Search results for: cell engineering

Authors: Leo Nnamdi Ozurumba-Dwight

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Messenger ribooxynucleic acid (mRNA) molecules are compositional, protein-based. These proteins, encoding mRNA molecules (which collectively connote the transcriptome), when analyzed by RNA sequencing (RNAseq), unveils the nature of gene expression in the RNA. The obtained gene expression provides clues of cellular traits and their dynamics in presentations. These can be studied in relation to function and responses. RNAseq is a practical concept in Genomics as it enables detection and quantitative analysis of mRNA molecules. Single cell and spatial transcriptomics both present varying avenues for expositions in genomic characteristics of single cells and pooled cells in disease conditions such as cancer, auto-immune diseases, hematopoietic based diseases, among others, from investigated biological tissue samples. Single cell transcriptomics helps conduct a direct assessment of each building unit of tissues (the cell) during diagnosis and molecular gene expressional studies. A typical technique to achieve this is through the use of a single-cell RNA sequencer (scRNAseq), which helps in conducting high throughput genomic expressional studies. However, this technique generates expressional gene data for several cells which lack presentations on the cells’ positional coordinates within the tissue. As science is developmental, the use of complimentary pre-established tissue reference maps using molecular and bioinformatics techniques has innovatively sprung-forth and is now used to resolve this set back to produce both levels of data in one shot of scRNAseq analysis. This is an emerging conceptual approach in methodology for integrative and progressively dependable transcriptomics analysis. This can support in-situ fashioned analysis for better understanding of tissue functional organization, unveil new biomarkers for early-stage detection of diseases, biomarkers for therapeutic targets in drug development, and exposit nature of cell-to-cell interactions. Also, these are vital genomic signatures and characterizations of clinical applications. Over the past decades, RNAseq has generated a wide array of information that is igniting bespoke breakthroughs and innovations in Biomedicine. On the other side, spatial transcriptomics is tissue level based and utilized to study biological specimens having heterogeneous features. It exposits the gross identity of investigated mammalian tissues, which can then be used to study cell differentiation, track cell line trajectory patterns and behavior, and regulatory homeostasis in disease states. Also, it requires referenced positional analysis to make up of genomic signatures that will be sassed from the single cells in the tissue sample. Given these two presented approaches to RNA transcriptomics study in varying quantities of cell lines, with avenues for appropriate resolutions, both approaches have made the study of gene expression from mRNA molecules interesting, progressive, developmental, and helping to tackle health challenges head-on.

Keywords: transcriptomics, RNA sequencing, single cell, spatial, gene expression.

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Authors: Guillaume Soubeyran, Fabrice Micoud, Benoit Morin, Jean-Philippe Poirot-Crouvezier, Magali Reytier

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Proton Exchange Membrane Fuel Cell (PEMFC) technology is considered as a solution for the reduction of CO2 emissions. However, this technology still meets several challenges for high-scale industrialization. In this context, the increase of durability remains a critical aspect for competitiveness of this technology. Fortunately, performance degradations in nominal operating conditions is partially reversible, meaning that if specific conditions are applied, a partial recovery of fuel cell performance can be achieved, while irreversible degradations can only be mitigated. Thus, it is worth studying the optimal conditions to rejuvenate these reversible degradations and assessing the long-term impact of such procedures on the performance of the cell. Reversible degradations consist mainly of anode Pt active sites poisoning by carbon monoxide at the anode, heterogeneities in water management during use, and oxidation/deactivation of Pt active sites at the cathode. The latter is identified as a major source of reversible performance loss caused by the presence oxygen, high temperature and high cathode potential that favor platinum oxidation, especially in high efficiency operating points. Hence, we studied here a recovery procedure aiming at reducing the platinum oxides by decreasing cathode potential during operation. Indeed, the application of short air starvation phase leads to a drop of cathode potential. Cell performances are temporarily increased afterwards. Nevertheless, local temperature and current heterogeneities within the cells are favored and shall be minimized. The consumption of fuel during the recovery phase shall also be considered to evaluate the global efficiency. Consequently, the purpose of this work is to find an optimal compromise between the recovery of reversible degradations by air starvation, the increase of global cell efficiency and the mitigation of irreversible degradations effects. Different operating parameters have first been studied such as cell voltage, temperature and humidity in single cell set-up. Considering the global PEMFC system efficiency, tests showed that reducing duration of recovery phase and reducing cell voltage was the key to ensure an efficient recovery. Recovery phase frequency was a major factor as well. A specific method was established to find the optimal frequency depending on the duration and voltage of the recovery phase. Then, long-term degradations have also been studied by applying FC-DLC cycles based on NEDC cycles on a 4-cell short stack by alternating test sequences with and without recovery phases. Depending on recovery phase timing, cell efficiency during the cycle was increased up to 2% thanks to a mean voltage increase of 10 mV during test sequences with recovery phases. However, cyclic voltammetry tests results suggest that the implementation of recovery phases causes an acceleration of the decrease of platinum active areas that could be due to the high potential variations applied to the cathode electrode during operation.

Keywords: durability, PEMFC, recovery procedure, reversible degradation

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Authors: Geovanna dos Santos Romeiro, Polintia Rayza Brito da Silva, Luis Henrique Moura, Izadora Moreira Do Amaral, Marília Vitória Pinto Milhomem

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Introduction: The nose is one of the most likely sites for the appearance of malignancy on the face. This can be associated with its unique position of exposure to environmental damage, lack of photoprotection and because it is an area susceptible to greater sun exposure. It is already known that the most common type of nasal tumor is basal cell carcinoma. Squamous cell carcinoma is less common but considerably more aggressive, with a tendency to grow rapidly and metastasize. Nasal skin cancer can have a good prognosis, regardless of the type of treatment chosen, i.e., surgery, radiotherapy or electrodissection. However, tumors that are not diagnosed and treated quickly can be harmful and have a greater chance of metastasizing. When curative surgery is performed, therapies and reconstructive surgical procedures are usually required. Objective: The objective is to review the literature on nasal skin tumors and their types and specific locations. Forty-four articles published in Pubmed related to the location of skin cancer in the specific nasal areas region were analyzed. Twelve were excluded for being prior to the year 2000, three with inconclusive results, and one with unbiased conclusions. Results and Conclusion: Regarding the prevalence of types of nasal tumors, basal cell carcinoma comprises the majority, occurring predominantly in the ala, tip and root; squamous cell carcinoma, on the other hand, is more common in the lateral borders and columella. Even so, 2 articles report that the prevalence of metastasis has a higher incidence in squamous cell carcinomas. All of this points to the importance of early location, including regions that are often overlooked in the examination if the patient is wearing glasses. This topic needs further investigation for a greater correlation between anatomy and clinical-surgical implications.

Keywords: skin cancer, melanoma, non-melanoma, surgery

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Authors: H. Oudira, A. Saifi

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The therapeutic utility of certain Auger emitters such as iodine-125 depends on their position within the cell nucleus . Or diagnostically, and to maintain as low as possible cell damage, it is preferable to have radionuclide localized outside the cell or at least the core. One solution to this problem is to consider markers capable of conveying anticancer drugs to the tumor site regardless of their location within the human body. The objective of this study is to simulate the impact of a complex such as bleomycin on single and double strand breaks in the DNA molecule. Indeed, this simulation consists of the following transactions: - Construction of BLM -Fe- DNA complex. - Simulation of the electron’s transport from the metastable state excitation of Fe 57 by the Monte Carlo method. - Treatment of chemical reactions in the considered environment by the diffusion equation. For physical, physico-chemical and finally chemical steps, the geometry of the complex is considered as a sphere of 50 nm centered on the binding site , and the mathematical method used is called step by step based on Monte Carlo codes.

Keywords: concentration, yield, radical species, bleomycin, excitation, DNA

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Authors: Pyi Phyo Maung

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In the study, the calculations of the permeability coefficient, values of the volume and porosity of a unit cell of a woven fabric before and after deformation based on the geometrical parameters are presented. Two types of carbon woven fabric structures were investigated: standard type, which integrated the filament, has a cross sectional shape of a cylinder and spread tow type, which has a rectangular cross sectional shape. The space antennas reflector, which distinctive feature is the presence of the surface of double curvature, is considered as the object of the research. Modeling of the kinetics of the process of impregnation of the reflector for the two types of carbon fabric’s unit cell structures was performed using software RAM-RTM. This work also investigated the influence of the grid angle between warp and welt of the unit cell on the duration of impregnation process. The results showed that decreasing the angle between warp and welt of the unit cell, the decreasing of the permeability values were occurred. Based on the results of calculation samples of the reflectors, their quality was determined. The comparisons of the theoretical and experimental results have been carried out. Comparison of the two textile structures (standard and spread tow) showed that the standard textiles with circular cross section were impregnated faster than spread tows, which have a rectangular cross section.

Keywords: vacuum assistant resin infusion, impregnation time, shear angle, reflector and modeling

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Authors: Sara Pereira-Nogueira, Tim Worbs, Marc Permanyer-Bosser, Reinhold Förster

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While the entry mechanism of lymphocytes into the lymph node via the blood are well described, it is still largely unknown how cells enter lymph nodes that arrive via afferent lymphatics. In order to address this, our group has established a micro-injection technique in mice through which cells are delivered directly into the lymphatic vessel immediately afferent to the popliteal lymph node. Injected cells can then be tracked via multi-colour fluorescence or 2-photon microscopy, and their localization can be analysed within the popliteal or downstream lymph nodes by immunohistology. Since naïve B cells express the chemokine receptor CXCR5 we intra-lymphatically co-injected B cells derived from wildtype and Cxcr5-deficient mice. While CXCR5 does not play a role in guiding B cells out of the subcapsular sinus, it affects their positioning within the lymph node parenchyma, since CXCR5-deficient B cells are impaired in migrating into the B cell follicle. The knowledge obtained by studying B-cell migration may prove beneficial in clinical settings regarding tumor metastasis or autoimmune diseases.

Keywords: afferent lymphatics, B cell migration, chemokine, intra-lymphatic injection

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Authors: Fatemeh Abbasi, Arne Hofemeier, Timo Betz

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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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Authors: Rima Harche, Abdelkader Mouheb

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The Algiers refinery as all the other refineries always suffers from the problem of stopping of the tubes of heat exchanger. For that a study experimental of this phenomenon was undertaken in site on the cell of heat exchangers E101 (E101 CBA and E101 EDF) intended for the heating of the crude before its fractionation, which are exposed to the problem of the fouling on the side tubes exchangers. It is of tube-calenders type with head floating. Each cell is made up of three heat exchangers, laid out in series.

Keywords: fouling, fluid temperatue , oil, tubular heat exchanger, fouling resistance, modeling, heat transfer coefficient

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Authors: Leo Nnamdi Ozurumba-Dwight

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Single-cell genomic analytical systems have proved to be a platform to isolate bulk cells into selected single cells for genomic, proteomic, and related metabolomic studies. This is enabling systematic investigations of the level of heterogeneity in a diverse and wide pool of cell populations. Single cell technologies, embracing techniques such as high parameter flow cytometry, single-cell sequencing, and high-resolution images are playing vital roles in these investigations on messenger ribonucleic acid (mRNA) molecules and related gene expressions in tracking the nature and course of disease conditions. This entails targeted molecular investigations on unit cells that help us understand cell behavoiur and expressions, which can be examined for their health implications on the health state of patients. One of the vital good sides of single-cell RNA sequencing (scRNA seq) is its probing capacity to detect deranged or abnormal cell populations present within homogenously perceived pooled cells, which would have evaded cursory screening on the pooled cell populations of biological samples obtained as part of diagnostic procedures. Despite conduction of just single-cell transcriptome analysis, scRNAseq now permits comparison of the transcriptome of the individual cells, which can be evaluated for gene expressional patterns that depict areas of heterogeneity with pharmaceutical drug discovery and clinical treatment applications. It is vital to strictly work through the tools of investigations from wet lab to bioinformatics and computational tooled analyses. In the precise steps for scRNAseq, it is critical to do thorough and effective isolation of viable single cells from the tissues of interest using dependable techniques (such as FACS) before proceeding to lysis, as this enhances the appropriate picking of quality mRNA molecules for subsequent sequencing (such as by the use of Polymerase Chain Reaction machine). Interestingly, scRNAseq can be deployed to analyze various types of biological samples such as embryos, nervous systems, tumour cells, stem cells, lymphocytes, and haematopoietic cells. In haematopoietic cells, it can be used to stratify acute myeloid leukemia patterns in patients, sorting them out into cohorts that enable re-modeling of treatment regimens based on stratified presentations. In immunotherapy, it can furnish specialist clinician-immunologist with tools to re-model treatment for each patient, an attribute of precision medicine. Finally, the good predictive attribute of scRNAseq can help reduce the cost of treatment for patients, thus attracting more patients who would have otherwise been discouraged from seeking quality clinical consultation help due to perceived high cost. This is a positive paradigm shift for patients’ attitudes primed towards seeking treatment.

Keywords: immunotherapy, transcriptome, re-modeling, mRNA, scRNA-seq

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Authors: Karl Kingsley

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Many mechanisms are involved in the control of cellular differentiation and growth, which are often dysregulated in many cancers. Many distinct pathways are involved in these mechanisms of control, including deoxyribonuclease (DNA) methyltransferase and histone deacetylase (HDAC) activation that controls both genetic and epigenetic modifications and micro ribonucleic acid (RNA) expression. Less is known about the expression of DNA methyltransferase (DNMT) and HDAC in oral cancers and the effect on microRNA expression. The primary objective of this study was to evaluate the expression of DNMT and HDAC family members in oral cancer and the concomitant expression of cancer-associated microRNAs. Using commercially available oral cancers, including squamous cell carcinoma (SCC)-4, SCC-9, SCC-15, and SCC-25, RNA was extracted and screened for DNMT, HDAC, and microRNA expression using highly-specific primers and quantitative polymerase chain reaction (qPCR). These data revealed low or absent expression of DNMT-1, which is associated with cellular differentiation but increased expression of DNMT-3a and DNMT-3b in all SCC cell lines compared with normal non-cancerous cell controls. In addition, no expression of HDAC1 and HDAC2 expression was found among the normal, non-cancerous cells but was highly expressed in each of the SCC cell lines examined. Differential expression of oncogenic and cancer-associated microRNAs was also observed among the SCC cell lines, including miR-21, miR-133, miR-149, miR-155, miR-365, and miR-720. These findings also appeared to vary according to observed growth rates among these cells. These data may be the first to demonstrate the expression and association between HDAC and DNMT3 family members among oral cancers. In addition, the differential expression of these epigenetic modifiers may be associated with the expression of specific microRNAs in these cancers, which have not previously been observed to the best of the author's knowledge. In addition, some associations and relationships may exist between the expression of these biomarkers and the rates of growth and proliferation, which may suggest that these expression patterns might represent potentially useful biomarkers to determine tumor aggressiveness and other phenotypic behaviors among oral cancers.

Keywords: oral cancer, DNA methyltransferase, histone deacetylase, microRNA

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Authors: Hilal T. Sasmazel, Seda Surucu

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Skin tissue engineering is a promising field for the treatment of skin defects using scaffolds. This approach involves the use of living cells and biomaterials to restore, maintain, or regenerate tissues and organs in the body by providing; (i) larger surface area for cell attachment, (ii) proper porosity for cell colonization and cell to cell interaction, and (iii) 3-dimensionality at macroscopic scale. Recent studies on this area mainly focus on fabrication of scaffolds that can closely mimic the natural extracellular matrix (ECM) for creation of tissue specific niche-like environment at the subcellular scale. Scaffolds designed as ECM-like architectures incorporating into the host with minimal scarring/pain and facilitate angiogenesis. This study is related to combining of synthetic PCL and natural chitosan polymers to form 3D PCL/Chitosan core-shell structures for skin tissue engineering applications. Amongst the polymers used in tissue engineering, natural polymer chitosan and synthetic polymer poly(ε-caprolactone) (PCL) are widely preferred in the literature. Chitosan has been among researchers for a very long time because of its superior biocompatibility and structural resemblance to the glycosaminoglycan of bone tissue. However, the low mechanical flexibility and limited biodegradability properties reveals the necessity of using this polymer in a composite structure. On the other hand, PCL is a versatile polymer due to its low melting point (60°C), ease of processability, degradability with non-enzymatic processes (hydrolysis) and good mechanical properties. Nevertheless, there are also several disadvantages of PCL such as its hydrophobic structure, limited bio-interaction and susceptibility to bacterial biodegradation. Therefore, it became crucial to use both of these polymers together as a hybrid material in order to overcome the disadvantages of both polymers and combine advantages of those. The scaffolds here were fabricated by using electrospinning technique and the characterizations of the samples were done by contact angle (CA) measurements, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-Ray Photoelectron spectroscopy (XPS). Additionally, gas permeability test, mechanical test, thickness measurement and PBS absorption and shrinkage tests were performed for all type of scaffolds (PCL, chitosan and PCL/chitosan core-shell). By using ImageJ launcher software program (USA) from SEM photographs the average inter-fiber diameter values were calculated as 0.717±0.198 µm for PCL, 0.660±0.070 µm for chitosan and 0.412±0.339 µm for PCL/chitosan core-shell structures. Additionally, the average inter-fiber pore size values exhibited decrease of 66.91% and 61.90% for the PCL and chitosan structures respectively, compare to PCL/chitosan core-shell structures. TEM images proved that homogenous and continuous bead free core-shell fibers were obtained. XPS analysis of the PCL/chitosan core-shell structures exhibited the characteristic peaks of PCL and chitosan polymers. Measured average gas permeability value of produced PCL/chitosan core-shell structure was determined 2315±3.4 g.m-2.day-1. In the future, cell-material interactions of those developed PCL/chitosan core-shell structures will be carried out with L929 ATCC CCL-1 mouse fibroblast cell line. Standard MTT assay and microscopic imaging methods will be used for the investigation of the cell attachment, proliferation and growth capacities of the developed materials.

Keywords: chitosan, coaxial electrospinning, core-shell, PCL, tissue scaffold

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Authors: Ya L. V., Benjamin Ong Wei Lin, Wanli Niu, Benjamin Seah Chin Tat

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This article introduces a methodology that improves reliability and cyclability of 2nd-life Li-ion battery system repurposed as an energy storage system (ESS). Most of the 2nd-life retired battery systems in the market have module/pack-level state-of-health (SOH) indicator, which is utilized for guiding appropriate depth-of-discharge (DOD) in the application of ESS. Due to the lack of cell-level SOH indication, the different degrading behaviors among various cells cannot be identified upon reaching retired status; in the end, considering end-of-life (EOL) loss and pack-level DOD, the repurposed ESS has to be oversized by > 1.5 times to complement the application requirement of reliability and cyclability. This proposed battery grading algorithm, using non-invasive methodology, is able to detect outlier cells based on historical voltage data and calculate cell-level historical maximum temperature data using semi-analytic methodology. In this way, the individual battery cell in the 2nd-life battery system can be graded in terms of SOH on basis of the historical voltage fluctuation and estimated historical maximum temperature variation. These grades will have corresponding DOD grades in the application of the repurposed ESS to enhance system reliability and cyclability. In all, this introduced battery grading algorithm is non-invasive, compatible with all kinds of retired Li-ion battery systems which lack of cell-level SOH indication, as well as potentially being embedded into battery management software for preventive maintenance and real-time cyclability optimization.

Keywords: battery grading algorithm, 2nd-life repurposing battery system, semi-analytic methodology, reliability and cyclability

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Authors: David A. Ullisch, Yantree D. Sankar-Thomas, Stefan Wilke, Thomas Selge, Matthias Pump, Thomas Leibold, Kai Schütte, Gilbert Gorr

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Plants have been considered as a source of natural substances for ages. Secondary metabolites from plants are utilized especially in medical applications but are more and more interesting as cosmetical ingredients and in the field of nutraceuticals. However, supply of compounds from natural harvest can be limited by numerous factors i.e. endangered species, low product content, climate impacts and cost intensive extraction. Especially in the pharmaceutical industry the ability to provide sufficient amounts of product and high quality are additional requirements which in some cases are difficult to fulfill by plant harvest. Whereas in many cases the complexity of secondary metabolites precludes chemical synthesis on a reasonable commercial basis, plant cells contain the biosynthetic pathway – a natural chemical factory – for a given compound. A promising approach for the sustainable production of natural products can be plant cell fermentation (PCF®). A thoroughly accomplished development process comprises the identification of a high producing cell line, optimization of growth and production conditions, the development of a robust and reliable production process and its scale-up. In order to address persistent, long lasting production, development of cryopreservation protocols and generation of working cell banks is another important requirement to be considered. So far the most prominent example using a PCF® process is the production of the anticancer compound paclitaxel. To demonstrate the power of plant suspension cultures here we present three case studies: 1) For more than 17 years Phyton produces paclitaxel at industrial scale i.e. up to 75,000 L in scale. With 60 g/kg dw this fully controlled process which is applied according to GMP results in outstanding high yields. 2) Thapsigargin is another anticancer compound which is currently isolated from seeds of Thapsia garganica. Thapsigargin is a powerful cytotoxin – a SERCA inhibitor – and the precursor for the derivative ADT, the key ingredient of the investigational prodrug Mipsagargin (G-202) which is in several clinical trials. Phyton successfully generated plant cell lines capable to express this compound. Here we present data about the screening for high producing cell lines. 3) The third case study covers ingenol-3-mebutate. This compound is found in the milky sap of the intact plants of the Euphorbiacae family at very low concentrations. Ingenol-3-mebutate is used in Picato® which is approved against actinic keratosis. Generation of cell lines expressing significant amounts of ingenol-3-mebutate is another example underlining the strength of plant cell culture. The authors gratefully acknowledge Inspyr Therapeutics for funding.

Keywords: Ingenol-3-mebutate, plant cell culture, sustainability, thapsigargin

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Authors: Jienny Lee, In-Soo Cho, Sang-Ho Cha

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Adult mesenchymal stem cells (MSCs) have been investigated using preclinical approaches for tissue regeneration. Porcine MSCs (pMSCs) are capable of growing and attaching to plastic with a fibroblast-like morphology and then differentiating into bone, adipose, and cartilage tissues in vitro. This study was conducted to investigate the proliferating abilities, differentiation potentials, and multipotency of miniature pig adipose tissue-derived MSCs (mpAD-MSCs) with or without long-term cryopreservation, considering that cryostorage has the potential for use in clinical applications. After confirming the characteristics of the mpAD-MSCs, we examined the effect of long-term cryopreservation (> 2 years) on expression of cell surface markers (CD34, CD90 and CD105), proliferating abilities (cumulative population doubling level, doubling time, colony-forming unit, and MTT assay) and differentiation potentials into mesodermal cell lineages. As a result, the expression of cell surface markers is similar between thawed and fresh mpAD-MSCs. However, long-term cryopreservation significantly lowered the differentiation potentials (adipogenic, chondrogenic, and osteogenic) of mpAD-MSCs. When compared with fresh mpAD-MSCs, thawed mpAD-MSCs exhibited lower expression of mesodermal cell lineage-related genes such as peroxisome proliferator-activated receptor-g2, lipoprotein lipase, collagen Type II alpha 1, osteonectin, and osteocalcin. Interestingly, long-term cryostoraged mpAD-MSCs exhibited significantly higher cell viability than the fresh mpAD-MSCs. Long-term cryopreservation induced a 30% increase in the cell viability of mpAD-MSCs when compared with the fresh mpAD-MSCs at 5 days after thawing. However, long-term cryopreservation significantly lowered expression of stemness markers such as Oct3/4, Sox2, and Nanog. Furthermore, long-term cryopreservation negatively affected expression of senescence-associated genes such as telomerase reverse transcriptase and heat shock protein 90 of mpAD-MSCs when compared with the fresh mpAD-MSCs. The results from this study might be important for the successful application of MSCs in clinical trials after long-term cryopreservation.

Keywords: mesenchymal stem cells, cryopreservation, stemness, senescence

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Authors: Vongsathorn Ngampuak, Yutachai Chookaew, Wipawee Dejtisakdi

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Dunaliella salina has great potential as a system for generating commercially valuable products, including beta-carotene, pharmaceuticals, and biofuels. Our goal is to improve this potential by enhancing growth rate and other properties of D. salina under optimal growth conditions. We used ethyl methane sulfonate (EMS) to generate random mutants in D. salina KU11, a strain classified in Thailand. In a preliminary experiment, we first treated D. salina cells with 0%, 0.8%, 1.0%, 1.2%, 1.44% and 1.66% EMS to generate a killing curve. After that, we randomly picked 30 candidates from approximately 300 isolated survivor colonies from the 1.44% EMS treatment (which permitted 30% survival) as an initial test of the mutant screen. Among the 30 survivor lines, we found that 2 strains (mutant #17 and #24) had significantly improved growth rates and cell number accumulation at stationary phase approximately up to 1.8 and 1.45 fold, respectively, 2 strains (mutant #6 and #23) had significantly decreased growth rates and cell number accumulation at stationary phase approximately down to 1.4 and 1.35 fold, respectively, while 26 of 30 lines had similar growth rates compared with the wild type control. We also analyzed cell size for each strain and found there was no significant difference comparing all mutants with the wild type. In addition, mutant #24 had shown an increase of biomass accumulation approximately 1.65 fold compared with the wild type strain on day 5 that was entering early stationary phase. From these preliminary results, it could be feasible to identify D. salina mutants with significant improved growth rate, cell accumulation and biomass production compared to the wild type for the further study; this makes it possible to improve this microorganism as a platform for biotechnology application.

Keywords: Dunaliella salina, ethyl methyl sulfonate, growth rate, biomass

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Authors: Iulianna Taritsa, Kuldeep Neote, Eric Fossel

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

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

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Authors: Olfat A. Fadali, Mohamed S. Mahmoud, Omnia H. Abdelraheem, Shimaa G. Mohammed

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The effect of electromagnetic field (EMF) on the removal of edible oil from oil-in-water emulsion by means of electrocoagulation was investigated in rectangular batch electrochemical cell with DC current. Iron (Fe) plate anodes and stainless steel cathodes were employed as electrodes. The effect of different magnetic field intensities (1.9, 3.9 and 5.2 tesla), three different positions of EMF (below, perpendicular and parallel to the electrocoagulation cell), as well as operating time; had been investigated. The application of electromagnetic field (5.2 tesla) raises percentage of oil removal from 72.4% for traditional electrocoagulation to 90.8% after 20 min.

Keywords: electrocoagulation, electromagnetic field, Oil-water emulsion, edible oil

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Authors: Md. M. Hossain, Regan Roat, Jenica Christopherson, Colette Free, Zhiguang Guo

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Long noncoding RNA (lncRNA) mediated post-transcriptional gene regulation, and their epigenetic landscapes have been shown to be involved in many human diseases. However, their regulation in diabetes through governing islet’s β-cell function and survival needs to be elucidated. Due to the technical and ethical constraints, it is difficult to study their role in β-cell function and survival in human under in vivo condition. In this study, humanized mice have been developed through transplanting human pancreatic islet under the kidney capsule of NOD.SCID mice and induced β-cell death leading to diabetes condition to study lncRNA mediated regulation. For this, human islets from 3 donors (3000 IEQ, purity > 80%) were transplanted under the kidney capsule of STZ induced diabetic NOD.scid mice. After at least 2 weeks of normoglycecemia, lymphocytes from diabetic NOD mice were adoptively transferred and islet grafts were collected once blood glucose reached > 200 mg/dl. RNA from human donor islets, islet grafts from humanized mice with either adoptive lymphocyte transfer (ALT) or PBS control (CTL) were ribodepleted; barcoded fragment libraries were constructed and sequenced on the Ion Proton sequencer. lncRNA expression in isolated human islets, islet grafts from humanized mice with and without induced β-cell death and their regulation in human islets function in vitro under glucose challenge, cytokine mediated inflammation and induced apoptotic condition were investigated. Out of 3155 detected lncRNAs, 299 that highly expressed in islets were found to be significantly downregulated and 224 upregulated in ALT compared to CTL. Most of these are found to be collocated within 5 kb upstream and 1 kb downstream of 788 up- and 624 down-regulated mRNAs. Genomic Regions Enrichment of Annotations Analysis revealed deregulated and collocated genes are related to pancreas endocrine development; insulin synthesis, processing, and secretion; pancreatitis and diabetes. Many of them, that found to be located within enhancer domains for islet specific gene activity, are associated to the deregulation of known islet/βcell specific transcription factors and genes that are important for β-cell differentiation, identity, and function. RNA sequencing analysis revealed aberrant lncRNA expression which is associated to the deregulated mRNAs in β-cell function as well as in molecular pathways related to diabetes. A distinct set of candidate lncRNA isoforms were identified as highly enriched and specific to human islets, which are deregulated in human islets from donors with different BMIs and with type 2 diabetes. These RNAs show an interesting regulation in cultured human islets under glucose stimulation and with induced β-cell death by cytokines. Aberrant expression of these lncRNAs was detected in the exosomes from the media of islets cultured with cytokines. Results of this study suggest that the islet specific lncRNAs are deregulated in human islet with β-cell death, hence important in diabetes. These lncRNAs might be important for human β-cell function and survival thus could be used as biomarkers and novel therapeutic targets for diabetes.

Keywords: β-cell, humanized mouse, pancreatic islet, LncRNAs

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Authors: Myoung-Jin Kim, Yeong-Seop Ahn, Hyun-Jee Yang, Hyoung-Kyu Song

Abstract:

This paper proposes the new cooperative communication scheme for the wireless communication system. When the receiver is located in the edge of coverage, the signal from the transmitter is distorted by the inter-cell interference (ICI) and power reduction by distance. In order to improve communication performance, the proposed scheme adds the relay. By using the relay, the receiver receives the signal from the transmitter and relay at the same time. Therefore, the new cooperative communication scheme obtains diversity gain and is improved by the relay.

Keywords: cooperative communication, diversity gain, OFDM, MIMO

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Authors: Neil Erick Q. Madariaga, Noel B. Linsangan

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Body Mass Index (BMI) is one of the different ways to monitor the health of a person. It is based on the height and weight of the person. This study aims to compute for the BMI using an Android tablet by obtaining the height of the person by using a camera and measuring the weight of the person by using a weighing scale or load cell. The height of the person was estimated by applying background subtraction to the image captured and applying different processes such as getting the vanishing point and applying Artificial Neural Network. The weight was measured by using Wheatstone bridge load cell configuration and sending the value to the computer by using Gizduino microcontroller and Bluetooth technology after the amplification using AD620 instrumentation amplifier. The application will process the images and read the measured values and show the BMI of the person. The study met all the objectives needed and further studies will be needed to improve the design project.

Keywords: body mass index, artificial neural network, vanishing point, bluetooth, wheatstone bridge load cell

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Authors: ZhenlinJu, Christopher P. Vellano, RehanAkbani, Yiling Lu, Gordon B. Mills

Abstract:

The epithelial–mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal characteristics, such as profound disruption of cell-cell junctions, loss of apical-basolateral polarity, and extensive reorganization of the actin cytoskeleton to induce cell motility and invasion. A hallmark of EMT is its capacity to promote metastasis, which is due in part to activation of several transcription factors and subsequent downregulation of E-cadherin. Unfortunately, current approaches have yet to uncover robust protein marker sets that can classify tumors as possessing strong EMT signatures. In this study, we utilize reverse phase protein array (RPPA) data and consensus clustering methods to successfully classify a subset of cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) tumors into an EMT protein signaling group (EMT group). The overall survival (OS) of patients in the EMT group is significantly worse than those in the other Hormone and PI3K/AKT signaling groups. In addition to a shrinkage and selection method for linear regression (LASSO), we applied training/test set and Monte Carlo resampling approaches to identify a set of protein markers that predicts the EMT status of CESC tumors. We fit a logistic model to these protein markers and developed a classifier, which was fixed in the training set and validated in the testing set. The classifier robustly predicted the EMT status of the testing set with an area under the curve (AUC) of 0.975 by Receiver Operating Characteristic (ROC) analysis. This method not only identifies a core set of proteins underlying an EMT signature in cervical cancer patients, but also provides a tool to examine protein predictors that drive molecular subtypes in other diseases.

Keywords: consensus clustering, TCGA CESC, Silhouette, Monte Carlo LASSO

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Authors: Neha Bhadauria, Indu Gaur, Shilpi Shilpi, Susmita Goswami, Prabir K. Paul

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The aerial surface of plants colonized by Human Enteric Pathogens ()has been implicated in outbreaks of enteric diseases in humans. Practice of organic farming primarily using animal dung as manure and sewage water for irrigation are the most significant source of enteric pathogens on the surface of leaves, fruits and vegetables. The present work aims to have an insight into the molecular mechanism of interaction of Human Enteric Pathogens or their metabolites with cell wall receptors in plants. Tomato plants grown under aseptic conditions at 12 hours L/D photoperiod, 25±1°C and 75% RH were inoculated individually with S. fonticola and K. pneumonia. The leaves from treated plants were sampled after 24 and 48 hours of incubation. The cell wall and cytoplasmic proteins were extracted and isocratically separated on 1D SDS-PAGE. The sampled leaves were also subjected to formaldehyde treatment prior to isolation of cytoplasmic proteins to study protein-protein interactions induced by Human Enteric Pathogens. Protein bands extracted from the gel were subjected to MALDI-TOF-TOF MS analysis. The foremost interaction of Human Enteric Pathogens on the plant surface was found to be cell wall bound receptors which possibly set ups a wave a critical protein-protein interaction in cytoplasm. The study revealed the expression and suppression of specific cytoplasmic and cell wall-bound proteins, some of them being important components of signaling pathways. The results also demonstrated HEP induced rearrangement of signaling pathways which possibly are crucial for adaptation of these pathogens to plant surface. At the end of the study, it can be concluded that controlling the over-expression or suppression of these specific proteins rearrange the signaling pathway thus reduces the outbreaks of food-borne illness.

Keywords: cytoplasmic protein, cell wall-bound protein, Human Enteric Pathogen (HEP), protein-protein interaction

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Authors: Seyedeh Mozhgan Seyed-Talebi, Weng-Kent Chan, Hsin-Yi Tiffany Chen

Abstract:

Lead-free perovskite photovoltaic (PV) technology employing non-toxic tin halide perovskite absorbers is pivotal for advancing perovskite solar cell (PSC) commercialization. Despite challenges posed by perovskite sensitivity to oxygen and humidity, our study utilizes DFT calculations using VASP and NanoDCAL software and SCAPS-1D simulations to elucidate the charge transport mechanism at the interface of CsSnI₃-FASnI₃ heterojunction. Results reveal how inherent electric fields facilitate efficient carrier transport, reducing recombination losses. We predict optimized power conversion efficiencies (PCEs) and highlight the potential of CsSnI3-FASnI3 heterojunctions for cost-effective and efficient charge transport layer-free (CTLF) photovoltaic devices. Our study provides insights into the future direction of recognizing more efficient, nontoxic heterojunction perovskite devices.

Keywords: charge transport layer free, CsSnI₃-FASnI₃ heterojunction, lead-free perovskite solar cell, tin halide perovskite., Charge transport layer free

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Authors: Marlon Gallo, Eduardo Miguel, Laura Oca, Eneko Gonzalez, Unai Iraola

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The heat generation of an energy storage system is an essential topic when designing a battery pack and its cooling system. Heat generation estimation is used together with thermal models to predict battery temperature in operation and adapt the design of the battery pack and the cooling system to these thermal needs guaranteeing its safety and correct operation. In the present work, a comparison between the use of a heat flux sensor (HFS) for indirect measurement of heat losses in a cell and the widely used and simplified version of Bernardi’s equation for estimation is presented. First, a Li-ion cell is thermally characterized with an HFS to measure the thermal parameters that are used in a first-order lumped thermal model. These parameters are the equivalent thermal capacity and the thermal equivalent resistance of a single Li-ion cell. Static (when no current is flowing through the cell) and dynamic (making current flow through the cell) tests are conducted in which HFS is used to measure heat between the cell and the ambient, so thermal capacity and resistances respectively can be calculated. An experimental platform records current, voltage, ambient temperature, surface temperature, and HFS output voltage. Second, an equivalent circuit model is built in a Matlab-Simulink environment. This allows the comparison between the generated heat predicted by Bernardi’s equation and the HFS measurements. Data post-processing is required to extrapolate the heat generation from the HFS measurements, as the sensor records the heat released to the ambient and not the one generated within the cell. Finally, the cell temperature evolution is estimated with the lumped thermal model (using both HFS and Bernardi’s equation total heat generation) and compared towards experimental temperature data (measured with a T-type thermocouple). At the end of this work, a critical review of the results obtained and the possible mismatch reasons are reported. The results show that indirectly measuring the heat generation with HFS gives a more precise estimation than Bernardi’s simplified equation. On the one hand, when using Bernardi’s simplified equation, estimated heat generation differs from cell temperature measurements during charges at high current rates. Additionally, for low capacity cells where a small change in capacity has a great influence on the terminal voltage, the estimated heat generation shows high dependency on the State of Charge (SoC) estimation, and therefore open circuit voltage calculation (as it is SoC dependent). On the other hand, with indirect measuring the heat generation with HFS, the resulting error is a maximum of 0.28ºC in the temperature prediction, in contrast with 1.38ºC with Bernardi’s simplified equation. This illustrates the limitations of Bernardi’s simplified equation for applications where precise heat monitoring is required. For higher current rates, Bernardi’s equation estimates more heat generation and consequently, a higher predicted temperature. Bernardi´s equation accounts for no losses after cutting the charging or discharging current. However, HFS measurement shows that after cutting the current the cell continues generating heat for some time, increasing the error of Bernardi´s equation.

Keywords: lithium-ion battery, heat flux sensor, heat generation, thermal characterization

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Authors: Xinxiu Li, Chuqian Zheng, Yanyan Qian, Hong Fan

Abstract:

Objectives: In hepatocellular carcinoma (HCC), oncogenes are continuously and robustly transcribed due to aberrant expression of essential components of the trans-acting super-enhancers (SE) complex. Preclinical and clinical trials are now being conducted on small-molecule inhibitors that target core-transcriptional components, including as transcriptional bromodomain protein 4 (BRD4) and cyclin-dependent kinase 7 (CDK7), in a number of malignant tumors. This study aims to explore whether co-overexpression of BRD4 and CDK7 is a potential marker of worse prognosis and a combined therapeutic target in HCC. Methods: The expression pattern of BRD4 and CDK7 and their correlation with prognosis in HCC were analyzed by RNA sequencing data and survival data of HCC patients from TCGA and GEO datasets. The protein levels of BRD4 and CDK7 were determined by immunohistochemistry (IHC), and survival data of patients were analyzed using the Kaplan-Meier method. The mRNA expression levels of genes in HCC cell lines were evaluated by quantitative PCR (q-PCR). CCK-8 and colony formation assays were conducted to assess cell proliferation of HCC upon treatment with BRD4 inhibitor JQ1 or/and CDK7 inhibitor THZ1. Results: It was shown that BRD4 and CDK7 were often overexpressed in HCCs and were associated with poor prognosis of HCC by analyzing the TCGA and GEO datasets. BRD4 or CDK7 overexpression was related to a lower survival rate. It's interesting to note that co-overexpression of CDK7 and BRD4 was a worse prognostic factor in HCC. Treatment with JQ1 or THZ1 alone had an inhibitory effect on cell proliferation; however, when JQ1 and THZ1 were combined, there was a more notable suppression of cell growth. At the same time, the combined use of JQ1 and THZ1 synergistically suppresses the expression of HCC driver genes. Conclusion: Our research revealed that BRD4 and CDK7 coupled can be a useful biomarker in HCC prognosis and the combination of JQ1 and THZ1 can be a promising therapeutic therapy against HCC.

Keywords: BRD4, CDK7, cell proliferation, combined inhibition

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Authors: Mohammad Kazem Mohammadi

Abstract:

The use of anticancer agents forms an important part of the treatment of cancer of various types. Uranyl Complexes with DPHMP ligand have been used for the prevention and treatment of cancers. U(IV) metal complexes prepared by reaction of uranyl salt UO2 (NO3)2.6H2O with DPHMP in dry acetonitrile. Characterization of the ligand and its complexes was made by microanalyses, FT-IR, 1H NMR, 13C NMR and UV–Visible spectroscopy. These new complex showed excellent antitumor activity against two kinds of cancer cells that that are HT29:Haman colon adenocarcinoma cell line and T47D:human breast adenocarcinoma cell line.

Keywords: uranyl complexes, DPHMP ligand, antitumor activity, HT29, T47D

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Authors: Mohd Ali Abbas Zaidi, Meenu Sachdeva, Jonaki Sen

Abstract:

In the vertebrate embryo, the forebrain anlagen develops from the anterior-most region of the neural tube which is the precursor of the central nervous system (CNS). The roof plate located at the dorsal midline region of the forebrain anlagen, acts as a source of several secreted molecules involved in patterning and morphogenesis of the forebrain. One such key morphogenetic event is the invagination of the forebrain roof plate which results in separation of the single forebrain vesicle into two cerebral hemispheres. Retinoic acid (RA) signaling plays a key role in this process. Blocking RA signaling at the dorsal forebrain midline inhibits dorsal invagination and results in the absence of certain key features of this region, such as thinning of the neuroepithelium and a lowering of cell proliferation. At present we are investigating the possibility of other signaling pathways acting in concert with RA signaling to regulate this process. We have focused on BMP signaling, which we found to be active in a mutually exclusive domain to that of RA signaling within the roof plate. We have also observed that there is a change in BMP signaling activity on modulation of RA signaling indicating an antagonistic relationship between the two. Moreover, constitutive activation of BMP signaling seems to completely inhibit thinning and partially affect invagination, leaving the lowering of cell proliferation in the midline unaffected. We are employing in-silico modeling as well as molecular manipulations to investigate the relative contribution if any, of regional differences in rates of cell proliferation and thinning of the neuroepithelium towards the process of invagination. We have found expression of certain cell adhesion molecules in forebrain roof-plate whose mRNA localization across the thickness of neuroepithelium is influenced by Bmp and RA signaling, giving regional rigidity to roof plate and assisting invagination. We also found expression of certain cytoskeleton modifiers in a localized small domains in invaginating forebrain roof plate suggesting that midline invagination is under control of many factors.

Keywords: bone morphogenetic signaling, cytoskeleton, cell adhesion molecules, forebrain roof plate, retinoic acid signaling

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Authors: Zahra Heydari

Abstract:

Introduction: One of the major clinical issues is acute renal failure, which is caused by ischemia-reperfusion of the kidney and is associated with high mortality. Despite advances in this area, important issues such as tissue necrosis, cell apoptosis, and so on in damaged tissue are suggestive for more researches and study on this subject. Objective: Evaluation of the potential utility of Ezetimibe in reducing injuries and cell death induced by kidney ischemia/ reperfusion through inducing expression changes of different cellular pathways in adult Sprague-Dawley rats. Materials and methods: Forty rats weighing 180-200g were divided into 4 groups. For this purpose, the first right kidneys of the rats were removed during surgery. After 20 days, the left renal artery was closed with a soft clamp and reperfusion was performed. After 24 hours, blood samples were collected and sent to the laboratory with kidneys to measure bax and bcl-2 by Western blotting and histopathological tests. Results: Quantitative damage reviews of Kidney tissue indicates damage Acute and severe tubular lesions were observed in the ischemia group. Also, the amount of injury was significantly reduced in the treatment group. There was also a significant difference between the ischemia and sham groups. In general, the results show that a single dose of 1.2 mg/kg of ezetimibe can reduce the bax/ bcl-2 ratio compared to the ischemia group. In general, the results showed Ezetimibe is effective in reducing cell damage and death due to ischemia/ reperfusion after renal ischemia through changes in the expression of various cellular pathways in rats.

Keywords: acute renal failure, renal ischemia-reperfusion injury, ezetimibe, apoptosis

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Authors: M. Miriam Hernández-Arroyo, Ivonne Caro-Gonzales, Miguel Ángel Plascencia-Espinosa, Sergio R. Trejo-Estrada

Abstract:

A large biodiversity of Lactobacillus strains has been detected in traditional foods and beverages from Mexico. A selected strain of Lactobacillus sp - PODI-20, used for the obtained from an artisanal fermented beverage was cultivated in different carbon sources in a complex medium, in order to define which carbon sourced induced more effectively the isomerization of arabinose by cell fractions obtained by fermentation. Four different carbon sources were tested in a medium containing peptone and yeast extract and mineral salts. Glucose, galactose, arabinose, and lactose were tested individually at three different concentrations: 3.5, 6, and 10% w/v. The biomass yield ranged from 1.72 to 17.6 g/L. The cell pellet was processed by mechanical homogenization. Both fractions, the cellular debris, and the lysis supernatant were tested for their ability to isomerize arabinose into ribulose. The highest yield of isomer was 12 % of isomerization in the supernatant fractions; whereas up to 9.3% was obtained by the use of cell debris. The isomerization of arabinose has great significance in the production of lactic acid by fermentation of complex carbohydrate hydrolysates.

Keywords: isomerase, tagatose, aguamiel, isomerization

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Authors: Katarína Koňariková, Georgios A. Perdikaris, Lucia Andrezálová, Zdeňka Ďuračková, Lucia Laubertová, Helena Gbelcová, Ingrid Žitňanová

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Introduction: The continuous demand for new anti-cancer drugs has stimulated chemotherapeutic research based on the use of essential metalloelements with the aim to develop potential drugs with lower toxicity and higher antiproliferative activity against tumors. Copper(II) and its complexes play an important role as suitable species for antiproliferative tests. Objectives: The central objective of the current study was to investigate the potential in vitro anti-proliferative effects of N-salicylidene-L-glutamato copper (II) complexes and molecular mechanism of apoptosis induced by tested complexes. In our project we tested N-salicylidene-L-glutamato copper (II) complexes ZK1 - [Cu(N-salicylidene-L-glutamato)(H2O)2].H2O; MK0 - ([Cu2(N-sal-D,L-glu)2(isoquinoline)2].2H2O); MK1 - [Cu(N-salicylidene-5-methyl-L-glutamato)(H2O)].H2O; MK3 - transbis(ethanol)tetrakis(imidazol)Cu(II)(2+)bis(N-salicylidene-D,L-glutamato-N,O)-KO:KO´-(imidazol); MK5 - [Cu(N-salicylidene-D,L- glutamato)(2-methylimidazol] at concentration range 0.001-100 µmol/L against human colon carcinoma cell line HT-29 and human breast carcinoma cell line MCF-7. Methods: Viability was assessed by direct counting of 0.4% trypan blue dye-excluding cells after 24, 48 and 72 hour cultivations with or without copper complex and by MTT assay. To analyze the type of cell death and its mechanism induced by our copper complex we used different methods. To distinguish apoptosis from necrosis we used electrophoretic analysis, to study the activity of caspases 8 and 9 – luminometric analysis and caspase activity 3 colorimetric assay. Results: The observed anti-proliferative effect of the copper complexes appeared to be dose-, time- and cell line- dependent. Human colon carcinoma cells HT-29 appeared to be more sensitive to the complex MK0 ([Cu2(N-sal-D,L-glu)2(isoquinoline)2].2H2O) than to ZK1 ([Cu(N-salicylidene-L-glutamato)(H2O)2].H2O) and MK1 ([Cu(N-salicylidene-5-methyl-L-glutamato)(H2O)].H2O)). Human colon carcinoma cells HT-29 appeared to be more sensitive to the complex than human breast carcinoma cells MCF-7. IC50 decreased with time of incubation (24, 48 and 72h) for HT-29, but increased for MCF-7. By electrophoresis we found apoptotic cell death induced by our copper complexes in HT-29 at concentrations 1, 10, 50 and 100 µmol/L after 48h (ZK1) and 72h (MK0, MK1) and in MCF-7 we did not find apoptosis. We also studied molecular mechanism of apoptosis in HT-29 induced by copper complexes. We found active caspase 9 in HT-29 after ZK1 ([Cu(N-salicylidene-L-glutamato)(H2O)2].H2O) and MK1 ([Cu(N-salicylidene-5-methyl-L-glutamato)(H2O)].H2O)) influence and active caspase 8 after MK0 ([Cu2(N-sal-D,L-glu)2(isoquinoline)2].2H2O) influence. Conclusion: Our copper complexes showed cytotoxic activities against human colon carcinoma cells HT-29 and breast cancer cell line MCF-7 in vitro. Apoptosis was activated by mitochondrial pathway (intrinsic pathway) in case of ZK1 [Cu(N-salicylidene-L-glutamato)(H2O)2].H2O; MK1 [Cu(N-salicylidene-5-methyl-L-glutamato)(H2O)].H2O; MK3 - transbis(ethanol)tetrakis(imidazol)Cu(II)(2+)bis(N-salicylidene-D,L-glutamato-N,O)-KO:KO´-(imidazol) and MK5 - [Cu(N-salicylidene-D,L- glutamato)(2-methylimidazol] copper complexes and by death receptors (extrinsic pathway) in case of MK0 [Cu2(N-sal-D,L-glu)2(isoquinoline)2].2H2O copper complex in HT-29.

Keywords: apoptosis, copper complex, cancer, carcinoma cell line

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