Search results for: insulin signaling

Authors: Farideh Tabatabaee Yazdi, Fereshteh Falah, Alireza Vasiee

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Introduction: Gamma-aminobutyric acid (GABA) is a non-protein amino acid that is widely present in organisms. GABA is a kind of pharmacological and biological component and its application is wide and useful. Several important physiological functions of GABA have been characterized, such as neurotransmission and induction of hypotension. GABA is also a strong secretagogue of insulin from the pancreas and effectively inhibits small airway-derived lung adenocarcinoma and tranquilizer. Many microorganisms can produce GABA, and lactic acid bacteria have been a focus of research in recent years because lactic acid bacteria possess special physiological activities and are generally regarded as safe. Among them, the Lb. Brevis produced the highest amount of GABA. The major factors affecting GABA production have been characterized, including carbon sources and glutamate concentration. The use of food industry waste to produce valuable products such as amino acids seems to be a good way to reduce production costs and prevent the waste of food resources. In a dairy factory, a high volume of sludge is produced from a separator that contains useful compounds such as growth factors, carbon, nitrogen, and organic matter that can be used by different microorganisms such as Lb.brevis as carbon and nitrogen sources. Therefore, it is a good source of GABA production. GABA is primarily formed by the irreversible α-decarboxylation reaction of L-glutamic acid or its salts, catalysed by the GAD enzyme. In the present study, this aim was achieved for the fast-growing of Lb.brevis and producing GABA, using the dairy industry sludge as a suitable growth medium. Lactobacillus Brevis strains obtained from Microbial Type Culture Collection (MTCC) were used as model strains. In order to prepare dairy sludge as a medium, sterilization should be done at 121 ° C for 15 minutes. Lb. Brevis was inoculated to the sludge media at pH=6 and incubated for 120 hours at 30 ° C. After fermentation, the supernatant solution is centrifuged and then, the GABA produced was analyzed by the Thin Layer chromatography (TLC) method qualitatively and by the high-performance liquid chromatography (HPLC) method quantitatively. By increasing the percentage of dairy sludge in the culture medium, the amount of GABA increased. Also, evaluated the growth of bacteria in this medium showed the positive effect of dairy sludge on the growth of Lb.brevis, which resulted in the production of more GABA. GABA-producing LAB offers the opportunity of developing naturally fermented health-oriented products. Although some GABA-producing LAB has been isolated to find strains suitable for different fermentations, further screening of various GABA-producing strains from LAB, especially high-yielding strains, is necessary. The production of lactic acid, bacterial gamma-aminobutyric acid, is safe and eco-friendly. The use of dairy industry waste causes enhanced environmental safety. Also provides the possibility of producing valuable compounds such as GABA. In general, dairy sludge is a suitable medium for the growth of Lactic Acid Bacteria and produce this amino acid that can reduce the final cost of it by providing carbon and nitrogen source.

Keywords: GABA, Lactobacillus, HPLC, dairy sludge

Procedia PDF Downloads 95

Authors: Margarita Ivanova, Julia Dao, Andrew Friedman, Neil Kasaci, Rekha Gopal, Ozlem Goker-Alpan

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In Fabry disease (FD), α-galactosidase A (α-Gal A) deficiency leads to the accumulation of globotriaosylceramide (Lyso-Gb3 and Gb3), triggering a pathologic cascade that causes the severity of organs damage. The heart is one of the several organs with high sensitivity to the α-Gal A deficiency. A subgroup of patients with significant residual of α-Gal A activity with primary cardiac involvement is occasionally referred to as “cardiac variant.” The cardiovascular complications are most frequently encountered, contributing substantially to morbidity, and are the leading cause of premature death in male and female patients with FD. The deposition of Lyso-Gb-3 and Gb-3 within the myocardium affects cardiac function with resultant progressive cardiovascular pathology. Gb-3 and Lyso-Gb-3 accumulation at the cellular level trigger a cascade of events leading to end-stage fibrosis. In the cardiac tissue, Lyso-Gb-3 deposition is associated with the increased release of inflammatory factors and transforming growth factors. Infiltration of lymphocytes and macrophages into endomyocardial tissue indicates that inflammation plays a significant role in cardiac damage. Moreover, accumulated data suggest that chronic inflammation leads to multisystemic FD pathology even under enzyme replacement therapy (ERT). NF-κB activation plays a subsequent role in the inflammatory response to cardiac dysfunction and advanced heart failure in the general population. TNFalpha/NF-κB signaling protects the myocardial evoking by ischemic preconditioning; however, this protective effect depends on the concentration of TNF-α. Thus, we hypothesize that TNF-α is a critical factor in determining the grade of cardio-pathology. Cardiac hypertrophy corresponds to the expansion of the coronary vasculature to maintain a sufficient supply of nutrients and oxygen. Coronary activation of angiogenesis and fibrosis plays a vital role in cardiac vascularization, hypertrophy, and tissue remodeling. We suggest that the interaction between the inflammatory pathways and cardiac vascularization is a bi-directional process controlled by secreted cytokines and growth factors. The co-coordination of these two processes has never been explored in FD. In a cohort of 40 patients with FD, biomarkers associated with inflammation and cardio hypertrophic remodeling were studied. FD patients were categorized into three groups based on LVmass/DSA, LVEF, and ECG abnormalities: FD with no cardio complication, FD with moderate cardio complication, and severe cardio complication. Serum levels of NF-kB, TNFalpha, Il-6, Il-2, MCP1, ING-gamma, VEGF, IGF-1, TGFβ, and FGF2 were quantified by enzyme-linked immunosorbent assays (ELISA). Among the biomarkers, MCP-1, INF-gamma, VEGF, TNF-alpha, and TGF-beta were elevated in FD patients. Some of these biomarkers also have the potential to correlate with cardio pathology in FD. Conclusion: The study provides information about the role of inflammatory pathways and biomarkers of cardio hypertrophic remodeling in FD patients. This study will also reveal the mechanisms that link intracellular accumulation of Lyso-GB-3 and Gb3 to the development of cardiomyopathy with myocardial thickening and resultant fibrosis.

Keywords: biomarkers, Fabry disease, inflammation, growth factors

Procedia PDF Downloads 51

Authors: B. Baran, S. N. Kaptanoglu, M. Ocal, Y. Kagnici, E. Esen, E. Siyez, D. M. Siyez

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Infertility is the fact that couples cannot have children despite 1 year of unprotected sexual life. Infertility can be considered as an important problem affecting not only sexual life but also social and psychological conditions of couples. Learning about information about preventable factors related to infertility during university years plays an important role in preventing a possible infertility case in older ages. The possibility to facilitate access to information with the internet has provided the opportunity to reach a broad audience in the diverse learning environments and educational environment. Moreover, the internet has become a basic resource for the 21st-century learners. Providing information about infertility over the internet will enable more people to reach in a short time. When studies conducted abroad about infertility are examined, interactive websites and online education programs come to the fore. In Turkey, while there is no comprehensive online education program for university students, it seems that existing studies are aimed to make more advertisements for doctors or hospitals. In this study, it was aimed to design and develop online infertility prevention education for university students. Mayer’s Multimedia Learning Theory made up the framework for the online learning environment in this study. The results of the needs analysis collected from the university students in Turkey who were selected with sampling to represent the audience for online learning contributed to the design phase. In this study, an infertility prevention online education environment designed as a 4-week education was developed by explaining the theoretical basis and needs analysis results. As a result; in the development of the online environment, different kind of visual aids that will increase teaching were used in the environment of online education according to Mayer’s principles of extraneous processing (coherence, signaling, spatial contiguity, temporal contiguity, redundancy, expectation principles), essential processing (segmenting, pre-training, modality principles) and generative processing (multimedia, personalization, voice principles). For example, the important points in reproductive systems’ expression were emphasized by visuals in order to draw learners’ attention, and the presentation of the information was also supported by the human voice. In addition, because of the limited knowledge of university students in the subject, the issue of female reproductive and male reproductive systems was taught before preventable factors related to infertility. Furthermore, 3D video and augmented reality application were developed in order to embody female and male reproductive systems. In conclusion, this study aims to develop an interactive Online Infertility Prevention Education in which university students can easily access reliable information and evaluate their own level of knowledge about the subject. It is believed that the study will also guide the researchers who want to develop online education in this area as it contains design-stage decisions of interactive online infertility prevention education for university students.

Keywords: infertility, multimedia learning theory, online education, reproductive health

Procedia PDF Downloads 139

Authors: Meitham Amereh, Mohsen Akbari, Ben Nadler

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Cancer has been recognized as one of the most challenging problems in biology and medicine. Aggressive tumors are a lethal type of cancers characterized by high genomic instability, rapid progression, invasiveness, and therapeutic resistance. Their behavior involves complicated molecular biology and consequential dynamics. Although tremendous effort has been devoted to developing therapeutic approaches, there is still a huge need for new insights into the dark aspects of tumors. As one of the key requirements in better understanding the complex behavior of tumors, mathematical modeling and continuum physics, in particular, play a pivotal role. Mathematical modeling can provide a quantitative prediction on biological processes and help interpret complicated physiological interactions in tumors microenvironment. The pathophysiology of aggressive tumors is strongly affected by the extracellular cues such as stresses produced by mechanical forces between the tumor and the host tissue. During the tumor progression, the growing mass displaces the surrounding extracellular matrix (ECM), and due to the level of tissue stiffness, stress accumulates inside the tumor. The produced stress can influence the tumor by breaking adherent junctions. During this process, the tumor stops the rapid proliferation and begins to remodel its shape to preserve the homeostatic equilibrium state. To reach this, the tumor, in turn, upregulates epithelial to mesenchymal transit-inducing transcription factors (EMT-TFs). These EMT-TFs are involved in various signaling cascades, which are often associated with tumor invasiveness and malignancy. In this work, we modeled the tumor as a growing hyperplastic mass and investigated the effects of mechanical stress from surrounding ECM on tumor invasion. The invasion is modeled as volume-preserving inelastic evolution. In this framework, principal balance laws are considered for tumor mass, linear momentum, and diffusion of nutrients. Also, mechanical interactions between the tumor and ECM is modeled using Ciarlet constitutive strain energy function, and dissipation inequality is utilized to model the volumetric growth rate. System parameters, such as rate of nutrient uptake and cell proliferation, are obtained experimentally. To validate the model, human Glioblastoma multiforme (hGBM) tumor spheroids were incorporated inside Matrigel/Alginate composite hydrogel and was injected into a microfluidic chip to mimic the tumor’s natural microenvironment. The invasion structure was analyzed by imaging the spheroid over time. Also, the expression of transcriptional factors involved in invasion was measured by immune-staining the tumor. The volumetric growth, stress distribution, and inelastic evolution of tumors were predicted by the model. Results showed that the level of invasion is in direct correlation with the level of predicted stress within the tumor. Moreover, the invasion length measured by fluorescent imaging was shown to be related to the inelastic evolution of tumors obtained by the model.

Keywords: cancer, invasion, mathematical modeling, microfluidic chip, tumor spheroids

Procedia PDF Downloads 86

Authors: Simone Raimondi De Souza, Glaucia Maria Moraes De Oliveira, Ronir Raggio Luiz, Glorimar Rosa

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Introduction: Obesity is among the main risk factors for cardiovascular disease (CVD). Genesis is multifactorial, including genetic, hormonal and environmental factors disorders, among which inadequate feeding pattern, for which nutritional counseling strategies have proven effective. The consumption of beta-glucans (soluble fibers that reportedly promote satiety) present in oat bran can be an effective strategy for preventing and treating obesity. Other benefits have been observed with oat bran consumption, such as reduction of hypercholesterolemia and hyperglycemia, two other risk factors for CVD. Objectives: To analyze the effect of oat bran consumption associated with nutritional counseling in reducing body mass index (BMI), blood cholesterol, glucose profile, waist and neck circumference in obese individuals, and to evaluate the change in eating pattern. Methods: clinical trial, randomized, double-blind, placebo-controlled, lasting 90 days with adults of both genders, with BMI ≥30kg/m2. The study was approved by the Ethics in Research involving human beings in a public institute of cardiology, in Rio de Janeiro, Brazil. Individuals were invited to participate and accepted formally by signing the Terms of Consent. Participants were randomized into oat bran group (gOB) or placebo group (gPCB) and received, respectively: morning prepared consisting of 40g oat bran, 30g of skimmed milk powder and 1g sweetener sucralose; refined flour 40g rice, 30g of milk powder and 1g sweetener sucralose. The Ten Steps to Healthy Eating, of Brazilian Ministry of Health were used to support the nutritional counseling. Variables analyzed: gender; age; BMI, waist circumference (WC) neck circumference (NC); systolic blood pressure (SBP); diastolic blood pressure (DBP); food consumption, total cholesterol (TC), LDL-cholesterol (LDL-c), HDL-cholesterol (HDL-c), non-HDL cholesterol (nHDLc), triglycerides (TG), fasting glucose (FG), fasting insulin (FI) and HOMA-IR. Dietary intake was assessed by 24-hour dietary recall. The Diet Quality Index revised for the Brazilian population (IQD-R) assessed quality of feeding pattern. Statistical analyzes were performed using SPSS version 21, considering statistically significant p-value less than 0.05. Results: A total of 38 participants were included, age = 50 ± 7,6years, 63% women. 19 subjects were placed in gOB and 19 in gPCB. After intervention, statistically significant reductions were observed in the following parameters: in gOB: IQD-R, TC, LDL-c, nHDL-c, FI, SBP, DBP, BMI, WC, NC; in gPCB: IQD-R, LDL-c, SBP, DBP, BMI, WC, NC. No statistically significant differences were observed in the results between groups. Conclusion: Our results reinforce nutritional counseling as important strategy for prevention and treatment of obesity and suggest that inclusion of oat bran in daily diet can bring additional benefits controlling risk factors for CVD. More studies are needed to establish all benefits of oat bran to human health as well as the ideal daily dose for consumption.

Keywords: oat bran, cardiovascular disease, nutritional counseling, obesity

Procedia PDF Downloads 202

Authors: Li Jiuen Ong, Magdalin Cheong, Sri Rahayu, Lek Alexander, Pei Ting Tan

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Background: Previous programme evaluations from the diabetes training programme conducted in Changi General Hospital revealed that healthcare professionals (HCPs) are keen to receive advance diabetes training and education, specifically in medical, nutritional therapy. HCPs also expressed a preference for interactive activities over didactic teaching methods to enhance their learning. Since the War on Diabetes was initiated by MOH in 2016, HCPs are challenged to be actively involved in continuous education to be better equipped to reduce the growing burden of diabetes. Hence, streamlining training to incorporate an element of fun is of utmost importance. Aim: The nutrition programme incorporates game play using an interactive board game that aims to provide a more conducive and less stressful environment for learning. The board game could be adapted for training of community HCPs, health ambassadors or caregivers to cope with the increasing demand of diabetes care in the hospital and community setting. Methodology: Stages for game’s conception (Jaffe, 2001) were adopted in the development of the interactive board game ‘Sweet Score™ ’ Nutrition concepts and topics in diabetes self-management are embedded into the game elements of varying levels of difficulty (‘Easy,’ ‘Medium,’ ‘Hard’) including activities such as a) Drawing/ sculpting (Pictionary-like) b)Facts/ Knowledge (MCQs/ True or False) Word definition) c) Performing/ Charades To study the effects of game play on knowledge acquisition and perceived experiences, participants were randomised into two groups, i.e., lecture group (control) and game group (intervention), to test the difference. Results: Participants in both groups (control group, n= 14; intervention group, n= 13) attempted a pre and post workshop quiz to assess the effectiveness of knowledge acquisition. The scores were analysed using paired T-test. There was an improvement of quiz scores after attending the game play (mean difference: 4.3, SD: 2.0, P<0.001) and the lecture (mean difference: 3.4, SD: 2.1, P<0.001). However, there was no significance difference in the improvement of quiz scores between gameplay and lecture (mean difference: 0.9, 95%CI: -0.8 to 2.5, P=0.280). This suggests that gameplay may be as effective as a lecture in terms of knowledge transfer. All the13 HCPs who participated in the game rated 4 out of 5 on the likert scale for the favourable learning experience and relevance of learning to their job, whereas only 8 out of 14 HCPs in the lecture reported a high rating in both aspects. 16. Conclusion: There is no known board game currently designed for diabetes training for HCPs.Evaluative data from future training can provide insights and direction to improve the game format and cover other aspects of diabetes management such as self-care, exercise, medications and insulin management. Further testing of the board game to ensure learning objectives are met is important and can assist in the development of awell-designed digital game as an alternative training approach during the COVID-19 pandemic. Learning through gameplay increases opportunities for HCPs to bond, interact and learn through games in a relaxed social setting and potentially brings more joy to the workplace.

Keywords: active learning, game, diabetes, nutrition

Procedia PDF Downloads 147

Authors: G. Cunningham, E. Cantor, X. Wu, F. Shen, G. Jiang, S. Philips, C. Bales, Y. Xiao, T. R. Cummins, J. C. Fehrenbacher, B. P. Schneider

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Background: Taxane-induced peripheral neuropathy (TIPN) is the most devastating survivorship issue for patients receiving therapy. Dose reductions due to TIPN in the curative setting lead to inferior outcomes for African American patients, as prior research has shown that this group is more susceptible to developing severe neuropathy. The mechanistic underpinnings of TIPN, however, have not been entirely elucidated. While it would be appealing to use primary tissue to study the development of TIPN, procuring nerves from patients is not realistically feasible, as nerve biopsies are painful and may result in permanent damage. Therefore, our laboratory has investigated paclitaxel-induced neuronal morphological and molecular changes using an ex vivo model of human-induced pluripotent stem cell (iPSC)-derived neurons. Methods: iPSCs are undifferentiated and endlessly dividing cells that can be generated from a patient’s somatic cells, such as peripheral blood mononuclear cells (PBMCs). We successfully reprogrammed PBMCs into iPSCs using the Erythroid Progenitor Reprograming Kit (STEMCell Technologiesᵀᴹ); pluripotency was verified by flow cytometry analysis. iPSCs were then induced into neurons using a differentiation protocol that bypasses the neural progenitor stage and uses selected small-molecule modulators of key signaling pathways (SMAD, Notch, FGFR1 inhibition, and Wnt activation). Results: Flow cytometry analysis revealed expression of core pluripotency transcription factors Nanog, Oct3/4 and Sox2 in iPSCs overlaps with commercially purchased pluripotent cell line UCSD064i-20-2. Trilineage differentiation of iPSCs was confirmed with immunofluorescent imaging with germ-layer-specific markers; Sox17 and ExoA2 for ectoderm, Nestin, and Pax6 for mesoderm, and Ncam and Brachyury for endoderm. Sensory neuron markers, β-III tubulin, and Peripherin were applied to stain the cells for the maturity of iPSC-derived neurons. Patch-clamp electrophysiology and calcitonin gene-related peptide (CGRP) release data supported the functionality of the induced neurons and provided insight into the timing for which downstream assays could be performed (week 4 post-induction). We have also performed a cell viability assay and fluorescence-activated cell sorting (FACS) using four cell-surface markers (CD184, CD44, CD15, and CD24) to select a neuronal population. At least 70% of the cells were viable in the isolated neuron population. Conclusion: We have found that these iPSC-derived neurons recapitulate mature neuronal phenotypes and demonstrate functionality. Thus, this represents a patient-derived ex vivo neuronal model to investigate the molecular mechanisms of clinical TIPN.

Keywords: chemotherapy, iPSC-derived neurons, peripheral neuropathy, taxane, paclitaxel

Procedia PDF Downloads 99

Authors: Soumia Hammadi, Imane Nouacer, Lamine Hamida, Younes A. Hammadi, Rachid Chaibi

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Aims and objective: In the present work, we investigate the impact of both acute and chronic diabetes mellitus induced by streptozotocin (STZ) on the hypothalamus of the small gerbil (Gerbillus gerbillus). In this purpose, we aimed to study the histologic structure of the gerbil’s hypothalamic supraoptic (NSO) and paraventricular nucleus (NPV) at two distinct time points: two days and 30 days after diabetes onset. Methods: We conducted our investigation using 19 adult male gerbils weighing 25 to 28 g, divided into three groups as follow: Group I: Control gerbils (n=6) received an intraperitoneal injection of citrate buffer. Group II: STZ-diabetic gerbils (n=8) received a single intraperitoneal injection of STZ at a dose of 165 mg/kg of body weight. Diabetes onset (D0) is considered with the first hyperglycemia level exceeding 2,5 g/L. This group was further divided into two subgroups: Group II-1: Experimental Gerbils, at acute state of diabetes (n=8) sacrificed after 02 days of diabetes onset, Group II-2: Experimental Gerbils at chronic state of diabetes (n=7) sacrificed after 30 days of diabetes onset. Two and 30 days after diabetes onset, gerbils had blood drawn from the retro-orbital sinus into EDTA tubes. After centrifugation at -4°C, plasma was frozen at -80°C for later measurement of Cortisol, ACTH, and insulin. Afterward, animals were decapitated; their brain was removed, weighed, fixed in aqueous bouin, and processed and stained with Toluidine Bleu stain for histo-stereological analysis. A comparison was done with control gerbils treated with citrate buffer. Results: Compared to control gerbils, at 02 Days post diabetes onset, the neuronal somata of the paraventricular (NPV) and supraoptic nuclei (NSO) expressed numerous vacuoles of various sizes, we distinct also a neuronal juxtaposition and several unidentifiable vacuolated profiles were also seen in the neuropile. At the same time, we revealed the presence of à shrunken and condensed nuclei, which seem to touch the parvocellular neurons ( NPV); this leads us to suggest the presence of an apoptotic process in the early stage of diabetes. At 30 days of diabetes mellitus, the NPV manifests a few neurons with a distant appearance, in addition the magnocellular neurons in both NPV and NSO were hypertrophied with a rich euchromatin nucleus, a well-defined nucleolus, and a granular cytoplasm. Despite the neuronal degeneration at this stage, unexpectedly, ACTH registers a continuous significant high level compared to the early stage of diabetes mellitus and to control gerbils. Conclusion: The results suggest that the induction of diabetes mellitus using STZ in the small gerbils lead to alterations in the structure and morphology of the hypothalamus and hyper-secretion of ACTH and cortisol, possibly indicating hyperactivity of the hypothalamo-pituitary adrenal axis (HPA) during both the early and later stages of the disease. The subsequent quantitative evaluation of CRH, immunehistochemical evaluation of apoptosis, and oxidative stress assessment could corroborate our results.

Keywords: diabetes type 1., streptozotocin., small gerbil., hypothalamus., paraventricular nucleus., supraoptic nucleus.

Procedia PDF Downloads 36

Authors: Javier Enciso-Benavides, Fredy Fabian, Carlos Castaneda, Luis Alfaro, Alex Choque, Aparicio Aguilar, Javier Enciso

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Background: The use of biomarkers in breast cancer diagnosis, therapy, and prognosis has gained increasing interest. Cancer stem cells (CSCs) are a subpopulation of tumor cells that can drive tumor initiation and may cause relapse. Therefore, due to the importance of diagnosis, therapy, and prognosis, several biomarkers that characterize CSCs have been identified; however, in treatment-naïve triple-negative breast tumors, there is an urgent need to identify new biomarkers and therapeutic targets. According to this, the aim of this study was to identify serum proteins associated with cancer stem cells and pluripotency in women with triple-negative breast tumors in order to subsequently identify a biomarker for this type of breast tumor. Material and Methods: Whole blood samples from 12 women with histopathologically diagnosed triple-negative breast tumors were used after obtaining informed consent from the patient. Blood serum was obtained by conventional procedure and frozen at -80ºC. Identification of cancer stem cell-associated proteins was performed by matrix-assisted laser desorption/ionisation-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS), protein analysis was obtained using the AB Sciex TOF/TOF™ 5800 system (AB Sciex, USA). Sequences not aligned by ProteinPilot™ software were analyzed by Protein BLAST. Results: The following proteins related to pluripotency and cancer stem cells were identified by MALDI TOF/TOF mass spectrometry: A-chain, Serpin A12 [Homo sapiens], AIEBP [Homo sapiens], Alpha-one antitrypsin, AT {internal fragment} [human, partial peptide, 20 aa] [Homo sapiens], collagen alpha 1 chain precursor variant [Homo sapiens], retinoblastoma-associated protein variant [Homo sapiens], insulin receptor, CRA_c isoform [Homo sapiens], Hydroxyisourate hydrolase [Streptomyces scopuliridis], MUCIN-6 [Macaca mulatta], Alpha-actinin-3 [Chrysochloris asiatica], Polyprotein M, CRA_d isoform, partial [Homo sapiens], Transcription factor SOX-12 [Homo sapiens]. Recommendations: The serum proteins identified in this study should be investigated in the exosome of triple-negative breast cancer stem cells and in the blood serum of women without breast cancer. Subsequently, proteins found only in the blood serum of women with triple-negative breast cancer should be identified in situ in triple-negative breast cancer tissue in order to identify a biomarker to study the evolution of this type of cancer, or that could be a therapeutic target. Conclusions: Eleven cancer stem cell-related serum proteins were identified in 12 women with triple-negative breast cancer, of which MUCIN-6, retinoblastoma-associated protein variant, transcription factor SOX-12, and collagen alpha 1 chain are the most representative and have not been studied so far in this type of breast tumor. Acknowledgement: This work was supported by Proyecto CONCYTEC–Banco Mundial “Mejoramiento y Ampliacion de los Servicios del Sistema Nacional de Ciencia Tecnología e Innovacion Tecnologica” 8682-PE (104-2018-FONDECYT-BM-IADT-AV).

Keywords: triple-negative breast cancer, MALDI TOF/TOF MS, serum proteins, cancer stem cells

Procedia PDF Downloads 184

Authors: Hirseland Andreas, Kerschbaumer Lukas

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In the case of Germany, there are about one million long-term unemployed – a figure that did not vary much during the past years. These long-term unemployed did not benefit from the prospering labor market while most short-term unemployed did. Instead, they are continuously dependent on welfare and sometimes precarious short-term employment, experiencing work poverty. Long-term unemployment thus turns into a main obstacle to become employed again, especially if it is accompanied by other impediments such as low-level education (school/vocational), poor health (especially chronical illness), advanced age (older than fifty), immigrant status, motherhood or engagement in care for other relatives. As can be shown by this current research project, in these cases the chance to regain employment decreases to near nil. Almost two-thirds of all welfare recipients have multiple impediments which hinder a successful transition from welfare back to sustainable and sufficient employment. Prospective employers are unlikely to hire long-term unemployed with additional impediments because they evaluate potential employees on their negative signaling (e.g. low-level education) and the implicit assumption of unproductiveness (e.g. poor health, age). Some findings of the panel survey “Labor market and social security” (PASS) carried out by the Institute of Employment Research (the research institute of the German Federal Labor Agency) spread a ray of hope, showing that unlikely does not necessarily mean impossible. The presentation reports on current research on these very scarce “success stories” of unlikely transitions from long-term unemployment to work and how these cases were able to perform this switch against all odds. The study is based on a mixed-method design. Within the panel survey (~15,000 respondents in ~10,000 households), only 66 cases of such unlikely transitions were observed. These cases have been explored by qualitative inquiry – in depth-interviews and qualitative network techniques. There is strong evidence that sustainable transitions are influenced by certain biographical resources like habits of network use, a set of informal skills and particularly a resilient way of dealing with obstacles, combined with contextual factors rather than by job-placement procedures promoted by Job-Centers according to activation rules or by following formal paths of application. On the employer’s side small and medium-sized enterprises are often found to give job opportunities to a wider variety of applicants, often based on a slow but steadily increasing relationship leading to employment. According to these results it is possible to show and discuss some limitations of (German) activation policies targeting the labor market and their impact on welfare dependency and long-term unemployment. Based on these findings, indications for more supportive small-scale measures in the field of labor-market policies are suggested to help long-term unemployed with multiple impediments to overcome their situation (e.g. organizing small-scale-structures and low-threshold services to encounter possible employers on a more informal basis like “meet and greet”).

Keywords: against-all-odds, mixed-methods, Welfare State, long-term unemployment

Procedia PDF Downloads 341

Authors: Priyanka Jain, Ashish K. Sharma, Esha Shukla, K. J. Mukherjee

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We propose a paradigm shift in our approach to design improved platforms for recombinant protein production, by addressing system level issues rather than the individual steps associated with recombinant protein synthesis like transcription, translation, etc. We demonstrate that by controlling and modulating the cellular stress response (CSR), which is responsible for feedback control of protein synthesis, we can generate hyper-producing strains. We did transcriptomic profiling of post-induction cultures, expressing different types of protein, to analyze the nature of this cellular stress response. We found significant down-regulation of substrate utilization, translation, and energy metabolism genes due to generation CSR inside the host cell. However, transcription profiling has also shown that many genes are up-regulated post induction and their role in modulating the CSR is unclear. We hypothesized that these up-regulated genes trigger signaling pathways, generating the CSR and concomitantly reduce the recombinant protein yield. To test this hypothesis, we knocked out the up-regulated genes, which did not have any downstream regulatees, and analyzed their impact on cellular health and recombinant protein expression. Two model proteins i.e., GFP and L-Asparaginase were chosen for this analysis. We observed a significant improvement in expression levels, with some knock-outs showing more than 7-fold higher expression compared to control. The 10 best single knock-outs were chosen to make 45 combinations of all possible double knock-outs. A further increase in expression was observed in some of these double knock- outs with GFP levels being highest in a double knock-out ΔyhbC + ΔelaA. However, for L-Asparaginase which is a secretory protein, the best results were obtained using a combination of ΔelaA+ΔcysW knock-outs. We then tested all the knock outs for their ability to enhance the expression of a 'difficult-to-express' protein. The Rubella virus E1 protein was chosen and tagged with sfGFP at the C-terminal using a linker peptide for easy online monitoring of expression of this fusion protein. Interestingly, the highest increase in Rubella-sGFP levels was obtained in the same double knock-out ΔelaA + ΔcysW (5.6 fold increase in expression yield compared to the control) which gave the highest expression for L-Asparaginase. However, for sfGFP alone, the ΔyhbC+ΔmarR knock-out gave the highest level of expression. These results indicate that there is a fair degree of commonality in the nature of the CSR generated by the induction of different proteins. Transcriptomic profiling of the double knock out showed that many genes associated with the translational machinery and energy biosynthesis did not get down-regulated post induction, unlike the control where these genes were significantly down-regulated. This confirmed our hypothesis of these genes playing an important role in the generation of the CSR and allowed us to design a strategy for making better expression hosts by simply knocking out key genes. This strategy is radically superior to the previous approach of individually up-regulating critical genes since it blocks the mounting of the CSR thus preventing the down-regulation of a very large number of genes responsible for sustaining the flux through the recombinant protein production pathway.

Keywords: cellular stress response, GFP, knock-outs, up-regulated genes

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Authors: Ewelina Wisnik, Zsolt Regdon, Kinga Chmielewska, Laszlo Virag, Agnieszka Robaszkiewicz

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Apart from protecting genome, PARP1 has been documented to regulate many intracellular processes inter alia gene transcription by physically interacting with chromatin bound proteins and by their ADP-ribosylation. Our recent findings indicate that expression of PARP1 decreases during the differentiation of human CD34+ hematopoietic stem cells to monocytes as a consequence of differentiation-associated cell growth arrest and formation of E2F4-RBL2-HDAC1-SWI/SNF repressive complex at the promoter of this gene. Since the RBL2 complexes repress genes in a E2F-dependent manner and are widespread in the genome in G0 arrested cells, we asked (a) if RBL2 directly contributes to defining monocyte phenotype and function by targeting gene promoters and (b) if RBL2 controls gene transcription indirectly by repressing PARP1. For identification of genes controlled by RBL2 and/or PARP1,we used primer libraries for surface receptors and TLR signaling mediators, genes were silenced by siRNA or shRNA, analysis of gene promoter occupation by selected proteins was carried out by ChIP-qPCR, while statistical analysis in GraphPad Prism 5 and STATISTICA, ChIP-Seq data were analysed in Galaxy 2.5.0.0. On the list of 28 genes regulated by RBL2, we identified only four solely repressed by RBL2-E2F4-HDAC1-BRM complex. Surprisingly, 24 out of 28 emerged genes controlled by RBL2 were co-regulated by PARP1 in six different manners. In one mode of RBL2/PARP1 co-operation, represented by MAP2K6 and MAPK3, PARP1 was found to associate with gene promoters upon RBL2 silencing, which was previously shown to restore PARP1 expression in monocytes. PARP1 effect on gene transcription was observed only in the presence of active EP300, which acetylated gene promoters and activated transcription. Further analysis revealed that PARP1 binding to MA2K6 and MAPK3 promoters enabled recruitment of EP300 in monocytes, while in proliferating cancer cell lines, which actively transcribe PARP1, this protein maintained EP300 at the promoters of MA2K6 and MAPK3. Genome-wide analysis revealed a similar distribution of PARP1 and EP300 around transcription start sites and the co-occupancy of some gene promoters by PARP1 and EP300 in cancer cells. Here, we described a new RBL2/PARP1/EP300 axis which controls gene transcription regardless of the cell type. In this model cell, cycle-dependent transcription of PARP1 regulates expression of some genes repressed by RBL2 upon cell cycle limitation. Thus, RBL2 may indirectly regulate transcription of some genes by controlling the expression of EP300-recruiting PARP1. Acknowledgement: This work was financed by Polish National Science Centre grants nr DEC-2013/11/D/NZ2/00033 and DEC-2015/19/N/NZ2/01735. L.V. is funded by the National Research, Development and Innovation Office grants GINOP-2.3.2-15-2016-00020 TUMORDNS, GINOP-2.3.2-15-2016-00048-STAYALIVE and OTKA K112336. AR is supported by Polish Ministry of Science and Higher Education 776/STYP/11/2016.

Keywords: retinoblastoma transcriptional co-repressor like 2 (RBL2), poly(ADP-ribose) polymerase 1 (PARP1), E1A binding protein p300 (EP300), monocytes

Procedia PDF Downloads 176

Authors: Marie-Helena Docherty, Sion Edryd Williams

Abstract:

The prevalence of diabetes mellitus is increasing worldwide. In Sub-Saharan Africa, type 2 diabetes represents over 90% of all types of diabetes with the number of diabetic patients expected to rise. This represents a huge economic burden in an area already contending with high rates of other significant diseases, including the highest worldwide prevalence of HIV. Diabetic complications considerably impact on morbidity and mortality. The epidemiological data for the region quotes high rates of retinopathy (7-63%), neuropathy (27-66%) and microalbuminuria (10-83%). It is therefore imperative that diabetic screening programmes are established. It is recognised that in many parts of the developing world the implementation and management of such programmes is limited by a lack of available resources. The International Diabetes Federation produced guidelines in 2012 taking these limitations into account suggesting that all diabetic patients should have access to basic screening. These guidelines are consistent with the national diabetic guidelines produced by the Lesotho Medical Council. However, diabetic care in Lesotho is delivered at the local level, with variable levels of quality. A cross sectional study was performed in the outpatient department of Maluti Hospital in Mapoteng, Lesotho, a busy rural hospital in the Berea district. Demographic data on gender, age and modality of treatment were collected over a six-week time period. Information regarding 3 basic screening parameters was obtained. These parameters included eye screening (defined as a documented ophthalmology review within the last 12 months), foot screening (defined as a documented foot health assessment by any health care professional within the last 12 months) and secondary prevention (defined as a documented blood pressure and lipid profile reading within the last 12 months). These parameters were selected on the basis of the absolute minimum level of resources in Maluti Hospital. Renal screening was excluded, as the hospital does not have access to reliable renal profile checks or urinalysis. There is however a fully functioning on-site ophthalmology department run by a senior ophthalmologist with the ability to provide retinal photography, retinal surgery and photocoagulation therapy. Data was collected on 183 type 2 diabetics. 112 patients were male and 71 were female. The average age was 43 years. 4 patients were diet controlled, 140 patients were on oral hypoglycaemic agents (metformin and/or glibenclamide), and 39 patients were on a combination of insulin and oral hypoglycaemics. In the preceding 12 months, 5 patients had undergone eye screening (3%), 24 patients had undergone foot screening (13%), and 31 patients had lipid profile testing (17%). All patients had a documented blood pressure reading (100%). Our results show that screening is poorly performed in the basic indicators suggested by the IDF and the Lesotho Medical Council. On the basis of these results, a screening programme was developed using the mnemonic SaFE; secondary prevention, foot and eye care. This is simple, memorable and transferable between healthcare professionals. In the future, the expectation would be to expand upon this current programme to include renal screening, and to further develop screening pertaining to secondary prevention.

Keywords: Africa, complications, rural, screening

Procedia PDF Downloads 260

Authors: Huan Peng, Chenye Zeng, Zhao Wang

Abstract:

Alzheimer’s disease (AD) is an age-related neurodegenerative disease that is a leading cause of dementia syndromes and has become a huge burden on society and families. The main pathological features of AD involve excessive deposition of β-amyloid (Aβ) and Tau proteins in the brain, resulting in loss of neurons, expansion of neuroinflammation, and cognitive dysfunction in patients. Researchers have found effective drugs to clear the brain of error-accumulating proteins or to slow the loss of neurons, but their direct administration has key bottlenecks such as single-drug limitation, rapid blood clearance rate, impenetrable blood-brain barrier (BBB), and poor ability to target tissues and cells. Therefore, we are committed to seeking a suitable and efficient delivery system. Inspired by the possibility that exosomes may be involved in the secretion and transport mechanism of many signaling molecules or proteins in the brain, exosomes have attracted extensive attention as natural nanoscale drug carriers. We selected exosomes derived from bone marrow mesenchymal stem cells (MSC-EXO) with low immunogenicity and exosomes derived from hippocampal neurons (HT22-EXO) that may have excellent homing ability to overcome the deficiencies of oral or injectable pathways and bypass the BBB through nasal administration and evaluated their delivery ability and effect on AD. First, MSC-EXO and HT22 cells were isolated and cultured, and MSCs were identified by microimaging and flow cytometry. Then MSC-EXO and HT22-EXO were obtained by gradient centrifugation and qEV SEC separation column, and a series of physicochemical characterization were performed by transmission electron microscope, western blot, nanoparticle tracking analysis and dynamic light scattering. Next, exosomes labeled with lipophilic fluorescent dye were administered to WT mice and APP/PS1 mice to obtain fluorescence images of various organs at different times. Finally, APP/PS1 mice were administered intranasally with two exosomes 20 times over 40 days and 20 μL each time. Behavioral analysis and pathological section analysis of the hippocampus were performed after the experiment. The results showed that MSC-EXO and HT22-EXO were successfully isolated and characterized, and they had good biocompatibility. MSC-EXO showed excellent brain enrichment in APP/PS1 mice after intranasal administration, could improve the neuronal damage and reduce inflammation levels in the hippocampus of APP/PS1 mice, and the improvement effect was significantly better than HT22-EXO. However, intranasal administration of the two exosomes did not cause depression and anxious-like phenotypes in APP/PS1 mice, nor significantly improved the short-term or spatial learning and memory ability of APP/PS1 mice, and had no significant effect on the content of Aβ plaques in the hippocampus, which also meant that MSC-EXO could use their own advantages in combination with other drugs to clear Aβ plaques. The possibility of realizing highly effective non-invasive synergistic treatment for AD provides new strategies and ideas for clinical research.

Keywords: Alzheimer’s disease, exosomes derived from mesenchymal stem cell, intranasal administration, therapy-carrier dual roles

Procedia PDF Downloads 19

Authors: Babak Forouraghi

Abstract:

A genetic circuit is a collection of interacting genes and proteins that enable individual cells to implement and perform vital biological functions such as cell division, growth, death, and signaling. In cell engineering, synthetic gene circuits are engineered networks of genes specifically designed to implement functionalities that are not evolved by nature. These engineered networks enable scientists to tackle complex problems such as engineering cells to produce therapeutics within the patient's body, altering T cells to target cancer-related antigens for treatment, improving antibody production using engineered cells, tissue engineering, and production of genetically modified plants and livestock. Construction of computational models to realize genetic circuits is an especially challenging task since it requires the discovery of flow of genetic information in complex biological systems. Building synthetic biological models is also a time-consuming process with relatively low prediction accuracy for highly complex genetic circuits. The primary goal of this study was to investigate the utility of a pre-trained bidirectional encoder transformer that can accurately predict gene expressions in genetic circuit designs. The main reason behind using transformers is their innate ability (attention mechanism) to take account of the semantic context present in long DNA chains that are heavily dependent on spatial representation of their constituent genes. Previous approaches to gene circuit design, such as CNN and RNN architectures, are unable to capture semantic dependencies in long contexts as required in most real-world applications of synthetic biology. For instance, RNN models (LSTM, GRU), although able to learn long-term dependencies, greatly suffer from vanishing gradient and low-efficiency problem when they sequentially process past states and compresses contextual information into a bottleneck with long input sequences. In other words, these architectures are not equipped with the necessary attention mechanisms to follow a long chain of genes with thousands of tokens. To address the above-mentioned limitations of previous approaches, a transformer model was built in this work as a variation to the existing DNA Bidirectional Encoder Representations from Transformers (DNABERT) model. It is shown that the proposed transformer is capable of capturing contextual information from long input sequences with attention mechanism. In a previous work on genetic circuit design, the traditional approaches to classification and regression, such as Random Forrest, Support Vector Machine, and Artificial Neural Networks, were able to achieve reasonably high R2 accuracy levels of 0.95 to 0.97. However, the transformer model utilized in this work with its attention-based mechanism, was able to achieve a perfect accuracy level of 100%. Further, it is demonstrated that the efficiency of the transformer-based gene expression classifier is not dependent on presence of large amounts of training examples, which may be difficult to compile in many real-world gene circuit designs.

Keywords: transformers, generative ai, gene expression design, classification

Procedia PDF Downloads 29

Authors: A. Newman-Tancredi, R. Depoortère, P. Gruca, E. Litwa, M. Lason, M. Papp

Abstract:

The N-methyl-D-aspartic acid (NMDA) receptor antagonist, ketamine, can elicit rapid-acting antidepressant (RAAD) effects in treatment-resistant patients, but it requires parenteral co-administration with a classical antidepressant under medical supervision. In addition, ketamine can also produce serious side effects that limit its long-term use, and there is much interest in identifying RAADs based on ketamine’s mechanism of action but with safer profiles. Ketamine elicits GABAergic interneuron inhibition, glutamatergic neuron stimulation, and, notably, activation of serotonin 5-HT1A receptors in the prefrontal cortex (PFC). Direct activation of the latter receptor subpopulation with selective ‘biased agonists’ may therefore be a promising strategy to identify novel RAADs and, consistent with this hypothesis, the prototypical cortical biased agonist, NLX-101, exhibited robust RAAD-like activity in the chronic mild stress model of depression (CMS). The present study compared the effects of a novel, selective 5-HT1A receptor-biased agonist, NLX-204, with those of ketamine and NLX-101. Materials and methods: CMS procedure was conducted on Wistar rats; drugs were administered either intraperitoneally (i.p.) or by bilateral intracortical microinjection. Ketamine: 10 mg/kg i.p. or 10 µg/side in PFC; NLX-204 and NLX-101: 0.08 and 0.16 mg/kg i.p. or 16 µg/side in PFC. In addition, interaction studies were carried out with systemic NLX-204 or NLX-101 (each at 0.16 mg/kg i.p.) in combination with intracortical WAY-100635 (selective 5-HT1A receptor antagonist; 2 µg/side) or muscimol (GABA-A receptor agonist, 12.5 ng/side). Anhedonia was assessed by CMS-induced decrease in sucrose solution consumption; anxiety-like behavior was assessed using the Elevated Plus Maze (EPM), and cognitive impairment was assessed by the Novel Object Recognition (NOR) test. Results: A single administration of NLX-204 was sufficient to reverse the CMS-induced deficit in sucrose consumption, similarly to ketamine and NLX-101. NLX-204 also reduced CMS-induced anxiety in the EPM and abolished CMS-induced NOR deficits. These effects were maintained (EPM and NOR) or enhanced (sucrose consumption) over a subsequent 2-week period of treatment. The anti-anhedonic response of the drugs was also maintained for several weeks Following treatment discontinuation, suggesting that they had sustained effects on neuronal networks. A single PFC administration of NLX-204 reversed deficient sucrose consumption, similarly to ketamine and NLX-101. Moreover, the anti-anhedonic activities of systemic NLX-204 and NLX 101 were abolished by coadministration with intracortical WAY-100635 or muscimol. Conclusions: (i) The antidepressant-like activity of NLX-204 in the rat CMS model was as rapid as that of ketamine or NLX-101, supporting targeting cortical 5-HT1A receptors with selective, biased agonists to achieve RAAD effects. (ii)The anti-anhedonic activity of systemic NLX-204 was mimicked by local administration of the compound in the PFC, confirming the involvement of cortical circuits in its RAAD-like effects. (iii) Notably, the effects of systemic NLX-204 and NLX-101 were abolished by PFC administration of muscimol, indicating that they act by (indirectly) eliciting a reduction in cortical GABAergic neurotransmission. This is consistent with ketamine’s mechanism of action and suggests that there are converging NMDA and 5-HT1A receptor signaling cascades in PFC underlying the RAAD-like activities of ketamine and NLX-204. Acknowledgements: The study was financially supported by NCN grant no. 2019/35/B/NZ7/00787.

Keywords: depression, ketamine, serotonin, 5-HT1A receptor, chronic mild stress

Procedia PDF Downloads 74

Authors: Babak Forouraghi

Abstract:

A genetic circuit is a collection of interacting genes and proteins that enable individual cells to implement and perform vital biological functions such as cell division, growth, death, and signaling. In cell engineering, synthetic gene circuits are engineered networks of genes specifically designed to implement functionalities that are not evolved by nature. These engineered networks enable scientists to tackle complex problems such as engineering cells to produce therapeutics within the patient's body, altering T cells to target cancer-related antigens for treatment, improving antibody production using engineered cells, tissue engineering, and production of genetically modified plants and livestock. Construction of computational models to realize genetic circuits is an especially challenging task since it requires the discovery of the flow of genetic information in complex biological systems. Building synthetic biological models is also a time-consuming process with relatively low prediction accuracy for highly complex genetic circuits. The primary goal of this study was to investigate the utility of a pre-trained bidirectional encoder transformer that can accurately predict gene expressions in genetic circuit designs. The main reason behind using transformers is their innate ability (attention mechanism) to take account of the semantic context present in long DNA chains that are heavily dependent on the spatial representation of their constituent genes. Previous approaches to gene circuit design, such as CNN and RNN architectures, are unable to capture semantic dependencies in long contexts, as required in most real-world applications of synthetic biology. For instance, RNN models (LSTM, GRU), although able to learn long-term dependencies, greatly suffer from vanishing gradient and low-efficiency problem when they sequentially process past states and compresses contextual information into a bottleneck with long input sequences. In other words, these architectures are not equipped with the necessary attention mechanisms to follow a long chain of genes with thousands of tokens. To address the above-mentioned limitations, a transformer model was built in this work as a variation to the existing DNA Bidirectional Encoder Representations from Transformers (DNABERT) model. It is shown that the proposed transformer is capable of capturing contextual information from long input sequences with an attention mechanism. In previous works on genetic circuit design, the traditional approaches to classification and regression, such as Random Forrest, Support Vector Machine, and Artificial Neural Networks, were able to achieve reasonably high R2 accuracy levels of 0.95 to 0.97. However, the transformer model utilized in this work, with its attention-based mechanism, was able to achieve a perfect accuracy level of 100%. Further, it is demonstrated that the efficiency of the transformer-based gene expression classifier is not dependent on the presence of large amounts of training examples, which may be difficult to compile in many real-world gene circuit designs.

Keywords: machine learning, classification and regression, gene circuit design, bidirectional transformers

Procedia PDF Downloads 33

Authors: Neelofar, Khursheed Alam, Jamal Ahmad

Abstract:

Protein modification in diabetes mellitus may lead to early glycation products (EGPs) or amadori product as well as advanced glycation end products (AGEs). Early glycation involves the reaction of glucose with N-terminal and lysyl side chain amino groups to form Schiff’s base which undergoes rearrangements to form more stable early glycation product known as Amadori product. After Amadori, the reactions become more complicated leading to the formation of advanced glycation end products (AGEs) that interact with various AGE receptors, thereby playing an important role in the long-term complications of diabetes. Millard reaction or nonenzymatic glycation reaction accelerate in diabetes due to hyperglycation and alter serum protein’s structure, their normal functions that lead micro and macro vascular complications in diabetic patients. In this study, Human Serum Albumin (HSA) with a constant concentration was incubated with different concentrations of glucose at 370C for a week. At 4th day, Amadori product was formed that was confirmed by colorimetric method NBT assay and TBA assay which both are authenticate early glycation product. Conformational changes in native as well as all samples of Amadori albumin with different concentrations of glucose were investigated by various biophysical and biochemical techniques. Main biophysical techniques hyperchromacity, quenching of fluorescence intensity, FTIR, CD and SDS-PAGE were used. Further conformational changes were observed by biochemical assays mainly HMF formation, fructoseamine, reduction of fructoseamine with NaBH4, carbonyl content estimation, lysine and arginine residues estimation, ANS binding property and thiol group estimation. This study find structural and biochemical changes in Amadori modified HSA with normal to hyperchronic range of glucose with respect to native HSA. When glucose concentration was increased from normal to chronic range biochemical and structural changes also increased. Highest alteration in secondary and tertiary structure and conformation in glycated HSA was observed at the hyperchronic concentration (75mM) of glucose. Although it has been found that Amadori modified proteins is also involved in secondary complications of diabetes as AGEs but very few studies have been done to analyze the conformational changes in Amadori modified proteins due to early glycation. Most of the studies were found on the structural changes in Amadori protein at a particular glucose concentration but no study was found to compare the biophysical and biochemical changes in HSA due to early glycation with a range of glucose concentration at a constant incubation time. So this study provide the information about the biochemical and biophysical changes occur in Amadori modified albumin at a range of glucose normal to chronic in diabetes. Although many implicates currently in use i.e. glycaemic control, insulin treatment and other chemical therapies that can control many aspects of diabetes. However, even with intensive use of current antidiabetic agents more than 50 % of diabetic patient’s type 2 suffers poor glycaemic control and 18 % develop serious complications within six years of diagnosis. Experimental evidence related to diabetes suggests that preventing the nonenzymatic glycation of relevant proteins or blocking their biological effects might beneficially influence the evolution of vascular complications in diabetic patients or quantization of amadori adduct of HSA by authentic antibodies against HSA-EGPs can be used as marker for early detection of the initiation/progression of secondary complications of diabetes. So this research work may be helpful for the same.

Keywords: diabetes mellitus, glycation, albumin, amadori, biophysical and biochemical techniques

Procedia PDF Downloads 239

Authors: Natalia Hordynska, Magdalena Szechynska-Hebda, Miroslaw Sobczak, Elzbieta Rozanska, Joanna Troczynska, Zofia Banaszak, Maria Wedzony

Abstract:

Albinism is a common problem encountered in wheat and triticale breeding programs, which require in vitro culture steps e.g. generation of doubled haploids via androgenesis process. Genetic factor is a major determinant of albinism, however, environmental conditions such as temperature and media composition influence the frequency of albino plant formation. Cold incubation of wheat and triticale spikes induced a switch from gametophytic to sporophytic development. Further, androgenic structures formed from anthers of the genotypes susceptible to androgenesis or treated with cold stress, had a pool of structurally primitive plastids, with small starch granules or swollen thylakoids. High temperature was a factor inducing andro-genesis of wheat and triticale, but at the same time, it was a factor favoring the formation of albino plants. In genotypes susceptible to albinism or after heat stress conditions, cells formed from anthers were vacuolated, and plastids were eliminated. Partial or complete loss of chlorophyll pigments and incomplete differentiation of chloroplast membranes result in formation of tissues or whole plant unable to perform photosynthesis. Indeed, susceptibility to the andro-genesis process was associated with an increase of total concentration of photosynthetic pigments in anthers, spikes and regenerated plants. The proper balance of the synthesis of various pigments, was the starting point for their proper incorporation into photosynthetic membranes. In contrast, genotypes resistant to the androgenesis process and those treated with heat, contained 100 times lower content of photosynthetic pigments. In particular, the synthesis of violaxanthin, zeaxanthin, lutein and chlorophyll b was limited. Furthermore, deregulation of starch and lipids synthesis, which led to the formation of very complex starch granules and an increased number of oleosomes, respectively, correlated with the reduction of the efficiency of androgenesis. The content of other sugars varied depending on the genotype and the type of stress. The highest content of various sugars was found for genotypes susceptible to andro-genesis, and highly reduced for genotypes resistant to androgenesis. The most important sugars seem to be glucose and fructose. They are involved in sugar sensing and signaling pathways, which affect the expression of various genes and regulate plant development. Sucrose, on the other hand, seems to have minor effect at each stage of the androgenesis. The sugar metabolism was related to metabolic activity of microspores. The genotypes susceptible to androgenesis process had much faster mitochondrium- and chloroplast-dependent energy conversion and higher heat production by tissues. Thus, the effectiveness of metabolic processes, their balance and the flexibility under the stress was a factor determining the direction of microspore development, and in the later stages of the androgenesis process, a factor supporting the induction of androgenic structures, chloroplast formation and the regeneration of green plants. The work was financed by Ministry of Agriculture and Rural Development within Program: ‘Biological Progress in Plant Production’, project no HOR.hn.802.15.2018.

Keywords: androgenesis, chloroplast, metabolism, temperature stress

Procedia PDF Downloads 235

Authors: Mohammadjavad Sotoudeheian, Navid Farahmandian

Abstract:

Kawasaki disease (KD) is the primary cause of acquired pediatric heart disease as an acute vasculitis. In children with prolonged fever, rash, and inflammation of the mucosa KD must be considered as a clinical diagnosis. There is a persuasive suggestion of immune-mediated damage as the pathophysiologic cascade of KD. For example, the invasion of cytotoxic T-cells supports a viral etiology and the inflammasome of the innate immune system is a critical component in the vasculitis formation in KD. Animal models of KD propose the cytokine profiles, such as increased IL-1 and GM-CSF, which cause vascular damage. CRP and IFN-γ elevated expression and the upregulation of IL-6, and IL-10 production are also described in previous studies. Untreated KD is a critical risk factor for coronary artery diseases and myocardial infarction. Vascular damage may encompass amplified T-cell activity. SMAD3 is an essential molecule in down-regulating T-cells and increasing expression of FoxP3. It has a critical effect in the differentiation of regulatory T-cells. The discrepancy of regulatory T-cells and pro-inflammatory Th17 has been studied in acute coronary syndrome during KD. However in the coronary artery damaged lymphocytes and IgA plasma cells are seen at the lesion locations, the major immune cells in the coronary lesions are monocytes/macrophages and neutrophils. These cells secrete TNF-α, and activates matrix metalloprotease (MMP)-9, reducing the integrity of vessels and prompting patients to arise aneurysm. MMPs can break down the components of the extracellular matrix and assist immune cell movement. IVIG as an effective form of treatment clarified the role of the immune system, which may target pathogenic antigens and regulate cytokine production. Several reports have revealed that in the coronary arteries, high expression of MMP-9 in monocyte/macrophage results in pathologic cascades. Curcumin is a potent antioxidant and anti-inflammatory molecule. Curcumin decreases the production of reactive oxygen and nitrogen species and inhibits transcription factors like AP-1 and NF-κB. Curcumin also contains the characteristics of inhibitory effects on MMPs, especially MMP-9. The upregulation of MMP-9 is an important cellular response. Curcumin treatment caused a reverse effect and down-regulates MMP-9 gene expression which may fund the anti-inflammatory effect. Curcumin inhibits MMP-9 expression via PKC and AMPK-dependent pathways in Human monocytes cells. Elevated expression and activity of MMP-9 are correlated with advanced vascular lesions. AMPK controls lipid metabolism and oxidation, and protein synthesis. AMPK is also necessary for the MMP-9 activity and THP-1 cell adhesion to endothelial cells. Curcumin was shown to inhibit the activation of AMPKα. Compound C (AMPK inhibitor) inhibits MMP-9 expression level. Therefore, through inactivating AMPKs and PKC, curcumin decreases the MMP-9 level, which results in inhibiting monocyte/macrophage differentiation. Compound C also suppress the phosphorylation of three major classes of MAP kinase signaling, suggesting that curcumin may suppress MMP-9 level by inactivation of MAPK pathways. MAPK cascades are activated to induce the expression of MMP-9. Curcumin inhibits MAPKs phosphorylation, which contributes to the down-regulation of MMP-9. This study demonstrated that the potential inhibitory properties of curcumin over MMP-9 lead to a therapeutic strategy to reduce the risk of coronary artery involvement during KD.

Keywords: MMP-9, coronary artery aneurysm, Kawasaki’s disease, curcumin, AMPK, immune system, NF-κB, MAPK

Procedia PDF Downloads 273

Authors: Rajeswari Raguraman, Sowmya Parameswaran, Krishnakumar Subramanian, Jagat Kanwar, Rupinder Kanwar

Abstract:

Background: Tumor microenvironment has been implicated in several cancers to regulate cell growth, invasion and metastasis culminating in outcome of therapy. Tumor stroma consists of multiple cell types that are in constant cross-talk with the tumor cells to favour a pro-tumorigenic environment. Not much is known about the existence of tumor microenvironment in the pediatric intraocular malignancy, Retinoblastoma (RB). In the present study, we aim to understand the multiple stromal cellular subtypes and tumor stromal interactions expressed in RB tumors. Materials and Methods: Immunohistochemistry for stromal cell markers CD31, CD68, alpha-smooth muscle (α-SMA), vimentin and glial fibrillary acidic protein (GFAP) was performed on formalin fixed paraffin embedded tissues sections of RB (n=12). The differential expression of stromal target molecules; fibroblast activation protein (FAP), tenascin-C (TNC), osteopontin (SPP1), bone marrow stromal antigen 2 (BST2), stromal derived factor 2 and 4 (SDF2 and SDF4) in primary RB tumors (n=20) and normal retina (n=5) was studied by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blotting. The differential expression was correlated with the histopathological features of RB. The interaction between RB cell lines (Weri-Rb-1, NCC-RbC-51) and Bone marrow stromal cells (BMSC) was also studied using direct co-culture and indirect co-culture methods. The functional effect of the co-culture methods on the RB cells was evaluated by invasion and proliferation assays. Global gene expression was studied by using Affymetrix 3’ IVT microarray. Pathway prediction was performed using KEGG and the key molecules were validated using qRT-PCR. Results: The immunohistochemistry revealed the presence of several stromal cell types such as endothelial cells (CD31+;Vim+/-); macrophages (CD68+;Vim+/-); Fibroblasts (Vim+; CD31-;CD68- );myofibroblasts (α-SMA+/ Vim+) and invading retinal astrocytes/ differentiated retinal glia (GFAP+; Vim+). A characteristic distribution of these stromal cell types was observed in the tumor microenvironment, with endothelial cells predominantly seen in blood vessels and macrophages near actively proliferating tumor or necrotic areas. Retinal astrocytes and glia were predominant near the optic nerve regions in invasive tumors with sparse distribution in tumor foci. Fibroblasts were widely distributed with rare evidence of myofibroblasts in the tumor. Both gene and protein expression revealed statistically significant (P<0.05) up-regulation of FAP, TNC and BST2 in primary RB tumors compared to the normal retina. Co-culture of BMSC with RB cells promoted invasion and proliferation of RB cells in direct and indirect contact methods respectively. Direct co-culture of RB cell lines with BMSC resulted in gene expression changes in ECM-receptor interaction, focal adhesion, IL-8 and TGF-β signaling pathways associated with cancer. In contrast, various metabolic pathways such a glucose, fructose and amino acid metabolism were significantly altered under the indirect co-culture condition. Conclusion: The study suggests that the close interaction between RB cells and the stroma might be involved in RB tumor invasion and progression which is likely to be mediated by ECM-receptor interactions and secretory factors. Targeting the tumor stroma would be an attractive option for redesigning treatment strategies for RB.

Keywords: gene expression profiles, retinoblastoma, stromal cells, tumor microenvironment

Procedia PDF Downloads 356

Authors: Michelle Maurer, Antonia Last, Mark S. Gresnigt, Bernhard Hube, Alexander S. Mosig

Abstract:

The intestinal epithelium forms an essential barrier to prevent translocation of microorganisms, toxins or other potentially harmful molecules into the bloodstream. In particular, dendritic cells of the intestinal epithelium orchestrate an adapted response of immune tolerance to commensals and immune defense against invading pathogens. Systemic inflammation is typically associated with a dysregulation of this adapted immune response and is accompanied by a disruption of the epithelial and endothelial gut barrier which enables dissemination of pathogens within the human body. To understand the pathophysiological mechanisms underlying the inflammation-associated gut barrier breakdown, it is crucial to elucidate the complex interplay of the host and the intestinal microbiome. A microfluidically perfused three-dimensional intestine-on-chip model was established to emulate these processes in the presence of immune cells, commensal bacteria, and facultative pathogens. Multi-organ tissue flow (MOTiF) biochips made from polystyrene were used for microfluidic perfusion of the intestinal tissue model. The biochips are composed of two chambers separated by a microporous membrane. Each chamber is connected to inlet and outlet channels allowing independent perfusion of the individual channels and application of microfluidic shear stress. Human umbilical vein endothelial cells (HUVECs), monocyte-derived macrophages and intestinal epithelial cells (Caco-2) were assembled on the biochip membrane. Following 7 – 14 days of growth in the presence of physiological flow conditions, the epithelium was colonized with the commensal bacterium Lactobacillus rhamnosus, while the endothelium was perfused with peripheral blood mononuclear cells (PBMCs). Additionally, L. rhamnosus was co-cultivated with the opportunistic fungal pathogen Candida albicans. Within one week of perfusion, the epithelial cells formed self-organized and well-polarized villus- and crypt-like structures that resemble essential morphological characteristics of the human intestine. Dendritic cells were differentiated in the epithelial tissue that specifically responds to bacterial lipopolysaccharide (LPS) challenge. LPS is well-tolerated at the luminal epithelial side of the intestinal model without signs of tissue damage or induction of an inflammatory response, even in the presence of circulating PBMC at the endothelial lining. In contrast, LPS stimulation at the endothelial side of the intestinal model triggered the release of pro-inflammatory cytokines such as TNF, IL-1β, IL-6, and IL-8 via activation of macrophages residing in the endothelium. Perfusion of the endothelium with PBMCs led to an enhanced cytokine release. L. rhamnosus colonization of the model was tolerated in the immune competent tissue model and was demonstrated to reduce damage induced by C. albicans infection. A microfluidic intestine-on-chip model was developed to mimic a systemic infection with a dysregulated immune response under physiological conditions. The model facilitates the colonization of commensal bacteria and co-cultivation with facultative pathogenic microorganisms. Both, commensal bacteria alone and facultative pathogens controlled by commensals, are tolerated by the host and contribute to cell signaling. The human intestine-on-chip model represents a promising tool to mimic microphysiological conditions of the human intestine and paves the way for more detailed in vitro studies of host-microbiota interactions under physiologically relevant conditions.

Keywords: host-microbiota interaction, immune tolerance, microfluidics, organ-on-chip

Procedia PDF Downloads 106

Authors: Chandra Shekhar Majumder, Md. Shamsul Haque, Khondaker Anower Hossain, Md. Rafiqul Islam

Abstract:

Introduction: Ocular helminths are parasites that infect the eye or its adnexa. They can be either motile worms or sessile worms that form cysts. These parasites require two hosts for their life cycle, a definite host (usually a human) and an intermediate host (usually an insect). While there have been reports of ocular helminths infecting various structures of the eye, including the anterior chamber and subconjunctival space, there is no previous record of such a case involving the lens. Research Aim: The aim of this case report is to present a rare case of ocular helminth infection in the lens and to contribute to the understanding of this unusual site of infection. Methodology: This study is a case report, presenting the details and findings of an 80-year-old retired policeman who presented with severe pain, redness, and vision loss in the left eye. The patient had a history of diabetes mellitus and hypertension. The examination revealed the presence of a thread-like helminth in the lens. The patient underwent treatment and follow-up, and the helminth specimen was sent for identification to the department of Parasitology. Case report: An 80-year-old retired policeman attended the OPD, Faridpur Medical College Hospital with the complaints of severe pain, redness and gross dimness of vision of the left eye for 5 days. He had a history of diabetes mellitus and hypertension for 3 years. On examination, L/E visual acuity was PL only, moderate ciliary congestion, KP 2+, cells 2+ and posterior synechia from 5 to 7 O’clock position was found. Lens was opaque. A thread like helminth was found under the anterior of the lens. The worm was moving and changing its position during examination. On examination of R/E, visual acuity was 6/36 unaided, 6/18 with pinhole. There was lental opacity. Slit-lamp and fundus examination were within normal limit. Patient was admitted in Faridpur Medical College Hospital. Diabetes mellitus was controlled with insulin. ICCE with PI was done on the same day of admission under depomedrol coverage. The helminth was recovered from the lens. It was thread like, about 5 to 6 mm in length, 1 mm in width and pinkish in colour. The patient followed up after 7 days, VA was HM, mild ciliary congestion, few KPs and cells were present. Media was hazy due to vitreous opacity. The worm was sent to the department of Parasitology, NIPSOM, Dhaka for identification. Findings: The findings of this case report highlight the presence of a helminth in the lens, which has not been previously reported. The helminth was successfully removed from the lens, but the patient experienced complications such as anterior uveitis and vitreous opacity. The exact mechanism by which the helminth enters the lens remains unclear. Theoretical Importance: This case report contributes to the existing literature on ocular helminth infections by reporting a unique case involving the lens. It highlights the need for further research to understand the pathogenesis and mechanism of entry of helminths in the lens. Data Collection and Analysis Procedures: The data for this case report were collected through clinical examination and medical records of the patient. The findings were described and presented in a descriptive manner. No statistical analysis was conducted. Question Addressed: This case report addresses the question of whether ocular helminth infections can occur in the lens, which has not been previously reported. Conclusion: To the best of our knowledge, this is the first reported case of ocular helminth infection in the lens. The presence of the helminth in the lens raises interesting questions regarding its pathogenesis and entry mechanism. Further study and research are needed to explore these aspects. Ophthalmologists and parasitologists should be aware of the possibility of ocular helminth infections in unusual sites like the lens.

Keywords: ocular, helminth, unsual site, lens

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Authors: Jayanwita Sarkar, Usha Chakraborty, Bishwanath Chakraborty

Abstract:

Plants are constantly interacting with various abiotic and biotic stresses. In changing climate scenario plants are continuously modifying physiological processes to adapt to changing environmental conditions which profoundly affect plant-pathogen interactions. Spot blotch in wheat is a fast-rising disease in the warmer plains of South Asia where the rise in minimum average temperature over most of the year already affecting wheat production. Hence, the study was undertaken to explore the role of elevated temperature in spot blotch disease development and modulation of antioxidative responses by plant growth promoting rhizobacteria (PGPR) for biocontrol of spot blotch at high temperature. Elevated temperature significantly increases the susceptibility of wheat plants to spot blotch causing pathogen Bipolaris sorokiniana. Two PGPR Bacillus safensis (W10) and Ochrobactrum pseudogrignonense (IP8) isolated from wheat (Triticum aestivum L.) and blady grass (Imperata cylindrical L.) rhizophere respectively, showing in vitro antagonistic activity against Bipolaris sorokiniana were tested for growth promotion and induction of resistance against spot blotch in wheat. GC-MS analysis showed that Bacillus safensis (W10) and Ochrobactrum pseudogrignonense (IP8) produced antifungal and antimicrobial compounds in culture. Seed priming with these two bacteria significantly increase growth, modulate antioxidative signaling and induce resistance and eventually reduce disease incidence in wheat plants at optimum as well as elevated temperature which was further confirmed by indirect immunofluorescence assay using polyclonal antibody raised against Bipolaris sorokiniana. Application of the PGPR led to enhancement in activities of plant defense enzymes- phenylalanine ammonia lyase, peroxidase, chitinase and β-1,3 glucanase in infected leaves. Immunolocalization of chitinase and β-1,3 glucanase in PGPR primed and pathogen inoculated leaf tissue was further confirmed by transmission electron microscopy using PAb of chitinase, β-1,3 glucanase and gold labelled conjugates. Activity of ascorbate-glutathione redox cycle related enzymes such as ascorbate peroxidase, superoxide dismutase and glutathione reductase along with antioxidants such as carotenoids, glutathione and ascorbate and osmolytes like proline and glycine betain accumulation were also increased during disease development in PGPR primed plant in comparison to unprimed plants at high temperature. Real-time PCR analysis revealed enhanced expression of defense genes- chalcone synthase and phenyl alanineammonia lyase. Over expression of heat shock proteins like HSP 70, small HSP 26.3 and heat shock factor HsfA3 in PGPR primed plants effectively protect plants against spot blotch infection at elevated temperature as compared with control plants. Our results revealed dynamic biochemical cross talk between elevated temperature and spot blotch disease development and furthermore highlight PGPR mediated array of antioxidative and molecular alterations responsible for induction of resistance against spot blotch disease at elevated temperature which seems to be associated with up-regulation of defense genes, heat shock proteins and heat shock factors, less ROS production, membrane damage, increased expression of redox enzymes and accumulation of osmolytes and antioxidants.

Keywords: antioxidative enzymes, defense enzymes, elevated temperature, heat shock proteins, PGPR, Real-Time PCR, spot blotch, wheat

Procedia PDF Downloads 139

Authors: Kuladip Jana

Abstract:

Benzo(a)pyrene [B(a)P] is an environmental toxicant present mostly in cigarette smoke and car exhaust, is an aryl hydrocarbon receptor (AhR) ligand that exerts its toxic effects on both male and female reproductive systems. In this study, the effect of B(a)P at different doses (0.1, 0.25, 0.5, 1 and 5 mg /kg body weight) was studied on male reproductive system of rat. A significant decrease in cauda epididymal sperm count and motility along with the presence of sperm head abnormalities and altered epididymal and testicular histology were documented following B(a)P treatment. B(a)P treatment resulted apoptotic sperm cells as observed by TUNEL and Annexin V-PI assay with increased ROS, altered sperm mitochondrial membrane potential (ΔΨm) with a simultaneous decrease in the activity of antioxidant enzymes and GSH status. TUNEL positive apoptotic cells also observed in testis as well as isolated germ and Leydig cells following B(a)P exposure. Western Blot analysis revealed the activation of p38MAPK, cytosolic translocation of cytochrome-c, up-regulation of Bax and inducible nitric oxide synthase (iNOS) with cleavage of PARP and down-regulation of BCl2 in testis upon B(a)P treatment. The protein and mRNA levels of testicular key steroidogenesis regulatory proteins like StAR, cytochrome P450 IIA1 (CYPIIA1), 3β HSD, 17β HSD showed a significant decrease in a dose dependent manner while an increase in the expression of cytochrome P450 1A1 (CYP1A1), Aryl hydrocarbon Receptor (AhR), active caspase- 9 and caspase- 3 following B(a)P exposure. We conclude that exposure of benzo(a)pyrene caused testicular gamatogenic and steroidogenic disorders by induction of oxidative stress, inhibition of StAR and other steroidogenic enzymes along with activation of p38MAPK and initiated caspase-3 mediated germ and Leydig cell apoptosis.The possible protective role of naturally occurring phytochemicals against B(a)P induced testicular toxicity needs immediate consideration. Curcumin and resveratrol separately were found to protect against B(a)P induced germ cell apoptosis, and their combinatorial effect was more significant. Our present study in isolated testicular germ cell population from adult male Wistar rats, highlighted their synergistic protective effect against B(a)P induced germ cell apoptosis. Curcumin-resveratrol co-treatment decreased the expression of pro-apoptotic proteins like cleaved caspase 3,8,9, cleaved PARP, Apaf1, FasL, tBid. Curcumin-resveratrol co-treatment decreased Bax/Bcl2 ratio, mitochondria to cytosolic translocation of cytochrome c and activated the survival protein Akt. Curcumin-resveratrol decreased the expression of p53 dependent apoptotic genes like Fas, FasL, Bax, Bcl2, Apaf1.Curcumin-resveratrol co-treatment thus prevented B(a)P induced germ cell apoptosis. B(a)P induced testicular ROS generation and oxidative stress were significantly ameliorated with curcumin and resveratrol. Curcumin-resveratrol co-treatment prevented B(a)P induced nuclear translocation of AhR and CYP1A1 production. The combinatorial treatment significantly inhibited B(a)P induced ERK 1/2, p38 MAPK and JNK 1/2 activation. B(a)P treatment increased the expression of p53 and its phosphorylation (p53 ser 15). Curcumin-resveratrol co-treatment significantly decreased p53 level and its phosphorylation (p53 ser 15). The study concludes that curcumin-resveratrol synergistically modulated MAPKs and p53, prevented oxidative stress, regulated the expression of pro and anti-apoptotic proteins as well as the proteins involved in B(a)P metabolism thus protected germ cells from B(a)P induced apoptosis.

Keywords: benzo(a)pyrene, germ cell, apoptosis, oxidative stress, resveratrol, curcumin

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Authors: Pallab Datta, Jyotirmoy Chatterjee, Santanu Dhara

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Over the past few years, phosphorous containing polymers have received widespread attention for applications such as high performance optical fibers, flame retardant materials, drug delivery and tissue engineering. Being pentavalent, phosphorous can exist in different chemical environments in these polymers which increase their versatility. In human biochemistry, phosphorous based compounds exert their functions both in soluble and insoluble form occurring as inorganic or as organophosphorous compounds. Specifically in case of biomacromolecules, phosphates are critical for functions of DNA, ATP, phosphoproteins, phospholipids, phosphoglycans and several coenzymes. Inspired by the role of phosphorous in functional biomacromolecules, design and synthesis of biomimetic materials are thus carried out by several authors to study macromolecular function or as substitutes in clinical tissue regeneration conditions. In addition, many regulatory signals of the body are controlled by phoshphorylation of key proteins present either in form of growth factors or matrix-bound scaffold proteins. This inspires works on synthesis of phospho-peptidomimetic amino acids for understanding key signaling pathways and this is extended to obtain molecules with potentially useful biological properties. Apart from above applications, phosphate groups bound to polymer backbones have also been demonstrated to improve function of osteoblast cells and augment performance of bone grafts. Despite the advantages of phosphate grafting, however, there is limited understanding on effect of degree of phosphorylation on macromolecular physicochemical and/or biological properties. Such investigations are necessary to effectively translate knowledge of macromolecular biochemistry into relevant clinical products since they directly influence processability of these polymers into suitable scaffold structures and control subsequent biological response. Amongst various techniques for fabrication of biomimetic scaffolds, nanofibrous scaffolds fabricated by electrospinning technique offer some special advantages in resembling the attributes of natural extracellular matrix. Understanding changes in physico-chemical properties of polymers as function of phosphorylation is therefore going to be crucial in development of nanofiber scaffolds based on phosphorylated polymers. The aim of the present work is to investigate the effect of phosphorous grafting on the electrospinning behavior of polymers with aim to obtain biomaterials for bone regeneration applications. For this purpose, phosphorylated derivatives of two polymers of widely different electrospinning behaviors were selected as starting materials. Poly(vinyl alcohol) is a conveniently electrospinnable polymer at different conditions and concentrations. On the other hand, electrospinning of chitosan backbone based polymers have been viewed as a critical challenge. The phosphorylated derivatives of these polymers were synthesized, characterized and electrospinning behavior of various solutions containing these derivatives was compared with electrospinning of pure poly (vinyl alcohol). In PVA, phosphorylation adversely impacted electrospinnability while in NMPC, higher phosphate content widened concentration range for nanofiber formation. Culture of MG-63 cells on electrospun nanofibers, revealed that degree of phosphate modification of a polymer significantly improves cell adhesion or osteoblast function of cultured cells. It is concluded that improvement of cell response parameters of nanofiber scaffolds can be attained as a function of controlled degree of phosphate grafting in polymeric biomaterials with implications for bone tissue engineering applications.

Keywords: bone regeneration, chitosan, electrospinning, phosphorylation

Procedia PDF Downloads 195

Authors: Ananya Pappu, Sneha Mishra

Abstract:

Introduction: The South Asian population experiences unique health challenges that predisposes this demographic to cardiometabolic diseases at lower BMIs. South Asians may therefore benefit from recommendations specific to their cultural needs. Here, we focus on current BMI guidelines for Asians with a discussion of South Asian dietary practices and culturally tailored interventions. By integrating traditional dietary practices with modern nutritional recommendations, this manuscript aims to highlight effective strategies to improving health outcomes among South Asians. Background: The South Asian community, including individuals from India, Pakistan, Bangladesh, and Sri Lanka, experiences high rates of cardiovascular diseases, cancers, diabetes, and strokes. Notably, the prevalence of diabetes and cardiovascular disease among Asians is elevated at BMIs below the WHO's standard overweight threshold. As it stands, a BMI of 25-30 kg/m² is considered overweight in non-Asians, while this cutoff is reduced to 23-27.4 kg/m² in Asians. This discrepancy can be attributed to studies which have shown different associations between BMI and health risks in Asians compared to other populations. Given these significant challenges, optimizing lifestyle management for cardiometabolic risk factors is crucial. Tailored interventions that consider cultural context seem to be the best approach for ensuring the success of both dietary and physical activity interventions in South Asian patients. Adopting a whole food, plant-based diet (WFPD) is one such strategy. The WFPD suggests that half of one meal should consist of non-starchy vegetables. In the South Asian diet, this includes traditional vegetables such as okra, tindora, eggplant, and leafy greens including amaranth, collards, chard, and mustards. A quarter of the meal should include plant-based protein sources like cooked beans, lentils, and paneer, with the remaining quarter comprising healthy grains or starches such as whole wheat breads, millets, tapioca, and barley. Adherence to the WFPD has been shown to improve cardiometabolic risk factors including weight, BMI, total cholesterol, HbA1c, and reduces the risk of developing non-alcoholic fatty liver disease (NAFLD). Another approach to improving dietary habits is timing meals. Many of the major cultures and religions in the Indian subcontinent incorporate religious fasting. Time-restricted eating (TRE), also known as intermittent fasting, is a practice akin to traditional fasting, which involves consuming all daily calories within a specific window. TRE has been shown to improve insulin resistance in prediabetic and diabetic patients. Common regimens include completing all meals within an 8-hour window, consuming a low-calorie diet every other day, and the 5:2 diet, which involves fasting twice weekly. These fasting practices align with the natural circadian rhythm, potentially enhancing metabolic health and reducing obesity and diabetes risks. Conclusion: South Asians develop cardiometabolic disease at lower BMIs; hence, it is important to counsel patients about lifestyle interventions that decrease their risk. Traditional South Asian diets can be made more nutrient-rich by incorporating vegetables, plant proteins like lentils and beans, and substituting refined grains for whole grains. Ultimately, the best diet is one to which a patient can adhere. It is therefore important to find a regimen that aligns with a patient’s cultural and traditional food practices.

Keywords: BMI, diet, obesity, South Asian, time-restricted eating

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Authors: Kuladip Jana, Bhaswati Banerjee, Parimal C. Sen

Abstract:

Benzo(a)pyrene [B(a)P] is an environmental toxicant present mostly in cigarette smoke and car exhaust, is an aryl hydrocarbon receptor (AhR) ligand that exerts its toxic effects on both male and female reproductive systems along with carcinogenesis in skin, prostate, ovary, lung and mammary glands. Our study was focused on elucidating the molecular mechanism of B(a)P induced male reproductive toxicity and its prevention with phytochemical like resveratrol. In this study, the effect of B(a)P at different doses (0.1, 0.25, 0.5, 1 and 5 mg /kg body weight) was studied on male reproductive system of Wistar rat. A significant decrease in cauda epididymal sperm count and motility along with the presence of sperm head abnormalities and altered epididymal and testicular histology were documented following B(a)P treatment. B(a)P treatment resulted apoptotic sperm cells as observed by TUNEL and Annexin V-PI assay with increased Reactive Oxygen Species (ROS), altered sperm mitochondrial membrane potential (ΔΨm) with a simultaneous decrease in the activity of antioxidant enzymes and GSH status. TUNEL positive apoptotic cells also observed in testis as well as isolated germ and Leydig cells following B(a)P exposure. Western Blot analysis revealed the activation of p38 mitogen activated protein kinase (p38MAPK), cytosolic translocation of cytochrome-c, upregulation of Bax and inducible nitric oxide synthase (iNOS) with cleavage of poly ADP ribose polymerase (PARP) and down regulation of BCl2 in testis upon B(a)P treatment. The protein and mRNA levels of testicular key steroidogenesis regulatory proteins like steroidogenic acute regulatory protein (StAR), cytochrome P450 IIA1 (CYPIIA1), 3β hydroxy steroid dehydrogenase (3β HSD), 17β hydroxy steroid dehydrogenase (17β HSD) showed a significant decrease in a dose dependent manner while an increase in the expression of cytochrome P450 1A1 (CYP1A1), Aryl hydrocarbon Receptor (AhR), active caspase- 9 and caspase- 3 following B(a)P exposure. We conclude that exposure of benzo(a)pyrene caused testicular gamatogenic and steroidogenic disorders by induction of oxidative stress, inhibition of StAR and other steroidogenic enzymes along with activation of p38MAPK and initiated caspase-3 mediated germ and Leydig cell apoptosis. Next we investigated the role of resveratrol on B(a)P induced male reproductive toxicity. Our study highlighted that resveratrol co-treatment with B(a)P maintained testicular redox potential, increased serum testosterone level and prevented steroidogenic dysfunction with enhanced expression of major testicular steroidogenic proteins (CYPIIA1, StAR, 3β HSD,17β HSD) relative to treatment with B(a)P only. Resveratrol suppressed B(a)P-induced testicular activation of p38 MAPK, ATF2, iNOS and ROS production; cytosolic translocation of Cytochome c and Caspase 3 activation thereby prevented oxidative stress of testis and inhibited apoptosis. Resveratrol co-treatment also decreased B(a)P-induced AhR protein level, its nuclear translocation and subsequent CYP1A1 promoter activation, thereby decreased protein and mRNA levels of testicular cytochrome P4501A1 (CYP1A1) and prevented BPDE-DNA adduct formation. Our findings cumulatively suggest that resveratrol prevents activation of B(a)P by modulating the transcriptional regulation of CYP1A1 and acting as an antioxidant thus prevents B(a)P-induced oxidative stress and testicular apoptosis.

Keywords: benzo(a)pyrene, resveratrol, testis, apoptosis, cytochrome P450 1A1 (CYP1A1), aryl hydrocarbon receptor (AhR), p38 MAPK/ATF2/iNOS

Procedia PDF Downloads 197

Authors: Yogesh Dalvi, Ruby Varghese, Nibu Varghese, C. K. Krishnan Nair

Abstract:

Introduction: The Genus Phellinus, locally known as Phansomba is a well-known traditional folk medicine. Especially, in Western Ghats of India, many tribes use several species of Phellinus for various ailments related to teeth, throat, tongue, stomach and even wound healing. It is one of the few mushrooms which play a pivotal role in Ayurvedic Dravyaguna. Aim: The present study focuses on to investigate phytochemical analysis, antioxidant, and antitumor (in vitro and in vivo) potential of Phellinus robinae from South India, Kerala Material and Methods: The present study explores the following: 1. Phellinus samples were collected from Ranni, Pathanamthitta district of Kerala state, India from Artocarpus heterophyllus Lam. and species were identified using rDNA region. 2. The fruiting body was shadow dried, powdered and extracted with 50% alcohol using water bath at 60°C which was further condensed by rotary evaporator and lyophilized at minus 40°C temperature. 3. Secondary metabolites were analyzed by using various phytochemical screening assay (Hager’s Test, Wagner’s Test, Sodium hydroxide Test, Lead acetate Test, Ferric chloride Test, Folin-ciocalteu Test, Foaming Test, Benedict’s test, Fehling’s Test and Lowry’s Test). 4. Antioxidant and free radical scavenging activity were analyzed by DPPH, FRAP and Iron chelating assay. 5. The antitumor potential of Water alcohol extract of Phellinus (PAWE) is evaluated through In vitro condition by Trypan blue dye exclusion method in DLA cell line and In vivo by murine model. Result and Discussion: Preliminary phytochemical screening by various biochemical tests revealed presence of a variety of active secondary molecules like alkaloids, flavanoids, saponins, carbohydrate, protein and phenol. In DPPH and FRAP assay PAWE showed significantly higher antioxidant activity as compared to standard Ascorbic acid. While, in Iron chelating assay, PAWE exhibits similar antioxidant activity that of Butylated Hydroxytoluene (BHT) as standard. Further, in the in vitro study, PAWE showed significant inhibition on DLA cell proliferation in dose dependent manner and showed no toxicity on mice splenocytes, when compared to standard chemotherapy drug doxorubicin. In vivo study, oral administration of PAWE showed dose dependent tumor regression in mice and also raised the immunogenicity by restoring levels of antioxidant enzymes in liver and kidney tissue. In both in vitro and in vivo gene expression studies PAWE up-regulates pro-apoptotic genes (Bax, Caspases 3, 8 and 9) and down- regulates anti-apoptotic genes (Bcl2). PAWE also down regulates inflammatory gene (Cox-2) and angiogenic gene (VEGF). Conclusion: Preliminary phytochemical screening revealed that PAWE contains various secondary metabolites which contribute to its antioxidant and free radical scavenging property as evaluated by DPPH, FRAP and Iron chelating assay. PAWE exhibits anti-proliferative activity by the induction of apoptosis through a signaling cascade of death receptor-mediated extrinsic (Caspase8 and Tnf-α), as well as mitochondria-mediated intrinsic (caspase9) and caspase pathways (Caspase3, 8 and 9) and also by regressing angiogenic factor (VEGF) without any inflammation or adverse side effects. Hence, PAWE serve as a potential antioxidant and antitumor agent.

Keywords: antioxidant, antitumor, Dalton lymphoma ascites (DLA), fungi, Phellinus robinae

Procedia PDF Downloads 276

Authors: Z. S. Haidar

Abstract:

Millions of teeth are removed annually, and dental extraction is one of the most commonly performed surgical procedures globally. Whether due to caries, periodontal disease, or trauma, exodontia and the ensuing wound healing and bone remodeling processes of the resultant socket (hole in the jaw bone) usually result in serious deformities of the residual alveolar osseous ridge and surrounding soft tissues (reduced height/width). Such voluminous changes render the placement of a proper conventional bridge, denture, or even an implant-supported prosthesis extremely challenging. Further, most extractions continue to be performed with no regard for preventing the onset of alveolar osteitis (also known as dry socket, a painful and difficult-to-treat/-manage condition post-exodontia). Hence, such serious resorptive morphological changes often result in significant facial deformities and a negative impact on the overall Quality of Life (QoL) of patients (and oral health-related QoL); alarming, particularly for the geriatric with compromised healing and in light of the thriving longevity statistics. Despite advances in tissue/wound grafting, serious limitations continue to exist, including efficacy and clinical outcome predictability, cost, treatment time, expertise, and risk of immune reactions. For cases of dry socket, specifically, the commercially available and often-prescribed home remedies are highly-lacking. Indeed, most are not recommended for use anymore. Alveogyl is a fine example. Hence, there is a great market demand and need for alternative solutions. Herein, SockGEL/PLUG (patent pending), an innovative, all-natural, drug-free, and injectable thermo-responsive hydrogel, was designed, formulated, characterized, and evaluated as an osteogenic, angiogenic, anti-microbial, and pain-soothing suture-free intra-alveolar dressing, safe and efficacious for use in fresh extraction sockets, immediately post-exodontia. It is composed of FDA-approved, biocompatible and biodegradable polymers, self-assembled electro-statically to formulate a scaffolding matrix to (1) prevent the on-set of alveolar osteitis via securing the fibrin-clot in situ and protecting/sealing the socket from contamination/infection; and (2) endogenously promote/accelerate wound healing and bone remodeling to preserve the volume of the alveolus. The intrinsic properties of the SockGEL/PLUG hydrogel were evaluated physical-chemical-mechanically for safety (cell viability), viscosity, rheology, bio-distribution, and essentially, capacity to induce wound healing and osteogenesis (small defect, in vivo) without any signaling cues from exogenous cells, growth factors or drugs. The proposed animal model of cranial critical-sized and non-vascularized bone defects shall provide new and critical insights into the role and mechanism of the employed natural bio-polymer blend and gel product in endogenous reparative regeneration of soft tissues and bone morphogenesis. Alongside, the fine-tuning of our modified formulation method will further tackle appropriateness, reproducibility, scalability, ease, and speed in producing stable, biodegradable, and sterilizable thermo-sensitive matrices (3-dimensional interpenetrating yet porous polymeric network) suitable for the intra-socket application. Findings are anticipated to provide sufficient evidence to translate into pilot clinical trials and validate the innovation before engaging the market for feasibility, acceptance, and cost-effectiveness studies.

Keywords: hydrogel, nanotechnology, bioengineering, bone regeneration, nanogel, drug delivery

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