Search results for: cellular automata (CA)

Authors: Ashima Sharma, Tapan K. Chaudhuri

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Human Serum Albumin (HSA) is one of the most demanded therapeutic protein with immense biotechnological applications. The current source of HSA is human blood plasma. Blood is a limited and an unsafe source as it possesses the risk of contamination by various blood derived pathogens. This issue led to exploitation of various hosts with the aim to obtain an alternative source for the production of the rHSA. But, till now no host has been proven to be effective commercially for rHSA production because of their respective limitations. Thus, there exists an indispensable need to promote non-animal derived rHSA production. Of all the host systems, Escherichia coli is one of the most convenient hosts which has contributed in the production of more than 30% of the FDA approved recombinant pharmaceuticals. E. coli grows rapidly and its culture reaches high cell density using inexpensive and simple substrates. The fermentation batch turnaround number for E. coli culture is 300 per year, which is far greater than any of the host systems available. Therefore, E. coli derived recombinant products have more economical potential as fermentation processes are cheaper compared to the other expression hosts available. Despite of all the mentioned advantages, E. coli had not been successfully adopted as a host for rHSA production. The major bottleneck in exploiting E. coli as a host for rHSA production was aggregation i.e. majority of the expressed recombinant protein was forming inclusion bodies (more than 90% of the total expressed rHSA) in the E. coli cytosol. Recovery of functional rHSA form inclusion body is not preferred because it is tedious, time consuming, laborious and expensive. Because of this limitation, E. coli host system was neglected for rHSA production for last few decades. Considering the advantages of E. coli as a host, the present work has targeted E. coli as an alternate host for rHSA production through resolving the major issue of inclusion body formation associated with it. In the present study, we have developed a novel and innovative method for enhanced soluble and functional production of rHSA in E.coli (~60% of the total expressed rHSA in the soluble fraction) through modulation of the cellular growth, folding and environmental parameters, thereby leading to significantly improved and enhanced -expression levels as well as the functional and soluble proportion of the total expressed rHSA in the cytosolic fraction of the host. Therefore, in the present case we have filled in the gap in the literature, by exploiting the most well studied host system Escherichia coli which is of low cost, fast growing, scalable and ‘yet neglected’, for the enhancement of functional production of HSA- one of the most crucial biomolecule for clinical and biotechnological applications.

Keywords: enhanced functional production of rHSA in E. coli, recombinant human serum albumin, recombinant protein expression, recombinant protein processing

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Authors: Gleb Fomin, Kairat Tabynov, Nurkeldy Turebekov, Dinara Turegeldiyeva, Rinat Islamov

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The level of major cytokines in the blood of patients with COVID-19 varies greatly depending on age, gender, duration and severity of infection, and comorbidity. There are two clinically significant cytokines, IL-6 and TNF-α, which increase in levels in patients with severe COVID-19. However, in a model of COVID-19 in hamsters, TNF-α levels are unchanged or reduced, while the expression of other cytokines reflects the profile of cytokines found in patients’ plasma. The aim of our study was to evaluate the relationship between the level of cytokines in the blood, lungs, and lung damage in the model of the Syrian hamster (Mesocricetus auratus) infected with the SARS-CoV-2 strain. The study used outbred female and male Syrian hamsters (n=36, 4 groups) weighing 80-110 g and 5 months old (protocol IACUC, #4, 09/22/2020). Animals were infected intranasally with the hCoV-19/Kazakhstan/KazNAU-NSCEDI-481/2020 strain and euthanized at 3 d.p.i. The level of cytokines IL-6, TNF-α, IFN-α, and IFN-γ was determined by ELISA MyBioSourse (USA) for hamsters. Lung samples were subjected to histological processing. The presence of pathological changes in histological preparations was assessed on a 3-point scale. The work was carried out in the ABSL-3 laboratory. The data were analyzed in GraphPad Prism 6.00 (GraphPad Software, La Jolla, California, USA). The work was supported by the MES RK grant (AP09259865). In the blood, the level of TNF-α increased in males (p=0.0012) and IFN-γ in males and females (p=0.0001). On the contrary, IFN-α production decreased (p=0.0006). Only TNF-α level increased in lung tissues (p=0.0011). Correlation analysis showed a negative relationship between the level of IL-6 in the blood and lung damage in males (r -0.71, p=0.0001) and females (r-0.57, p=0.025). On the contrary, in males, the level of IL-6 in the lungs and score is positively correlated (r 0.80, p=0.01). The level of IFN-γ in the blood (r -0.64, p=0.035) and lungs (r-0.72, p=0.017) in males has a negative correlation with lung damage. No links were found for TNF-α and IFN-α. The study showed a positive association between lung injury and tissue levels of IL-6 in male hamsters. It is known that in humans, high concentrations of IL-6 in the lungs are associated with suppression of cellular immunity and, as a result, with an increase in the severity of COVID-19. TNF-α and IFN-γ play a key role in the pathogenesis of COVID-19 in hamsters. However, the mechanisms of their activity require more detailed study. IFN-α plays a lesser role in direct lung injury in a Syrian hamster model. We have shown the significance of tissue IL-6 and IFN-γ as predictors of the severity of lung damage in COVID-19 in the Syrian hamster model. Changes in the level of cytokines in the blood may not always reflect pathological processes in the lungs with COVID-19.

Keywords: syrian hamster, COVID-19, cytokines, biological model

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Authors: Yuliya Huseva

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To investigate the contents of tears to determine latent blood. Methods: Tear samples from 72 women were studied with the microscopy of tears aspirated with a capillary and stained by Nocht and with a chemical method of test strips with chromogen. Statistical data processing was carried out using statistical packages Statistica 10.0 for Windows, calculation of Pearson's chi-square test, Yule association coefficient, the method of determining sensitivity and specificity. Results:, In 30.6% (22) of tear samples erythrocytes were revealed microscopically. Correlations between the presence of erythrocytes in the tear and the phase of the menstrual cycle has been discovered. In the follicular phase of the cycle, erythrocytes were found in 59.1% (13) people, which is significantly more (x2=4.2, p=0.041) compared to the luteal phase - in 40.9% (9) women. In the first seven days of the follicular phase of the menstrual cycle the erythrocytes were predominanted of in the tears of women examined testifies in favour of the vicarious bleeding from the mucous membranes of extragenital organs in sync with menstruation. Of the other cellular elements in tear samples with latent haemolacria, neutrophils prevailed - in 45.5% (10), while lymphocytes were less common - in 27.3% (6), because neutrophil exudation is accompanied by vasodilatation of the conjunctiva and the release of erythrocytes into the conjunctival cavity. It was found that the prognostic significance of the chemical method was 0.53 of the microscopic method. In contrast to microscopy, which detected blood in tear samples from 30.6% (22) of women, blood was detected chemically in tears of 16.7% (12). An association between latent haemolacria and endometriosis was found (k=0.75, p≤0.05). Microscopically, in the tears of patients with endometriosis, erythrocytes were detected in 70% of cases, while in healthy women without endometriosis - in 25% of cases. The proportion of women with erythrocytes in tears, determined by a chemical method, was 41.7% among patients with endometriosis, which is significantly more (x2=6.5, p=0.011) than 11.7% among women without endometriosis. The data obtained can be explained by the etiopathogenesis of the extragenital endometriosis which is caused by hematogenous spread of endometrial tissue into the orbit. In endometriosis, erythrocytes are found against the background of accumulations of epithelial cells. In the tear samples of 4 women with endometriosis, glandular cuboidal epithelial cells, morphologically similar to endometrial cells, were found, which may indicate a generalization of the disease. Conclusions: Single erythrocytes can normally be found in the tears, their number depends on the phase of the menstrual cycle, increasing in the follicular phase. Erythrocytes found in tears against the background of accumulations of epitheliocytes and their glandular atypia may indicate a manifestation of extragenital endometriosis. Both used methods (microscopic and chemical) are informative in revealing latent haemolacria. The microscopic method is more sensitive, reveals intact erythrocytes, and besides, it provides information about other cells. At the same time, the chemical method is faster and technically simpler, it determines the presence of haemoglobin and its metabolic products, and can be used as a screening.

Keywords: tear, blood, microscopy, epitheliocytes

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Authors: Ramin Mehdiabadi, Neda Menbari, Mohammad Nazir Menbari

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As a pressing public health concern, breast cancer stands as the predominant oncological diagnosis and principal cause of cancer-related mortality among women globally, accounting for 11.7% of new cancer incidences and 6.9% of cancer-related deaths. The annual figures indicate that approximately 230,480 women are diagnosed with breast cancer in the United States alone, with 39,520 succumbing to the disease. While developed economies have reported a deceleration in both incidence and mortality rates across various forms of cancer, including breast cancer, emerging and low-income economies manifest a contrary escalation, largely attributable to lifestyle-mediated risk factors such as tobacco usage, physical inactivity, and high caloric intake. Breast cancer is distinctly characterized by molecular heterogeneity, manifesting in specific subtypes delineated by biomarkers—Estrogen Receptors (ER), Progesterone Receptors (PR), and Human Epidermal Growth Factor Receptor 2 (HER2). These subtypes, comprising Luminal A, Luminal B, HER2-enriched, triple-negative/basal-like, and normal-like, necessitate nuanced, subtype-specific therapeutic regimens, thereby challenging the applicability of generalized treatment protocols. Within this molecular complexity, the transcription factor Forkhead Box M1 (FoxM1) has garnered attention as a significant driver of cellular proliferation, tumorigenesis, metastatic progression, and treatment resistance in a spectrum of human malignancies, including breast cancer. Concurrently, microRNAs (miRs), specifically miR-216b-5p, have been identified as post-transcriptional gene expression regulators and potential tumor suppressors. The overarching objective of this academic investigation is to explicate the multifaceted interrelationship between FoxM1 and miR-216b-5p across the disparate molecular subtypes of breast cancer. Employing a methodologically rigorous, interdisciplinary research design that incorporates cutting-edge molecular biology techniques, sophisticated bioinformatics analytics, and exhaustive meta-analyses of extant clinical data, this scholarly endeavor aims to unveil novel biomarker-specific therapeutic pathways. By doing so, this research is positioned to make a seminal contribution to the advancement of personalized, efficacious, and minimally toxic treatment paradigms, thus profoundly impacting the global efforts to ameliorate the burden of breast cancer.

Keywords: breast cancer, fox m1, microRNAs, mir-216b-5p, gene expression

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Authors: África Vicario, Fernando J. Álvarez, Felipe Parralejo, Fernando Aranda

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The irrigation systems of traditional agriculture, such as gravity-fed irrigation, produce a great waste of water because, generally, there is no control over the amount of water supplied in relation to the water needed. The AIRON Project tries to solve this problem by implementing an IoT-based system to sensor the irrigation plots so that the state of the crops and the amount of water used for irrigation can be known remotely. The IoT system consists of a sensor network that measures the humidity of the soil, the weather conditions (temperature, relative humidity, wind and solar radiation) and the irrigation water flow. The communication between this network and a central gateway is conducted by means of long-range wireless communication that depends on the characteristics of the irrigation plot. The main objective of the AIRON project is to deploy an IoT sensor network in two different plots of the irrigation community of Aranjuez in the Spanish region of Madrid. The first plot is 2 km away from the central gateway, so LoRa has been used as the base communication technology. The problem with this plot is the absence of mains electric power, so devices with energy-saving modes have had to be used to maximize the external batteries' use time. An ESP32 SOC board with a LoRa module is employed in this case to gather data from the sensor network and send them to a gateway consisting of a Raspberry Pi with a LoRa hat. The second plot is located 18 km away from the gateway, a range that hampers the use of LoRa technology. In order to establish reliable communication in this case, the long-term evolution (LTE) standard is used, which makes it possible to reach much greater distances by using the cellular network. As mains electric power is available in this plot, a Raspberry Pi has been used instead of the ESP32 board to collect sensor data. All data received from the two plots are stored on a proprietary server located at the irrigation management company's headquarters. The analysis of these data by means of machine learning algorithms that are currently under development should allow a short-term prediction of the irrigation water demand that would significantly reduce the waste of this increasingly valuable natural resource. The major finding of this work is the real possibility of deploying a remote sensing system for irrigated plots by using Commercial-Off-The-Shelf (COTS) devices, easily scalable and adaptable to design requirements such as the distance to the control center or the availability of mains electrical power at the site.

Keywords: internet of things, irrigation water control, LoRa, LTE, smart farming

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Authors: S. E. Mohmad-Saberi, W. Song, N. Oliver, M. Adrian, T.C. Hsu, A. Darbyshire

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Biodegradable polyurethane (PU) has emerged as a potential material to promote repair and regeneration of damaged/diseased tissues in heart valve regeneration due to its excellent biomechanical profile. Understanding the effects of sterilization on their properties is vital since they are more sensitive and more critical of porous structures compared to bulk ones. In this study, the effects of low concentration of hydrogen peroxide (H₂O₂) solution sterilization has been investigated to determine whether the procedure would be efficient and non-destructive to porous three-dimensional (3D) elastomeric nanocomposite, polyhedral oligomeric silsesquioxane-terminated poly (ethylene-diethylene glycol succinate-sebacate) urea-urethane (POSS-EDSS-PU) scaffold. All the samples were tested for sterility following sterilization using phosphate buffer saline (PBS) as control and 5 % v/v H₂O₂ solution. The samples were incubated in tryptic soy broth for the cultivation of microorganisms under agitation at 37˚C for 72 hours. The effects of the 5 % v/v H₂O₂ solution sterilization were evaluated in terms of morphology, chemical and mechanical properties using scanning electron microscopy (SEM), Fourier transform infrared (FTIR) and tensile tester apparatus. Toxicity effects of the 5 % v/v H₂O₂ solution decontamination were studied by in vitro cytotoxicity test, where the cellular responses of human dermal fibroblast (HDF) were examined. A clear, uncontaminated broth using 5 % v/v H₂O₂ solution method indicated efficient sterilization after 3 days, while the non-sterilized control shows clouding broth indicated contamination. The morphology of 3D POSS-EDSS-PU scaffold appeared to have similar morphology after sterilization with 5 % v/v H₂O₂ solution regarding of pore size and surface. FTIR results show that the sterilized samples and non-sterilized control share the same spectra pattern, confirming no significant alterations over the surface chemistry. For the mechanical properties of the H₂O₂ solution-treated scaffolds, the tensile strain was not significantly decreased, however, become significantly stiffer after the sterilization. No cytotoxic effects were observed after the 5 % v/v H₂O₂ solution sterilization as confirmed by cell viability assessed by Alamar Blue assay. The results suggest that low concentration of 5 % v/v hydrogen peroxide solution can be used as an alternative method for sterilizing biodegradable 3D porous scaffold with micro/nano-architecture without structural deformation. This study provides the understanding of the sterilization effects on biomechanical profile and cell proliferation of 3D POSS-EDSS-PU scaffolds.

Keywords: biodegradable, hydrogen peroxide solution, POSS-EDSS-PU, sterilization

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Authors: Pedro Batista, André Vinha, Filipe Castelo, Bárbara Costa, Ricardo Sousa, Raquel Ricardo, André Pinto

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Background: Septic arthritis is a diagnosis that must be considered in any patient presenting with acute joint swelling and fever. Among the several risk factors for septic arthritis, such as age, rheumatoid arthritis, recent surgery, or skin infection, diabetes mellitus can sometimes be the main risk factor. Staphylococcus aureus is the most common pathogen isolated in septic arthritis; however, it is uncommon in monomicrobial necrotizing fasciitis. Objectives: A case report of concomitant septic arthritis and necrotizing fasciitis in a patient with undiagnosed diabetes based on clinical history. Study Design & Methods: We report a case of a 58-year-old Portuguese previously healthy man who presented to the emergency department with fever and left knee swelling and pain for two days. The blood work revealed ketonemia of 6.7 mmol/L and glycemia of 496 mg/dL. The vital signs were significant for a temperature of 38.5 ºC and 123 bpm of heart rate. The left knee had edema and inflammatory signs. Computed tomography of the left knee showed diffuse edema of the subcutaneous cellular tissue and soft tissue air bubbles. A diagnosis of septic arthritis and necrotising fasciitis was made. He was taken to the operating room for surgical debridement. The samples collected intraoperatively were sent for microbiological analysis, revealing infection by multi-sensitive Staphylococcus aureus. Given this result, the empiric flucloxacillin (500 mg IV) and clindamycin (1000 mg IV) were maintained for 3 weeks. On the seventh day of hospitalization, there was a significant improvement in subcutaneous and musculoskeletal tissues. After two weeks of hospitalization, there was no purulent content and partial closure of the wounds was possible. After 3 weeks, he was switched to oral antibiotics (flucloxacillin 500 mg). A week later, a urinary infection by Pseudomonas aeruginosa was diagnosed and ciprofloxacin 500 mg was administered for 7 days without complications. After 30 days of hospital admission, the patient was discharged home and recovered. Results: The final diagnosis of concomitant septic arthritis and necrotizing fasciitis was made based on the imaging findings, surgical exploration and microbiological tests results. Conclusions: Early antibiotic administration and surgical debridement are key in the management of septic arthritis and necrotizing fasciitis. Furthermore, risk factors control (euglycemic blood glucose levels) must always be taken into account given the crucial role in the patient's recovery.

Keywords: septic arthritis, Necrotizing fasciitis, diabetes, Staphylococcus Aureus

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Authors: T. Ferreira, A. Kulkarni, C. Bretscher, K. Richter, M. Ehrlich, A. Marchini

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H-1 protoparvovirus (H-1PV) is a virus with inherent oncolytic and oncosuppressive activities while remaining non-pathogenic in humans. H-1PV was the first oncolytic parvovirus to undergo clinical testing. Results from trials in patients with glioblastoma or pancreatic carcinoma showed an excellent safety profile and first signs of efficacy. H-1PV infection is vastly dependent on cellular factors, from cell attachment and entry to viral replication and egress. Hence, we believe that the characterisation of the parvovirus life cycle would ultimately help further improve H-1PV clinical outcome. In the present study, we explored the entry pathway of H-1PV in cervical HeLa and glioma NCH125 cancer cell lines. Electron and confocal microscopy showed viral particles associated with clathrin-coated pits and vesicles, providing the first evidence that H-1PV cell entry occurs through clathrin-mediated endocytosis. Accordingly, we observed that by blocking clathrin-mediated endocytosis with hypertonic sucrose, chlorpromazine, or pitstop 2, H-1PV transduction was markedly decreased. Accordingly, siRNA-mediated knockdown of AP2M1, which retains a crucial role in clathrin-mediated endocytosis, verified the reliance of H-1PV on this route to enter HeLa and NCH125 cancer cells. By contrast, we found no evidence of viral entry through caveolae-mediated endocytosis. Indeed, pre-treatment of cells with nystatin or methyl-β-cyclodextrin, both inhibitors of caveolae-mediated endocytosis, did not affect viral transduction levels. Unexpectedly, siRNA-mediated knockdown of caveolin-1, the main driver of caveolae-mediated endocytosis, increased H-1PV transduction, suggesting caveolin-1 is a negative modulator of H-1PV infection. We also show that H-1PV entry is dependent on dynamin, a protein responsible for mediating the scission of vesicle neck and promoting further internalisation. Furthermore, since dynamin inhibition almost completely abolished H-1PV infection, makes it unlikely that H-1PV uses macropinocytosis as an alternative pathway to enter cells. After viral internalisation, H-1PV passes through early to late endosomes as observed by confocal microscopy. Inside these endocytic compartments, the acidic environment proved to be crucial for a productive infection. Inhibition of acidification of pH dramatically reduced H-1PV transduction. Besides, a fraction of H-1PV particles was observed inside LAMP1-positive lysosomes, most likely following a non-infectious route. To the author's best knowledge, this is the first study to characterise the cell entry pathways of H-1PV. Along these lines, this work will further contribute to understand H-1PV oncolytic properties as well as to improve its clinical potential in cancer virotherapy.

Keywords: clathrin-mediated endocytosis, H-1 parvovirus, oncolytic virus, virus entry

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Authors: Carolina Schencke, Cristian Sandoval, Belgica Vasquez, Mariano Del Sol

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Introduction: In a burn injury, the successful repair requires not only the participation of various cells, such as granulocytes and fibroblasts, but also of collagen, which plays a crucial role as a structural and regulatory molecule of scar tissue. Since honey and ascorbic acid have presented a great therapeutic potential to cellular and structural level, experimental studies have proposed its combination in the treatment of wounds. Aim: To evaluate stereological and morphometric parameters of healing wounds, caused by burns, treated with honey Ulmo (Eucryphia cordifolia) unsupplemented, comparing its effect with Ulmo honey supplemented with ascorbic acid. Materials and Methods: Fifteen healthy adult guinea pigs (Cavia porcellus) were used, of both sexes, average weight 450 g from the Centro de Excelencia en Estudios Morfológicos y Quirúrgicos (CEMyQ) at the Universidad de La Frontera, Chile. The animals were divided at random into three groups: positive control (C+), honey only (H) and supplemented honey (SH) and were fed on pellets supplemented with ascorbic acid and water ad libitum, under ambient conditions controlled for temperature, ambient noise and a cycle of 12h light–darkness. The protocol for the experiment was approved by the Scientific Ethics Committee of the Universidad de La Frontera, Chile. The parameters measured were number density per area (NA), volume density (VV), and surface density (SV) of fibroblast; NA and VV of polymorphonuclear cells (PMN) and, evaluation of the content of collagen fibers in the scar dermis. One-way ANOVA was used for statistics analysis and its respective Post hoc tests. Results: The ANOVA analysis for NA, VV and SV of fibroblasts, NA and VV of PMN, and evaluation of collagen content, type I and III, showed that at least one group differs from other (P≤ 0.001). There were differences (P= 0.000) in NA of fibroblast between the groups [C+= 3599.560 mm-2 (SD= 764.461), H= 3355.336 mm-2 (SD= 699.443) and SH= 4253.025 mm-2 (SD= 1041.751)]. The VV and SV of fibroblast increased (P= 0.000) in the SH group [20.400% (SD= 5.897) and 100.876 mm2/mm3 (SD= 29.431), respectively], compared to the C+ [16.324% (SD= 7.719) and 81.676 mm2/mm3 (SD= 28.884), respectively). The mean values of NA and VV of PMN were higher (P= 0.000) in the H [756.875 mm-2 (SD= 516.489) and 2.686% (SD= 2.380), respectively) group. Regarding to the evaluation of the content of collagen fibers, type I and III, the one-way analysis of ANOVA showed a statistically significant difference (P< 0.05). The content of collagen fibers type I was higher in C+ (1988.292 μm2; SD= 1312.379), while the content of collagen fibers type III was higher in SH (1967.163 μm2; SD= 1047.944 μm2) group. Conclusions: The stereological results were correlated with the stage of healing observed for each group. These results suggest that the combination of honey with ascorbic acid potentiate the healing effect, where both participated synergistically.

Keywords: ascorbic acid, morphometry, stereology, Ulmo honey

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Authors: Martyna Chotomska, Aleksandra Studzinska, Marta Lisowska, Justyna Szubert, Aleksandra Tabis, Jacek Bania, Arkadiusz Miazek

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Objectives: Assessing antigen-specific T cell responses is of utmost importance for the pre-clinical testing of prototype vaccines against intracellular pathogens and tumor antigens. Mainly two types of in vitro assays are used for this purpose 1) enzyme-linked immunospot (ELISpot) and 2) intracellular cytokine staining (ICS). Both are time-consuming, relatively expensive, and require manual dexterity. Here, we assess if a straightforward detection of luciferase activity in blood samples of transgenic reporter mice expressing a secreted Lucia luciferase under the transcriptional control of IFN-γ promoter parallels the sensitivity of IFNγ ELISpot assay. Methods: IFN-γ-LUCIA mouse strain carrying multiple copies of Lucia luciferase transgene under the transcriptional control of IFNγ minimal promoter were generated by pronuclear injection of linear DNA. The specificity of transgene expression and mobilization was assessed in vitro using transgenic splenocytes exposed to various mitogens. The IFN-γ-LUCIA mice were immunized with 50mg of ovalbumin (OVA) emulsified in incomplete Freund’s adjuvant three times every two weeks by subcutaneous injections. Blood samples were collected before and five days after each immunization. Luciferase activity was assessed in blood serum. Peripheral blood mononuclear cells were separated and assessed for frequencies of OVA-specific IFNγ-secreting T cells. Results: We show that in vitro cultured splenocytes of IFN-γ-LUCIA mice respond by 2 and 3 fold increase in secreted luciferase activity to T cell mitogens concanavalin A and phorbol myristate acetate, respectively but fail to respond to B cell-stimulating E.coli lipopolysaccharide. Immunization of IFN-γ-LUCIA mice with OVA leads to over 4 fold increase in luciferase activity in blood serum five days post-immunization with a barely detectable increase in OVA-specific, IFNγ-secreting T cells by ELISpot. Second and third immunizations, further increase the luciferase activity and coincidently also increase the frequencies of OVA-specific T cells by ELISpot. Conclusions: We conclude that minimally invasive monitoring of luciferase secretions in blood serum of IFN-γ-LUCIA mice constitutes a sensitive method for evaluating primary and memory Th1 responses to protein antigens. As such, this method may complement existing methods for rapid immunogenicity assessment of prototype vaccines.

Keywords: ELISpot, immunogenicity, interferon-gamma, reporter mice, vaccines

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Authors: Qimin Shi, Yi Sun, Constantinus Politis, Shoufeng Yang

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In-situ preparation of particle-reinforced aluminium matrix composites (PRAMCs) by laser powder bed fusion (LPBF) additive manufacturing is a promising strategy to strengthen traditional Al-based alloys. The laser-driven thermite reaction can be a practical mechanism to in-situ synthesize PRAMCs. However, introducing oxygen elements through adding Fe₂O₃ makes the powder mixture highly sensitive to form porosity and Al₂O₃ film during LPBF, bringing challenges to producing dense Al-based materials. Therefore, this work develops a processing strategy combined with consecutive high-energy laser melting scanning and low-energy laser remelting scanning to prepare PRAMCs from a Fe₂O₃/AlSi12 powder mixture. The powder mixture consists of 5 wt% Fe₂O₃ and the remainder AlSi12 powder. The addition of 5 wt% Fe₂O₃ aims to achieve balanced strength and ductility. A high relative density (98.2 ± 0.55 %) was successfully obtained by optimizing laser melting (Emelting) and laser remelting surface energy density (Eremelting) to Emelting = 35 J/mm² and Eremelting = 5 J/mm². Results further reveal the necessity of increasing Emelting, to improve metal liquid’s spreading/wetting by breaking up the Al₂O₃ films surrounding the molten pools; however, the high-energy laser melting produced much porosity, including H₂₋, O₂₋ and keyhole-induced pores. The subsequent low-energy laser remelting could close the resulting internal pores, backfill open gaps and smoothen solidified surfaces. As a result, the material was densified by repeating laser melting and laser remelting layer by layer. Although with two-times laser scanning, the microstructure still shows fine cellular Si networks with Al grains inside (grain size of about 370 nm) and in-situ nano-precipitates (Al₂O₃, Si, and Al-Fe(-Si) intermetallics). Finally, the fine microstructure, nano-structured dispersion strengthening, and high-level densification strengthened the in-situ PRAMCs, reaching yield strength of 426 ± 4 MPa and tensile strength of 473 ± 6 MPa. Furthermore, the results can expect to provide valuable information to process other powder mixtures with severe porosity/oxide-film formation potential, considering the evidenced contribution of laser melting/remelting strategy to densify material and obtain good mechanical properties during LPBF.

Keywords: densification, laser powder bed fusion, metal matrix composites, microstructures, mechanical properties

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Authors: Simona Perga, Chiara Beltramo, Floriana Fruscione, Isabella Martini, Federica Cavallo, Federica Riccardo, Paolo Buracco, Selina Iussich, Elisabetta Razzuoli, Katia Varello, Lorella Maniscalco, Elena Bozzetta, Angelo Ferrari, Paola Modesto

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Introduction: Human and canine melanoma have common clinical, histologic characteristics making dogs a good model for comparative oncology. The identification of specific genes and a better understanding of the genetic landscape, signaling pathways, and tumor–microenvironmental interactions involved in the cancer onset and progression is essential for the development of therapeutic strategies against this tumor in both species. In the present study, the differential expression of genes in spontaneously occurring canine melanoma and in paired normal tissue was investigated by targeted RNAseq. Material and Methods: Total RNA was extracted from 17 canine malignant melanoma (CMM) samples and from five paired normal tissues stored in RNA-later. In order to capture the greater genetic variability, gene expression analysis was carried out using two panels (Qiagen): Human Immuno-Oncology (HIO) and Mouse-Immuno-Oncology (MIO) and the miSeq platform (Illumina). These kits allow the detection of the expression profile of 990 genes involved in the immune response against tumors in humans and mice. The data were analyzed through the CLCbio Genomics Workbench (Qiagen) software using the Canis lupus familiaris genome as a reference. Data analysis were carried out both comparing the biologic group (tumoral vs. healthy tissues) and comparing neoplastic tissue vs. paired healthy tissue; a Fold Change greater than two and a p-value less than 0.05 were set as the threshold to select interesting genes. Results and Discussion: Using HIO 63, down-regulated genes were detected; 13 of those were also down-regulated comparing neoplastic sample vs. paired healthy tissue. Eighteen genes were up-regulated, 14 of those were also down-regulated comparing neoplastic sample vs. paired healthy tissue. Using the MIO, 35 down regulated-genes were detected; only four of these were down-regulated, also comparing neoplastic sample vs. paired healthy tissue. Twelve genes were up-regulated in both types of analysis. Considering the two kits, the greatest variation in Fold Change was in up-regulated genes. Dogs displayed a greater genetic homology with humans than mice; moreover, the results have shown that the two kits are able to detect different genes. Most of these genes have specific cellular functions or belong to some enzymatic categories; some have already been described to be correlated to human melanoma and confirm the validity of the dog as a model for the study of molecular aspects of human melanoma.

Keywords: animal model, canine melanoma, gene expression, spontaneous tumors, targeted RNAseq

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Authors: Ghada Al-Kafaji, Mohamed Sabry

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Mitochondria are the site of cellular respiration and produce energy in the form of adenosine triphosphate (ATP) via oxidative phosphorylation. They are the major source of intracellular reactive oxygen species (ROS) and are also direct target to ROS attack. Oxidative stress and ROS-mediated disruptions of mitochondrial function are major components involved in the pathogenicity of diabetic complications. In this work, the changes in mitochondrial DNA (mtDNA) copy number, biogenesis, gene expression of mtDNA-encoded subunits of electron transport chain (ETC) complexes, and mitochondrial function in response to hyperglycemia-induced ROS and the effect of direct inhibition of ROS on mitochondria were investigated in an in vitro model of diabetic nephropathy using human renal mesangial cells. The cells were exposed to normoglycemic and hyperglycemic conditions in the presence and absence of Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) or catalase for 1, 4 and 7 days. ROS production was assessed by the confocal microscope and flow cytometry. mtDNA copy number and PGC-1a, NRF-1, and TFAM, as well as ND2, CYTB, COI, and ATPase 6 transcripts, were all analyzed by real-time PCR. PGC-1a, NRF-1, and TFAM, as well as ND2, CYTB, COI, and ATPase 6 proteins, were analyzed by Western blotting. Mitochondrial function was determined by assessing mitochondrial membrane potential and adenosine triphosphate (ATP) levels. Hyperglycemia-induced a significant increase in the production of mitochondrial superoxide and hydrogen peroxide at day 1 (P < 0.05), and this increase remained significantly elevated at days 4 and 7 (P < 0.05). The copy number of mtDNA and expression of PGC-1a, NRF-1, and TFAM as well as ND2, CYTB, CO1 and ATPase 6 increased after one day of hyperglycemia (P < 0.05), with a significant reduction in all those parameters at 4 and 7 days (P < 0.05). The mitochondrial membrane potential decreased progressively at 1 to 7 days of hyperglycemia with the parallel progressive reduction in ATP levels over time (P < 0.05). MnTBAP and catalase treatment of cells cultured under hyperglycemic conditions attenuated ROS production reversed renal mitochondrial oxidative stress and improved mtDNA, mitochondrial biogenesis, and function. These results show that hyperglycemia-induced ROS caused an early increase in mtDNA copy number, mitochondrial biogenesis and mtDNA-encoded gene expression of the ETC subunits in human mesangial cells as a compensatory response to the decline in mitochondrial function, which precede the mtDNA defect and mitochondrial dysfunction with a progressive oxidative response. Protection from ROS-mediated damage to renal mitochondria induced by hyperglycemia may be a novel therapeutic approach for the prevention/treatment of DN.

Keywords: diabetic nephropathy, hyperglycemia, reactive oxygen species, oxidative stress, mtDNA, mitochondrial dysfunction, manganese superoxide dismutase, catalase

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Authors: Zain Ul Abadeen, Muhammad Zargham Khan, Muhammad Kashif Saleemi, Ahrar Khan, Ijaz Javed Hassan, Aisha Khatoon, Qasim Altaf

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Aflatoxins are secondary metabolites produced by toxigenic fungi, and there are four types of aflatoxins include AFB1, AFB2, AFG1 and AFG2. Aflatoxin B1 (AFB1) is considered as most toxic form. It is mainly responsible for the contamination of poultry feed and produces a condition called aflatoxicosis leads to immunosuppression in poultry birds. Saccharomyces cerevisiae is a single cell microorganism and acts as a source of growth factors, minerals and amino acids which improve the immunity and digestibility in poultry birds as probiotics. Saccharomyces cerevisiae is well recognized to cause the biological degradation of mycotoxins (toxin binder) because its cell wall contains β-glucans and mannans which specifically bind with aflatoxins and reduce their absorption or transfer them to some non-toxic compounds. The present study was designed to investigate the immunosuppressive effects of aflatoxins in broiler chicks and the reduction of severity of these effects by the use of Baker’s Yeast (Saccharomyces cerevisiae). One-day-old broiler chicks were procured from local hatchery and were divided into various groups (A-I). These groups were treated with different levels of AFB1 @ 400 µg/kg and 600 µg/kg along with different levels of Baker’s Yeast (Saccharomyces cerevisiae) 0.1% and 0.5 % in the feed. The total duration of the experiment was six weeks and different immunological parameters including the cellular immune response by injecting PHA-P (Phytohemagglutinin-P) in the skin of the birds, phagocytic function of mononuclear cells by Carbon clearance assay from blood samples and humoral immune response against intravenously injected sheep RBCs from the serum samples were determined. The birds from each group were slaughtered at the end of the experiment to determine the presence of gross lesions in the immune organs and these tissues were fixed in 10% neutral buffered formalin for histological investigations. The results showed that AFB1 intoxicated groups had reduced body weight gain, feed intake, organs weight and immunological responses compared to the control and Baker’s Yeast (Saccharomyces cerevisiae) treated groups. Different gross and histological degenerative changes were recorded in the immune organs of AFB1 intoxicated groups compared to control and Baker’s Yeast (Saccharomyces cerevisiae) treated groups. The present study concluded that Baker’s Yeast (Saccharomyces cerevisiae) addition in the feed helps to ameliorate the immunotoxigenic effects produced by AFB1 in broiler chicks.

Keywords: aflatoxins, body weight gain, feed intake, immunological response, toxigenic effect

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Authors: Sarah F. M. Pilati, Carolina S. Flausino, Guilherme F. Hoffmeister, Davi R. Tames, Telmo J. Mezadri

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The effects that may be related to narghile smoking in the tissues of the oral cavity, trachea and lung and associated inflammation has been the question raised lately. The objective of this study was to identify histopathological changes and the presence of inflammation through the exposure of mice to narghile smoking through a whole-body study. The animals were divided in 4 groups with 5 animals in each group, being: one control group, one with 7 days of exposure, 15 days and the last one with 30 days. The animals were exposed to the conventional hookah smoke from Mizo brand with 0.5% percentage of unwashed tobacco and the EcOco brand coconut fiber having a dimension of 2cm × 2cm. The duration of the session was 30 minutes / day per 7, 15 and 30 days. The tobacco smoke concentration at which test animals were exposed was 35 ml every two seconds while the remaining 58 seconds were pure air. Afterward, the mice were sacrificed and submitted to histological evaluation through slices. It was found in the tongue of the 7-day group the presence in epithelium areas with acanthosis, hyperkeratosis and epithelial projections. In-depth, more intense inflammation was observed. All alteration processes increased significantly as the days of exposure increased. In trachea, with the 7-day group, there was a decrease in thickening of the pseudostratified epithelium and a slight decrease in lashes, giving rise to the metaplasia process, a process that was established in the 31-day sampling when the epithelium became stratified. In the conjunctive tissue, it was observed the presence of defense cells and formation of new vessels, evidencing the chronic inflammatory process, which decreased in the course of the samples due to the deposition of collagen fibers as seen in the 15 and 31 days groups. Among the structures of the lung, the study focused on the bronchioles and alveoli. From the 7-day group, intra-alveolar septum thickness increased, alveolar space decreased, inflammatory infiltrate with mononuclear and defense cells and new vessels formation were observed, increasing the number of red blood cells in the region. The results showed that with the passing of the days a progressive increase of the signs of changes in the region was observed, a factor that shows that narghile smoking stimulates alterations mainly in the alveoli (place where gas exchanges occur that should not present alterations) calling attention to the harmful and aggressive effect of narghile smoking. These data also highlighted the harmful effect of smoking, since the presence of acanthosis, hyperkeratosis, epithelial projections and inflammation evidences the cellular alteration process for the tongue tissue protection. Also, the narghile smoking stimulates both epithelial and inflammatory changes in the trachea, in addition to a process of metaplasia, a factor that reinforces the harmful effect and the carcinogenic potential of the narghile smoking.

Keywords: metaplasia, inflammation, pathological constriction, hyperkeratosis

Procedia PDF Downloads 173

Authors: Richa Sharma, Uma Nahar, Sadhna Sharma, Indu Verma

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Counteracting the deadly pathogen Mycobacterium tuberculosis (M. tb) effectively is still a global challenge. Scrutinizing alternative weapons like antimicrobial peptides to strengthen existing tuberculosis artillery is urgently required. Considering the antimycobacterial potential of Human Beta Defensin 1 (HBD-1) along with isoniazid, the present study was designed to explore the ability of HBD-1 to act against active and dormant M. tb. HBD-1 was screened in silico using antimicrobial peptide prediction servers to identify its short antimicrobial motif. The activity of both HBD-1 and its selected motif (Pep B) was determined at different concentrations against actively growing M. tb in vitro and ex vivo in monocyte derived macrophages (MDMs). Log phase M. tb was grown along with HBD-1 and Pep B for 7 days. M. tb infected MDMs were treated with HBD-1 and Pep B for 72 hours. Thereafter, colony forming unit (CFU) enumeration was performed to determine activity of both peptides against actively growing in vitro and intracellular M. tb. The dormant M. tb models were prepared by following two approaches and treated with different concentrations of HBD-1 and Pep B. Firstly, 20-22 days old M. tbH37Rv was grown in potassium deficient Sauton media for 35 days. The presence of dormant bacilli was confirmed by Nile red staining. Dormant bacilli were further treated with rifampicin, isoniazid, HBD-1 and its motif for 7 days. The effect of both peptides on latent bacilli was assessed by colony forming units (CFU) and most probable number (MPN) enumeration. Secondly, human PBMC granuloma model was prepared by infecting PBMCs seeded on collagen matrix with M. tb(MOI 0.1) for 10 days. Histopathology was done to confirm granuloma formation. The granuloma thus formed was incubated for 72 hours with rifampicin, HBD-1 and Pep B individually. Difference in bacillary load was determined by CFU enumeration. The minimum inhibitory concentrations of HBD-1 and Pep B restricting growth of mycobacteria in vitro were 2μg/ml and 20μg/ml respectively. The intracellular mycobacterial load was reduced significantly by HBD-1 and Pep B at 1μg/ml and 5μg/ml respectively. Nile red positive bacterial population, high MPN/ low CFU count and tolerance to isoniazid, confirmed the formation of potassium deficienybaseddormancy model. HBD-1 (8μg/ml) showed 96% and 99% killing and Pep B (40μg/ml) lowered dormant bacillary load by 68.89% and 92.49% based on CFU and MPN enumeration respectively. Further, H&E stained aggregates of macrophages and lymphocytes, acid fast bacilli surrounded by cellular aggregates and rifampicin resistance, indicated the formation of human granuloma dormancy model. HBD-1 (8μg/ml) led to 81.3% reduction in CFU whereas its motif Pep B (40μg/ml) showed only 54.66% decrease in bacterial load inside granuloma. Thus, the present study indicated that HBD-1 and its motif are effective antimicrobial players against both actively growing and dormant M. tb. They should be further explored to tap their potential to design a powerful weapon for combating tuberculosis.

Keywords: antimicrobial peptides, dormant, human beta defensin 1, tuberculosis

Procedia PDF Downloads 263

Authors: Sihem Bazid, Meriem El Kolli, Aicha Medjahed

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Proteins and polysaccharides are the two types of biopolymers most frequently used in the food industry to control the mechanical properties and structural stability and organoleptic properties of the products. The textural and structural properties of these two types of blend polymers depend on their interaction and their ability to form organized structures. From an industrial point of view, a better understanding of mixtures protein / polysaccharide is an important issue since they are already heavily involved in processed food. It is in this context that we have chosen to work on a model system composed of a fibrous protein mixture (gelatin)/anionic polysaccharide (sodium carboxymethylcellulose). Gelatin, one of the most popular biopolymers, is widely used in food, pharmaceutical, cosmetic and photographic applications, because of its unique functional and technological properties. Sodium Carboxymethylcellulose (NaCMC) is an anionic linear polysaccharide derived from cellulose. It is an important industrial polymer with a wide range of applications. The functional properties of this anionic polysaccharide can be modified by the presence of proteins with which it might interact. Another factor may also manage the interaction of protein-polysaccharide mixtures is the triple helix of the gelatin. Its complex synthesis method results in an extracellular assembly containing several levels. Collagen can be in a soluble state or associate into fibrils, which can associate in fiber. Each level corresponds to an organization recognized by the cellular and metabolic system. Gelatin allows this approach, the formation of gelatin gel has triple helical folding of denatured collagen chains, this gel has been the subject of numerous studies, and it is now known that the properties depend only on the rate of triple helices forming the network. Chemical modification of this system is quite controlled. Observe the dynamics of the triple helix may be relevant in understanding the interactions involved in protein-polysaccharides mixtures. Gelatin is central to any industrial process, understand and analyze the molecular dynamics induced by the triple helix in the transitions gelatin, can have great economic importance in all fields and especially the food. The goal is to understand the possible mechanisms involved depending on the nature of the mixtures obtained. From a fundamental point of view, it is clear that the protective effect of NaCMC on gelatin and conformational changes of the α helix are strongly influenced by the nature of the medium. Our goal is to minimize the maximum the α helix structure changes to maintain more stable gelatin and protect against denaturation that occurs during such conversion processes in the food industry. In order to study the nature of interactions and assess the properties of mixtures, polarimetry was used to monitor the optical parameters and to assess the rate of helicity gelatin.

Keywords: gelatin, sodium carboxymethylcellulose, interaction gelatin-NaCMC, the rate of helicity, polarimetry

Procedia PDF Downloads 312

Authors: Iliyasu Musa Omoyine, Musa Sunday Abraham, Oladele Sunday Blessing, Iliya Ibrahim Abdullahi, Ibegbu Augustine Oseloka, Nuhu Nana-Hawau, Animoku Abdulrazaq Amoto, Yusuf Abdullateef Onoruoiza, Sambo Sohnap James, Akpulu Steven Peter, Ajayi Abayomi

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The neuroprotective role of inflammation from detrimental intrinsic and extrinsic factors has been reported. However, the overactivation of astrocytes and microglia due to lead toxicity produce excessive pro-inflammatory cytokines, mediating neurodegenerative diseases. The present study investigated the mitigatory effects of quercetin on neuroinflammation, correlating with memory function in lead-exposed rats. In this study, Wistar rats were administered orally with Quercetin (Q: 60 mg/kg) and Succimer as a standard drug (S: 10 mg/kg) for 21 days after lead exposure (Pb: 125 mg/kg) of 21 days or in combination with Pb, once daily for 42 days. Working and reference memory was assessed using an Eight-arm radial water maze (8-ARWM). The changes in brain lead level, the neuronal nitric oxide synthase (nNOS) activity, and the level of neuroinflammatory markers such as tumour necrosis factor-alpha (TNF-α) and Interleukin 1 Beta (IL-1β) were determined. Immunohistochemically, astrocyte expression was evaluated. The results showed that the brain level of lead was increased significantly in lead-exposed rats. The expression of astrocytes increased in the CA3 and CA1 regions of the hippocampus, and the levels of brain TNF-α and IL-1β increased in lead-exposed rats. Lead impaired reference and working memory by increasing reference memory errors and working memory incorrect errors in lead-exposed rats. However, quercetin treatment effectively improved memory and inhibited neuroinflammation by reducing astrocytes’ expression and the levels of TNF-α and IL-1β. The expression of astrocytes and the levels of TNF-α and IL-1β correlated with memory function. The possible explanation for quercetin’s anti-neuroinflammatory effect is that it modulates the activity of cellular proteins involved in the inflammatory response; inhibits the transcription factor of nuclear factor-kappa B (NF-κB), which regulates the expression of proinflammatory molecules; inhibits kinases required for the synthesis of Glial fibrillary acidic protein (GFAP) and modifies the phosphorylation of some proteins, which affect the structure and function of intermediate filament proteins; and, lastly, induces Cyclic-AMP Response Element Binding (CREB) activation and neurogenesis as a compensatory mechanism for memory deficits and neuronal cell death. In conclusion, the levels of neuroinflammatory markers negatively correlated with memory function. Thus, quercetin may be a promising therapy in neuroinflammation and memory dysfunction in populations prone to lead exposure.

Keywords: lead, quercetin, neuroinflammation, memory

Procedia PDF Downloads 53

Authors: Shilpee Jain, Sachin Latiyan, Kaushik Suneet

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Unlike traditional robots, medical microbots are not only smaller in size, but they also possess various unique properties, for example, biocompatibility, stability in the biological fluids, navigation opposite to the bloodstream, wireless control over locomotion, etc. The idea behind their usage in the medical field was to build a minimally invasive method for addressing the post-operative complications, including longer recovery time, infection eruption and pain. Herein, the present study demonstrates the fabrication of dual nature magneto-conducting Fe3O4 magnetic nanoparticles (MNPs) and SU8 derived carbon-based Janus microbots for the efficient intracellular delivery of biomolecules. The low aspect ratio with feature size 2-5 μm microbots were fabricated by using a photolithography technique. These microbots were pyrolyzed at 900°C, which converts SU8 into amorphous carbon. The pyrolyzed microbots have dual properties, i.e., the half part is magneto-conducting and another half is only conducting for sufficing the therapeutic payloads efficiently with the application of external electric/magnetic field stimulations. For the efficient intracellular delivery of the microbots, the size and aspect ratio plays a significant role. However, on a smaller scale, the proper control over movement is difficult to achieve. The dual nature of Janus microbots allowed to control its maneuverability in the complex fluids using external electric as well as the magnetic field. Interestingly, Janus microbots move faster with the application of an external electric field (44 µm/s) as compared to the magnetic field (18 µm/s) application. Furthermore, these Janus microbots exhibit auto-fluorescence behavior that will help to track their pathway during navigation. Typically, the use of MNPs in the microdevices enhances the tendency to agglomerate. However, the incorporation of Fe₃O₄ MNPs in the pyrolyzed carbon reduces the chances of agglomeration of the microbots. The biocompatibility of the medical microbots, which is the essential property of any biosystems, was determined in vitro using HeLa cells. The microbots were found to compatible with HeLa cells. Additionally, the intracellular uptake of microbots was higher in the presence of an external electric field as compared to without electric field stimulation. In summary, the cytocompatible Janus microbots were fabricated successfully. They are stable in the biological fluids, wireless controllable navigation with the help of a few Guess external magnetic fields, their movement can be tracked because of autofluorescence behavior, they are less susceptible to agglomeration and higher cellular uptake could be achieved with the application of the external electric field. Thus, these carriers could offer a versatile platform to suffice the therapeutic payloads under wireless actuation.

Keywords: amorphous carbon, electric/magnetic stimulations, Janus microbots, magnetic nanoparticles, minimally invasive procedures

Procedia PDF Downloads 125

Authors: Stefania Fortino, Petr Hradil, Timo Avikainen

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Timber bridges have an excellent environmental performance, are economical, relatively easy to build and can have a long service life. However, the durability of these bridges is the main problem because of their exposure to outdoor climate conditions. The moisture content accumulated in wood for long periods, in combination with certain temperatures, may cause conditions suitable for timber decay. In addition, moisture content variations affect the structural integrity, serviceability and loading capacity of timber bridges. Therefore, the monitoring of the moisture content in wood is important for the durability of the material but also for the whole superstructure. The measurements obtained by the usual sensor-based techniques provide hygro-thermal data only in specific locations of the wood components. In this context, the monitoring can be assisted by numerical modelling to get more information on the hygro-thermal response of the bridges. This work presents a hygro-thermal model based on a multi-phase moisture transport theory to predict the distribution of moisture content, relative humidity and temperature in wood. Below the fibre saturation point, the multi-phase theory simulates three phenomena in cellular wood during moisture transfer, i.e., the diffusion of water vapour in the pores, the sorption of bound water and the diffusion of bound water in the cell walls. In the multi-phase model, the two water phases are separated, and the coupling between them is defined through a sorption rate. Furthermore, an average between the temperature-dependent adsorption and desorption isotherms is used. In previous works by some of the authors, this approach was found very suitable to study the moisture transport in uncoated and coated stress-laminated timber decks. Compared to previous works, the hygro-thermal fluxes on the external surfaces include the influence of the absorbed solar radiation during the time and consequently, the temperatures on the surfaces exposed to the sun are higher. This affects the whole hygro-thermal response of the timber component. The multi-phase model, implemented in a user subroutine of Abaqus FEM code, provides the distribution of the moisture content, the temperature and the relative humidity in a volume of the timber deck. As a case study, the hygro-thermal data in wood are collected from the ongoing monitoring of the stress-laminated timber deck of Tapiola Bridge in Finland, based on integrated humidity-temperature sensors and the numerical results are found in good agreement with the measurements. The proposed model, used to assist the monitoring, can contribute to reducing the maintenance costs of bridges, as well as the cost of instrumentation, and increase safety.

Keywords: moisture content, multi-phase models, solar radiation, timber decks, FEM

Procedia PDF Downloads 175

Authors: Mumuni Sumaila, Viness Pillay, Yahya E. Choonara, Pradeep Kumar, Pierre P. Kondiah

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Tuberculosis (TB) remains a serious challenge to public health globally, despite every effort put together to curb the disease. Current TB therapeutics available have proven to be inefficient due to a multitude of drawbacks that range from serious adverse effects/drug toxicity to inconsistent bioavailability, which ultimately contributes to the emergence of drug-resistant TB. An effective ‘cargo’ system designed to cleverly deliver therapeutic doses of anti-TB drugs to infection sites and in a sustained-release manner may provide a better therapeutic choice towards winning the war against TB. In the current study, we investigated mannose-functionalized lipopolysaccharide hybrid nanoparticles for safety and efficacy towards macrophage-targeted simultaneous delivery of the two first-line anti-TB drugs, rifampicin (RF) and isoniazid (IS). RF-IS-loaded lipopolysaccharide hybrid nanoparticles were fabricated using the solvent injection technique (SIT), incorporating soy lecithin (SL) and low molecular weight chitosan (CS) as the lipid and polysaccharide components, respectively. Surface-functionalized nanoparticles were obtained through the reaction of the aldehyde group of mannose with free amine functionality present at the surface of the nanoparticles. The functionalized nanocarriers were spherical with average particle size and surface charge of 107.83 nm and +21.77 mV, respectively, and entrapment efficiencies (EE) were 53.52% and 69.80% for RF and IS, respectively. FTIR spectrum revealed high-intensity bands between 1663 cm⁻¹ and 1408 cm⁻¹ wavenumbers (absent in non-functionalized nanoparticles), which could be attributed to the C=N stretching vibration produced by the formation of Schiff’s base (–N=CH–) during the mannosylation reaction. In vitro release studies showed a sustained-release profile for RF and IS, with less than half of the total payload released over a 48-hour period. The nanocarriers were biocompatible and safe, with more than 80% cell viability achieved when incubated with RAW 264.7 cells at concentrations 30 to 500 μg/mL over a 24-hour period. Cellular uptake studies (after a 24-hour incubation period with the murine macrophage cells, RAW 264.7) revealed a 13- and a 9-fold increase in intracellular accumulation of RF and IS, respectively, when compared with the unformulated RF+IS solution. A 6- and a 3-fold increase in intracellular accumulation of RF and IS, respectively, were observed when compared with the non-functionalized nanoparticles. Furthermore, fluorescent microscopy images showed nanoparticle internalization and accumulation within the RAW 264.7 cells, which was more significant in the mannose-functionalized system compared to the non-functionalized nanoparticles. The overall results suggested that the fabricated mannose-functionalized lipopolysaccharide nanoparticles are a safe and promising platform for macrophage-targeted delivery of anti-TB therapeutics. However, in vivo pharmacokinetic/pharmacodynamics studies are required to further substantiate the therapeutic efficacy of the nanosystem.

Keywords: anti-tuberculosis therapeutics, hybrid nanosystem, lipopolysaccharide nanoparticles, macrophage-targeted delivery

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Authors: Tiffany Baetens, Sophie Halliez, Luc Buée, Emiliano Pallecchi, Vincent Thomy, Steve Arscott

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We demonstrate here a viable stretchable bio-microelectromechanical systems (BioMEMS) technology for use with biological studies concerned with the effect of high mechanical strains on living cells. An example of this is traumatic brain injury (TBI) where neurons are damaged with physical force to the brain during, e.g., accidents and sports. Robust, miniaturized integrated systems are needed by biologists to be able to study the effect of TBI on neuron cells in vitro. The major challenges in this area are (i) to develop micro, and nanofabrication processes which are based on stretchable substrates and to (ii) create systems which are robust and performant at very high mechanical strain values—sometimes as high as 100%. At the time of writing, such processes and systems were rapidly evolving subject of research and development. The BioMEMS which we present here is composed of an elastomer substrate (low Young’s modulus ~1 MPa) onto which is patterned robust electrodes and insulators. The patterning of the thin films is achieved using standard photolithography techniques directly on the elastomer substrate—thus making the process generic and applicable to many materials’ in based systems. The chosen elastomer used is commercial ‘Sylgard 184’ polydimethylsiloxane (PDMS). It is spin-coated onto a silicon wafer. Multistep ultra-violet based photolithography involving commercial photoresists are then used to pattern robust thin film metallic electrodes (chromium/gold) and insulating layers (parylene) on the top of the PDMS substrate. The thin film metals are deposited using thermal evaporation and shaped using lift-off techniques The BioMEMS has been characterized mechanically using an in-house strain-applicator tool. The system is composed of 12 electrodes with one reference electrode transversally-orientated to the uniaxial longitudinal straining of the system. The electrical resistance of the electrodes is observed to remain very stable with applied strain—with a resistivity approaching that of evaporated gold—up to an interline strain of ~50%. The mechanical characterization revealed some interesting original properties of such stretchable BioMEMS. For example, a Poisson effect induced electrical ‘self-healing’ of cracking was identified. Biocompatibility of the commercial photoresist has been studied and is conclusive. We will present the results of the BioMEMS, which has also characterized living cells with a commercial Multi Electrode Array (MEA) characterization tool (Multi Channel Systems, USA). The BioMEMS enables the cells to be strained up to 50% and then characterized electrically and optically.

Keywords: BioMEMS, elastomer, electrical impedance measurements of living cells, high mechanical strain, microfabrication, stretchable systems, thin films, traumatic brain injury

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Authors: Joanna Gerszon, Aleksandra Rodacka

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In the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, protein and peptide aggregation processes play a vital role in contributing to the formation of intracellular and extracellular protein deposits. One of the major components of these deposits is the oxidatively modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Therefore, the purpose of this research was to answer the question whether piceatannol, a stilbene derivative, counteracts and/or slows down oxidative stress-induced GAPDH aggregation. The study also aimed to determine if this natural occurring compound prevents unfavorable nuclear translocation of GAPDH in hippocampal cells. The isothermal titration calorimetry (ITC) analysis indicated that one molecule of GAPDH can bind up to 8 molecules of piceatannol (7.3 ± 0.9). As a consequence of piceatannol binding to the enzyme, the loss of activity was observed. Parallel with GAPDH inactivation the changes in zeta potential, and loss of free thiol groups were noted. Nevertheless, the ligand-protein binding does not influence the secondary structure of the GAPDH. Precise molecular docking analysis of the interactions inside the active center allowed to presume that these effects are due to piceatannol ability to assemble a covalent binding with nucleophilic cysteine residue (Cys149) which is directly involved in the catalytic reaction. Molecular docking also showed that simultaneously 11 molecules of ligand can be bound to dehydrogenase. Taking into consideration obtained data, the influence of piceatannol on level of GAPDH aggregation induced by excessive oxidative stress was examined. The applied methods (thioflavin-T binding-dependent fluorescence as well as microscopy methods - transmission electron microscopy, Congo Red staining) revealed that piceatannol significantly diminishes level of GAPDH aggregation. Finally, studies involving cellular model (Western blot analyses of nuclear and cytosolic fractions and confocal microscopy) indicated that piceatannol-GAPDH binding prevents GAPDH from nuclear translocation induced by excessive oxidative stress in hippocampal cells. In consequence, it counteracts cell apoptosis. These studies demonstrate that by binding with GAPDH, piceatannol blocks cysteine residue and counteracts its oxidative modifications, that induce oligomerization and GAPDH aggregation as well as it prevents hippocampal cells from apoptosis by retaining GAPDH in the cytoplasm. All these findings provide a new insight into the role of piceatannol interaction with GAPDH and present a potential therapeutic strategy for some neurological disorders related to GAPDH aggregation. This work was supported by the by National Science Centre, Poland (grant number 2017/25/N/NZ1/02849).

Keywords: glyceraldehyde-3-phosphate dehydrogenase, neurodegenerative disease, neuroprotection, piceatannol, protein aggregation

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Authors: Chang-Hui Shen, Paulina Konarzewska

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

Keywords: vacuole, phospholipid, homeostasis, Ino2p, FAB1

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Authors: Mustafa M. Donma, Orkide Donma

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Zinc is a vital element required for growth and development. This fact makes zinc important, particularly for children. It maintains normal cellular structure and functions. This essential element appears to have protective effects against coronary artery disease and cardiomyopathy. Higher serum zinc levels are associated with lower risk of cardiovascular diseases (CVDs). There is a significant association between low serum zinc levels and heart failure. Zinc may be a potential biomarker of cardiovascular health. High sensitive cardiac troponin T (hs-cTnT) and cardiac myosin binding protein C (cMyBP-C) are new generation markers used for prediagnosis, diagnosis, and prognosis of CVDs. The aim of this study is to determine zinc as well as new generation cardiac markers profiles in children with normal body mass index (N-BMI), obese (OB), morbid obese (MO) children, and children with metabolic syndrome (MetS) findings. The association among them will also be investigated. Four study groups were constituted. The study protocol was approved by the institutional Ethics Committee of Tekirdag Namik Kemal University. Parents of the participants filled informed consent forms to participate in the study. Group 1 is composed of 44 children with N-BMI. Group 2 and Group 3 comprised 43 OB and 45 MO children, respectively. Forty-five MO children with MetS findings were included in Group 4. World Health Organization age- and sex-adjusted BMI percentile tables were used to constitute groups. These values were 15-85, 95-99, and above 99 for N-BMI, OB, and MO, respectively. Criteria for MetS findings were determined. Routine biochemical analyses, including zinc, were performed. High sensitive-cTnT and cMyBP-C concentrations were measured by kits based on enzyme-linked immunosorbent assay principle. Appropriate statistical tests within the scope of SPSS were used for the evaluation of the study data. p<0.05 was accepted as statistically significant. Four groups were matched for age and gender. Decreased zinc concentrations were measured in Groups 2, 3, and 4 compared to Group 1. Groups did not differ from one another in terms of hs-cTnT. There were statistically significant differences between cMyBP-C levels of MetS group and N-BMI as well as OB groups. There was an increasing trend going from N-BMI group to MetS group. There were statistically significant negative correlations between zinc and hs-cTnT as well as cMyBP-C concentrations in MetS group. In conclusion, inverse correlations detected between zinc and new generation cardiac markers (hs-TnT and cMyBP-C) have pointed out that decreased levels of this physiologically essential trace element accompany increased levels of hs-cTnT as well as cMyBP-C in children with MetS. This finding emphasizes that both zinc and these new generation cardiac markers may be evaluated as biomarkers of cardiovascular health during severe childhood obesity precipitated with MetS findings and also suggested as the messengers of the future risk in the adulthood periods of children with MetS.

Keywords: cardiac myosin binding protein-C, cardiovascular diseases, children, high sensitive cardiac troponin T, obesity

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Authors: Milu T. Cherian, Sergio C. Chai, Morgan A. Casal, Taosheng Chen

Abstract:

This study aims to use CINPA1, a recently discovered small-molecule inhibitor of the xenobiotic receptor CAR (constitutive androstane receptor) for understanding the binding modes of CAR and to guide CAR-mediated gene expression profiling studies in human primary hepatocytes. CAR and PXR are xenobiotic sensors that respond to drugs and endobiotics by modulating the expression of metabolic genes that enhance detoxification and elimination. Elevated levels of drug metabolizing enzymes and efflux transporters resulting from CAR activation promote the elimination of chemotherapeutic agents leading to reduced therapeutic effectiveness. Multidrug resistance in tumors after chemotherapy could be associated with errant CAR activity, as shown in the case of neuroblastoma. CAR inhibitors used in combination with existing chemotherapeutics could be utilized to attenuate multidrug resistance and resensitize chemo-resistant cancer cells. CAR and PXR have many overlapping modulating ligands as well as many overlapping target genes which confounded attempts to understand and regulate receptor-specific activity. Through a directed screening approach we previously identified a new CAR inhibitor, CINPA1, which is novel in its ability to inhibit CAR function without activating PXR. The cellular mechanisms by which CINPA1 inhibits CAR function were also extensively examined along with its pharmacokinetic properties. CINPA1 binding was shown to change CAR-coregulator interactions as well as modify CAR recruitment at DNA response elements of regulated genes. CINPA1 was shown to be broken down in the liver to form two, mostly inactive, metabolites. The structure-activity differences of CINPA1 and its metabolites were used to guide computational modeling using the CAR-LBD structure. To rationalize how ligand binding may lead to different CAR pharmacology, an analysis of the docked poses of human CAR bound to CITCO (a CAR activator) vs. CINPA1 or the metabolites was conducted. From our modeling, strong hydrogen bonding of CINPA1 with N165 and H203 in the CAR-LBD was predicted. These residues were validated to be important for CINPA1 binding using single amino-acid CAR mutants in a CAR-mediated functional reporter assay. Also predicted were residues making key hydrophobic interactions with CINPA1 but not the inactive metabolites. Some of these hydrophobic amino acids were also identified and additionally, the differential coregulator interactions of these mutants were determined in mammalian two-hybrid systems. CINPA1 represents an excellent starting point for future optimization into highly relevant probe molecules to study the function of the CAR receptor in normal- and pathophysiology, and possible development of therapeutics (for e.g. use for resensitizing chemoresistant neuroblastoma cells).

Keywords: antagonist, chemoresistance, constitutive androstane receptor (CAR), multi-drug resistance, structure activity relationship (SAR), xenobiotic resistance

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Authors: Karthyayani Rajamani, Yi-Chun Lin, Tung-Chou Wen, Jeanne Hsieh, Yi-Maun Subeq, Jen-Wei Liu, Po-Cheng Lin, Horng-Jyh Harn, Shinn-Zong Lin, Tzyy-Wen Chiou

Abstract:

Assuring cell quality is an essential parameter for the success of stem cell therapy, utilization of various components to improve this potential has been the primary goal of stem cell research. The aim of this study was not only to demonstrate the capacity of trans-cinnamaldehyde (TC) to reverse stress-induced senescence but also improve the therapeutic abilities of stem cells. Because of the availability and the promising application potential in regenerative medicine, adipose-derived stem cells (ADSCs) were chosen for the study. We found that H2O2 treatment resulted in the expression of senescence characteristics in the ADSCs, including decreased proliferation rate, increased senescence-associated- β-galactosidase (SA-β-gal) activity, decreased SIRT1 (silent mating type information regulation 2 homologs) expression and decreased telomerase activity. However, TC treatment was sufficient to rescue or reduce the effects of H2O2 induction, ultimately leading to an increased proliferation rate, a decrease in the percentage of SA-β-gal positive cells, upregulation of SIRT1 expression, and increased telomerase activity of the senescent ADSCs at the cellular level. Further recently it was observed that the ADSCs were treated with TC without induction of senescence, all the before said positives were observed. Moreover, a chemically induced liver fibrosis animal model was used to evaluate the functionality of these rescued cells in vivo. Liver dysfunction was established by injecting 200 mg/kg thioacetamide (TAA) intraperitoneally into Wistar rats every third day for 60 days. The experimental rats were separated into groups; normal group (rats without TAA induction), sham group (without ADSC transplantation), positive control group (transplanted with normal ADSCs); H2O2 group (transplanted with H2O2 -induced senescent ADSCs), H2O2+TC group (transplanted with ADSCs pretreated with H2O2 and then further treated with TC) and TC group (ADSC treated with TC without H2O2 treatment). In the transplantation group, 1 × 106 human ADSCs were introduced into each rat via direct liver injection. Based on the biochemical analysis and immunohistochemical staining results, it was determined that the therapeutic effects on liver fibrosis by the induced senescent ADSCs (H2O2 group) were not as significant as those exerted by the normal ADSCs (the positive control group). However, the H2O2+TC group showed significant reversal of liver damage when compared to the H2O2 group 1 week post-transplantation. Further ADSCs without H2O2 treatment but with just TC treatment performed much better than all the groups. These data confirmed that the TC treatment had the potential to improve the therapeutic effect of ADSCs. It is therefore suggested that TC has potential applications in maintaining stem cell quality and could possibly aid in the treatment of senescence-related disorders.

Keywords: senescence, SIRT1, adipose derived stem cells, liver fibrosis

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Authors: Mahdi Farajzadeh Ajirlou

Abstract:

Prior evidence of normal cardiac anatomy is desirable to relieve the anxiety of cases with a family history of congenital heart disease or to offer the option of early gestation termination or close follow-up should a cardiac anomaly be proved. Fetal heart discovery plays an important part in the opinion of the fetus, and it can reflect the fetal heart function of the fetus, which is regulated by the central nervous system. Acquisition of ventricular volume and inflow data would be useful to quantify more valve regurgitation and ventricular function to determine the degree of cardiovascular concession in fetal conditions at threat for hydrops fetalis. This study discusses imaging the fetal heart with transvaginal ultrasound, Doppler ultrasound, three-dimensional ultrasound (3DUS) and four-dimensional (4D) ultrasound, spatiotemporal image correlation (STIC), glamorous resonance imaging and cardiac catheterization. Doppler ultrasound (DUS) image is a kind of real- time image with a better imaging effect on blood vessels and soft tissues. DUS imaging can observe the shape of the fetus, but it cannot show whether the fetus is hypoxic or distressed. Spatiotemporal image correlation (STIC) enables the acquisition of a volume of data concomitant with the beating heart. The automated volume accession is made possible by the array in the transducer performing a slow single reach, recording a single 3D data set conforming to numerous 2D frames one behind the other. The volume accession can be done in a stationary 3D, either online 4D (direct volume scan, live 3D ultrasound or a so-called 4D (3D/ 4D)), or either spatiotemporal image correlation-STIC (off-line 4D, which is a circular volume check-up). Fetal cardiovascular MRI would appear to be an ideal approach to the noninvasive disquisition of the impact of abnormal cardiovascular hemodynamics on antenatal brain growth and development. Still, there are practical limitations to the use of conventional MRI for fetal cardiovascular assessment, including the small size and high heart rate of the mortal fetus, the lack of conventional cardiac gating styles to attend data accession, and the implicit corruption of MRI data due to motherly respiration and unpredictable fetal movements. Fetal cardiac MRI has the implicit to complement ultrasound in detecting cardiovascular deformations and extracardiac lesions. Fetal cardiac intervention (FCI), minimally invasive catheter interventions, is a new and evolving fashion that allows for in-utero treatment of a subset of severe forms of congenital heart deficiency. In special cases, it may be possible to modify the natural history of congenital heart disorders. It's entirely possible that future generations will ‘repair’ congenital heart deficiency in utero using nanotechnologies or remote computer-guided micro-robots that work in the cellular layer.

Keywords: fetal, cardiac MRI, ultrasound, 3D, 4D, heart disease, invasive, noninvasive, catheter

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Authors: Kirti Hira, A. Sajeli Begum, S. Mahibalan, Poorna Chandra Rao

Abstract:

Introduction: Cancer is one of the leading causes of death worldwide. Although existing therapy effectively kills cancer cells, they do affect normal growing cells leading to many undesirable side effects. Hence there is need to develop effective as well as safe drug molecules to combat cancer, which is possible through phyto-research. The currently available plant-derived blockbuster drugs are the example for this. In view of this, an investigation was done to identify cytotoxic lead molecules from Hedyotis umbellata (Family Rubiaceae), a widely distributed weed in India. Materials and Methods: The methanolic extract of the whole plant of H. umbellata (MHU), prepared through Soxhlet extraction method was further fractionated with diethyl ether and n-butanol, successively. MHU, ether fraction (EMHU) and butanol fraction (BMHU) were lyophilized and were tested for the cytotoxic effect using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay against non-small cell lung cancer (NSCLC) A549 cell lines. The potentially active EMHU was subjected to chromatographic purification using normal-phase silica columns, in order to isolate the responsible bioactive compounds. The isolated pure compounds were tested for their cytotoxic effect by MTT assay against A549 cells. Compound-3, which was found to be most active, was characterized using IR, 1H- and 13C-NMR and MS analysis. The study was further extended to decipher the mechanism of action of cytotoxicity of compound-3 against A549 cells through various in vitro cellular models. Cell cycle analysis was done using flow cytometry following PI (Propidium Iodide) staining. Protein analysis was done using Western blot technique. Results: Among MHU, EMHU, and BMHU, the non-polar fraction EMHU demonstrated a significant dose-dependent cytotoxic effect with IC50 of 67.7μg/ml. Chromatography of EMHU yielded seven compounds. MTT assay of isolated compounds explored compound-3 as potentially active one, which inhibited the growth of A549 cells with IC50value of 14.2μM. Further, compound-3 was identified as cedrelopsin, a coumarin derivative having molecular weight of 260. Results of in vitro mechanistic studies explained that cedrelopsin induced cell cycle arrest at G2/M phase and down-regulated the expression of G2/M regulatory proteins such as cyclin B1, cdc2, and cdc25C, dose dependently. This is the first report that explores the cytotoxic mechanism of cedrelopsin. Conclusion: Thus a potential small lead molecule, cedrelopsin isolated from H. umbellata, showing antiproliferative effect mediated by G2/M arrest in A549 cells was discovered. The effect of cedrelopsin against other cancer cell lines followed by in vivo studies can be performed in future to develop a new drug candidate.

Keywords: A549, cedrelopsin, G2/M phase, Hedyotis umbellata

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Authors: J. A. N. Sandamali, R. P. Hewawasam, K. A. P. W. Jayatilaka, L. K. B. Mudduwa

Abstract:

Introduction: Doxorubicin is widely used in the treatment of solid organ tumors and hematological malignancies, but the dose-dependent cardiotoxicity due to free radical formation compromises its clinical utility. Therapeutic strategies which enhance cellular endogenous defense systems have been identified as promising approaches to combat oxidative stress-associated conditions. Cinnamomum zeylanicum (Ceylon cinnamon) has a number antioxidant compounds, which can effectively scavenge reactive oxygen including superoxide anions, hydroxyl radicals and as well as other free radicals. Therefore, the objective of the study was to elucidate the most effective dose of Cinnamomum bark extract which ameliorates doxorubicin-induced cardiotoxicity. Materials and methods: Wistar rats were divided into seven groups of 10 animals in each. Group 1: normal control (distilled water, orally, for 14 days, 10 mL/kg saline, ip, after 16 hours fast on the 11th day); Group 2: doxorubicin control (distilled water, orally, for 14 days, 18 mg/kg doxorubicin, ip, after 16 hour fast on the 11th day); Groups 3-7: five doses of freeze dried aqueous bark extracts (0.125, 0.25, 0.5, 1.0, 2.0g/kg, orally, daily for 14 days, 18 mg/kg doxorubicin, ip, after 16 hours fast on the 11th day). Animals were sacrificed on the 15th day and blood was collected for the estimation of cardiac troponin I (cTnI), AST and LDH concentrations and myocardial tissues were collected for histopathological assessment of myocardial damage and irreversible changes were graded by developing a score. Results: cTnI concentration of groups 1-7 were 0, 161.9, 128.6, 95.9, 38, 19.41 & 12.36 pg/mL showing significant differences (p<0.05) between group 2 and groups 4-7. In groups 1-7, serum AST concentration were 26.82, 68.1, 37.18, 36.23, 26.8, 26.62 & 22.43U/L and LDH concentrations were 1166.13, 2428.84, 1658.35, 1474.34, 1277.58, 1110.21 & 974.40U/L and a significant difference (p<0.05) was observed between group 2 and groups 3-7. The maximum score for myocardial necrosis was observed in group 2. Parallel to the increase of the dosage of plant extract, a gradual reduction of the score for myocardial necrosis was observed in groups 3-7. Reversible histological changes such as vacuolation, congestion were observed in group 2 and all plant treated groups. Haemorrhages, inflammatory cell infiltrations, and interstitial oedema were observed in group 2, but absent in groups treated with higher doses of the plant extract. Discussion & Conclusion: According to the in vitro antioxidant assays performed, Cinnamomum zeylanicum (Ceylon cinnamon) bark possesses high amounts of polyphenolic substances and high antioxidant activity. The present study showed that Cinnamomum zeylanicum extract at 2.0 g/kg possesses the most significant cardioprotective effect against doxorubicin-induced cardiotoxicity. It can be postulated that pretreatment with Cinnamomum bark extract may replenish the cardiomyocytes with antioxidants that are needed for the defense against oxidative stress induced by doxorubicin.

Keywords: cardioprotection, Cinnamomum zeylanicum, doxorubicin, free radicals

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