Search results for: human cells line

Authors: Maddalena Arigoni, Maria Luisa Ratto, Raffaele A. Calogero, Luca Alessandri

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The presence of tumor heterogeneity, where distinct cancer cells exhibit diverse morphological and phenotypic profiles, including gene expression, metabolism, and proliferation, poses challenges for molecular prognostic markers and patient classification for targeted therapies. Understanding the causes and progression of cancer requires research efforts aimed at characterizing heterogeneity, which can be facilitated by evolving single-cell sequencing technologies. However, analyzing single-cell data necessitates computational methods that often lack objective validation. Therefore, the establishment of benchmarking datasets is necessary to provide a controlled environment for validating bioinformatics tools in the field of single-cell oncology. Benchmarking bioinformatics tools for single-cell experiments can be costly due to the high expense involved. Therefore, datasets used for benchmarking are typically sourced from publicly available experiments, which often lack a comprehensive cell annotation. This limitation can affect the accuracy and effectiveness of such experiments as benchmarking tools. To address this issue, we introduce omics benchmark experiments designed to evaluate bioinformatics tools to depict the heterogeneity in single-cell tumor experiments. We conducted single-cell RNA sequencing on six lung cancer tumor cell lines that display resistant clones upon treatment of EGFR mutated tumors and are characterized by driver genes, namely ROS1, ALK, HER2, MET, KRAS, and BRAF. These driver genes are associated with downstream networks controlled by EGFR mutations, such as JAK-STAT, PI3K-AKT-mTOR, and MEK-ERK. The experiment also featured an EGFR-mutated cell line. Using 10XGenomics platform with cellplex technology, we analyzed the seven cell lines together with a pseudo-immunological microenvironment consisting of PBMC cells labeled with the Biolegend TotalSeq™-B Human Universal Cocktail (CITEseq). This technology allowed for independent labeling of each cell line and single-cell analysis of the pooled seven cell lines and the pseudo-microenvironment. The data generated from the aforementioned experiments are available as part of an online tool, which allows users to define cell heterogeneity and generates count tables as an output. The tool provides the cell line derivation for each cell and cell annotations for the pseudo-microenvironment based on CITEseq data by an experienced immunologist. Additionally, we created a range of pseudo-tumor tissues using different ratios of the aforementioned cells embedded in matrigel. These tissues were analyzed using 10XGenomics (FFPE samples) and Curio Bioscience (fresh frozen samples) platforms for spatial transcriptomics, further expanding the scope of our benchmark experiments. The benchmark experiments we conducted provide a unique opportunity to evaluate the performance of bioinformatics tools for detecting and characterizing tumor heterogeneity at the single-cell level. Overall, our experiments provide a controlled and standardized environment for assessing the accuracy and robustness of bioinformatics tools for studying tumor heterogeneity at the single-cell level, which can ultimately lead to more precise and effective cancer diagnosis and treatment.

Keywords: single cell omics, benchmark, spatial transcriptomics, CITEseq

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Authors: Julia Kapr, Andrea Rossi, Haribaskar Ramachandran, Marius Pollet, Ilka Egger, Selina Dangeleit, Katharina Koch, Jean Krutmann, Ellen Fritsche

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It is widely acknowledged that animal models do not always represent human disease. Especially human brain development is difficult to model in animals due to a variety of structural and functional species-specificities. This causes significant discrepancies between predicted and apparent drug efficacies in clinical trials and their subsequent failure. Emerging alternatives based on 3D in vitro approaches, such as human brain spheres or organoids, may in the future reduce and ultimately replace animal models. Here, we present a human induced pluripotent stem cell (hiPSC)-based 3D neural in a vitro disease model for the Cockayne Syndrome B (CSB). CSB is a rare hereditary disease and is accompanied by severe neurologic defects, such as microcephaly, ataxia and intellectual disability, with currently no treatment options. Therefore, the aim of this study is to investigate the molecular and cellular defects found in neural hiPSC-derived CSB models. Understanding the underlying pathology of CSB enables the development of treatment options. The two CSB models used in this study comprise a patient-derived hiPSC line and its isogenic control as well as a CSB-deficient cell line based on a healthy hiPSC line (IMR90-4) background thereby excluding genetic background-related effects. Neurally induced and differentiated brain sphere cultures were characterized via RNA Sequencing, western blot (WB), immunocytochemistry (ICC) and multielectrode arrays (MEAs). CSB-deficiency leads to an altered gene expression of markers for autophagy, focal adhesion and neural network formation. Cell migration was significantly reduced and electrical activity was significantly increased in the disease cell lines. These data hint that the cellular pathologies is possibly underlying CSB. By induction of autophagy, the migration phenotype could be partially rescued, suggesting a crucial role of disturbed autophagy in defective neural migration of the disease lines. Altered autophagy may also lead to inefficient mitophagy. Accordingly, disease cell lines were shown to have a lower mitochondrial base activity and a higher susceptibility to mitochondrial stress induced by rotenone. Since mitochondria play an important role in neurotransmitter cycling, we suggest that defective mitochondria may lead to altered electrical activity in the disease cell lines. Failure to clear the defective mitochondria by mitophagy and thus missing initiation cues for new mitochondrial production could potentiate this problem. With our data, we aim at establishing a disease adverse outcome pathway (AOP), thereby adding to the in-depth understanding of this multi-faced disorder and subsequently contributing to alternative drug development.

Keywords: autophagy, disease modeling, in vitro, pluripotent stem cells

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Authors: Mohd. A. M. Rahim, Yahaya Hassan, Mathumalar L. Fahrni

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Second line antiretroviral therapy (ART) regimen is used when patients fail their first line regimen. There are many factors such as non-adherence, drug resistance as well as virological and immunological failure that lead to second line highly active antiretroviral therapy (HAART) regimen treatment failure. This study was aimed at determining predictor factors to treatment failure with second line HAART and analyzing median survival time. An observational, retrospective study was conducted in Sungai Buloh Hospital (HSB) to assess current status of HIV patients treated with second line HAART regimen. Convenience sampling was used and 104 patients were included based on the study’s inclusion and exclusion criteria. Data was collected for six months i.e. from July until December 2013. Data was then analysed using SPSS version 18. Kaplan-Meier and Cox regression analyses were used to measure median survival times and predictor factors for treatment failure. The study population consisted mainly of male subjects, aged 30-45 years, who were heterosexual, and had HIV infection for less than 6 years. The most common second line HAART regimen given was lopinavir/ritonavir (LPV/r)-based combination. Kaplan-Meier analysis showed that patients on LPV/r demonstrated longer median survival times than patients on indinavir/ritonavir (IDV/r) based combination (p<0.001). The commonest reason for a treatment to fail with second line HAART was non-adherence. Based on Cox regression analysis, other predictor factors for treatment failure with second line HAART regimen were age and mode of HIV transmission.

Keywords: adherence, antiretroviral therapy, second line, treatment failure

Procedia PDF Downloads 238

Authors: Regina M. Chiechio, Rémi Leguevél, Helene Solhi, Marie Madeleine Gueguen, Stephanie Dutertre, Xavier, Jean-Pierre Bazureau, Olivier Mignen, Pascale Even-Hernandez, Paolo Musumeci, Maria Jose Lo Faro, Valerie Marchi

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Thanks to their ultra-small size, high electron density, and low toxicity, gold nanoclusters (Au NCs) have unique photoelectrochemical and luminescence properties that make them very interesting for diagnosis bio-imaging and theranostics. These applications require control of their delivery and interaction with cells; for this reason, the surface chemistry of Au NCs is essential to determine their interaction with the targeted biological objects. Here we demonstrate their ability as markers of pancreatic tumor cells. By functionalizing the surface of the NCs with a recognition peptite (U11), the nanostructures are able to preferentially bind to pancreatic cancer cells via a receptor (uPAR) overexpressed by these cells. Furthermore, the NCs can mark even the nucleus without the need of fixing the cells. These nanostructures can therefore be used as a non-toxic, multivalent luminescent platform, capable of selectively recognizing tumor cells for bioimaging, drug delivery, and radiosensitization.

Keywords: gold nanoclusters, luminescence, biomarkers, pancreatic cancer, biomedical applications, bioimaging, fluorescent probes, drug delivery

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Authors: Niklas Ravn-Boess, Nainita Bhowmick, Takamitsu Hattori, Shohei Koide, Christopher Park, Dimitris Placantonakis

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Background: Glioblastoma (GBM) is the most common and deadly primary brain malignancy in adults. Tumor propagation, brain invasion, and resistance to therapy critically depend on GBM stem-like cells (GSCs); however, the mechanisms that regulate GSC self-renewal are incompletely understood. Given the aggressiveness and poor prognosis of GBM, it is imperative to find biomarkers that could also translate into novel drug targets. Along these lines, we have identified a cell surface antigen, CD97 (ADGRE5), an adhesion G protein-coupled receptor (GPCR), that is expressed on GBM cells but is absent from non-neoplastic brain tissue. CD97 has been shown to promote invasiveness, angiogenesis, and migration in several human cancers, but its frequency of expression and functional role in regulating GBM growth and survival, and its potential as a therapeutic target has not been investigated. Design: We assessed CD97 mRNA and protein expression in patient derived GBM samples and cell lines using publicly available RNA-sequencing datasets and flow cytometry, respectively. To assess CD97 function, we generated shRNA lentiviral constructs that target a sequence in the CD97 extracellular domain (ECD). A scrambled shRNA (scr) with no predicted targets in the genome was used as a control. We evaluated CD97 shRNA lentivirally transduced GBM cells for Ki67, Annexin V, and DAPI. We also tested CD97 KD cells for their ability to self-renew using clonogenic tumorsphere formation assays. Further, we utilized synthetic Abs (sAbs) generated against the ECD of CD97 to test for potential antitumor effects using patient-derived GBM cell lines. Results: CD97 mRNA expression was expressed at high levels in all GBM samples available in the TCGA cohort. We found high levels of surface CD97 protein expression in 6/6 patient-derived GBM cell cultures, but not human neural stem cells. Flow cytometry confirmed downregulation of CD97 in CD97 shRNA lentivirally transduced cells. CD97 KD induced a significant reduction in cell growth in 3 independent GBM cell lines representing mesenchymal and proneural subtypes, which was accompanied by reduced (~20%) Ki67 staining and increased (~30%) apoptosis. Incubation of GBM cells with sAbs (20 ug/ ml) against the ECD of CD97 for 3 days induced GSC differentiation, as determined by the expression of GFAP and Tubulin. Using three unique GBM patient derived cultures, we found that CD97 KD attenuated the ability of GBM cells to initiate sphere formation by over 300 fold, consistent with an impairment in GSC self-renewal. Conclusion: Loss of CD97 expression in patient-derived GBM cells markedly decreases proliferation, induces cell death, and reduces tumorsphere formation. sAbs against the ECD of CD97 reduce tumorsphere formation, recapitulating the phenotype of CD97 KD, suggesting that sAbs that inhibit CD97 function exhibit anti-tumor activity. Collectively, these findings indicate that CD97 is necessary for the proliferation and survival of human GBM cells and identify CD97 as a promising therapeutically targetable vulnerability in GBM.

Keywords: adhesion GPCR, CD97, GBM stem cell, glioblastoma

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Authors: K. Yadamma, K. Rudrama Devi

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The protective effect of Ginger Extract against cyclophosphamide induced cytotoxicity was evaluated in in vivo animal model using analysis of chromosomal aberrations in somatic cells of mice. Three doses of Ginger Extract (150mg/kg, 200mg/kg, and 250mg/kg body weight) were selected for modulation and given to animals after priming. The animals were sacrificed 24, 48, 72 hrs after the treatment and slides were prepared for the incidence of chromosomal aberrations in bone marrow cells of mice. When animals were treated with cyclophosphamide 50mg/kg, showed cytogenetic damage in somatic cells. However, a significant decrease was observed in the percentage of chromosomal aberrations when animals were primed with various doses of Ginger Extract. The present results clearly indicate the protective nature of Ginger Extract against cyclophosphamide induced genetic damage in mouse bone marrow cells.

Keywords: ginger extract, protection, bone marrow cells, swiss albino mice

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Authors: A. Kojah, A. Nacaroğlu

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Single phase transmission lines are used to transfer data or energy between two users. Transient conditions such as switching operations and short circuit faults cause the generation of the fluctuation on the waveform to be transmitted. Spatial voltage distribution on the single phase transmission line may change owing to the position and duration of the short circuit fault in the system. In this paper, the state space representation of the single phase transmission line for short circuit fault and for various types of terminations is given. Since the transmission line is modeled in time domain using distributed parametric elements, the mathematical representation of the event is given in state space (time domain) differential equation form. It also makes easy to solve the problem because of the time and space dependent characteristics of the voltage variations on the distributed parametrically modeled transmission line.

Keywords: energy transmission, transient effects, transmission line, transient voltage, RLC short circuit, single phase

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Authors: ShiQing Gao, XingYi Zhang, YouHe Zhou

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When a floating tilted cylinder pierces a fluid interface, the fulfilment of constant-contact-angle condition along the cylinder results in shift, stretch and distortion of the contact line, thus leading to a capillary interaction. We perform an investigation of the scaling dependence of tilt angle, contact angle, and cylinder radius on the contact line profile and the corresponding capillary interaction by numerical simulation and experiment. Characterized by three characteristic parameters respectively, the dependences for each deformation mode are systematically analyzed. Both the experiment and simulation reveals an invariant structure that is independent of contact angle and radius to characterize the stretch of the contact line for every tilted case. Based on this observation, we then propose a general capillary force scaling law to incredibly grasp all the simulated results, by simply approximating the contact line profile as tilted ellipse.

Keywords: gas-liquid/liquid-fluid interface, colloidal particle, contact line shape, capillary interaction, surface evolver (SE)

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Authors: Priyanka Sharma, Niti Puri

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Anemia develops when blood lacks enough healthy erythrocytes. Past studies indicated that anemia, inflammatory process, and oxidative stress are interconnected. Erythrocytes are continuously exposed to reactive oxygen species (ROS) during circulation, due to normal aerobic cellular metabolism and also pathology of inflammatory diseases. Systemic mastocytosis and genetic depletion of mast cells have been shown to affect anaemia. In the present study, we attempted to reveal whether mast cells have a direct role in clearance or erythrophagocytosis of normal or oxidatively damaged erythrocytes. Murine erythrocytes were treated with tert-butyl hydroperoxidase (t-BHP), an agent that induces oxidative damage and mimics in vivo oxidative stress. Normal and oxidatively damaged erythrocytes were labeled with carboxyfluorescein succinimidyl ester (CFSE) to track erythrophagocytosis. We show, for the first time, direct erythrophagocytosis of oxidatively damaged erythrocytes in vitro by RBL-2H3 mast cells as well as in vivo by murine peritoneal mast cells. Also, activated mast cells, as may be present in inflammatory conditions, showed a significant increase in the uptake of oxidatively damaged erythrocytes than resting mast cells. This suggests the involvement of mast cells in erythrocyte clearance during oxidative stress or inflammatory disorders. Partial inhibition of phagocytosis by various inhibitors indicated that this process may be controlled by several pathways. Hence, our study provides important evidence for involvement of mast cells in severe anemia due to inflammation and oxidative stress and might be helpful to circumvent the adverse anemic disorders.

Keywords: mast cells, anemia, erythrophagocytosis, oxidatively damaged erythrocytes

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Authors: Ozgu Hafizoglu

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Analogy is an essential tool of human cognition that enables connecting diffuse and diverse systems with attributional, deep structural, casual relations that are essential to learning, to innovation in artificial worlds, and to discovery in science. Cognitive Model of Analogy (CMA) leads and creates information pattern transfer within and between domains and disciplines in science. This paper demonstrates the Cognitive Model of Analogy (CMA) as an evolutionary approach to scientific research. The model puts forward the challenges of deep uncertainty about the future, emphasizing the need for flexibility of the system in order to enable reasoning methodology to adapt to changing conditions. In this paper, the model of analogical reasoning is created based on brain cells, their fractal, and operational forms within the system itself. Visualization techniques are used to show correspondences. Distinct phases of the problem-solving processes are divided thusly: encoding, mapping, inference, and response. The system is revealed relevant to brain activation considering each of these phases with an emphasis on achieving a better visualization of the brain cells: glial cells, axons, axon terminals, and neurons, relative to matching conditions of analogical reasoning and relational information. It’s found that encoding, mapping, inference, and response processes in four-term analogical reasoning are corresponding with the fractal and operational forms of brain cells: glial, axons, and neurons.

Keywords: analogy, analogical reasoning, cognitive model, brain and glials

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Authors: Nancy M. El-Baz, Laila Ziko, Rania Siam, Wael Mamdouh

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Cancer is one of the most devastating diseases, and has over than 10 million new cases annually worldwide. Despite the effectiveness of chemotherapeutic agents, their systemic toxicity and non-selective anticancer actions represent the main obstacles facing cancer curability. Due to the effective enhanced permeability and retention (EPR) effect of nanomaterials, nanoparticles (NPs) have been used as drug nanocarriers providing targeted cancer drug delivery systems. In addition, several inorganic nanoparticles such as silver (AgNPs) nanoparticles demonstrated a potent anticancer activity against different cancers. The present study aimed at formulating core-shell inorganic NPs-based combinatorial therapy based on combining the anticancer activity of AgNPs along with doxorubicin (DOX) and evaluating their cytotoxicity on MCF-7 breast cancer cells. These inorganic NPs-based combinatorial therapies were designed to (i) Target and kill cancer cells with high selectivity, (ii) Have an improved efficacy/toxicity balance, and (iii) Have an enhanced therapeutic index when compared to the original non-modified DOX with much lower dosage The in-vitro cytotoxicity studies demonstrated that the NPs-based combinatorial therapy achieved the same efficacy of non-modified DOX on breast cancer cell line, but with 96% reduced dose. Such reduction in DOX dose revealed that the combination between DOX and NPs possess a synergic anticancer activity against breast cancer. We believe that this is the first report on a synergic anticancer effect at very low dose of DOX against MCF-7 cells. Future studies on NPs-based combinatorial therapy may aid in formulating novel and significantly more effective cancer therapeutics.

Keywords: nanoparticles-based combinatorial therapy, silver nanoparticles, doxorubicin, breast cancer

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Authors: Sihyun Chae, Ryoto Arai, Waldo Concepcion, Paula Popescu

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The kidneys perform an important role in the human hormones that regulate the blood pressure, produce an active form of vitamin D and control the production of red blood cells. Kidney disease can cause health problems, such as heart disease. Also, increase the chance of having a stroke or heart attack. There are mainly to types of treatments for kidney disease, dialysis, and kidney transplant. For a better quality of life, the kidney transplant is desirable. However, kidney transplant can cause antibody reaction and patients’ body would be attacked by immune system of their own. For solving that issue, patients with transplanted kidney always take immunosuppressive drugs which can hurt kidney as side effects. Patients willing to do a kidney transplant have a waiting time of 3.6 years in average searching to find an appropriate kidney, considering there are almost 96,380 patients waiting for kidney transplant. There is a promising method to solve these issues: bioartificial kidney. Our membrane is specially designed with unique perforations capable to filter the blood cells separating the white blood cells from red blood cells. White blood cells will not pass through the encapsulated kidney preventing the immune system to attack the new organ and eliminating the need of a matching donor. It is possible to construct life-time long encapsulation without needing pumps or a power supply on the cell’s separation method preventing futures surgeries due the Cross-Channel Flow inside the device. This technology allows the possibility to use an animal kidney, prevent cancer cells to spread through the body, arm and leg transplants in the future. This project aims to improve the quality of life of patients with kidney disease.

Keywords: kidney encapsulation, immunosuppression filter, leukocyte filter, leukocyte

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Authors: Sue-Yi Chen, Wei-Chun Hsu, Jon-Yiew Gan

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Si-based double-junction tandem solar cells have become a popular research topic because of the advantages of low manufacturing cost and high energy conversion efficiency. However, there is no set of calculations to select the appropriate top cell materials. Therefore, this paper will propose a simple but practical selection method. First of all, we calculate the S-Q limit and explain the reasons for developing tandem solar cells. Secondly, we calculate the theoretical energy conversion efficiency of the double-junction tandem solar cells while combining the commercial monocrystalline Si and materials' practical efficiency to consider the actual situation. Finally, we conservatively conclude that if considering 75% performance of the theoretical energy conversion efficiency of the top cell, the suitable bandgap energy range will fall between 1.38eV to 2.5eV. Besides, we also briefly describe some improvements of several proper materials, CZTS, CdSe, Cu2O, ZnTe, and CdS, hoping that future research can select and manufacture high-efficiency Si-based tandem solar cells based on this paper successfully. Most importantly, our calculation method is not limited to silicon solely. If other materials’ performances match or surpass silicon's ability in the future, researchers can also apply this set of deduction processes.

Keywords: high-efficiency solar cells, material selection, Si-based double-junction solar cells, Tandem solar cells, photovoltaics.

Procedia PDF Downloads 90

Authors: Dalita G. S. M. Cavalcante, Elton A. P. dos Reis, Andressa S. Gomes, Caroline S. Danna, Leandra Ernest Kerche-Silva, Eidi Yoshihara, Aldo E. Job

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In order to minimize environmental impacts, a composite was developed from mixture of leather shavings (LE) with natural rubber (NR), which patent is already deposited. The new material created can be used in applications such as floors e heels for shoes. Besides these applications, the aim is to use this new material for the production of products for the textile industry, such as boots, gloves and bags. But the question arises, as to biocompatibility of this new material. This is justified because the structure of the leather shavings has chrome. The trivalent chromium is usually not toxic, but the hexavalent chromium can be highly toxic and genotoxic for living beings, causing damage to the DNA molecule and contributing to the formation of cancer. Based on this, the objective of this study is evaluate the possible genotoxic effects of the new composite, using as system - test two cell lines (MRC-5 and CHO-K1) by comet assay. For this, the production of the composite was performed in three proportions: for every 100 grams of NR was added 40 (E40), 50 (E50) or 60 (E60) grams of LE. The latex was collected from the rubber tree (Hevea brasiliensis). For vulcanization of the NR, activators and accelerators were used. The two cell lines were exposed to the new composite in its three proportions using elution method, that is, cells exposed to liquid extracts obtained from the composite for 24 hours. For obtaining the liquid extract, each sample of the composite was crushed into pieces and mixed with an extraction solution. The quantification of total chromium and hexavalent chromium in the extracts were performed by Optical Emission Spectrometry by Inductively Coupled Plasma (ICP-OES). The levels of DNA damage in cells exposed to both extracts were monitored by alkaline version of the comet assay. The results of the quantification of metals in ICP-OES indicated the presence of total chromium in different extracts, but were not detected presence of hexavalent chromium in any extract. Through the comet assay were not found DNA damage of the CHO-K1 cells exposed to both extracts. As for MRC-5, was found a significant increase in DNA damage in cells exposed to E50 and E60. Based on the above data, it can be asserted that the extracts obtained from the composite were highly genotoxic for MRC-5 cells. These biological responses do not appear to be related to chromium metal, since there was a predominance of trivalent chromium in the extracts, indicating that during the production process of the new composite, there was no formation of hexavalent chromium. In conclusion it can infer that the leather shavings containing chromium can be reused, thereby reducing the environmental impacts of this waste. Already on the composite indicates to its incorporation in applications that do not aim at direct contact with the human skin, and it is suggested the chain of composite production be studied, in an attempt to make it biocompatible so that it may be safely used by the textile industry.

Keywords: cell line, chrome, genotoxicity, leather, natural rubber

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Authors: Tao Yin, Zeqiang Zhang, Wei Liang, Yanqing Zeng, Yu Zhang

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To accelerate the remanufacturing process of electronic waste products, this study designs a partial disassembly line with the multi-robotic station to effectively dispose of excessive wastes. The multi-robotic partial disassembly line is a technical upgrade to the existing manual disassembly line. Balancing optimization can make the disassembly line smoother and more efficient. For partial disassembly line balancing with the multi-robotic station (PDLBMRS), a mixed-integer programming model (MIPM) considering the robotic efficiency differences is established to minimize cycle time, energy consumption and hazard index and to calculate their optimal global values. Besides, an enhanced NSGA-II algorithm (HNSGA-II) is proposed to optimize PDLBMRS efficiently. Finally, MIPM and HNSGA-II are applied to an actual mixed disassembly case of two types of computers, the comparison of the results solved by GUROBI and HNSGA-II verifies the correctness of the model and excellent performance of the algorithm, and the obtained Pareto solution set provides multiple options for decision-makers.

Keywords: waste disposal, disassembly line balancing, multi-robot station, robotic efficiency difference, HNSGA-II

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Authors: Małgorzata Drzewiecka, Tomasz Śliwiński, Maciej Radek

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The inhibition of histone deacetyles (HDACs) holds promise as a potential anti-cancer therapy because histone and non-histone protein acetylation is frequently disrupted in cancer, leading to cancer initiation and progression. Additionally, histone deacetylase inhibitors (HDACi) such as class I HDAC inhibitor - valproic acid (VPA) have been shown to enhance the effectiveness of DNA-damaging factors, such as cisplatin or radiation. In this study, we found that, using of VPA in combination with talazoparib (BMN-637 – PARP1 inhibitor – PARPi) and/or Dacarabazine (DTIC - alkylating agent) resulted in increased DNA double strand break (DSB) and reduced survival (while not affecting primary melanocytes )and proliferation of melanoma cells. Furthermore, pharmacologic inhibition of class I HDACs sensitizes melanoma cells to apoptosis following exposure to DTIC and BMN-637. In addition, inhibition of HDAC caused sensitization of melanoma cells to dacarbazine and BMN-637 in melanoma xenografts in vivo. At the mRNA and protein level histone deacetylase inhibitor downregulated RAD51 and FANCD2. This study provides that combining HDACi, alkylating agent and PARPi could potentially enhance the treatment of melanoma, which is known for being one of the most aggressive malignant tumors. The findings presented here point to a scenario in which HDAC via enhancing the HR-dependent repair of DSBs created during the processing of DNA lesions, are essential nodes in the resistance of malignant melanoma cells to methylating agent-based therapies.

Keywords: melanoma, hdac, parp inhibitor, valproic acid

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Authors: A. Maria G. Melero, A. M. Senna, J. A. Domingues, M. A. Hausen, E. Aparecida R. Duek, V. R. Botaro

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A hydrogel from cellulose acetate cross linked with ethylenediaminetetraacetic dianhydride (HAC-EDTA) was synthesized by our research group, and submitted to characterization and biological tests. Cytocompatibility analysis was performed by confocal microscopy using human adipocyte derived stem cells (ASCs). The FTIR analysis showed characteristic bands of cellulose acetate and hydroxyl groups and the tensile tests evidence that HAC-EDTA present a Young’s modulus of 643.7 MPa. The confocal analysis revealed that there was cell growth at the surface of HAC-EDTA. After one day of culture the cells presented spherical morphology, which may be caused by stress of the sequestration of Ca²⁺ and Mg²⁺ ions at the cell medium by HAC-EDTA, as demonstrated by ICP-MS. However, after seven days and 14 days of culture, the cells present fibroblastoid morphology, phenotype expected by this cellular type. The results give efforts to indicate this new material as a potential biomaterial for tissue engineering, in the future in vivo approach.

Keywords: cellulose acetate, hydrogel, biomaterial, cellular growth

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Authors: A. A. M. Azoddein, Y. Nuratri, A. B. Bustary, F. A. M. Azli, S. C. Sayuti

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Pseudomonas putida is a potential strain in biological treatment to remove mercury contained in the effluent of petrochemical industry due to its mercury reductase enzyme that able to reduce ionic mercury to elementary mercury. Freeze-dried P. putida allows easy, inexpensive shipping, handling and high stability of the product. This study was aimed to freeze dry P. putida cells with addition of lyoprotectant. Lyoprotectant was added into the cells suspension prior to freezing. Dried P. putida obtained was then mixed with synthetic mercury. Viability of recovery P. putida after freeze dry was significantly influenced by the type of lyoprotectant. Among the lyoprotectants, tween 80/ sucrose was found to be the best lyoprotectant. Sucrose able to recover more than 78% (6.2E+09 CFU/ml) of the original cells (7.90E+09CFU/ml) after freeze dry and able to retain 5.40E+05 viable cells after 4 weeks storage in 4oC without vacuum. Polyethylene glycol (PEG) pre-treated freeze dry cells and broth pre-treated freeze dry cells after freeze-dry recovered more than 64% (5.0 E+09CFU/ml) and >0.1% (5.60E+07CFU/ml). Freeze-dried P. putida cells in PEG and broth cannot survive after 4 weeks storage. Freeze dry also does not really change the pattern of growth P. putida but extension of lag time was found 1 hour after 3 weeks of storage. Additional time was required for freeze-dried P. putida cells to recover before introduce freeze-dried cells to more complicated condition such as mercury solution. The maximum mercury reduction of PEG pre-treated freeze-dried cells after freeze dry and after storage 3 weeks was 56.78% and 17.91%. The maximum of mercury reduction of tween 80/sucrose pre-treated freeze-dried cells after freeze dry and after storage 3 weeks were 26.35% and 25.03%. Freeze dried P. putida was found to have lower mercury reduction compare to the fresh P. putida that has been growth in agar. Result from this study may be beneficial and useful as initial reference before commercialize freeze-dried P. putida.

Keywords: Pseudomonas putida, freeze-dry, PEG, tween80/Sucrose, mercury, cell viability

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Authors: Mohammad Parvaresh, Mahdi Babaee, Bahareh Arghand, Davood Nourmohammadi

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Environmental degradation is caused by social inequalities and the inappropriate use of nature and a factor in the violation of human rights. Indeed, the right to a safe, healthy and ecologically-balanced environment is an independent human right. Therefore, the relationship between human rights and environmental protection is crucial for the study of social justice and sustainable development, and environmental problems are a result of the failure to realize social and economic justice. In this regard, 'article 50 of the constitution of the Islamic Republic of Iran as a general principle have many of the concepts of sustainable development, including: the growth and improvement of human life, the rights of present and future generations, and the integrity of the inner and outer generation, the prohibition of any environmental degradation'. Also, Charter on Citizen’s Rights, which was conveyed by the President of the Islamic Republic of Iran, Mr. Rouhani refers to the right to a healthy environment and sustainable development. In this regard in 2013, Tehran Province Water and Wastewater Co. defined a plan called 'Tehran’s Health Line' was includes Western and Eastern part by about 26 kilometers of water transferring pipelines varied 1000 to 2000 mm diameters. This project aims to: (1) Transfer water from the northwest water treatment plant to the southwest areas, which suffer from qualitative and quantitative water, in order to mix with the improper wells’ water; (2) Reducing the water consumption provided by harvesting from wells which results in improving the underground water resources, causing the large settlements and stopping the immigrating slums into the center or north side of the city. All of the financial resources accounted for 53,000,000 US$ which is mobilized by Tehran Province Water and Wastewater Co. to expedite the work. The present study examines the Tehran Health Line plan and the purpose of implementation of this plan to achieve environmental protection, environmental justice and citizenship rights for all people who live in Tehran.

Keywords: environmental justice, international environmental law, erga omnes, charter on citizen's rights, Tehran health line

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Authors: Heba Khader, Victor Solodushko, Brian Fouty

Abstract:

Hyperglycemia is a hallmark of uncontrolled diabetes and causes vascular endothelial dysfunction. An increase in glucose uptake and metabolism by vascular endothelial cells is the presumed trigger for this hyperglycemia-induced dysfunction. Glucose uptake into vascular endothelial cells is mediated largely by Glut-1. Glut-1 is an equilibrative glucose transporter with a Km value of 2 mM. At physiologic glucose concentrations, Glut-1 is almost saturated and, therefore, increasing glucose concentration does not increase glucose uptake unless Glut-1 is upregulated. However, hyperglycemia downregulates Glut-1 and decreases rather than increases glucose uptake in vascular endothelial cells. This apparent discrepancy necessitates further study on the effect of increasing glucose uptake on the oxidative state and function of vascular endothelial cells. To test this, a Tet-on system was generated to conditionally regulate Glut-1 expression in endothelial cells by the addition and removal of doxycycline. Glut-1 overexpression was confirmed by Western blot and radiolabeled glucose uptake measurements. Upregulation of Glut-1 resulted in a 4-fold increase in glucose uptake into endothelial cells as determined by 3H deoxy-D-glucose uptake. Increased glucose uptake through Glut-1 did not induce an oxidative stress nor did it cause endothelial dysfunction in rat pulmonary microvascular endothelial cells determined by monolayer resistance, cell proliferation or advanced glycation end product formation. Increased glucose uptake through Glut-1did not lead to an increase in glucose metabolism, due in part to inhibition of hexokinase in Glut-1 overexpressing cells. In summary, this study demonstrates that increasing glucose uptake and intracellular glucose by overexpression of Glut-1 does not alter the oxidative state of rat pulmonary microvascular endothelial cells or cause endothelial cell dysfunction. These results conflict with the current paradigm that hyperglycemia leads to oxidative stress and endothelial dysfunction in vascular endothelial cells through an increase in glucose uptake.

Keywords: endothelial cells, glucose uptake, Glut1, hyperglycemia

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Authors: P. I. Bobyleva, E. R. Andreeva, I. V. Andrianova, E. V. Maslova, L. B. Buravkova

Abstract:

This work studied the ability of adipose tissue-derived mesenchymal stromal cells (MSCs) to form stroma for expansion of cord blood hematopoietic cells. We showed that 72-hour interaction of MSCs with cord blood mononuclear cells (MNCs) in vitro at atmospheric (20%) and low (5%) O2 conditions increased the expression of ICAM-1, HCAM (at the beginning of interaction) on MSCs. Viability of MSCs and MNCs were maintained at high level. Adhesion of MNCs to MSCs was faster at 20% O2. MSCs promoted the proliferation of adhered MNCs to form the suspension containing great number of hematopoietic colony-forming units, and this effect was more pronounced at 5% O2. Thus, adipose-derived MSCs supplied sufficient stromal support to cord blood MNCs both at 20% and 5% О2, providing their adhesion with further expansion of new generation of different hematopoietic lineages.

Keywords: hematopoietic stem and progenitor cells, mesenchymal stromal cells, tissue-related oxygen, adipose tissue

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Authors: Yung-Chih Kuo, I-Hsuan Lee

Abstract:

Solid lipid nanoparticles (SLNs) conjugated with aprotinin (Apr) and melanotransferrin antibody (Anti-MTf) were used to carry doxorubicin (Dox) across the blood–brain barrier (BBB) for glioblastoma multiforme (GBM) chemotherapy. Dox-entrapped SLNs with grafted Apr and Anti-MTf (Apr-Anti-MTf-Dox-SLNs) were applied to a cultured monolayer comprising human brain-microvascular endothelial cells (HBMECs) with regulation of human astrocyte (HAs) and to a proliferated colony of U87MG cells. Based on the average particle diameter, zeta potential, entrapping efficiency of Dox, and grafting efficiency of Apr and Anti-MTf, we found that 40% (w/w) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in lipids were appropriate for fabricating Apr-Anti-MTf-Dox-SLNs. In addition, Apr-Anti-MTf-Dox-SLNs could prevent Dox from fast dissolution and did not induce a serious cytotoxicity to HBMECs and HAs when compared with free Dox. Moreover, the treatments with Apr-Anti-MTf-Dox-SLNs enhanced the ability of Dox to infuse the BBB and to inhibit the growth of GBM. The current Apr-Anti-MTf-Dox-SLNs can be a promising pharmacotherapeutic preparation to penetrate the BBB for malignant brain tumor treatment.

Keywords: solid lipid nanoparticle, glioblastoma multiforme, blood–brain barrier, doxorubicin

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Authors: Vytautas Galvanauskas, Vykantas Grincas, Rimvydas Simutis

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This paper investigates a possible practical solution for reasonable oxygen supply during the pluripotent stem cells expansion processes, where the stem cells propagate as aggregates in stirred-suspension bioreactors. Low glucose and low oxygen concentrations are preferred for efficient proliferation of pluripotent stem cells. However, strong oxygen limitation, especially inside of cell aggregates, can lead to cell starvation and death. In this research, the oxygen concentration profile inside of stem cell aggregates in a stem cell expansion process was predicted using a modified oxygen diffusion model. This profile can be realized during the stem cells cultivation process by manipulating the oxygen concentration in inlet gas or inlet gas flow. The proposed approach is relatively simple and may be attractive for installation in a real pluripotent stem cell expansion processes.

Keywords: aggregated stem cells, dissolved oxygen profiles, modeling, stirred-tank, 3D expansion

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Authors: Zayedali Shaikh

Abstract:

Increasingly now more than ever, UV damage brings with it a litany of minor deformities that can range from mild lesions and discoloring to cataracts and blindness. Pluripotent stem cells in planaria and human skin can be used to treat wounds and skin damage, with the primary limitations being inadequate growth factors. Photobiomodulation therapy in the form of low-intensity red light therapy has been proven to provide helpful benefits in the healing of skin that displays some of the symptoms of UV damage, such as burns and lesions, along with stimulating the proliferation of stem cells in recellularizing tissue. This paper puts forth an alternate means by which to treat the effects of UV damage using the freshwater planarian model system, Dugesia dorotocephala, known for its regenerative abilities and abundance of pluripotent stem cells, which allow for the rapid growth and repair of missing or damaged structures. Our work consisted of exposing planaria to different types of light: red light, blue light, white light, darkness, red and blue light together, UV light, and finally, red and UV light together. The primary focus of this research was on the red and UV lights, with six controls acting as metrics to compare our findings. Through computer-assisted morphological analysis, the results show that there is no significant difference in the rates of regeneration of planaria treated with simultaneous exposure to red and UV light versus planaria in darkness (p > .05), a representation of their preferred natural habitat. Our research suggests the viability of red-light therapy in actively combating UV damage and expediting the growth of epidermal stem cells by acting as another growth factor.

Keywords: regenerative medicine, stem cells, planaria, photobiomodulation

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Authors: C. F. Kumru, C. Kocatepe, O. Arikan

Abstract:

In this study, electric field distribution analyses for three pylon models are carried out by a Finite Element Method (FEM) based software. Analyses are performed in both stationary and time domains to observe instantaneous values along with the effective ones. Considering the results of the study, different line geometries is considerably affecting the magnitude and distribution of electric field although the line voltages are the same. Furthermore, it is observed that maximum values of instantaneous electric field obtained in time domain analysis are quite higher than the effective ones in stationary mode. In consequence, electric field distribution analyses should be individually made for each different line model and the limit exposure values or distances to residential buildings should be defined according to the results obtained.

Keywords: electric field, energy transmission line, finite element method, pylon

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Authors: Chien Y. Lin, Jung Y. Huang, Leu-Wei Lo

Abstract:

A growing body of data reveals that the membrane cholesterol molecules can alter the signaling pathways of living cells. However, the understanding about how membrane cholesterol modulates receptor proteins is still lacking. Single-molecule tracking can effectively probe into the microscopic environments and thermal fluctuations of receptor proteins in a living cell. In this study we applies single-molecule optical tracking on ligand-induced dimerization process of EGFRs in the plasma membranes of two cancer cell lines (HeLa and A431) and one normal endothelial cell line (MCF12A). We tracked individual EGFR and dual receptors, diffusing in a correlated manner in the plasma membranes of live cells. We developed an energetic model by integrating the generalized Langevin equation with the Cahn-Hilliard equation to help extracting important information from single-molecule trajectories. From the study, we discovered that ligand-bound EGFRs move from non-raft areas into lipid raft domains. This ligand-induced motion is a common behavior in both cancer and normal cells. By manipulating the total amount of membrane cholesterol with methyl-β-cyclodextrin and the local concentration of membrane cholesterol with nystatin, we further found that the amount of cholesterol can affect the stability of EGFR dimers. The EGFR dimers in the plasma membrane of normal cells are more sensitive to the local concentration changes of cholesterol than EGFR dimers in the cancer cells. Our method successfully captures dynamic interactions of receptors at the single-molecule level and provides insight into the functional architecture of both the diffusing EGFR molecules and their local cellular environment.

Keywords: membrane proteins, single-molecule tracking, Cahn-Hilliard equation, EGFR dimers

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Authors: Md. Mizanur Rahman, Anquan Liu, Anna Frostegård, Johan Frostegård

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The role of the human immune system is essential in cardiovascular diseases and atherosclerosis. Activated cells in atherosclerosis produce abundant amounts of cytokines, but the exact mechanisms involved in the effects of these inflammatory cytokines are not clear in atherosclerosis. In a large clinical cohort, we have previously determined that antibodies against phosphorylcholine (anti-PC) are negatively and independently associated with both development of atherosclerosis and also a low risk of cardiovascular disease. Further, we reported that rheumatoid arthritis patients who were non-responders to TNF-inhibitors, where those with low anti-PC levels. Upon anti-TNF treatment, anti-PC levels increased. We, therefore, hypothesised that proinflammatory cytokines such as TNF could play a role in anti-PC regulation. Peripheral blood mononuclear cells (PBMC) were cultured with or without TNF and anti-TNF. The cell supernatants were collected after six days for ELISA measurements. In separate experiments, cells were cultured for 24 hours in both polystyrene plates and ELISPOT plates under a similar condition for ELISA and ELISPOT assays respectively. Total RNA was extracted after 6 hours of cell culture to perform RT-qPCR. Cell viability was confirmed by trypan blue staining and MTT assays. ELISA measurements detected less than 40% of anti-PC in TNF-treated cells, in comparison to control cells, whereas anti-PC production was recovered by anti-TNF treatment. ELISPOT assays showed that TNF suppresses anti-PC production by inhibiting anti-PC producing B-cells. In addition, RT-qPCR and ELISA showed that TNF also has effects also on B-cell activation as BAFF expression was inhibited by TNF treatment. Atherosclerosis is a major cause of cardiovascular diseases, but anti-PC is a protection marker for atherosclerosis development. Our findings show that TNF is a negative regulator of anti-PC production. Immune modulation and rising of anti-PC could be of major significance for the patients.

Keywords: anti-PC, Anti-TNF, atherosclerosis, cardiovascular diseases, phosphorylecholine

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Authors: Arzu Karahan, Loriano Ballarin, Baruch Rinkevich

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Marine invertebrates, the highly diverse phyla of multicellular organisms, represent phenomena that are either not found or highly restricted in the vertebrates. These include phenomena like budding, fission, a fusion of ramets, and high regeneration power, such as the ability to create whole new organisms from either tiny parental fragment, many of which are controlled by totipotent, pluripotent, and multipotent stem cells. Thus, there is very much that can be learned from these organisms on the practical and evolutionary levels, further resembling Darwin's words, “It is not the strongest of the species that survives, nor the most intelligent, but the one most responsive to change”. The ‘stem cell’ notion highlights a cell that has the ability to continuously divide and differentiate into various progenitors and daughter cells. In vertebrates, adult stem cells are rare cells defined as lineage-restricted (multipotent at best) with tissue or organ-specific activities that are located in defined niches and further regulate the machinery of homeostasis, repair, and regeneration. They are usually categorized by their morphology, tissue of origin, plasticity, and potency. The above description not always holds when comparing the vertebrates with marine invertebrates’ stem cells that display wider ranges of plasticity and diversity at the taxonomic and the cellular levels. While marine/aquatic invertebrates stem cells (MISC) have recently raised more scientific interest, the know-how is still behind the attraction they deserve. MISC, not only are highly potent but, in many cases, are abundant (e.g., 1/3 of the entire animal cells), do not locate in permanent niches, participates in delayed-aging and whole-body regeneration phenomena, the knowledge of which can be clinically relevant. Moreover, they have massive hidden potential for the discovery of new bioactive molecules that can be used for human health (antitumor, antimicrobial) and biotechnology. The MARISTEM COST action (Stem Cells of Marine/Aquatic Invertebrates: From Basic Research to Innovative Applications) aims to connect the European fragmented MISC community. Under this scientific umbrella, the action conceptualizes the idea for adult stem cells that do not share many properties with the vertebrates’ stem cells, organizes meetings, summer schools, and workshops, stimulating young researchers, supplying technical and adviser support via short-term scientific studies, making new bridges between the MISC community and biomedical disciplines.

Keywords: aquatic/marine invertebrates, adult stem cell, regeneration, cell cultures, bioactive molecules

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Authors: Amelia J. McFarland, Andrew K. Davey, Shailendra Anoopkumar-Dukie

Abstract:

Microglia are the resident macrophage population of the central nervous system (CNS), contributing to both innate and adaptive immune response, and brain homeostasis. Activation of microglia occurs in response to a multitude of pathogenic stimuli in their microenvironment; this induces morphological and functional changes, resulting in a state of acute neuroinflammation which facilitates injury resolution. Adequate microglial function is essential for the health of the neuroparenchyma, with microglial dysfunction implicated in numerous CNS pathologies. Given the critical role that these macrophage-derived cells play in CNS homeostasis, there is a high demand for microglial models suitable for use in neuroscience research. The isolation of primary human microglia, however, is both difficult and costly, with microglial activation an unwanted but inevitable result of the extraction process. Consequently, there is a need for the development of alternative experimental models which exhibit morphological, biochemical and functional characteristics of human microglia without the difficulties associated with primary cell lines. In this study, our aim was to evaluate whether THP-1 human peripheral blood monocytes would display microglial-like qualities following an induced differentiation, and, therefore, be suitable for use as surrogate microglia. To achieve this aim, THP-1 human peripheral blood monocytes from acute monocytic leukaemia were differentiated with a range of phorbol 12-myristate 13-acetate (PMA) concentrations (50-200 nM) using two different protocols: a 5-day continuous PMA exposure or a 3-day continuous PMA exposure followed by a 5-day rest in normal media. In each protocol and at each PMA concentration, microglial-like cell morphology was assessed through crystal violet staining and the presence of CD-14 microglial / macrophage cell surface marker. Lipopolysaccharide (LPS) from Escherichia coli (055: B5) was then added at a range of concentrations from 0-10 mcg/mL to activate the PMA-differentiated THP-1 cells. Functional microglial-like behavior was evaluated by quantifying the release of prostaglandin (PG)-E2 and pro-inflammatory cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α using mediator-specific ELISAs. Furthermore, production of global reactive oxygen species (ROS) and nitric oxide (NO) were determined fluorometrically using dichlorodihydrofluorescein diacetate (DCFH-DA) and diaminofluorescein diacetate (DAF-2-DA) respectively. Following PMA-treatment, it was observed both differentiation protocols resulted in cells displaying distinct microglial morphology from 10 nM PMA. Activation of differentiated cells using LPS significantly augmented IL-1β, TNF-α and PGE2 release at all LPS concentrations under both differentiation protocols. Similarly, a significant increase in DCFH-DA and DAF-2-DA fluorescence was observed, indicative of increases in ROS and NO production. For all endpoints, the 5-day continuous PMA treatment protocol yielded significantly higher mediator levels than the 3-day treatment and 5-day rest protocol. Our data, therefore, suggests that the differentiation of THP-1 human monocyte cells with PMA yields a homogenous microglial-like population which, following stimulation with LPS, undergo activation to release a range of pro-inflammatory mediators associated with microglial activation. Thus, the use of PMA-differentiated THP-1 cells represents a suitable microglial model for in vitro research.

Keywords: differentiation, lipopolysaccharide, microglia, monocyte, neuroscience, THP-1

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Authors: Fatemeh Abbasi, Arne Hofemeier, Timo Betz

Abstract:

Muscle growth and regeneration critically depend on satellite cells (SCs) which are muscle stem cells located between the basal lamina and myofibres. Upon damage, SCs become activated, enter the cell cycle, and give rise to myoblasts that form new myofibres, while a sub-population self-renew and re-populate the muscle stem cell niche. In aged muscle as well as in certain muscle diseases such as muscular dystrophy, some of the SCs lose their regenerative ability. Although it is demonstrated that the chemical composition of SCs quiescent niche is different from the activated niche, the mechanism initially activated in the SCs remains unknown. While extensive research efforts focused on potential chemical activation, no such factor has been identified to the author’s best knowledge. However, it is substantiated that niche mechanics affects SCs behaviors, such as stemness and engraftment. We hypothesize that mechanical stress in the healthy niche (homeostasis) is different from the regenerative niche and that this difference could serve as an early signal activating SCs upon fiber damage. To investigate this hypothesis, we develop a biomimetic system to reconstitute both, the mechanical and the chemical environment of the SC niche. Cells will be confined between two elastic polyacrylamide (PAA) hydrogels with controlled elastic moduli and functionalized surface chemistry. By controlling the distance between the PAA hydrogel surfaces, we vary the compression forces exerted by the substrates on the cells, while the lateral displacement of the upper hydrogel will create controlled shear forces. To establish such a system, a simplified system is presented. We engineered a sandwich-like configuration of two elastic PAA layer with stiffnesses between 1 and 10 kPa and confined a precursor myoblast cell line (C2C12) in between these layers. Our initial observations in this sandwich model indicate that C2C12 cells show different behaviors under mechanical compression if compared to a control one-layer gel without compression. Interestingly, this behavior is stiffness-dependent. While the shape of C2C12 cells in the sandwich consisting of two stiff (10 kPa) layers was much more elongated, showing almost a neuronal phenotype, the cell shape in a sandwich situation consisting of one stiff and one soft (1 kPa) layer was more spherical. Surprisingly, even in proliferation medium and at very low cell density, the sandwich situation stimulated cell differentiation with increased striation and myofibre formation. Such behavior is commonly found for confluent cells in differentiation medium. These results suggest that mechanical changes in stiffness and applied pressure might be a relevant stimulation for changes in muscle cell behavior.

Keywords: C2C12 cells, compression, force, satellite cells, skeletal muscle

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