adverse effects. Both the green-lipped mussel (GLM) and fucoidan derived from brown seaweed are rich in bioactivities. These two, mussel and fucoidan, have not been previously formulated together. This study aims to combine GLM oil from Perna canaliculus with low molecular weight fucoidan (LMWF) extracted from Undaria pinnatifida to investigate the unique mixture’s anti-inflammatory and antioxidant properties. The cytotoxicity of individual compounds and combinations was assessed using the MTT assay in (THP-1 and RAW264.7) cell lines. The anti-inflammatory activity of mussel-fucoidan was evaluated by treating LPS-stimulated human monocyte and macrophage (THP1-1) cells. Subsequently, the inflammatory cytokines released into the supernatant of these cell lines were quantified via ELISA. Antioxidant activity was determined by using the free radical scavenging assay (DPPH). DPPH assay demonstrated that the radical scavenging activity of the combinations, particularly at concentrations exceeding 1 mg/ml, showed a significantly higher percentage of inhibition when compared to the individual component. This suggests an enhancement effect when the two compounds are combined, leading to increased antioxidant activity. In terms of immunomodulatory activity, the individual compounds exhibited distinct behaviors. GLM oil displayed a higher ability to suppress the cytokine TNF- compared to LMWF. Interestingly, the LMWF fraction, when used individually, did not demonstrate TNF- suppression. However, when combined with GLM, the TNF- suppression (anti-inflammatory) activity of the combination was better than GLM or LWMF alone. This observation underscores the potential for enhancement interactions between the two components in terms of anti-inflammatory properties. This study revealed that each individual compound, LMWF, and GLM, possesses unique and notable bioactivity. The combination of these two individual compounds results in an enhancement effect, where the bioactivity of each is enhanced, creating a superior combination. This suggests that the combination of LMWF and GLM has the potential to offer a more potent and multifaceted therapeutic effect, particularly in the context of antioxidant and anti-inflammatory activities. These findings hold promise for the development of novel therapeutic interventions or supplements that harness the enhancement effects.

Keywords: formation damage, porosity loses, pore throat, quartz cement

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Authors: Simona Dostalova, Pavel Kopel, Marketa Vaculovicova, Vojtech Adam, Rene Kizek

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Protein apoferritin seems to be a very promising structure for use as a nanocarrier. It is prepared from intracellular ferritin protein naturally found in most organisms. The role of ferritin proteins is to store and transport ferrous ions. Apoferritin is a hollow protein cage without ferrous ions that can be prepared from ferritin by reduction with thioglycolic acid or dithionite. The structure of apoferritin is composed of 24 protein subunits, creating a sphere with 12 nm in diameter. The inner cavity has a diameter of 8 nm. The drug encapsulation process is based on the response of apoferritin structure to the pH changes of surrounding solution. In low pH, apoferritin is disassembled into individual subunits and its structure is “opened”. It can then be mixed with any desired cytotoxic drug and after adjustment of pH back to neutral the subunits are reconnected again and the drug is encapsulated within the apoferritin particles. Excess drug molecules can be removed by dialysis. The receptors for apoferritin, SCARA5 and TfR1 can be found in the membrane of both healthy and cancer cells. To enhance the specific targeting of apoferritin nanocarrier, it is possible to modify its surface with targeting moieties, such as antibodies. To ensure sterically correct complex, we used a a peptide linker based on a protein G with N-terminus affinity towards Fc region of antibodies. To connect the peptide to the surface of apoferritin, the C-terminus of peptide was made of cysteine with affinity to gold. The surface of apoferritin with encapsulated doxorubicin (ApoDox) was coated either with gold nanoparticles (ApoDox-Nano) or gold (III) chloride hydrate reduced with sodium borohydride (ApoDox-HAu). The applied amount of gold in form of gold (III) chloride hydrate was 10 times higher than in the case of gold nanoparticles. However, after removal of the excess unbound ions by electrophoretic separation, the concentration of gold on the surface of apoferritin was only 6 times higher for ApoDox-HAu in comparison with ApoDox-Nano. Moreover, the reduction with sodium borohydride caused a loss of doxorubicin fluorescent properties (excitation maximum at 480 nm with emission maximum at 600 nm) and thus its biological activity. Fluorescent properties of ApoDox-Nano were similar to the unmodified ApoDox, therefore it was more suited for the intended use. To evaluate the specificity of apoferritin modified with antibodies, we used ELISA-like method with the surface of microtitration plate wells coated by the antigen (goat anti-human IgG antibodies). To these wells, we applied ApoDox without targeting antibodies and ApoDox-Nano modified with targeting antibodies (human IgG antibodies). The amount of unmodified ApoDox on antigen after incubation and subsequent rinsing with water was 5 times lower than in the case of ApoDox-Nano modified with targeting antibodies. The modification of non-gold ApoDox with antibodies caused no change in its targeting properties. It can therefore be concluded that the demonstrated procedure allows us to create nanocarrier with enhanced targeting properties, suitable for nanomedicine.

Keywords: apoferritin, doxorubicin, nanocarrier, targeting antibodies

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