Search results for: extracellular enzymes

Authors: Simon Grossemy, Peggy P. Y. Chan, Pauline M. Doran

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Nerve tissue engineering is the main field of research aimed at finding an alternative to autografts as a treatment for nerve injuries. Scaffolds are used as a support to enhance nerve regeneration. In order to successfully design novel scaffolds and in vitro cell culture systems, a deep understanding of the factors affecting nerve regeneration processes is needed. Physical and biological parameters associated with the culture environment have been identified as potentially influential in nerve cell differentiation, including electrical stimulation, exposure to extracellular-matrix (ECM) proteins, dynamic medium conditions and co-culture with glial cells. The mechanisms involved in driving the cell to differentiation in the presence of these factors are poorly understood; the complexity of each of them raises the possibility that they may strongly influence each other. Some questions that arise in investigating nerve regeneration include: What are the best protein coatings to promote neural cell attachment? Is the scaffold design suitable for providing all the required factors combined? What is the influence of dynamic stimulation on cell viability and differentiation? In order to study these effects, scaffolds adaptable to bioreactor culture conditions were designed to allow electrical stimulation of cells exposed to ECM proteins, all within a dynamic medium environment. Gold coatings were used to make the surface of viscose rayon microfiber scaffolds (VRMS) conductive, and poly-L-lysine (PLL) and laminin (LN) surface coatings were used to mimic the ECM environment and allow the attachment of rat PC12 neural cells. The robustness of the coatings was analyzed by surface resistivity measurements, scanning electron microscope (SEM) observation and immunocytochemistry. Cell attachment to protein coatings of PLL, LN and PLL+LN was studied using DNA quantification with Hoechst. The double coating of PLL+LN was selected based on high levels of PC12 cell attachment and the reported advantages of laminin for neural differentiation. The underlying gold coatings were shown to be biocompatible using cell proliferation and live/dead staining assays. Coatings exhibiting stable properties over time under dynamic fluid conditions were developed; indeed, cell attachment and the conductive power of the scaffolds were maintained over 2 weeks of bioreactor operation. These scaffolds are promising research tools for understanding complex neural cell behavior. They have been used to investigate major factors in the physical culture environment that affect nerve cell viability and differentiation, including electrical stimulation, bioreactor hydrodynamic conditions, and combinations of these parameters. The cell and tissue differentiation response was evaluated using DNA quantification, immunocytochemistry, RT-qPCR and functional analyses.

Keywords: bioreactor, electrical stimulation, nerve differentiation, PC12 cells, scaffold

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Authors: Evelyn Osehontue Uroro, Richard Bright, Jing Yang Quek, Krasimir Vasilev

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Bacterial infections have been a challenge both in the public and private sectors. The colonization of bacteria often occurs in medical devices such as catheters, heart valves, respirators, and orthopaedic implants. When biomedical devices are inserted into patients, the deposition of macromolecules such as fibrinogen and immunoglobin on their surfaces makes it easier for them to be prone to bacteria colonization leading to the formation of biofilms. The formation of biofilms on medical devices has led to a series of device-related infections which are usually difficult to eradicate and sometimes cause the death of patients. These infections require surgical replacements along with prolonged antibiotic therapy, which would incur additional health costs. It is, therefore, necessary to prevent device-related infections by inhibiting the formation of biofilms using intelligent technology. Antibiotic resistance of bacteria is also a major threat due to overuse. Different antimicrobial agents have been applied to microbial infections. They include conventional antibiotics like rifampicin. The use of conventional antibiotics like rifampicin has raised concerns as some have been found to have hepatic and nephrotoxic effects due to overuse. Hence, there is also a need for proper delivery of these antibiotics. Different techniques have been developed to encapsulate and slowly release antimicrobial agents, thus reducing host cytotoxicity. Examples of delivery systems are solid lipid nanoparticles, hydrogels, micelles, and polymeric nanoparticles. The different ways by which drugs are released from polymeric nanoparticles include diffusion-based release, elution-based release, and chemical/stimuli-responsive release. Polymeric nanoparticles have gained a lot of research interest as they are basically made from biodegradable polymers. An example of such a biodegradable polymer is polycaprolactone (PCL). PCL degrades slowly by hydrolysis but is often sensitive and responsive to stimuli like enzymes to release encapsulants for antimicrobial therapy. This study presents the synthesis of PCL nanoparticles loaded with rifampicin and the on-demand release of rifampicin for treating staphylococcus aureus infections.

Keywords: enzyme, Staphylococcus aureus, PCL, rifampicin

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Authors: Radheshyam Yadav, Ramakrishna Wusirika

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Plant Growth Promoting Bacteria (PGPB) and Arbuscular Mycorrhizal (AM) Fungi are ubiquitous in soil and often very critical for crop yield and agriculture sustainability, and this has motivated the agricultural practices to support and promote PGPB and AM Fungi in agriculture. PGPB can be involved in a range of processes that affect Nitrogen (N) and Phosphorus (P) transformations in soil and thus influence nutrient availability and uptake to the plants. A field study with two wheat cultivars, HD-3086, and HD-2967 was performed in Malwa region, Bathinda of Punjab, India, to evaluate the effect of native and non-native PGPB alone and in combination with AM fungi as an inoculant on wheat grain yield, nutrient uptake and soil health parameters (dehydrogenase, urease, β‐glucosidase). Our results showed that despite an early insignificant increase in shoot length, plants treated with PGPB (Bacillus sp.) and AM Fungi led to a significant increase in shoot growth at maturity, aboveground biomass, nitrogen (45% - 40%) and phosphorus (40% - 34%) content in wheat grains relative to untreated control plants. Similarly, enhanced grain yield and nutrients uptake i.e. copper (27.15% - 36.25%) iron (43% - 53%) and zinc (44% - 47%) was recorded in PGPB and AM Fungi treated plants relative to untreated control. Overall, inoculation with native PGPB alone and in combination with AM Fungi provided benefits to enhance grain yield, wheat biofortification, and improved soil fertility, despite this effect varied depending on different PGPB isolates and wheat cultivars. These field study results provide evidence of the benefits of agricultural practices involving native PGPB and AM Fungi to the plants. These native strains and AM Fungi increased accumulations of copper, iron, and zinc in wheat grains, enhanced grain yield, and soil fertility.

Keywords: AM Fungi, biofortification, PGPB, soil microbial enzymes

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Authors: Onuoha Ogbonnaya Gideon, Ishaq Hafsah Yusuf

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Introduction: Meat is a perishable food of good nutrition. Meat ranks among the most significant, nutritious, and favored food items available to most locals. It is a good source of protein (17-19%), depending on sources, and contains appreciable amounts of fat and moisture. However, it has a very short shelf life due mainly to its high moisture, fat, and other nutrient contents. Meat spoilage can result from microbial proliferation as well as inherent enzymes in the meat tissues. Bacteria contamination and permeability to both oxygen and water vapor are major concerns associated with spoilage of meat and its storage. Packaging is fundamental in the preservation and presentation of food. Red clay is a very common substance; hydrous aluminum phyllosilicate, sometimes with varying amounts of iron, magnesium, alkali metals, alkaline earth, and cation formed from sedimentary rocks. Furthermore, red clay is an extremely absorbent material and develops plasticity when wet due to the molecular film of water surrounding the clay particles but can become hard, impervious, brittle, and non-brittle and non-plastic when dry. In developing countries, the high cost of refrigeration technologies and most other methods of preserving meat are exorbitant and thus can be substituted with the less expensive and readily available red clay for the preservation of meat. Methodology: 1000g of lean meat was diced into cubes of 10g each. The sample was then divided into four groups labelled raw meat (RMC); raw in 10% brine solution (RMB), boiled meat (BMC), and fried meat (FMC). It was then encapsulated with 2mm thick red clay and then heated in a muffle furnace at a temperature of 600OC for 30min. The samples were kept on a bench top for 30 days, and a storage study was carried out. Results: Our findings showed a decrease in value during storage for the physiochemical properties of all the sample; pH values decreased [RMC (7.05-7.6), RMB (8.46-7.0), BMC (6.0-5.0), FMC (4.08-3.9)]; free fatty acid content decreased with storage time [RMC (32.6%-31%), RMB (30.2%-28.6%), BMC (30.5%-27.4%), FMC (25.6%-23.8%)]; total soluble solid value decreased [RMC16.20-15.07, RMB (17.22-16.04), BMC (17.05-15.54), FMC (15.3-14.9)]. Conclusion: This result shows that encapsulation with red clay reduced all the values analyzed and thus has the potential to extend the shelf life of stored meat.

Keywords: red clay, encapsulating, shelf life, physicochemical properties, lean meat

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

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

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

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Authors: Maciej Sobczak, Julita Pietrzak, Tomasz Płoszaj, Agnieszka Robaszkiewicz

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Brg1, a member of SWI/SNF complex, plays a role in chromatin remodeling, therefore, regulates expression of many genes. Brg1 is an ATPase of SWI/SNF complex, thus its activity requires ATP. Through its bromodomain recognizes acetylated histone residues and evicts them, thus promoting transcriptionally active state of chromatin. One of the enzymes that is responsible for acetylation of histone residues is Ep300. It was previously shown in the literature that cooperation of Brg1 and Ep300 occurs at the promoter regions that have binding sites for E2F-family transcription factors as well as CpG islands. According to literature, approximately 20% of human cancer possess mutation in Brg1 or any other crucial SWI/SNF subunit. That phenomenon makes Brg1-Ep300 a very promising target for anti-cancer therapy. Therefore in our study, we investigated if physical interaction between Brg1 and Ep300 exists and what impact those two proteins have on key for breast cancer cells processes such as DNA damage repair and cell proliferation. Bioinformatical analysis pointed out, that genes involved in cell proliferation and DNA damage repair are overexpressed in MCF7 and MDA-MB-231 cells. Moreover, promoter regions of these genes are highly acetylated, which suggests high transcriptional activity of those sites. Notably, many of those gene possess within their promoters an E2F, Brg1 motives, as well as CpG islands and acetylated histones. Our data show that Brg1 physically interacts with Ep300, and together they regulate expression of genes involved in DNA damage repair and cell proliferation. Upon inhibiting Brg1 or Ep300, expression of vital for cancer cell survival genes such as CDK2/4, BRCA1/2, PCNA, and XRCC1 is decreased in MDA-MB-231 and MCF7 cells. Moreover, inhibition or silencing of either Brg1 or Ep300 leads to cell cycle arrest in G1. After inhibition of BRG1 or Ep300 on tested gene promoters, the repressor complex including Rb, HDAC1, and EZH2 is formed, which inhibits gene expression. These results highlight potentially significant target for targeted anticancer therapy to be introduced as a supportive therapy.

Keywords: brg1, ep300, breast cancer, epigenetics

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

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

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

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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: Boris Bajer, Andrea Havranova, Miroslav Vlcek, Richard Imrich, Adela Penesova

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Lifestyle interventions can prevent the deterioration of impaired glucose tolerance to manifest type 2 diabetes, and also prevent cardiovascular diseases, as it showed many studies (the Finnish Diabetes Prevention Study, Diabetes Prevention Program (DPP), . the China Da Qing Diabetes Prevention Study, etc.) Therefore the aim of our study was to compare the effect of intensified lifestyle intervention on cardiometabolic parameters. Methods: It is an ongoing randomized interventional clinical study (NCT02325804) focused on the reduction of body weight/fat. Intervention: hypocaloric diet (30% restriction of calories) and physical activity 150 minutes/week. Before and after 8 weeks of intervention all patients underwent complete medical examination (measurement of physical fitness, resting metabolic rate (RMR), body composition analysis, oral glucose tolerance test, parameters of lipid metabolism, and other cardiometabolic risk factors. Results: So far 39 patients finished the intervention. The average reduction of body weight was 6,8 + 4,9 kg (0-15 kg; p=0,0006), accompanied with significant reduction of body fat percentage (p ≤ 0,0001), amount of fat mass (p=0,03), waist circumference (p=0.02). Amount of lean mass and RMR remained unchanged. Heart rate (p=0,02), systolic and diastolic blood pressure was reduced (p=0,01 p=0,02 resp.) as well as insulin sensitivity was improved. Lipid parameters also changed - cholesterol, LDL decreased (p=0,05, p=0,04 resp.), while triglycerides showed tendency to decrease (p=0,055). Liver function improved, alanine aminotrasnferase (ALT) were reduced (p=0,01). Physical fitness significantly improved (as measure VO2 max (p=0,02). Conclusion: Results of our study are in line with previous results about the beneficial effect of intensive lifestyle changes on the reduction of cardiometabolic risk factors and improvement of liver function. Supported by grants APVV 15-0228; VEGA 2/0161/16

Keywords: obesity, weight loss, diet lipids, blood pressure, liver enzymes

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Authors: L. Musak, J. Valachova, T. Vasicko, O. Osina

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Stainless steel is resistant to electrochemical corrosion by passivation. Welders are greatly exposed to welding fumes of toxic metals, which added to this steel. The content of chromium (Cr) is above 11.5%, Ni and Mo from 2 to 6.5%. The aim of the study was the evaluation of occupational exposure to Cr, chromosome analysis and valuation of individual susceptibility polymorphism of gene CCND1 c.870 G>A. The exposed group was consisted from 117 welders of stainless steels. The average age was 38.43 years and average exposure time 7.14 years. Smokers represented 40.17%. The control group consisted of 123 non-exposed workers with an average age of 39.74 years and time employment 16.67 years. Smokers accounted for 22.76%. Analysis of Cr in blood and urine was performed by atomic absorption spectrophotometry (AAS Varian SpectraAA 30P) with electrothermal decomposition of the sample in the graphite furnace. For the evaluation of chromosomal aberrations (CA) cytogenetic analysis of peripheral blood lymphocytes was used. Gene polymorphism was determined by PCR-RFLP reaction using appropriate primers and restriction enzymes. For statistic analysis the Mann-Whitney U-test was used. The mean Cr level in blood of exposed group was 0.095 µmol/l (0.019 min - max 0.504). No value exceeds the average normal value. The mean value Cr in urine was 7.9 µmol/mol creatinine (min 0.026 to max 19.26). The total number of CA was 1.86% in compared to 1.70% controls. (CTA-type 0.90% vs. 0.80% and CSA-type 0.96% vs. 0.90%). In the number of total CA statistical difference was observed between smokers and non-smokers of exposed group (S-1.57% vs. NS-2.04%, P<0.05). In CCND1 gene polymorphisms was observed the increasing of the total CA with wild-type allele (WT) via heterozygous to the VAR genotype (1.44% <1.82% <2.13%). A statistically higher incidence of CTA-type aberrations in variant genotypes between exposed and control groups was observed (1.22% vs. 0.59%, P <0.05). The work place is usually higher source of exposure to harmful factors. Workers need consistent and frequent health control. In assessing the risk of adverse effects of metals it is important to consider their persistence, behavior and bioavailability. Prolonged exposure to carcinogens may not manifest symptoms of poisoning, but delayed effects may occur, which resulted in a higher incidence of malignant tumors.

Keywords: CCND1, genotoxicity, polymorphism, stainless steel, welders

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Authors: Ali Mirtaghavi, Andy Baldwin, Rajendarn Muthuraj, Jack Luo

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Recent advances in biomaterials have led to utilizing biopolymers to develop 3D scaffolds in tissue regeneration. One of the major challenges of designing biomaterials for 3D scaffolds is to mimic the building blocks similar to the extracellular matrix (ECM) of the native tissues. Biopolymer based aerogels obtained by freeze-drying have shown to provide structural similarities to the ECM owing to their 3D format and a highly porous structure with interconnected pores, similar to the ECM. Gelatin (GEL) is known to be a promising biomaterial with inherent regenerative characteristics owing to its chemical similarities to the ECM in native tissue, biocompatibility abundance, cost-effectiveness and accessible functional groups, which makes it facile for chemical modifications with other biomaterials to form biocomposites. Despite such advantages, gelatin offers poor mechanical properties, sensitive enzymatic degradation and high viscosity at room temperature which limits its application and encourages its use to develop biocomposites. Hydrophilic biomass-based cellulose nanofibrous (CNF) has been explored to use as suspension for biocomposite aerogels for the development of 3D porous structures with excellent mechanical properties, biocompatibility and slow enzymatic degradation. In this work, CNF biocomposite aerogels with various ratios of CNF:GEL) (90:10, 70:30 and 50:50) were prepared by freeze-drying technique, and their properties were investigated in terms of physicochemical, mechanical and biological characteristics. Epichlorohydrin (EPH) was used to investigate the effect of chemical crosslinking on the molecular interaction of CNF: GEL, and its effects on physicochemical, mechanical and biological properties of the biocomposite aerogels. Ultimately, chemical crosslinking helped to improve the mechanical resilience of the resulting aerogels. Amongst all the CNF-GEL composites, the crosslinked CNF: GEL (70:30) biocomposite was found to be favourable for cell attachment and viability. It possessed highly porous structure (porosity of ~93%) with pore sizes ranging from 16-110 µm, adequate mechanical properties (compression modulus of ~47 kPa) and optimal biocompatibility both in-vitro and in-vivo, as well as controlled enzymatic biodegradation, high water penetration, which could be considered a suitable option for wound healing application. In-vivo experiments showed improvement on inflammation and foreign giant body cell reaction for the crosslinked CNF: GEL (70:30) compared to the other samples. This could be due to the superior interaction of CNF with gelatin through chemical crosslinking, resulting in more optimal in-vivo improvement. In-vitro cell culture investigation on human dermal fibroblasts showed satisfactory 3D cell attachment over time. Overall, it has been observed that the developed CNF: GEL aerogel can be considered as a potential scaffold for soft tissue regeneration application.

Keywords: 3D scaffolds, aerogels, Biocomposites , tissue engineering

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Authors: Sneha Singh, Abhimanyu Dev, Vinod Nigam

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The major issue which decides the impending use of gold nanoparticles (AuNPs) in nanobiotechnological applications is their particle size and stability. Often the AuNPs obtained biomimetically are considered useless owing to their instability in the aqueous medium and thereby limiting the widespread acceptance of this facile green synthesis procedure. So, the use of nontoxic surfactants is warranted to stabilize the biogenic nanoparticles (NPs). But does the surfactant only play a role in stabilizing by being adsorbed to the NPs surface or can it have any other significant contribution in synthesis process and controlling their size as well as shape? Keeping this idea in mind, AuNPs were synthesized by using surfactant treated (lechate) and untreated (cell lysate supernatant) Bacillus licheniformis cell extract. The cell extracts mediated reduction of chloroauric acid (HAuCl 4) in the presence of non-ionic surfactant, Tween 20 (TW20), and its effect on the AuNPs stability was studied. Interestingly, the surfactant used in the study served as potential alternative to harvest cellular enzymes involved in bioreduction process in a hassle free condition. The surfactants ability to solubilize/leach membrane proteins and simultaneously stabilizing the AuNPs could have advantage from process point of view as it will reduce the time and economics involve in the nanofabrication of biogenic NPs. The synthesis was substantiated with UV-Vis spectroscopy, Dynamic light scattering study, FTIR spectroscopy, and Transmission electron microscopy. The Zeta potential of AuNPs solutions was measured routinely to corroborate the stability observations recorded visually. Highly stable, ultra-small AuNPs of 2.6 nm size were obtained from the study. Further, the biological efficacy of the obtained AuNPs as potential antibacterial agent was evaluated against Bacilllus subtilis, Pseudomonas aeruginosa, and Escherichia coli by observing the zone of inhibition. This potential of AuNPs of size < 3 nm as antibacterial agent could pave way for development of new antimicrobials and overcoming the problems of antibiotics resistance

Keywords: antibacterial, bioreduction, nanoparticles, surfactant

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Authors: Sheraz Gul

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The requirements for progressing a compound to clinical trials is well established and relies on the results from in-vitro and in-vivo animal tests to indicate that it is likely to be safe and efficacious when testing in humans. The typical data package required will include demonstrating compound safety, toxicity, bioavailability, pharmacodynamics (potential effects of the compound on body systems) and pharmacokinetics (how the compound is potentially absorbed, distributed, metabolised and eliminated after dosing in humans). If the desired criteria are met and the compound meets the clinical Candidate criteria and is deemed worthy of further development, a submission to regulatory bodies such as the US Food & Drug Administration for an exploratory Investigational New Drug Study can be made. The purpose of this study is to collect data to establish that the compound will not expose humans to unreasonable risks when used in limited, early-stage clinical studies in patients or normal volunteer subjects (Phase I). These studies are also designed to determine the metabolism and pharmacologic actions of the drug in humans, the side effects associated with increasing doses, and, if possible, to gain early evidence on their effectiveness. In order to reach the above goals, we have developed a pre-clinical high throughput Absorption, Distribution, Metabolism and Excretion–Toxicity (ADME–Toxicity) panel of assays to identify compounds that are likely to meet the Lead and Candidate compound acceptance criteria. This panel includes solubility studies in a range of biological fluids, cell viability studies in cancer and primary cell-lines, mitochondrial toxicity, off-target effects (across the kinase, protease, histone deacetylase, phosphodiesterase and GPCR protein families), CYP450 inhibition (5 different CYP450 enzymes), CYP450 induction, cardio-toxicity (hERG) and gene-toxicity. This panel of assays has been applied to multiple compound series developed in a number of projects delivering Lead and clinical Candidates and examples from these will be presented.

Keywords: absorption, distribution, metabolism and excretion–toxicity , drug discovery, food and drug administration , pharmacodynamics

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Authors: Yasir Bashawri, Vincent N. Chigor James McDonald, Merfyn Williams, Davey Jones, A. Prysor Williams

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Resistance to antibiotics has become a threat to public health. As a result of their misuse and overuse, bacteria have become resistant to many common antibiotics. Βeta lactam (β-lactam) antibiotics are one of the most significant classes of antimicrobials in providing therapeutic benefits for the treatment of bacterial infections in both human and veterinary medicine, for approximately 60% of all antibiotics are used. In particular, some Enterobacteriaceae produce Extend Spectrum Beta Lactamases (ESBLs) that enable them to some break down multi-groups of antibiotics. CTX-M enzymes have rapidly become the most important ESBLs, with increases in mainly CTX-M 15 in many countries during the last decade. Global travel by intercontinental medical ‘tourists’, migrant employees and overseas students could theoretically be a risk factor for spreading antibiotic resistance genes in different parts of the world. Bangor city, North Wales, is subject to sudden demographic changes due to a large proportion (>25%) of the population being students, most of which arrive over a space of days. This makes it a suitable location to study the impacts of large demographic change on the presence of ESBLs. The aim of this study is to monitor the presence of ESBLs in Escherichia coli and faecal coliform bacteria isolated from Bangor wastewater treatment plant, before, during and after the arrival week of students to Bangor University. Over a five-week period, water samples were collected twice a week, from the influent, primary sedimentation tank, aeration tank and the final effluent. Isolation and counts for Escherichia coli and other faecal coliforms were done on selective agar (primary UTI agar). ESBL presence will be confirmed by phenotypic and genotypic methods. Sampling at all points of the tertiary treatment stages will indicate the effectiveness of wastewater treatment in reducing the spread of ESBLs genes. The study will yield valuable information to help tackle a problem which many regard to be the one of the biggest threats to modern-day society.

Keywords: extended spectrum β-lactamase, enterobacteriaceae, international travel, wastewater treatment plant

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Authors: Mohamed M. Ibrahim, Gaber A. M. Mersal, Salih Al-Juaid, Samir A. El-Shazly

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A series of mixed ligand complexes, viz., [Cu(BPy)(Gly)X]Y {X = Cl (1), Y = 0; X = 0, Y = ClO4- (2); X = H2O, Y = NO3- (3); X = H2O, Y = CH3COO- (4); and [Cu(BPy)(Gly)-(H2O)]2(SO4) (5) have been synthesized. Their structures and properties were characterized by elemental analysis, thermal analaysis, IR, UV–vis, and ESR spectroscopy, as well as electrochemical measurements including cyclic voltammetry, electrical molar conductivity, and magnetic moment measurements. Complexes 1 and 2 formed slightly distorted square-pyramidal coordination geometries of CuN3OCl and CuN3O2, respectively in which the N,O-donor glycine and N,N-donor bipyridyl bind at the basal plane with chloride ion or water as the axial ligand. Complex 3 shows square planar CuN3O coordination geometry, which exhibits chemically significant hydrogen bonding interactions besides showing coordination polymer formation. The superoxide dismutase and catalase-like activities of all complexes were tested and were found to be promising candidates as durable electron-transfer catalyst being close to the efficiency of the mimicking enzymes displaying either catalase or tyrosinase activity to serve for complete reactive oxygen species (ROS) detoxification, both with respect to superoxide radicals and related peroxides. The DNA binding interaction with super coiled pGEM-T plasmid DNA was investigated by using spectral (absorption and emission) titration and electrochemical techniques. The results revealed that DNA intercalate with complexes 1 and 2 through the groove binding mode. The calculated intrinsic binding constant (Kb) of 1 and 2 were 4.71 and 2.429 × 105 M−1, respectively. Gel electrophoresis study reveals the fact that both complexes cleave super coiled pGEM-T plasmid DNA to nicked and linear forms in the absence of any additives. On the other hand, the interaction of both complexes with DNA, the quasi-reversible CuII/CuI redox couple slightly improves its reversibility with considerable decrease in current intensity. All the experimental results indicate that the bipyridyl mixed copper(II) complex (1) intercalate more effectively into the DNA base pairs.

Keywords: enzyme mimics, mixed ligand complexes, X-ray structures, antioxidant, DNA-binding, DNA cleavage

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Authors: Amna Shoaib, Sidrah Hanif, Rashid Mehmood

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Current research work was carried out to check influence of farmyard manure (FYM) in Lycopersicon esculentum L. against Fusarium oxysporum f. sp. lycopersici (FO) in copper polluted soil. Silt-loam soil naturally enriched with 70 ppm of Cu was inoculated with 1 x 106 spore suspensions of FO and incorporated with 0%, 1%, 1.5% or 2% FYM. The multilateral interaction of host-pathogen-metal-organic amendment was assessed in terms of morphology, growth, yield, physiology, biochemistry and metal uptake in tomato plant after 30 and 60 days of sowing. When soil was inoculated with FO, plant growth and biomass were significantly increased during vegetative stage, while declining during flowering stage with substantial increase in productivity over control. Infected plants exhibited late wilting and disease severity was found on 26-50% of plant during reproductive stage. Incorporation of up to 1% FYM suppressed disease severity, improved plant growth and biomass, while it decreased yield. Rest of manure doses was found ineffective in suppressing disease. Content of total chlorophyll, sugar and protein were significantly declined in FO inoculated plants and incorporation of FYM caused significant reduction or no influence on sugar and chlorophyll content, and no pronounced difference among different FYM doses were observed. On the other hand, proline, peroxidase, catalase and nitrate reductase activity were found to be increased in infected plants and incorporation of 1-2% FYM further enhanced the activity of these enzymes. Tomato plant uptake of 30-40% of copper naturally present in the soil and incorporation of 1-2% FYM markedly decreased plant uptake of metal by 15-30%, while increased Cu retention in soil. Present study concludes that lower dose (1%) of FYM could be used to manage disease, increase growth and biomass, while being ineffective for yield and productivity in Cu-polluted soil. Altered physiology/biochemistry of plant in response to any treatment could be served as basis for resistant against pathogen and metal homeostasis in plants.

Keywords: Lycopersicon esculentum, copper, Fusarium wilt, farm yard manure

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Authors: Fawzyah Albaldi

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Urinary tract infections (UTIs) are the most prevalent of infectious diseases and > 80% are caused by uropathogenic E. coli (UPEC). Infection occurs following adhesion to urothelial plaques on bladder epithelial cells, whose major protein constituent are the uroplakins (UPs). Two of the four uroplakins (UPIa and UPIb) are members of the tetraspanin superfamily. The UPEC adhesin FimH is known to interact directly with UPIa. Tetraspanins are a diverse family of transmembrane proteins that generally act as “molecular organizers” by binding different proteins and lipids to form tetraspanin enriched microdomains (TEMs). Previous work by our group has shown that TEMs are involved in the adhesion of many pathogenic bacteria to human cells. Adhesion can be blocked by tetraspanin-derived synthetic peptides, suggesting that tetraspanins may be valuable drug targets. In this study, we investigate the role of tetraspanins in UPEC adherence to bladder epithelial cells. Human bladder cancer cell lines (T24, 5637, RT4), commonly used as in-vitro models to investigate UPEC infection, along with primary human bladder cells, were used in this project. The aim was to establish a model for UPEC adhesion/infection with the objective of evaluating the impact of tetraspanin-derived reagents on this process. Such reagents could reduce the progression of UTI, particularly in patients with indwelling catheters. Tetraspanin expression on the bladder cells was investigated by q-PCR and flow cytometry, with CD9 and CD81 generally highly expressed. Interestingly, despite these cell lines being used by other groups to investigate FimH antagonists, uroplakin proteins (UPIa, UPIb and UPIII) were poorly expressed at the cell surface, although some were present intracellularly. Attempts were made to differentiate the cell lines, to induce cell surface expression of these UPs, but these were largely unsuccessful. Pre-treatment of bladder epithelial cells with anti-CD9 monoclonal antibody significantly decreased UPEC infection, whilst anti-CD81 had no effects. A short (15aa) synthetic peptide corresponding to the large extracellular region (EC2) of CD9 also significantly reduced UPEC adherence. Furthermore, we demonstrated specific binding of that fluorescently tagged peptide to the cells. CD9 is known to associate with a number of heparan sulphate proteoglycans (HSPGs) that have also been implicated in bacterial adhesion. Here, we demonstrated that unfractionated heparin (UFH)and heparin analogs significantly inhibited UPEC adhesion to RT4 cells, as did pre-treatment of the cells with heparinases. Pre-treatment with chondroitin sulphate (CS) and chondroitinase also significantly decreased UPEC adherence to RT4 cells. This study may shed light on a common pathogenicity mechanism involving the organisation of HSPGs by tetraspanins. In summary, although we determined that the bladder cell lines were not suitable to investigate the role of uroplakins in UPEC adhesion, we demonstrated roles for CD9 and cell surface proteoglycans in this interaction. Agents that target these may be useful in treating/preventing UTIs.

Keywords: UTIs, tspan, uroplakins, CD9

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Authors: Lina Paola Orozco Marin, Yuliet Montoya Osorio, John Bustamante Osorno

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Ischemic events can culminate in acute myocardial infarction by irreversible cardiac lesions that cannot be restored due to the limited regenerative capacity of the heart. Cell therapy seeks to replace these injured or necrotic cells by transplanting healthy and functional cells. The therapeutic alternatives proposed by tissue engineering and cardiovascular regenerative medicine are the use of biomaterials to mimic the native extracellular medium, which is full of proteins, proteoglycans, and glycoproteins. The selected biomaterials must provide structural support to the encapsulated cells to avoid their migration and death in the host tissue. In this context, the present research work focused on developing a natural thermosensitive hydrogel, its physical and chemical characterization, and the determination of its biocompatibility in vitro. The hydrogel was developed by mixing hydrolyzed bovine and porcine collagen at 2% w/v, chitosan at 2.5% w/v, and beta-glycerolphosphate at 8.5% w/w and 10.5% w/w in magnetic stirring at 4°C. Once obtained, the thermosensitivity and gelation time were determined, incubating the samples at 37°C and evaluating them through the inverted tube method. The morphological characterization of the hydrogels was carried out through scanning electron microscopy. Chemical characterization was carried out employing infrared spectroscopy. The biocompatibility was determined using the MTT cytotoxicity test according to the ISO 10993-5 standard for the hydrogel’s precursors using the fetal human ventricular cardiomyocytes cell line RL-14. The RL-14 cells were also seeded on the top of the hydrogels, and the supernatants were subculture at different periods to their observation under a bright field microscope. Four types of thermosensitive hydrogels were obtained, which differ in their composition and concentration, called A1 (chitosan/bovine collagen/beta-glycerolphosphate 8.5%w/w), A2 (chitosan/porcine collagen/beta-glycerolphosphate 8.5%), B1 (chitosan/bovine collagen/beta-glycerolphosphate 10.5%) and B2 (chitosan/porcine collagen/beta-glycerolphosphate 10.5%). A1 and A2 had a gelation time of 40 minutes, and B1 and B2 had a gelation time of 30 minutes at 37°C. Electron micrographs revealed a three-dimensional internal structure with interconnected pores for the four types of hydrogels. This facilitates the exchange of nutrients, oxygen, and the exit of metabolites, allowing to preserve a microenvironment suitable for cell proliferation. In the infrared spectra, it was possible to observe the interaction that occurs between the amides of polymeric compounds with the phosphate groups of beta-glycerolphosphate. Finally, the biocompatibility tests indicated that cells in contact with the hydrogel or with each of its precursors are not affected in their proliferation capacity for a period of 16 days. These results show the potential of the hydrogel to increase the cell survival rate in the cardiac cell therapies under investigation. Moreover, the results lay the foundations for its characterization and biological evaluation in both in vitro and in vivo models.

Keywords: cardiac cell therapy, cardiac ischemia, natural polymers, thermosensitive hydrogel

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Authors: Sweta Agrawal, Sachin Chaugule, Gargi Rane, Shashank More, Madhavi Indap

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Angiogenesis and metastasis are two of the most important hallmarks of cancer. Hence, most of the cancer therapies nowadays are multi-targeted so as to reduce resistance and have better efficacy. As synthetic molecules arise with a burden of their toxicities and side-effects, more and more research is being focussed on exploiting the vast natural resources of drugs, in the form of plants and animals. Although, the idea of using marine organisms as a source of pharmaceuticals is not new, the pace at which marine drugs are being discovered, has definitely up surged! In the present study, we have assessed the anti-angiogenic and in vitro anti-metastatic activity of aqueous fraction from the extract of marine gastropod Euchelus asper. The soft body of Euchelus Asper was extracted with methanol and named EAME. Partition chromatography of EAME gave three fractions EAME I, II and III. Biochemical analysis revealed the presence of proteins in EAME III. Preliminary analysis had revealed the anti-angiogenic activity was exhibited by EAME III out of the three fractions. Hereafter, EAME III (concentration 25µg/ml-400µg/ml) was tested on chick chorioallantoic membrane (CAM) model for the detailed analysis of its potential anti-angiogenic effect. In vitro testing of the fraction (concentration 0.25µg/ml - 1µg/ml), involved cytotoxicity by SRB assay, cell cycle analysis by flow cytometry and anti-proliferative effect by scratch wound healing assay on A549 lung carcinoma cells. Apart from this, a portion of treated CAM as well as conditioned medium from treated A549 were subjected to gelatin zymography for assessment of matrix metalloproteinases MMP-2 and MMP-9 levels. Our results revealed that EAME III exhibited significant anti-angiogenic activity on CAM which was also supported by histological observations. During histological studies of CAM, it was found that EAME III caused reduction in angiogenesis by altering the extracellular matrix of the CAM membrane. In vitro analysis disclosed that EAME III exhibited moderate cytotoxic effect on A549 cells and its effect was not dose-dependent. The results of flow cytometry confirmed that EAME III caused cell cycle arrest in A549 cell line as almost all of the treated cells were found in G1 phase. Further, the migration and proliferation of A549 was significantly reduced by EAME III as observed from the scratch wound assay. Moreover, Gelatin zymography analysis revealed that EAME III caused suppression of MMP-2 in CAM membrane and reduced MMP-9 and MMP-2 expression in A549 cells. This verified that the anti-angiogenic and anti-metastatic effects of EAME III were correlated with the suppression of MMP-2 and -9. To conclude, EAME III shows dual anti-tumour action by reducing angiogenesis and exerting anti-metastatic effect on lung cancer cells, thus it has the potential to be used as an anti-cancer agent against lung carcinoma.

Keywords: angiogenesis, anti-cancer, marine drugs, matrix metalloproteinases

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Authors: Michael P. Mannino, Gerald W. Hart

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

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

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Authors: Kathryn Nderitu, Atunga Nyachieo, Ezekiel Mecha

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Introduction: Solanum nigrum , also known as black nightshade, biosynthesizes various phytochemical compounds with various pharmacological activities, including treating cardiovascular diseases and type 2 diabetes, among others. Materials and Methods: To assess the anti-obesity effects of Solanum nigrum using high-fat-fed diet rats, Sprague Dawley male rats (n = 35) of weights 160–180 g were assigned randomly into seven groups comprising n = 5 rats each. Each group was fed for 11 weeks as follows: normal group (normal chow rat feed); high-fat diet control (HFD); HFD and standard drug (Orlistat 30 mg/kg bw); HFD and methanolic extract 150 mg/kgbw; HFD and methanolic extract 300 mg/kgbw; HFD and dichloromethane extract 150 mg/kgbw; HFD and dichloromethane extract 300 mg/kgbw. Body mass index and food intake were monitored per week, and an oral glucose tolerance test was measured in weeks 5 and 10. Lipid profiles, liver function tests, adipose tissue, liver weights, and phytochemical analysis of Solanum nigrum were later carried out. Results: High-fat diet control group rats exhibited a significant increase in body mass index (BMI), while rats administered with leaf extracts of Solanum nigrum showed a reduction in BMI. Both low doses of dichloromethane (150 mg/kgbw) and high doses of methanol extracts (300 mg/kgbw) showed a better reduction in BMI than the other treatment groups. A significant decrease (p <0.05) in low-density lipoprotein-cholesterol, triglycerides, and cholesterol was observed among the rats administered with Solanum nigrum extracts compared to those of HFD control. Moreover, the HFD control group significantly increased liver and adipose tissue weights compared to other treatment groups (p<0.05). Solanum nigrum also decreased glycemic levels and normalized the hepatic enzymes of HFD control. However, food intake among the groups showed no significant difference (p>0.05). Qualitative analysis of Solanum nigrum leaf extracts indicated the presence of various bioactive compounds associated with anti-obesity. Conclusion: These results validate the use of Solanum nigrum in controlling obesity.

Keywords: solanum nigrum, High fat diet, phytocompounds, obesity

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Authors: Jie Lin, Yiwen Pan, Li Zhang, Zhangyong Xia

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Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipids, immune cells, and extracellular matrix in the arterial walls. This pathological process can lead to the formation of plaques that can obstruct blood flow and trigger various cardiovascular diseases such as heart attack and stroke. The underlying molecular mechanisms still remain unclear, although many studies revealed the dysfunction of endothelial cells, recruitment and activation of monocytes and macrophages, and the production of pro-inflammatory cytokines and chemokines in atherosclerosis. This study aimed to identify hub genes involved in the progression of atherosclerosis and to analyze their biological function in silico, thereby enhancing our understanding of the disease’s molecular mechanisms. Through the analysis of microarray data, we examined the gene expression in media and neo-intima from plaques, as well as distant macroscopically intact tissue, across a cohort of 32 hypertensive patients. Initially, 112 differentially expressed genes (DEGs) were identified. Subsequent immune infiltration analysis indicated a predominant presence of 27 immune cell types in the atherosclerosis group, particularly noting an increase in monocytes and macrophages. In the Weighted gene co-expression network analysis (WGCNA), 10 modules with a minimum of 30 genes were defined as key modules, with blue, dark, Oliver green and sky-blue modules being the most significant. These modules corresponded respectively to monocyte, activated B cell, and activated CD4 T cell gene patterns, revealing a strong morphological-genetic correlation. From these three gene patterns (modules morphology), a total of 2509 key genes (Gene Significance >0.2, module membership>0.8) were extracted. Six hub genes (CD36, DPP4, HMOX1, PLA2G7, PLN2, and ACADL) were then identified by intersecting 2509 key genes, 102 DEGs with lipid-related genes from the Genecard database. The bio-functional analysis of six hub genes was estimated by a robust classifier with an area under the curve (AUC) of 0.873 in the ROC plot, indicating excellent efficacy in differentiating between the disease and control group. Moreover, PCA visualization demonstrated clear separation between the groups based on these six hub genes, suggesting their potential utility as classification features in predictive models. Protein-protein interaction (PPI) analysis highlighted DPP4 as the most interconnected gene. Within the constructed key gene-drug network, 462 drugs were predicted, with ursodeoxycholic acid (UDCA) being identified as a potential therapeutic agent for modulating DPP4 expression. In summary, our study identified critical hub genes implicated in the progression of atherosclerosis through comprehensive bioinformatic analyses. These findings not only advance our understanding of the disease but also pave the way for applying similar analytical frameworks and predictive models to other diseases, thereby broadening the potential for clinical applications and therapeutic discoveries.

Keywords: atherosclerosis, hub genes, drug prediction, bioinformatics

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Authors: Rawan Ashraf, Ahmed E. Gomaa, Gehan Safwat, Ayman Diab

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Background: Three-dimensional (3D) bio-printing has become a versatile and powerful method for generating a variety of biological constructs, including bone or extracellular matrix scaffolds endo- or epithelial, muscle tissue, as well as organoids. Aim of the study: Fabricate a low cost DIY 3D bio-printer to produce 3D bio-printed products such as anti-microbial packaging or multi-organs on chips. We demonstrate the alignment between two types of 3D printer technology (3D Bio-printer and DLP) on Multi-organ-on-a-chip (multi-OoC) devices fabrication. Methods: First, Design and Fabrication of the Syringe Unit for Modification of an Off-the-Shelf 3D Printer, then Preparation of Hydrogel based on natural polymers Sodium Alginate and Gelatin, followed by acquisition of the cell suspension, then modeling the desired 3D structure. Preparation for 3D printing, then Cell-free and cell-laden hydrogels went through the printing process at room temperature under sterile conditions and finally post printing curing process and studying the printed structure regards physical and chemical characteristics. The hard scaffold of the Organ on chip devices was designed and fabricated using the DLP-3D printer, following similar approaches as the Microfluidics system fabrication. Results: The fabricated Bio-Ink was based onHydrogel polymer mix of sodium alginate and gelatin 15% to 0.5%, respectively. Later the 3D printing process was conducted using a higher percentage of alginate-based hydrogels because of it viscosity and the controllable crosslinking, unlike the thermal crosslinking of Gelatin. The hydrogels were colored to simulate the representation of two types of cells. The adaption of the hard scaffold, whether for the Microfluidics system or the hard-tissues, has been acquired by the DLP 3D printers with fabricated natural bioactive essential oils that contain antimicrobial activity, followed by printing in Situ three complex layers of soft-hydrogel as a cell-free Bio-Ink to simulate the real-life tissue engineering process. The final product was a proof of concept for a rapid 3D cell culturing approaches that uses an engineered hard scaffold along with soft-tissues, thus, several applications were offered as products of the current prototype, including the Organ-On-Chip as a successful integration between DLP and 3D bioprinter. Conclusion: Multiple designs for the organ-on-a-chip (multi-OoC) devices have been acquired in our study with main focus on the low cost fabrication of such technology and the potential to revolutionize human health research and development. We describe circumstances in which multi-organ models are useful after briefly examining the requirement for full multi-organ models with a systemic component. Following that, we took a look at the current multi-OoC platforms, such as integrated body-on-a-chip devices and modular techniques that use linked organ-specific modules.

Keywords: 3d bio-printer, hydrogel, multi-organ on chip, bio-inks

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Authors: Ifigeneia V. Mavragani, Zacharenia Nikitaki, George Kalantzis, George Iliakis, Alexandros G. Georgakilas

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Complex DNA damage consisting of a combination of DNA lesions, such as Double Strand Breaks (DSBs) and non-DSB base lesions occurring in a small volume is considered as one of the most important biological endpoints regarding ionizing radiation (IR) exposure. Strong theoretical (Monte Carlo simulations) and experimental evidence suggests an increment of the complexity of DNA damage and therefore repair resistance with increasing linear energy transfer (LET). Experimental detection of complex (clustered) DNA damage is often associated with technical deficiencies limiting its measurement, especially in cellular or tissue systems. Our groups have recently made significant improvements towards the identification of key parameters relating to the efficient detection of complex DSBs and non-DSBs in human cellular systems exposed to IR of varying quality (γ-, X-rays 0.3-1 keV/μm, α-particles 116 keV/μm and 36Ar ions 270 keV/μm). The induction and processing of DSB and non-DSB-oxidative clusters were measured using adaptations of immunofluorescence (γH2AX or 53PB1 foci staining as DSB probes and human repair enzymes OGG1 or APE1 as probes for oxidized purines and abasic sites respectively). In the current study, Relative Biological Effectiveness (RBE) values for DSB and non-DSB induction have been measured in different human normal (FEP18-11-T1) and cancerous cell lines (MCF7, HepG2, A549, MO59K/J). The experimental results are compared to simulation data obtained using a validated microdosimetric fast Monte Carlo DNA Damage Simulation code (MCDS). Moreover, this simulation approach is implemented in two realistic clinical cases, i.e. prostate cancer treatment using X-rays generated by a linear accelerator and a pediatric osteosarcoma case using a 200.6 MeV proton pencil beam. RBE values for complex DNA damage induction are calculated for the tumor areas. These results reveal a disparity between theory and experiment and underline the necessity for implementing highly precise and more efficient experimental and simulation approaches.

Keywords: complex DNA damage, DNA damage simulation, protons, radiotherapy

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Authors: Eiji Nakamachi, Ryota Sakiyama, Koji Yamamoto, Yusuke Morita, Hidetoshi Sakamoto

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The regeneration of injured central nerve network caused by the cerebrovascular accidents is difficult, because of poor regeneration capability of central nerve system composed of the brain and the spinal cord. Recently, new regeneration methods such as transplant of nerve cells and supply of nerve nutritional factor were proposed and examined. However, there still remain many problems with the canceration of engrafted cells and so on and it is strongly required to establish an efficacious treating method of a central nerve system. Blackman proposed the electromagnetic stimulation method to enhance the axonal nerve extension. In this study, we try to design and fabricate a new three-dimensional (3D) bio-reactor, which can load a uniform AC magnetic field stimulation on PC12 cells in the extracellular environment for enhancement of an axonal nerve extension and 3D nerve network generation. Simultaneously, we measure the morphology of PC12 cell bodies, axons, and dendrites by the multiphoton excitation fluorescence microscope (MPM) and evaluate the effectiveness of the uniform AC magnetic stimulation to enhance the axonal nerve extension. Firstly, we designed and fabricated the uniform AC magnetic field stimulation bio-reactor. For the AC magnetic stimulation system, we used the laminated silicon steel sheets for a yoke structure of 3D chamber, which had a high magnetic permeability. Next, we adopted the pole piece structure and installed similar specification coils on both sides of the yoke. We searched an optimum pole piece structure using the magnetic field finite element (FE) analyses and the response surface methodology. We confirmed that the optimum 3D chamber structure showed a uniform magnetic flux density in the PC12 cell culture area by using FE analysis. Then, we fabricated the uniform AC magnetic field stimulation bio-reactor by adopting analytically determined specifications, such as the size of chamber and electromagnetic conditions. We confirmed that measurement results of magnetic field in the chamber showed a good agreement with FE results. Secondly, we fabricated a dish, which set inside the uniform AC magnetic field stimulation of bio-reactor. PC12 cells were disseminated with collagen gel and could be 3D cultured in the dish. The collagen gel were poured in the dish. The collagen gel, which had a disk shape of 6 mm diameter and 3mm height, was set on the membrane filter, which was located at 4 mm height from the bottom of dish. The disk was full filled with the culture medium inside the dish. Finally, we evaluated the effectiveness of the uniform AC magnetic field stimulation to enhance the nurve axonal extension. We confirmed that a 6.8 increase in the average axonal extension length of PC12 under the uniform AC magnetic field stimulation at 7 days culture in our bio-reactor, and a 24.7 increase in the maximum axonal extension length. Further, we confirmed that a 60 increase in the number of dendrites of PC12 under the uniform AC magnetic field stimulation. Finally, we confirm the availability of our uniform AC magnetic stimulation bio-reactor for the nerve axonal extension and the nerve network generation.

Keywords: nerve regeneration, axonal extension , PC12 cell, magnetic field, three-dimensional bio-reactor

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Authors: L. Musak, J. Valachova, T. Vasicko, O. Osina

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Introduction: Stainless steel is resistant to electrochemical corrosion by passivation. Welders are greatly exposed to welding fumes of toxic metals, which added to this steel. The content of chromium (Cr) in steel was above 11.5%, Ni and Mo from 2 to 6.5%. The aim of the study was the evaluation of occupational exposure to Cr, chromosome analysis and valuation of individual susceptibility polymorphism of gene CCND1 c.870 G>A. Materials and Methods: The exposed group was consisted from 117 welders of stainless steels. The average age was 38.43 years and average exposure time 7.14 years. Smokers represented 40.17%. The control group consisted of 123 non-exposed workers with an average age of 39.74 years and time employment 16.67 years. Smokers accounted for 22.76%. Analysis of Cr in blood and urine was performed by atomic absorption spectrophotometry (AAS Varian SpectraAA 30P) with electrothermal decomposition of the sample in the graphite furnace. For the evaluation of chromosomal aberrations (CA) was used cytogenetic analysis of peripheral blood lymphocytes, gene polymorphism was determined by PCR-RFLP reaction using appropriate primers and restriction enzymes. For statistical analysis was used the Mann-Whitney U-test. Results: The mean Cr level in exposed group was 0.095 mmol/l (0.019 min-max 0.504). No value does exceed the average normal value. The average value Cr in urine was 7.9 mmol/mol creatinine (min 0.026 to max 19.26). The total number of CA was 1.86% in compared to 1.70% controls. (CTA-type 0.90% vs 0.80% and CSA-type 0.96% vs 0.90%). In the number of total CA was observed statistical difference between smokers and non-smokers of exposed group (S-1.57% vs. NS-2.04%, P<0.05). In CCND1 gene polymorphisms was observed the increasing of the total CA with wild-type allele (WT) via heterozygous to the VAR genotype (1.44%<1.82%<2.13%). There was observed a statistically higher incidence of CTA-type aberrations in variant genotypes between exposed and control groups (1.22% vs. 0.59%, P<0.05). Discussion and conclusions: The work place is usually higher source of exposure to harmful factors. Workers need consistently and checked frequently health control. In assessing the risk of adverse effects of metals is important to consider their persistence, behavior and bioavailability. Prolonged exposure to carcinogens may not manifest symptoms of poisoning, but delayed effects may occur, which resulted in a higher incidence of malignant tumors.

Keywords: genotoxicity, chromium, stainless steels, welders

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Authors: Mwafaq Ibdah, Mossab, Yahyaa, Dor Rachmany, Yoram Gerchman, Doron Holland, Liora Shaltiel-Harpaz

Abstract:

Pear Psylla is the most important pest of pear in all pear-growing regions, in Asian, European, and the USA. Pear psylla damages pears in several ways: high-density populations of these insects can cause premature leaf and fruit drop, diminish plant growth, and reduce fruit size. In addition, their honeydew promotes sooty mold on leaves and russeting on fruit. Pear psyllas are also considered vectors of pear pathogens such as Candidatus Phytoplasma pyri causing pear decline that can lead to loss of crop and tree vigor, and sometimes loss of trees. Psylla control is a major obstacle to efficient integrated pest management. Recently we have identified two naturally resistance pear accessions (Py.760-261 and Py.701-202) in the Newe Ya’ar live collection. GC-MS volatile metabolic profiling identified several volatile compounds common in these accessions but lacking, or much less common, in a sensitive accession, the commercial Spadona variety. Among these volatiles were styrene and its derivatives. When the resistant accessions were used as inter-stock, the volatile compounds appear in commercial Spadona scion leaves, and it showed reduced susceptibility to pear psylla. Laboratory experiments and applications of some of these volatile compounds were very effective against psylla eggs, nymphs, and adults. The genes and enzymes involved in the specific reactions that lead to the biosynthesis of styrene in plant are unknown. We have identified a phenolic acid decarboxylase that catalyzes the formation of p-hydroxystyrene, which occurs as a styrene analog in resistant pear genotypes. The His-tagged and affinity chromatography purified E. coli-expressed pear PyPAD1 protein could decarboxylate p-coumaric acid and ferulic acid to p-hydroxystyrene and 3-methoxy-4-hydroxystyrene. In addition, PyPAD1 had the highest activity toward p-coumaric acid. Expression analysis of the PyPAD gene revealed that its expressed as expected, i.e., high when styrene levels and psylla resistance were high.

Keywords: pear Psylla, volatile, GC-MS, resistance

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Authors: O. Gsib, U. Peirera, C. Egles, S. A. Bencherif

Abstract:

In skin regenerative medicine, one of the critical issues is to produce a three-dimensional scaffold with optimized porosity for dermal fibroblast infiltration and neovascularization, which exhibits high mechanical properties and displays sufficient wound healing characteristics. In this study, we report on the synthesis and characterization of macroporous sequential interpenetrating polymer networks (IPNs) combining skin wound healing properties of fibrin with the excellent physical properties of polyethylene glycol (PEG). Fibrin fibers serve as a provisional biologically active network to promote cell adhesion and proliferation while PEG provides the mechanical stability to maintain the entire 3D construct. After having modified both PEG and Serum Albumin (used for promoting enzymatic degradability) by adding methacrylate residues (PEGDM and SAM, respectively), Fibrin/PEGDM-SAM sequential IPNs were synthesized as follows: Macroporous sponges were first produced from PEGDM-SAM hydrogels by a freeze-drying technique and then rehydrated by adding the fibrin precursors. Environmental Scanning Electron Microscopy (ESEM) and Confocal Laser Scanning Microscopy (CLSM) were used to characterize their microstructure. Human dermal fibroblasts were cultivated during one week in the constructs and different cell culture parameters (viability, morphology, proliferation) were evaluated. Subcutaneous implantations of the scaffolds were conducted on five-week old male nude mice to investigate their biocompatibility in vivo. We successfully synthesized interconnected and macroporous Fibrin/PEGDM-SAM sequential IPNs. The viability of primary dermal fibroblasts was well maintained (above 90%) after 2 days of culture. Cells were able to adhere, spread and proliferate in the scaffolds suggesting the suitable porosity and intrinsic biologic properties of the constructs. The fibrin network adopted a spider web shape that covered partially the pores allowing easier cell infiltration into the macroporous structure. To further characterize the in vitro cell behavior, cell proliferation (EdU incorporation, MTS assay) is being studied. Preliminary histological analysis of animal studies indicated the persistence of hydrogels even after one-month post implantation and confirmed the absence of inflammation response, good biocompatibility and biointegration of our scaffolds within the surrounding tissues. These results suggest that our Fibrin/PEGDM-SAM IPNs could be considered as potential candidates for dermis regenerative medicine. Histological analysis will be completed to further assess scaffold remodeling including de novo extracellular matrix protein synthesis and early stage angiogenesis analysis. Compression measurements will be conducted to investigate the mechanical properties.

Keywords: fibrin, hydrogels for dermal reconstruction, polyethylene glycol, semi-interpenetrating polymer network

Procedia PDF Downloads 209

Authors: Reham Khalaf-Nazzal, Imad Dweikat, Paula Gimenez, Iker Oyenarte, Alfonso Martinez-Cruz, Domonik Muller

Abstract:

Metal cation transport mediator (CNNM) gene family comprises 4 isoforms that are expressed in various human tissues. Structurally, CNNMs are complex proteins that contain an extracellular N-terminal domain preceding a DUF21 transmembrane domain, a ‘Bateman module’ and a C-terminal cNMP-binding domain. Mutations in CNNM2 cause familial dominant hypomagnesaemia. Growing evidence highlights the role of CNNM2 in neurodevelopment. Mutations in CNNM2 have been implicated in epilepsy, intellectual disability, schizophrenia, and others. In the present study, we aim to elucidate the function of CNNM2 in the developing brain. Thus, we present the genetic origin of symptoms in two family cohorts. In the first family, three siblings of a consanguineous Palestinian family in which parents are first cousins, and consanguinity ran over several generations, presented a varying degree of intellectual disability, cone-rod dystrophy, and autism spectrum disorder. Exome sequencing and segregation analysis revealed the presence of homozygous pathogenic mutation in the CNNM2 gene, the parents were heterozygous for that gene mutation. Magnesium blood levels were normal in the three children and their parents in several measurements. They had no symptoms of hypomagnesemia. The CNNM2 mutation in this family was found to locate in the CBS1 domain of the CNNM2 protein. The crystal structure of the mutated CNNM2 protein was not significantly different from the wild-type protein, and the binding of AMP or MgATP was not dramatically affected. This suggests that the CBS1 domain could be involved in pure neurodevelopmental functions independent of its magnesium-handling role, and this mutation could have affected a protein partner binding or other functions in this protein. In the second family, another autosomal dominant CNNM2 mutation was found to run in a large family with multiple individuals over three generations. All affected family members had hypomagnesemia and hypermagnesuria. Oral supplementation of magnesium did not increase the levels of magnesium in serum significantly. Some affected members of this family have defects in fine motor skills such as dyslexia and dyslalia. The detected mutation is located in the N-terminal part, which contains a signal peptide thought to be involved in the sorting and routing of the protein. In this project, we describe heterogenous clinical phenotypes related to CNNM2 mutations and protein functions. In the first family, and up to the authors’ knowledge, we report for the first time the involvement of CNNM2 in retinal photoreceptor development and function. In addition, we report the presence of a neurophenotype independent of magnesium status related to the CNNM2 protein mutation. Taking into account the different modes of inheritance and the different positions of the mutations within CNNM2 and its different structural and functional domains, it is likely that CNNM2 might be involved in a wide spectrum of neuropsychiatric comorbidities with considerable varying phenotypes.

Keywords: magnesium transport, autosomal recessive, autism, neurodevelopment, CBS domain

Procedia PDF Downloads 124