Search results for: stem cells

Authors: P. Kazymbet, G. Abildinova, K.Makhambetov, M. Bakhtin, D. Rybalkina, K. Zhumadilov

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Conducted cytogenetic research in workers Stepnogorsk Mining-Chemical Combine (Akmola region) with the study of 26341 chromosomal metaphase. Using a regression analysis with program DataFit, version 5.0, dependence between exposure dose and the following cytogenetic exponents has been studied: frequency of aberrant cells, frequency of chromosomal aberrations, frequency of the amounts of dicentric chromosomes, and centric rings. Experimental data on calibration curves "dose-effect" enabled the development of a mathematical model, allowing on data of the frequency of aberrant cells, chromosome aberrations, the amounts of dicentric chromosomes and centric rings calculate the absorbed dose at the time of the study. In the dose range of 0.1 Gy to 5.0 Gy dependence cytogenetic parameters on the dose had the following equation: Y = 0,0067е^0,3307х (R2 = 0,8206) – for frequency of chromosomal aberrations; Y = 0,0057е^0,3161х (R2 = 0,8832) –for frequency of cells with chromosomal aberrations; Y =5 Е-0,5е^0,6383 (R2 = 0,6321) – or frequency of the amounts of dicentric chromosomes and centric rings on cells. On the basis of cytogenetic parameters and regression equations calculated absorbed dose in workers of uranium production at the time of the study did not exceed 0.3 Gy.

Keywords: Stepnogorsk, mathematical modeling, cytogenetic, dicentric chromosomes

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Authors: Kave Moloudi, Heidi Abrahamse, Blassan P. George

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Radiotherapy (RT) and photodynamic therapy (PDT) are both forms of cancer treatment that aim to kill cancer cells while minimizing damage to healthy tissue. The similarity between RT and PDT lies in their mechanism of action. Both treatments use energy to damage cancer cells. RT uses high-energy radiation to damage the DNA of cancer cells, while PDT uses light energy to activate a photosensitizing agent, which produces reactive oxygen species (ROS) that damage the cancer cells. Both treatments require careful planning and monitoring to ensure the correct dose is delivered to the tumor while minimizing damage to surrounding healthy tissue. They are also often used in combination with other treatments, such as surgery or chemotherapy, to improve overall outcomes. However, there are also significant differences between RT and PDT. For example, RT is a non-invasive treatment that can be delivered externally or internally, while PDT requires the injection of a photosensitizing agent and the use of a specialized light source to activate it. Additionally, the side effects and risks associated with each treatment can vary. In this review, we focus on generalizing the 6Rs of radiobiology in PDT, which can open a window for the clinical application of Radio-photodynamic therapy with minimum side effects. Furthermore, this review can open new insight to work on and design new radio-photosensitizer agents in Radio-photodynamic therapy.

Keywords: radiobiology, photodynamic therapy, radiotherapy, 6Rs in radiobiology, ROS, DNA damages, cellular and molecular mechanism, clinical application.

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Authors: Sara R. Knigge, Sugat R. Tuladhar, Alexander Becker, Tobias Schilling, Birgit Glasmacher

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Commercially available mechanical or biological heart valve prostheses both tend to fail long-term due to thrombosis, calcific degeneration, infection, or immunogenic rejection. Moreover, these prostheses are non-viable and do not grow with the patients, which is a problem for young patients. As a result, patients often need to undergo redo-operations. Tissue-engineered (TE) heart valves based on degradable electrospun fiber scaffolds represent a promising approach to overcome these limitations. Such scaffolds need sufficient mechanical properties to withstand the hydrodynamic stress of intracardiac hemodynamics. Additionally, the scaffolds should be colonized by autologous or homologous cells to facilitate the in vivo remodeling of the scaffolds to a viable structure. This study investigates how process parameters of electrospinning and degradation affect the mechanical properties of electrospun scaffolds made of FDA-approved, biodegradable polymer polycaprolactone (PCL). Fiber mats were produced from a PCL/tetrafluoroethylene solution by electrospinning. The e-spinning process was varied in terms of scaffold thickness, fiber diameter, fiber orientation, and fiber interconnectivity. The morphology of the fiber mats was characterized with a scanning electron microscope (SEM). The mats were degraded in different solutions (cell culture media, SBF, PBS and 10 M NaOH-Solution). At different time points of degradation (2, 4 and 6 weeks), tensile and cyclic loading tests were performed. Fresh porcine pericardium and heart valves served as a control for the mechanical assessment. The progression of polymer degradation was quantified by SEM and differential scanning calorimetry (DSC). Primary Human aortic endothelial cells (HAECs) and Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) were seeded on the fiber mats to investigate the cell colonization potential. The results showed that both the electrospinning parameters and the degradation significantly influenced the mechanical properties. Especially the fiber orientation has a considerable impact and leads to a pronounced anisotropic behavior of the scaffold. Preliminary results showed that the polymer became strongly more brittle over time. However, the embrittlement can initially only be detected in the mechanical test. In the SEM and DSC investigations, neither morphological nor thermodynamic changes are significantly detectable. Live/Dead staining and SEM imaging of the cell-seeded scaffolds showed that HAECs and iPSC-ECs were able to grow on the surface of the polymer. In summary, this study's results indicate a promising approach to the development of a TE aortic heart valve based on an electrospun scaffold.

Keywords: electrospun scaffolds, long-term polymer degradation, mechanical behavior of electrospun PCL, tissue engineered aortic heart valve

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Authors: Hager Elsheikh, Matthias Sendler, Juliana Glaubnitz

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In pancreatitis, an inflammatory reaction occurs in the pancreatic secretory cells due to premature activation of proteases, leading to pancreatic self-digestion and necrotic cell death of acinar cells. Acute pancreatitis in patients is characterized by a severe immune reaction that could lead to serious complications, such as organ failure or septic shock, if left untreated. Chronic pancreatitis is a recurrence of episodes of acute pancreatitis resulting in a fibro-inflammatory immune response, in which the type 2 immune response is primarily driven by AAMs in the pancreas. One of the most important signaling pathways for M2 macrophage activation is the IL-4/STAT6 pathway. Pancreatic fibrosis is induced by the hyperactivation of pancreatic stellate cells by dysregulation in the inflammatory response, leading to further damage, autodigestion and possibly necrosis of pancreatic acinar cells. The aim of this research is to investigate the effect of STAT6 knockout in disease severity and development of fibrosis wound healing in the presence of different macrophage populations, regulated by the type 2 immune response, after inducing chronic and/or acute pancreatitis in mice models via cerulean injection. We further investigate the influence of the JAK/STAT6 signaling pathway on the balance of fibrosis and regeneration in STAT6 deficient and wild-type mice. The characterization of resident and recruited macrophages will provide insight into the influence of the JAK/STAT6 signaling pathway on infiltrating cells and, ultimately, tissue fibrosis and disease severity.

Keywords: acute and chronic pancreatitis, tissue regeneration, macrophage polarization, Gastroenterology

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Authors: Arindam Pramanik, Parimal Karmakar

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We have explored the synergistic anti-cancer activity of copper ion and acetylacetone complex containing 1,3 diketone group (like curcumin) in metallorganic compound “Copper acetylacetonate” (CuAA). The cytotoxicity mechanism of CuAA complex was evaluated on various cancer cell lines in vitro. Among these, reactive oxygen species (ROS), glutathione level (GSH) in the cell was found to increase. Further mitochondrial membrane damage was observed. The fate of cell death was found to be induced by apoptosis. For application purpose, we have developed a novel biodegradable, non-toxic polymer-based nanoparticle which has hydrophobically modified core for loading of the CuAA. Folic acid is conjugated on the surface of the polymer (chitosan) nanoparticle for targeting to cancer cells for minimizing toxicity to normal cells in-vivo. Thus, this novel drug CuAA has an efficient anticancer activity which has been targeted specifically to cancer cells through polymer nanoparticle.

Keywords: anticancer, apoptosis, copper nanoparticle, targeted drug delivery

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Authors: Leila Karshenas, Hamid Reza Khodaei, Behnaz Mahdavi

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We know that nitric oxide (NO) plays an important role in all sexual activities of animals. It is made in body from NO synthase enzyme and L-arginin molecule. NO can bound with sulfur-iron complexes and because production of steroid sexual hormones is related to enzymes which have this complex, NO can change the activity of these enzymes. NO affects many cells including endothelial cells of veins, macrophages and mast cells. These cells are found in testis leydig cells and therefore are important source of NO in testis tissue. Minimizing damages to sperm at the time of sperm freezing and thawing is really important. The goal of this study was to determine the function of NO before freezing and its effects on quality and viability of sperms after thawing and incubation. 4 Holstein bulls were selected from the age of 4, and artificial insemination was done for 3 weeks (2 times a week). Treatments were 0, 10, 50 and 100 nm of sodium nitroprusside (SNP). Data analysis was performed by SAS98 program. Also, mean comparison was done using Duncan's multiple ranges test (P<0.05). Concentrations used was found to increase motility and viability of spermatozoa at 1, 2 and 3 hours after thawing significantly (P<0.05), but there was no significant difference at zero time. SNP levels reduced the amount of lipid peroxidation in sperm membrane, increased acrosome health and improved sample membranes especially in 50 and 100 nm treatments. According to results, adding SNP to semen diluents increases motility and viability of spermatozoa. Also, it reduces lipid peroxidation in sperm membrane and improves sperm function.

Keywords: sperm motility, nitric oxide, lipid peroxidation, spermatozoa

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Authors: Tianjiao Chu, Carla Rios Luci, Christy Maksoudian, Ara Sargsian, Bella B. Manshian, Stefaan J. Soenen

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Nanomaterials have gained high interest in their use as potent anticancer agents. Apart from delivering chemotherapeutic agents in order to reduce off-target effects, molecular agents have also been widely explored. The advances in our understanding of cell biology and cell death mechanisms1 has generated a broad library of potential therapeutic targets by siRNA, mRNA, or pDNA complexes. In the present study, we explore the ability of pDNA-polyplexes to induce tumor-specific necroptosis. This results in a cascade of effects, where immunogenic cell death potentiates anti-tumor immune responses and results in an influx of dendritic cells and cytotoxic T cells, rendering the tumor more amenable to immune checkpoint inhibition. This study aims to explore whether the induction of necroptosis in a subpopulation of tumor cells can be used to potentiate immune checkpoint inhibition studies.

Keywords: nanoparticle, MLKL, necroptosis, immunotherapy

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Authors: Dian Muzdalifah, Anastasia F. Devi, Zatil A. Athaillah, Linar Z. Udin

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Tempe is a functional food prepared from soybeans through Rhizopus spp fermentation. It is well known as functional food, originated from Indonesia. Most studies on tempe functionalities refer to ripe (48 h fermentation) tempe and only limited studies discuss overripe tempe while longer fermentation time possibly increased tempe health benefit. Hence, the present study was performed to investigate the cytotoxic activity againts MCF-7 breast cancer cells and antioxidant property of tempe prepared from 0–156 h of fermentation. Tempe samples were dried and extracted with acetone and ethanol, respectively. Their extracts were used for subsequent analysis. The cytotoxic activity was assessed on MCF 7 breast cancer cells using Alamar Blue method. The antioxidant activity was determined by DPPH free radical scavenging assay. The results indicated that acetone extracts of 108 h tempe had a potent cytotoxic activity against MCF-7 breast cancer cells (IC50 = 2.54 ± 0,30 μg/mL). Ethanol extracts of 108 h tempe also showed the potency, but at slightly higher IC50 (5.20 ± 1.01 μg/mL). Both acetone and ethanol extracts of 108 and 120 h tempe showed high antioxidant activity expressed as percent inhibition with no significant difference. However, acetone extracts of 120 h tempe (81.31 ± 3.70 %) had better ability to inhibit oxidation reaction than that of ethanol extracts (75.77 ± 6.00 %). It can be concluded that the cytotoxic activity of tempe from 0–156 h of fermentation is positively correlated to their corresponding antioxidant property. Longer fermentation time, up to 108 h, increased the ability of tempe to inhibit the growth of MCF-7 breast cancer cells and oxidative reaction. But extended fermentation time, up to 156 h, tends to decrease its ability. Further studies are encouraged to identify the active components contained in each extract.

Keywords: antioxidant property, cytotoxic activity, extracts, overripe tempeh

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Authors: Naila Sher, Mushtaq Ahmed, Nadia Mushtaq, Rahmat Ali Khan

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In cancer therapy, nanoparticles (NPs) shall be applied possibly by inoculation in the veins of humans. This action will connect them with white (WBCs) and red blood cells (RBCs) in the bloodstream before they reach their main targeted cancer cells. However, possible effects of silver, gold, and silver/gold bimetallic NPs (Ag, Au, and Ag/Au BNPs) on baby hamster kidney-21 (BHK-21) cells were studied by MTT assay. Here, Ag, Au, and their Ag/Au BNPs (bimetallic nanoparticles) were synthesized by using Hippeastrum hybridum (HH) extract. These NPs were characterized by UV-visible spectroscopy, FT-IR, XRD, and EDX, and SEM analysis. XRD analysis conferring the crystal structure with an average size of 13.3, 10.72, and 8.34nm of Ag, Au, and Ag/Au BNPs, respectively. SEM showed that Ag, Au, and Ag/Au BNPs had irregular morphologies, with nano measures calculated sizes of 40, 30, and 20 nm respectively. EDX spectrometers confirmed the presence of elemental Ag signal of the AgNPs with 22.75%, Au signal of the AuNPs with 48.08%, Ag signal with 12%, and Au signal with 38.26% of the Ag/Au BNPs. The BHK-21cells were incubated in the existence of doxorubicin, plant extract, Ag, Au, and Ag/Au BNPs. The cytotoxic effects could be observed in a dose-dependent mode; doxorubicin and Ag/Au BNPs were more toxic than plant extract, Ag, and Au NPs. It is demonstrated that NPs interact with BHK-21cells and significantly reduce cell viability in a concentration-dependent manner. However, to reduce the potential threats of NPs further studies are recommended.

Keywords: hippeastrum hybridum, nanoparticle, BHK-21cells

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Authors: Richard Harrison

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Developing competency in mathematics and related critical thinking skills is essential to the education of undergraduate students of Science, Technology, Engineering and Mathematics (STEM). Recently, the HE sector has been impacted by a seemingly widening disconnect between the mathematical competency of incoming first-year STEM students and their entrance qualification tariffs. Despite relatively high grades in A-Level Mathematics, students may initially lack fundamental skills in key areas such as algebraic manipulation and have limited capacity to apply problem solving strategies. Compounded by compensatory measures applied to entrance qualifications during the pandemic, there has been an associated decline in student performance on introductory university mathematics modules. In the UK, a number of online resources have been developed to help scaffold the transition to university mathematics. However, in general, these do not offer a structured learning journey focused on individual developmental needs, nor do they offer an experience coherent with the teaching and learning characteristics of the destination institution. In order to address some of these issues, a bespoke framework has been designed and implemented on our VLE in the Faculty of Engineering & Physical Sciences (FEPS) at the University of Surrey. Called the FEPS Maths Support Framework, it was conceived to scaffold the mathematical development of individuals prior to entering the university and during the early stages of their transition to undergraduate studies. More than 90% of our incoming STEM students voluntarily participate in the process. Students complete a set of initial diagnostic questions in the late summer. Based on their performance and feedback on these questions, they are subsequently guided to self-select specific mathematical topic areas for review using our proprietary resources. This further assists students in preparing for discipline related diagnostic tests. The framework helps to identify students who are mathematically weak and facilitates early intervention to support students according to their specific developmental needs. This paper presents a summary of results from a rich data set captured from the framework over a 3-year period. Quantitative data provides evidence that students have engaged and developed during the process. This is further supported by process evaluation feedback from the students. Ranked performance data associated with seven key mathematical topic areas and eight engineering and science discipline areas reveals interesting patterns which can be used to identify more generic relative capabilities of the discipline area cohorts. In turn, this facilitates evidence based management of the mathematical development of the new cohort, informing any associated adjustments to teaching and learning at a more holistic level. Evidence is presented establishing our framework as an effective early intervention strategy for addressing the sector-wide issue of supporting the mathematical development of STEM students transitioning to HE

Keywords: competency, development, intervention, scaffolding

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Authors: Fumio Kasai, Noriko Hirayama, Jorge Pereira, Azusa Ohtani, Masashi Iemura, Malcolm A. Ferguson Smith, Arihiro Kohara

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The VERO cell line was established in 1962 from normal tissue of an African green monkey, Chlorocebus aethiops (2n=60), and has been commonly used worldwide for screening for toxins or as a cell substrate for the production of viral vaccines. The VERO genome was sequenced in 2014; however, its cytogenetic features have not been fully characterized as it contains several chromosome abnormalities and different karyotypes coexist in the cell line. In this study, the VERO cell line (JCRB0111) was compared with one of the sublines. In contrast to 59 chromosomes as the modal chromosome number in the VERO cell line, the subline had two peaks of 56 and 58 chromosomes. M-FISH analysis using human probes revealed that the VERO cell line was characterized by a translocation t(2;25) found in all metaphases, which was absent in the subline. Different abnormalities detected only in the subline show that the cell line is heterogeneous, indicating that the subline has the potential to change its genomic characteristics during cell culture. The various alterations in the two independent lineages suggest that genomic changes in both VERO cells can be accounted for by progressive rearrangements during their evolution in culture. Both t(5;X) and t(8;14) observed in all metaphases of the two cell lines might have a key role in VERO cells and could be used as genetic markers to identify VERO cells. The flow karyotype shows distinct differences from normal. Further analysis of sorted abnormal chromosomes may uncover other characteristics of VERO cells. Because of the absence of STR data, cytogenetic data are important in characterizing animal cell lines and can be an indicator of their quality control.

Keywords: VERO, cell culture passage, chromosome rearrangement, heterogeneous cells

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Authors: Ashit Syngle, Inderjit Verma, Pawan Krishan

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Aim: Endothelial progenitor cells (EPCs) are unique populations which have reparative potential in overcoming the endothelial damage and reducing cardiovascular risk. Patients with ankylosing spondylitis (AS) have increased risk of cardiovascular morbidity and mortality. The aim of this study was to investigate the endothelial progenitor cell population in AS patients and its potential relationships with disease variables. Methods: Endothelial progenitor cells were measured in peripheral blood samples from 20 AS and 20 healthy controls by flow cytometry on the basis of CD34 and CD133 expression. Disease activity was evaluated by using Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). Functional ability was monitored by using Bath Ankylosing Spondylitis Functional Index (BASFI). Results: EPCs were depleted in AS patients as compared to the healthy controls (CD34+/CD133+: 0.027 ± 0.010 % vs. 0.044 ± 0.011 %, p<0.001). EPCs depletion were significantly associated with disease duration (r=-0.52, p=0.01) and BASDAI (r=-0.45, p=0.04). Conclusion: This is the first study to demonstrate endothelial progenitor cells depletion in AS patients. EPCs depletion inversely correlates with disease duration and disease activity, suggesting the pivotal role of inflammation in depletion of EPCs. EPC would possibly also serve as a therapeutic target for preventing cardiovascular disease in AS.

Keywords: ankylosing spondylitis, endothelial progenitor cells, inflammation, vascular damage

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Authors: Busra Gundede, Ozal Mutlu, Nagihan Gulsoy

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Background: Over the years, material research has led to the development of numerous metals and alloys for using in biomedical applications. One of the major tasks of biomaterial research is the functionalization of the material surface to improve the biocompatibility according to a specific application. 316L and 316L alloys are excellent for various bio-applications. This research was investigated the effect of titanium (Ti), niobium (Nb), and zirconium (Zr) additives on injection molded austenitic grade 316L stainless steels in vitro biocompatibility. For this purpose, cytotoxic tests were performed to evaluate the potential biocompatibility of the specimens. Materials and Methods: 3T3 fibroblast were cultivated in DMEM supplemented with 10% fetal bovine serum and %1 penicillin-streptomycin at 37°C with 5% CO2 and 95%humidity. Trypsin/EDTA solution was used to remove cells from the culture flask. Cells were reseeded at a density of 1×105cell in 25T flasks. The medium change took place every 3 days. The trypan blue assay was used to determine cell viability. Cell viability is calculated as the number of viable cells divided by the total number of cells within the grids on the cell counter machine counted the number of blue staining cells and the number of total cells. Cell viability should be at least 95% for healthy log-phase cultures. MTT assay was assessed for 96-hours. Cells were cultivated in 6-well flask within 5 ml DMEM and incubated as same conditions. 0,5mg/ml MTT was added for 4-hours and then acid-isoprohanol was added for solubilize to formazan crystals. Cell morphology after 96h was investigated by SEM. The medium was removed, samples were washed with 0.15 M PBS buffer and fixed for 12h at 4- 8°C with %2,5 gluteraldehyte. Samples were treated with 1% osmium tetroxide. Samples were then dehydrated and dried, mounted on appropriate stubs with colloidal silver and sputter-coated with gold. Images were collected using a scanning electron microscope. ROS assay is a cell viability test for in vitro studies. Cells were grown for 96h, ROS solution added on cells in 6 well plate flask and incubated for 1h. Fluorescence signal indicates ROS generation by cells. Results: Trypan Blue exclusion assay results were 96%, 92%, 95%, 90%, 91% for negative control group, 316L, 316L-Ti, 316L-Nb and 316L-Zr, respectively. Results were found nearly similar to each other when compared with control group. Cell viability from MTT analysis was found to be 100%, 108%, 103%, 107%, and 105% for the control group, 316L, 316L-Ti, 316L-Nb and 316L-Zr, respectively. Fluorescence microscopy analysis indicated that all test groups were same as the control group in ROS assay. SEM images demonstrated that the attachment of 3T3 cells on biomaterials. Conclusion: We, therefore, concluded that Ti, Nb and Zr additives improved physical properties of 316L stainless. In our in vitro experiments showed that these new additives did not modify the cytocompatibility of stainless steel and these additives on 316L might be useful for biomedical applications.

Keywords: 316L stainles steel, biocompatibility, cell culture, Ti, Nb, Zr

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Authors: Sibele A. F. Leite, Bernno S. Leite, José Vicente H. D´Angelo, Ana Teresa P. Dell’Isola, Julio CéSar Souza

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The generation of bioenergy is one of the oldest and simplest biomass applications and is one of the safest options for minimizing emissions of greenhouse gasses and replace the use of fossil fuels. In addition, the increasing development of technologies for energy biomass conversion parallel to the advancement of research in biotechnology and engineering has enabled new opportunities for exploitation of biomass. Agricultural residues offer great potential for energy use, and Brazil is in a prominent position in the production and export of agricultural products such as banana and rice. Despite the economic importance of the growth prospects of these activities and the increasing of the agricultural waste, they are rarely explored for energy and production of new materials. Brazil products almost 10.5 million tons/year of rice husk and 26.8 million tons/year of banana stem. Thereby, the aim of this study was to analysis the potential of biomass residues for energy production and applications in new materials. Rice husk (specify the type) and banana stem (specify the type) were characterized by physicochemical analyses using the following parameters: organic carbon, nitrogen (NTK), proximate analyses, FT-IR spectroscopy, thermogravimetric analyses (TG), calorific values and silica content. Rice husk and banana stem presented attractive superior calorific (from 11.5 to 13.7MJ/kg), and they may be compared to vegetal coal (21.25 MJ/kg). These results are due to the high organic matter content. According to the proximate analysis, biomass has high carbon content (fixed and volatile) and low moisture and ash content. In addition, data obtained by Walkley–Black method point out that most of the carbon present in the rice husk (50.5 wt%) and in banana stalk (35.5 wt%) should be understood as organic carbon (readily oxidizable). Organic matter was also detected by Kjeldahl method which gives the values of nitrogen (especially on the organic form) for both residues: 3.8 and 4.7 g/kg of rice husk and banana stem respectively. TG and DSC analyses support the previous results, as they can provide information about the thermal stability of the samples allowing a correlation between thermal behavior and chemical composition. According to the thermogravimetric curves, there were two main stages of mass-losses. The first and smaller one occurred below 100 °C, which was suitable for water losses and the second event occurred between 200 and 500 °C which indicates decomposition of the organic matter. At this broad peak, the main loss was between 250-350 °C, and it is because of sugar decomposition (components readily oxidizable). Above 350 °C, mass loss of the biomass may be associated with lignin decomposition. Spectroscopic characterization just provided qualitative information about the organic matter, but spectra have shown absorption bands around 1030 cm-1 which may be identified as species containing silicon. This result is expected for the rice husk and deserves further investigation to the stalk of banana, as it can bring a different perspective for this biomass residue.

Keywords: rice husk, banana stem, bioenergy, renewable feedstock

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Authors: Ponchang Apollos Wuyep, Alice Njolke Mafe, Longchi Satkat Zacheaus, Dogun Ojochogu, Dabot Ayuba Yakubu

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Fungi causes huge losses when infections occur both in plants and animals. Synthetic Antifungal drugs are mostly very expensive and highly cytotoxic when taken. This study was aimed at determining the in vitro and in vivo antifungal activities of the leaves and stem extracts of Terminalia mantaly (Umbrella tree)H. Perrier on Aspergillus species in a bid to identify potential sources of cheap starting materials for the synthesis of new drugs to address the growing antimicrobial resistance. T. mantaly leave and stem powdered plant was extracted by fractionation using the method of solvent partition co-efficient in their graded form in the order n-hexane, Ethyl acetate, methanol and distilled water and phytochemical screening of each fraction revealed the presence of alkaloids, saponins, Tannins, flavonoids, carbohydrates, steroids, anthraquinones, cardiac glycosides and terpenoids in varying degrees. The Agar well diffusion technique was used to screen for antifungal activity of the fractions on clinical isolates of Aspergillus species (Aspergillus flavus and Aspergillus fumigatus). Minimum inhibitory concentration (MIC50) of the most active extracts was determined by the broth dilution method. The fractions test indicated a high antifungal activity with zones of inhibition ranging from 6 to 26 mm and 8 to 30mm (leave fractions) and 10mm to 34mm and 14mm to36mm (stem fractions) on A. flavus and A. fumigatus respectively. All the fractions indicated antifungal activity in a dose response relationship at concentrations of 62.5mg/ml, 125mg/ml, 250mg/ml and 500mg/ml. Better antifungal efficacy was shown by the Ethyl acetate, Hexane and Methanol fractions in the in vitro as the most potent fraction with MIC ranging from 62.5 to 125mg/ml. There was no statistically significant difference (P>0.05) in the potency of the Eight fractions from leave and stem (Hexane, Ethyl acetate, methanol and distilled water, antifungal (fluconazole), which served as positive control and 10% DMSO(Dimethyl Sulfoxide)which served as negative control. In the in vivo investigations, the ingestion technique was used for the infectious studies Female Drosophilla melanogaster(UAS-Diptericin)normal flies(positive control),infected and not treated flies (negative control) and infected flies with A. fumigatus and placed on normal diet, diet containing fractions(MSM and HSM each at concentrations of 10mg/ml 20mg/ml, 30mg/ml, 40mg/ml, 50mg/ml, 60mg/ml, 70mg/ml, 80mg/ml, 90mg/ml and 100mg/ml), diet containing control drugs(fluconazole as positive control)and infected flies on normal diet(negative control), the flies were observed for fifteen(15) days. Then the total mortality of flies was recorded each day. The results of the study reveals that the flies were susceptible to infection with A. fumigatus and responded to treatment with more effectiveness at 50mg/ml, 60mg/ml and 70mg/ml for both the Methanol and Hexane stem fractions. Therefore, the Methanol and Hexane stem fractions of T. mantaly contain therapeutically useful compounds, justifying the traditional use of this plant for the treatment of fungal infections.

Keywords: Terminalia mantaly, Aspergillus fumigatus, cytotoxic, Drosophila melanogaster, antifungal

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Authors: Ji Hye Im, Nguyen T. T. Phuong, Keon Wook Kang

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Estrogens are important for the development and growth of estrogen receptor (ER)-positive breast cancer, for which anti-estrogen therapy is one of the most effective treatments. However, its efficacy can be limited by either de novo or acquired resistance. Aromatase is a key enzyme for the biosynthesis of estrogens, and inhibition of this enzyme leads to profound hypoestrogenism. Here, we found that the basal expression and activity of aromatase were significantly increased in tamoxifen (TAM)-resistant human breast cancer (TAMR-MCF-7) cells compared to control MCF-7 cells. We further revealed that aromatase immunoreactivity in tumor tissues was increased in recurrence group after TAM therapy compared to non-recurrence group after TAM therapy. Phosphorylation of Akt, extracellular signal-regulated kinase (ERK), and p38 kinase were all increased in TAMR-MCF-7 cells. Inhibition of phosphoinositide 3-kinase (PI3K) suppressed the transactivation of the aromatase gene and its enzyme activity. Furthermore, we have also shown that PI3K/Akt-dependent cAMP-response element binding protein (CREB) activation was required for the enhanced expression of aromatase in TAMR-MCF-7 cells. Our findings suggest that aromatase expression is up-regulated in TAM-resistant breast cancer via PI3K/Akt-dependent CREB activation.

Keywords: TAMR-MCF-7, CREB, estrogen receptor, aromatase

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Authors: S. I. Nwadioha, M. S. Odimayo, G. T. A. Jombo, E. O. P. Nwokedi

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Aims and Objectives: To determine the baseline CD4 positive T lymphocytes count of HIV/AIDS treatment naïve adults clients presenting for the first time treatment in Benue State University Teaching Hospital. Subjects and Methods: A total of 700 subjects age between 18 years to 70 years, were recruited for the study, comprising 600 HIV sero-positive patients and 100 healthy controls in Benue State University Teaching Hospital, Makurdi from 2013 to 2014. The CD4 counts of the subjects were evaluated using a Partec flow cytometer. Results: CD4 count of 200-299 cells/μl peaked with 25% (n=150/600)[control; 0%( n= 0/100)]. The study also showed that 44% (266/600) of HIV subjects had acquired immunodeficiency syndrome as defined by low CD4 counts below 200 cells/μl. Seventy-five per cent (n=451/600)of our patients would require to be placed on antiretroviral therapy with CD4 count of less than 350 cells/μl. At CD4 350 baseline criterion, age group 20-29 years had the highest demand 35%(160/451) for ARV followed by age groups 30-39 and 40-49 years with 28%(128/451) and 22%(98/451) respectively. Conclusion: There is a high prevalence of acquired immunodeficiency syndrome as defined by CD4 counts below 200 cells/μl, among the young active productive age group. The strict adopting of the ART WHO 2010 scale- up criteria doubles the number of the HIV clients that would qualify for ART with its attendant health benefits on the long run.

Keywords: CD4 counts, HIV patients, young age group, Nigeria

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Authors: Bounar Nedjemeddine

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Materials with the pyrochlore lattice structure have attracted much recent attention due to their wide applications in ceramic thermal barrier coatings, high-permittivity dielectrics, and potential solid electrolytes in solid-oxide fuel cells. The work described in this paper is devoted to the synthesis and characterization of a pyrochlore structure based on lanthanum (La₂O₃) and tin (SnO₂) oxides of general formula La₂Sn₂O₇, substituted by Sr at the site La. Their structures were determined from X-ray powder diffraction using CELFER analysis. All the compositions present the space group Fd-3m. The substitution of La by Sr in the La₂Sn₂O₇ compound causes a variation of the cell parameters. The difference in charge between La³⁺ and Sr²⁺ and the difference in size cause the cell parameters to decrease from a=10.7165 A° to a=10.6848 A° for the substitution rates (x = 0.05, 0.1, 0.15 ...), which leads to a decrease in the volume of the mesh. For a substitution rate x = 0.25, there is an increase in the cell parameters (a=10.7035A°), which can be explained by a competitiveness of the size effect and the presence of a gap in the structure which go in the opposite direction.

Keywords: solid-oxide fuel cells, structure, pyrochlore, X-ray diffraction

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Authors: Ashit Syngle, Inderjit Verma, Pawan Krishan

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Objective: Endothelial progenitor cells (EPCs) have reparative potential in overcoming the endothelial dysfunction and reducing cardiovascular risk. EPC depletion has been demonstrated in the setting of established atherosclerotic diseases. With this background, we evaluated whether reduced EPCs population are associated with endothelial dysfunction, subclinical atherosclerosis and inflammatory markers in ankylosing spondylitis (AS) patients without any known traditional cardiovascular risk factor in AS patients. Methods: Levels of circulating EPCs (CD34+/CD133+), brachial artery flow-mediated dilatation, carotid intima-media thickness (CIMT) and inflammatory markers i.e erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), tissue necrosis factor (TNF)–α, interleukin (IL)-6, IL-1 were assessed in 30 AS patients (mean age33.41 ± 10.25; 11 female and 19 male) who fulfilled the modified New York diagnostic criteria with 25 healthy volunteers (mean age 29.36± 8.64; 9 female and 16 male) matched for age and sex. Results: EPCs (CD34+/CD133+) cells were significantly (0.020 ± 0.001% versus 0.040 ± 0.010%, p<0.001) reduced in patients with AS compared to healthy controls. Endothelial function (7.35 ± 2.54 versus 10.27 ±1.73, p=0.002), CIMT (0.63 ± 0.01 versus 0.35 ± 0.02, p < 0.001) and inflammatory markers were also significantly (p < 0.01) altered as compared to healthy controls. Specifically, CD34+CD133+cells were inversely multivariate correlated with CRP and TNF-α and endothelial dysfunction was positively correlated with reduced number of EPC. Conclusion: Depletion of EPCs population is an independent predictor of endothelial dysfunction and early atherosclerosis in AS patients and may provide additional information beyond conventional risk factors and inflammatory markers.

Keywords: endothelial progenitor cells, atherosclerosis, ankylosing spondylitis, cardiovascular

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Authors: Okyu Kwon, Dongho Kang, Byungsik Kim, Seungkwon Jung

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We propose a technique to measure the impact of loss of traffic function in a particular area to surrounding areas. The proposed method is applied to the city of Seoul, which is the capital of South Korea, with a population of about ten million. Based on the actual road network in Seoul, we construct an abstract road network between 1kmx1km grid cells. The link weight of the abstract road network is re-adjusted considering traffic volume measured at several survey points. On the modified abstract road network, we evaluate the traffic vulnerability by calculating a network measure of betweenness centrality (BC) for every single grid cells. This study analyzes traffic impacts caused by road dysfunction due to heavy rainfall in urban areas. We could see the change of the BC value in all other grid cells by calculating the BC value once again when the specific grid cell lost its traffic function, that is, when the node disappeared on the grid-based road network. The results show that it is appropriate to use the sum of the BC variation of other cells as the influence index of each lattice cell on traffic. This research was supported by a grant (2017-MOIS31-004) from Fundamental Technology Development Program for Extreme Disaster Response funded by Korean Ministry of Interior and Safety (MOIS).

Keywords: vulnerability, road network, beweenness centrality, heavy rainfall, road impact

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Authors: Thayse Marques Passos, Brid Quilty, Mary Pryce

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The interest in developing alternative techniques to obtain safe water, free from pathogens and hazardous substances, is growing in recent times. The photodynamic inactivation of microorganisms (PDI) is a promising ecologically-friendly and multi-target approach for water disinfection. It uses visible light as an energy source combined with a photosensitiser (PS) to transfer energy/electrons to a substrate or molecular oxygen generating reactive oxygen species, which cause cidal effects towards cells. PDI has mainly been used in clinical studies and investigations on its application to disinfect water is relatively recent. The majority of studies use planktonic cells. However, in their natural environments, bacteria quite often do not occur as freely suspended cells (planktonic) but in cell aggregates that are either freely floating or attached to surfaces as biofilms. Microbes can form aggregates and biofilms as a strategy to protect them from environmental stress. As aggregates, bacteria have a better metabolic function, they communicate more efficiently, and they are more resistant to biocide compounds than their planktonic forms. Among the bacteria that are able to form aggregates are members of the genus Pseudomonas, they are a very diverse group widely distributed in the environment. Pseudomonas species can form aggregates/biofilms in water and can cause particular problems in water distribution systems. The aim of this study was to evaluate the effectiveness of photodynamic inactivation in killing a range of planktonic cells including Escherichia coli DSM 1103, Staphylococcus aureus DSM 799, Shigella sonnei DSM 5570, Salmonella enterica and Pseudomonas putida DSM 6125, and aggregating cells of Pseudomonas fluorescens DSM 50090, Pseudomonas aeruginosa PAO1. The experiments were performed in glass Petri dishes, containing the bacterial suspension and the photosensitiser, irradiated with a multi-LED (wavelengths 430nm and 660nm) for different time intervals. The responses of the cells were monitored using the pour plate technique and confocal microscopy. The study showed that bacteria belonging to Pseudomonads group tend to be more tolerant to PDI. While E. coli, S. aureus, S. sonnei and S. enterica required a dosage ranging from 39.47 J/cm2 to 59.21 J/cm2 for a 5 log reduction, Pseudomonads needed a dosage ranging from 78.94 to 118.42 J/cm2, a higher dose being required when the cells aggregated.

Keywords: bacterial aggregation, photoinactivation, Pseudomonads, water disinfection

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Authors: Emad Heydarnia, Aref Sepasi, Nika Asefi, Sara Khakshournia, Javad Mohammadnejad

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Background: Despite breakthrough therapeutics in breast cancer, it is one of the main causes of mortality among women worldwide. Thus, drug therapies for treating breast cancer have recently been developed by scientists. Metformin and Sorafenib are well-known therapeutic in breast cancer. In the present study, we combined Sorafenib and PCL-sorafenib with metformin to improve drug absorption and promote therapeutic efficiency. Methods: The MCF-7 cells were treated with Metformin, Sorafenib, or PCL-sorafenib. The growth inhibitory effect of these drugs and cell viability were assessed using MTT and flow cytometry assays, respectively. The expression of targeted genes involved in cell proliferation, signaling, and the cell cycle was measured by Real-time PCR. Results: The results showed that MCF-7 cells treated with Metformin/Sorafenib and PCL-sorafenib/Metformin co-treatment contributed to 50% viability compared to untreated group. Moreover, PI and Annexin V staining tests showed that the cells viability for Metformin/Sorafenib and PCL-sorafenib/Metformin was 38% and 17%, respectively. Furthermore, Sorafenib/Metformin and PCL-sorafenib/Metformin leads to p53 gene expression increase by which they can increase ROS, thereby decreasing GPX4 gene expression. In addition, they affected the expression of BCL2, and BAX genes and altered the cell cycle. Conclusion: Together, the combination of PCL-sorafenib/Metformin and Sorafenib/Metformin increased Sorafenib absorption at lower doses and also leads to apoptosis and oxidative stress increases in MCF-7 cells.

Keywords: breast cancer, metformin, nanotechnology, sorafenib

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Authors: Seyedeh Nasim Mirbahari, Taha Azad, Mehdi Totonchi

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Oncolytic viruses, which only replicate in tumor cells, are being extensively studied for their use in cancer therapy. A particular virus known as the vaccinia virus, a member of the poxvirus family, has demonstrated oncolytic abilities glioma. Treating Glioma with traditional methods such as chemotherapy and radiotherapy is quite challenging. Even though oncolytic viruses have shown immense potential in cancer treatment, their effectiveness in glioblastoma treatment is still low. Therefore, there is a need to improve and optimize immunotherapies for better results. In this study, we have designed oncoVV-TT, which can more effectively target tumor cells while minimizing replication in normal cells by replacing the thymidine kinase gene with a luc-p2a-GFP gene expression cassette. Human glioblastoma cell line U251 MG, rat glioblastoma cell line C6, and non-tumor cell line HFF were plated at 105 cells in a 12-well plates in 2 mL of DMEM-F2 medium with 10% FBS added to each well. Then incubated at 37°C. After 16 hours, the cells were treated with oncoVV-TT at an MOI of 0.01, 0.1 and left in the incubator for a further 24, 48, 72 and 96 hours. Viral replication assay, fluorescence imaging and viability tests, including trypan blue and crystal violet, were conducted to evaluate the cytotoxic effect of oncoVV-TT. The finding shows that oncoVV-TT had significantly higher cytotoxic activity and proliferation rates in tumor cells in a dose and time-dependent manner, with the strongest effect observed in U251 MG. To conclude, oncoVV-TT has the potential to be a promising oncolytic virus for cancer treatment, with a more cytotoxic effect in human glioblastoma cells versus rat glioma cells. To assess the effectiveness of vaccinia virus-mediated viral therapy, we have tested U251mg and C6 tumor cell lines taken from human and rat gliomas, respectively. The study evaluated oncoVV-TT's ability to replicate and lyse cells and analyzed the survival rates of the tested cell lines when treated with different doses of oncoVV-TT. Additionally, we compared the sensitivity of human and mouse glioma cell lines to the oncolytic vaccinia virus. All experiments regarding viruses were conducted under biosafety level 2. We engineered a Vaccinia-based oncolytic virus called oncoVV-TT to replicate specifically in tumor cells. To propagate the oncoVV-TT virus, HeLa cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 10 MOI virus was added. After 48 h, cells were harvested by scraping, and viruses were collected by 3 sequential freezing and thawing cycles followed by removal of cell debris by centrifugation (1500 rpm, 5 min). The supernatant was stored at −80 ◦C for the following experiments. To measure the replication of the virus in Hela, cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 5 MOI virus or equal dilution of PBS was added. At the treatment time of 0 h, 24 h, 48 h, 72 h and 96 h, the viral titers were determined under the fluorescence microscope (BZ-X700; Keyence, Osaka, Japan). Fluorescence intensity was quantified using the imagej software according to the manufacturer’s protocol. For the isolation of single-virus clones, HeLa cells seeded in six-well plates (5×105 cells/well). After 24 h (100% confluent), the cells were infected with a 10-fold dilution series of TianTan green fluorescent protein (GFP)virus and incubated for 4 h. To examine the cytotoxic effect of oncoVV-TT virus ofn U251mg and C6 cell, trypan blue and crystal violet assay was used.

Keywords: oncolytic virus, immune therapy, glioma, vaccinia virus

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Authors: F. Kashanian, M. M. Masoudi, A. Akbari, A. Shamloo, M. R. Zand, S. S. Salehi

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Nano-sized materials present new opportunities in biology and medicine and they are used as biomedical tools for investigation, separation of molecules and cells. To achieve more effective cancer therapy, it is essential to select cancer cells exactly. This research suggests that using the antibody-functionalized nontoxic Arginine-doped magnetic nanoparticles (A-MNPs), has been prosperous in detection, capture, and magnetic separation of circulating tumor cells (CTCs) in tumor tissue. In this study, A-MNPs were synthesized via a simple precipitation reaction and directly immobilized Ep-CAM EBA-1 antibodies over superparamagnetic A-MNPs for Mucin BCA-225 in breast cancer cell. The samples were characterized by vibrating sample magnetometer (VSM), FT-IR spectroscopy, Tunneling Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). These antibody-functionalized nontoxic A-MNPs were used to capture breast cancer cell. Through employing a strong permanent magnet, the magnetic separation was achieved within a few seconds. Antibody-Conjugated nontoxic Arginine-doped Fe₃O₄ nanoparticles have the potential for the future study to capture CTCs which are released from tumor tissue and for drug delivery, and these results demonstrate that the antibody-conjugated A-MNPs can be used in magnetic hyperthermia techniques for cancer treatment.

Keywords: tumor tissue, antibody, magnetic nanoparticle, CTCs capturing

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Authors: Saman Khan, Imrana Naseem

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Copper is an essential trace element required for pro-angiogenic co-factors including vascular endothelial growth factor (VEGF). Elevated levels of copper are found in various types of cancer including prostrate, colon, breast, lung and liver for angiogensis and metastasis. Therefore, targeting copper via copper-specific chelators in cancer cells can be developed as effective anticancer treatment strategy. In continuation of our pursuit to design and synthesize copper chelators, herein we opted for a reaction to incorporate di-(2-picolyl) amine in 3-(bromoacetyl) coumarin (parent backbone) for the synthesis of complex 1. We evaluated lipid peroxidation, protein carbonylation, ROS generation, DNA damage and consequent apoptosis by complex 1 in exogenously added Cu(II) in human peripheral lymphocytes (simulate malignancy condition). Results showed that Cu(II)-complex 1 interaction leads to cell proliferation inhibition, apoptosis, ROS generation and DNA damage in human lymphocytes, and these effects were abrogated by cuprous chelator neocuproine and ROS scavengers (thiourea, catalase, SOD). This indicates that complex 1 cytotoxicity is due to redox cycling of copper to generate ROS which leads to pro-oxidant cell death in cancer cells. To further confirm our hypothesis, using the rat model of diethylnitrosamine (DEN) induced hepatocellular carcinoma; we showed that complex 1 mediates DNA breakage and cell death in isolated carcinoma cells. Membrane permeant copper chelator, neocuproine, and ROS scavengers inhibited the complex 1-mediated cellular DNA degradation and apoptosis. In summary, complex 1 anticancer activity is due to its copper chelation capability. These results will provide copper chelation as an effective targeted cancer treatment strategy for selective cytotoxic action against malignant cells without affecting normal cells.

Keywords: cancer treatment, copper chelation, ROS generation, DNA damage, redox cycling, apoptosis

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Authors: Seungmin Shin, Jin-Baek Kim

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Hematite (α-Fe2O3) has very good semiconducting properties with a band gap of 2.1 eV and is antiferromagnetic. Due to its electrochemical stability, low toxicity, wide abundance, and low-cost, hematite, it is a particularly attractive material for photoelectrochemical cells. Additionally, hematite has also found applications in gas sensing, field emission, heterogeneous catalysis, and lithium-ion battery electrodes. Here, we discovered a new universal top-down method for the synthesis of one-dimensional hematite nanowire arrays. Various shapes and lengths of hematite nanowire have been easily fabricated over large areas by sequential processes. The obtained hematite nanowire arrays are promising candidates as photoanodes in photoelectrochemical solar cells.

Keywords: hematite, lithography, nanowire, top-down process

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Authors: Svetlana Guryeva, Inna Kornienko, Andrey Usanov, Dmitry Usanov, Elena Petersen

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Every day cells in our organism are exposed to various factors: chemical agents, reactive oxygen species, ionizing radiation, and others. These factors can cause damage to DNA, cellular membrane, intracellular compartments, and proteins. The fate of cells depends on the exposure intensity and duration. The prolonged and intense exposure causes the irreversible damage accumulation, which triggers the permanent cell cycle arrest (cellular senescence) or cell death programs. In the case of low dose of impacts, it can lead to cell renovation and to cell functional state improvement. Therefore, it is a pivotal question to investigate the factors and doses that result in described positive effects. In order to estimate the influence of different agents, the proliferation index and levels of cell death markers (annexin V/propidium iodide), senescence-associated β-galactosidase, and lipofuscin were measured. The experiments were conducted on primary human fibroblasts of the 8th passage. According to the levels of mentioned markers, these cells were defined as senescent cells. The effect of low-frequency magnetic field was investigated. Different modes of magnetic field exposure were tested. The physical agents were compared with chemical agents: metformin (10 mM) and taurine (0.8 mM and 1.6 mM). Cells were incubating with chemicals for 5 days. The highest decrease in the level of senescence-associated β-galactosidase (21%) and lipofuscin (17%) was observed in the primary senescent fibroblasts after 5 days after double treatments with 48 h intervals with low-frequency magnetic field. There were no significant changes in the proliferation index after magnetic field application. The cytotoxic effect of magnetic field was not observed. The chemical agent taurine (1.6 mM) decreased the level of senescence-associated β-galactosidase (23%) and lipofuscin (22%). Metformin improved the activity of senescence-associated β-galactosidase on 15% and the level of lipofuscin on 19% in this experiment. According to these results, the effect of double treatment with 48 h interval with low-frequency magnetic field and the effect of taurine (1.6 mM) were comparable to the effect of metformin, for which anti-aging properties are proved. In conclusion, this study can become the first step towards creation of the standardized system for the investigation of different effects on senescent cells.

Keywords: biomarkers, magnetic field, metformin, primary fibroblasts, senescence, taurine

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Authors: Dace Grauda, Inta Belogrudova, Alexei Katashev, Linda Lancere, Isaak Rashal

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The goal of study was the elaboration of easy applicable flow cytometry method for detection of influence of abiotic stress factors on plants, which could be useful for detection of environmental stresses in urban areas. The lime tree Tillia vulgaris H. is a popular tree species used for urban landscaping in Europe and is one of the main species of street greenery in Riga, Latvia. Tree decline and low vitality has observed in the central part of Riga. For this reason lime trees were select as a model object for the investigation. During the period of end of June and beginning of July 12 samples from different urban environment locations as well as plant material from a greenhouse were collected. BD FACSJazz® cell sorter (BD Biosciences, USA) with flow cytometer function was used to test viability of plant cells. The method was based on changes of relative fluorescence intensity of cells in blue laser (488 nm) after influence of stress factors. SpheroTM rainbow calibration particles (3.0–3.4 μm, BD Biosciences, USA) in phosphate buffered saline (PBS) were used for calibration of flow cytometer. BD PharmingenTM PBS (BD Biosciences, USA) was used for flow cytometry assays. The mean fluorescence intensity information from the purified cell suspension samples was recorded. Preliminary, multiple gate sizes and shapes were tested to find one with the lowest CV. It was found that low CV can be obtained if only the densest part of plant cells forward scatter/side scatter profile is analysed because in this case plant cells are most similar in size and shape. The young pollen cells in one nucleus stage were found as the best for detection of influence of abiotic stress. For experiments only fresh plant material was used– the buds of Tillia vulgaris with diameter 2 mm. For the cell suspension (in vitro culture) establishment modified protocol of microspore culture was applied. The cells were suspended in the MS (Murashige and Skoog) medium. For imitation of dust of urban area SiO2 nanoparticles with concentration 0.001 g/ml were dissolved in distilled water. Into 10 ml of cell suspension 1 ml of SiO2 nanoparticles suspension was added, then cells were incubated in speed shaking regime for 1 and 3 hours. As a stress factor the irradiation of cells for 20 min by UV was used (Hamamatsu light source L9566-02A, L10852 lamp, A10014-50-0110), maximum relative intensity (100%) at 365 nm and at ~310 nm (75%). Before UV irradiation the suspension of cells were placed onto a thin layer on a filter paper disk (diameter 45 mm) in a Petri dish with solid MS media. Cells without treatment were used as a control. Experiments were performed at room temperature (23-25 °C). Using flow cytometer BS FACS Software cells plot was created to determine the densest part, which was later gated using oval-shaped gate. Gate included from 95 to 99% of all cells. To determine relative fluorescence of cells logarithmic fluorescence scale in arbitrary fluorescence units were used. 3x103 gated cells were analysed from the each sample. The significant differences were found among relative fluorescence of cells from different trees after treatment with SiO2 nanoparticles and UV irradiation in comparison with the control.

Keywords: flow cytometry, fluorescence, SiO2 nanoparticles, UV irradiation

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Authors: Kangana Sengar, Sanjay Deb, Ramesh Dawar

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Introduction: Ki-67 can be a useful marker in determining proliferative activity in patients with multiple myeloma (MM). However, using Ki-67 alone results in the erroneous inclusion of non-myeloma cells leading to false high counts. We have used Dual IHC (immunohistochemistry) staining with Ki-67 and CD138 to enhance specificity in assessing proliferative activity of bone marrow plasma cells. Aims and objectives: To estimate the proportion of proliferating (Ki-67 expressing) plasma cells in patients with MM and correlation of Ki-67 with other known prognostic parameters. Materials and Methods: Fifty FFPE (formalin fixed paraffin embedded) blocks of trephine biopsies of cases diagnosed as MM from 2010 to 2015 are subjected to H & E staining and Dual IHC staining for CD 138 and Ki-67. H & E staining is done to evaluate various histological parameters like percentage of plasma cells, pattern of infiltration (nodular, interstitial, mixed and diffuse), routine parameters of marrow cellularity and hematopoiesis. Clinical data is collected from patient records from Medical Record Department. Each of CD138 expressing cells (cytoplasmic, red) are scored as proliferating plasma cells (containing a brown Ki¬67 nucleus) or non¬proliferating plasma cells (containing a blue, counter-stained, Ki-¬67 negative nucleus). Ki-67 is measured as percentage positivity with a maximum score of hundred percent and lowest of zero percent. The intensity of staining is not relevant. Results: Statistically significant correlation of Ki-67 in D-S Stage (Durie & Salmon Stage) I vs. III (p=0.026) and ISS (International Staging System) Stage I vs. III (p=0.019), β2m (p=0.029) and percentage of plasma cells (p < 0.001) is seen. No statistically significant correlation is seen between Ki-67 and hemoglobin, platelet count, total leukocyte count, total protein, albumin, S. calcium, S. creatinine, S. LDH, blood urea and pattern of infiltration. Conclusion: Ki-67 index correlated with other known prognostic parameters. However, it is not determined routinely in patients with MM due to little information available regarding its relevance and paucity of studies done to correlate with other known prognostic factors in MM patients. To the best of our knowledge, this is the first study in India using Dual IHC staining for Ki-67 and CD138 in MM patients. Routine determination of Ki-67 will help to identify patients who may benefit with more aggressive therapy. Recommendation: In this study follow up of patients is not included, and the sample size is small. Studying with larger sample size and long follow up is advocated to prognosticate Ki-67 as a marker of survival in patients with multiple myeloma.

Keywords: bone marrow, dual IHC, Ki-67, multiple myeloma

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Authors: Olivier de Backer, Alexis Khelfi, Olivier Svensek, Axelle Nolmans, Dominique Desnoeck

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The SMC5-SMC6 complex helps replication and repair of DNA double-strand breaks. Nse1, Nse3 and Nse4 are non-SMC components of the complex in which Nse3 stimulates the E3 ubiquitin ligase activity of Nse1 and is required for recruiting the complex on DNA. In most eukaryotes, Nse3 is a single protein, but in eutherians (placental mammals), it belongs to a large family of proteins called MAGE (Melanoma antigen) that share a conserved domain of about 200 aa known as MHD (Mage homology domain). MAGE assembles specific RING and HECT ubiquitin ligases and determines new substrates for ubiquitination. The MHD is required for the interaction with the cognate E3 ligase. Some MAGEs (referred to as Type I) are exclusively expressed in germ cells of the testis but are often expressed ectopically in cancer cells as the result of epigenetic modifications. The 12 MAGE-A genes belong to this category. Serval MAGE-A proteins could promote tumorigenesis by targeting tumor suppressor proteins (including p53) for ubiquitination and degradation. We showed that depletion of MAGE-A proteins in melanoma cells results in impaired DNA damage response and increased double-strand breaks after exposure to camptothecin. Moreover, it was shown that other actors of the DNA Damage Response were impacted when cells were depleted of MAGEA proteins.

Keywords: DNA damage response, melanoma, camptothecin, new role, MAGEA

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