Search results for: transitional cell carcinoma

Authors: Beste Ozcan, Valerio Sperati, Laura Romano, Tania Moretta, Simone Scaffaro, Noemi Faedda, Federica Giovannone, Carla Sogos, Vincenzo Guidetti, Gianluca Baldassarre

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+me is an experimental interactive toy with the appearance of a soft, pillow-like, panda. Shape and consistency are designed to arise emotional attachment in young children: a child can wear it around his/her neck and treat it as a companion (i.e. a transitional object). When caressed on paws or head, the panda emits appealing, interesting outputs like colored lights or amusing sounds, thanks to embedded electronics. Such sensory patterns can be modified through a wirelessly connected tablet: by this, an adult caregiver can adapt +me responses to a child's reactions or requests, for example, changing the light hue or the type of sound. The toy control is therefore shared, as it depends on both the child (who handles the panda) and the adult (who manages the tablet and mediates the sensory input-output contingencies). These features make +me a potential tool for therapy with children with Neurodevelopmental Disorders (ND), characterized by impairments in the social area, like Autism Spectrum Disorders (ASD) and Language Disorders (LD): as a proposal, the toy could be used together with a therapist, in rehabilitative play activities aimed at encouraging simple social interactions and reinforcing basic relational and communication skills. +me was tested in two pilot experiments, the first one involving 15 Typically Developed (TD) children aged in 8-34 months, the second one involving 7 children with ASD, and 7 with LD, aged in 30-48 months. In both studies a researcher/caregiver, during a one-to-one, ten-minute activity plays with the panda and encourages the child to do the same. The purpose of both studies was to ascertain the general acceptability of the device as an interesting toy that is an object able to capture the child's attention and to maintain a high motivation to interact with it and with the adult. Behavioral indexes for estimating the interplay between the child, +me and caregiver were rated from the video recording of the experimental sessions. Preliminary results show how -on average- participants from 3 groups exhibit a good engagement: they touch, caress, explore the panda and show enjoyment when they manage to trigger luminous and sound responses. During the experiments, children tend to imitate the caregiver's actions on +me, often looking (and smiling) at him/her. Interesting behavioral differences between TD, ASD, and LD groups are scored: for example, ASD participants produce a fewer number of smiles both to panda and to a caregiver with respect to TD group, while LD scores stand between ASD and TD subjects. These preliminary observations suggest that the interactive toy +me is able to raise and maintain the interest of toddlers and therefore it can be reasonably used as a supporting tool during therapy, to stimulate pivotal social skills as imitation, turn-taking, eye contact, and social smiles. Interestingly, the young age of participants, along with the behavioral differences between groups, seem to suggest a further potential use of the device: a tool for early differential diagnosis (the average age of a child

Keywords: autism spectrum disorders, interactive toy, social interaction, therapy, transitional wearable companion

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Authors: Messaoud Eljamai, Sami Hidouri

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Massive multiple-input multiple-output (MIMO) is an emerging technology for new cellular networks such as 5G systems. Its principle is to use many antennas per cell in order to maximize the network's spectral efficiency. Inter-cellular interference remains a fundamental problem. The use of massive MIMO will not derogate from the rule. It improves performances only when the number of antennas is significantly greater than the number of users. This, considerably, limits the networks spectral efficiency. In this paper, a coordinated detector for an uplink massive MIMO system is proposed in order to mitigate the inter-cellular interference. The proposed scheme combines the coordinated multipoint technique with an interference-cancelling algorithm. It requires the serving cell to send their received symbols, after processing, decision and error detection, to the interfered cells via a backhaul link. Each interfered cell is capable of eliminating intercellular interferences by generating and subtracting the user’s contribution from the received signal. The resulting signal is more reliable than the original received signal. This allows the uplink massive MIMO system to improve their performances dramatically. Simulation results show that the proposed detector improves system spectral efficiency compared to classical linear detectors.

Keywords: massive MIMO, COMP, interference canceling algorithm, spectral efficiency

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Authors: Aliyu Maje Bello, Yaowaluck Maprang Roshorm

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Hand, foot, and mouth disease (HFMD) is a highly contagious viral infection affecting mostly infants and children. Although the Enterovirus A71 (EV71) is usually the major causative agent of HFMD, other enteroviruses such as coxsackievirus A16, A10, and A6 are also found in some of the recent outbreaks. The commercially available vaccines have demonstrated their effectiveness against only EV71 infection but no protection against other enteroviruses. To address the limitation of the monovalent EV71 vaccine, the present study thus designed a tetravalent vaccine against the four major enteroviruses causing HFMD and primarily evaluated the designed vaccine using an immunoinformatics approach. The immunogen was designed to contain the EV71 VP1 protein and multiple reported epitopes from all four distinct enteroviruses and thus designated a tetravalent vaccine. The 3D structure of the designed tetravalent vaccine was modeled, refined, and validated. Epitope screening showed the presence of B-cell, CTL, CD4 T cell, and IFN epitopes with vast application among the Asian population. Docking analysis confirmed the stable and strong binding interactions between the immunogen and immune receptor B-cell receptor (BCR). In silico cloning and immune simulation analyses guaranteed high efficiency and sufficient expression of the vaccine candidate in humans. Overall, the promising results obtained from the in-silico studies of the proposed tetravalent vaccine make it a potential candidate worth further experimental validation.

Keywords: enteroviruses, coxsackieviruses, hand foot and mouth disease, immunoinformatics, tetravalent vaccine

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Authors: Huyen T. Pham, Nhue P. Nguyen, Tien Q. Phi, Phuong T. Dang, Hy G. Le

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Since the marine environmental conditions are extremely different from the other ones, so that marine actinomycetes might produce novel bioactive compounds. Therefore, actinomycete strains were screened from marine water and sediment samples collected from the coastal areas of Northern Vietnam. Ninety-nine actinomycete strains were obtained on starch-casein agar media by dilution technique, only seven strains, named HP112, HP12, HP411, HPN11, HP 11, HPT13 and HPX12, showed significant antibacterial activity against both gram-positive and gram-negative bacteria (Bacillus subtilis ATCC 6633, Staphylococcus epidemidis ATCC 12228, Escherichia coli ATCC 11105). Further studies were carried out with the most active HP411strain against Candida albicans ATCC 10231. This strain could grow rapidly on starch casein agar and other media with high salt containing 7-10% NaCl at 28-30oC. Spore-chain of HP411 showed an elongated and circular shape with 10 to 30 spores/chain. Identification of the strain was carried out by employing the taxonomical studies including the 16S rRNA sequence. Based on phylogenetic and phenotypic evidence it is proposed that HP411 to be belongs to species Streptomyces variabilis. The potent of the crude extract of fermentation broth of HP411that are effective against wide range of pathogens: both gram-positive, gram-negative and fungi. Further studies revealed that the crude extract HP411 could obtain the anticancer activity for cancer cell lines: Hep-G2 (liver cancer cell line); RD (cardiac and skeletal muscle letters cell line); FL (membrane of the uterus cancer cell line). However, the actinomycetes from marine ecosystem will be useful for the discovery of new drugs in the furture.

Keywords: marine actinomycetes, antibacterial, anticancer, Streptomyces variabilis

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Authors: Norimichi Nagano, Yoshio Hirano, Tsutomu Yasukawa

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Mesenchymal stem cells are thought to confer neuroprotection, facilitate tissue regeneration and exert their effects on retinal degenerative diseases, however, adverse events such as proliferative vitreoretinopathy and preretinal membrane disease associated with cell suspension transplantation have also been reported. We have recently developed human (allogeneic) adipose tissue-derived mesenchymal stem cell (adMSC) sheets through our proprietary sheet transformation technique, which could potentially mitigate these adverse events. To clarify the properties of our adMSC sheets named PAL-222, we performed in vitro studies such as viability testing, cytokine secretions by ELISA, immunohistochemical study, and migration assay. The viability of the cells exceeded 70%. Vascular Endothelial Growth Factor (VEGF) and Pigment Epithelium-Derived Factor (PEDF), which are quite important cytokines for the retinal area, were observed. PAL-222 expressed type I collagen, a strength marker, type IV collagen, a marker of the basement membrane, and elastin, an elasticity marker. Finally, the migration assay was performed and showed negative, which means that PAL-222 is stably kept in the topical area and does not come to pieces. Next, to evaluate the efficacy in vivo, we transplanted PAL-222 into the subretinal space of the eye of Royal College of Surgeons rats with congenital retinal degeneration and assessed it for three weeks after transplantation. We confirmed that PAL-222 suppressed the decrease in the thickness of the outer nuclear layer, which means that the photoreceptor protective effect treated with PAL-222 was significantly higher than that in the sham group. (p < 0.01). This finding demonstrates that PAL-222 showed their retinoprotective effect in a model of congenital retinal degeneration. As the study suggested the efficacy of PAL-222 in both in vitro and in vivo studies, we are presently engaged in clinical trials of PAL-222 for myopic chorioretinal atrophy, which is one of the retinal degenerative diseases, for the purpose of regenerative medicine.

Keywords: cell sheet, clinical trial, mesenchymal stem cell, myopic chorioretinal atrophy

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Authors: Osama Elsarrar, Marjorie Darrah, Richard Devin

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This paper discusses the idea of capturing an expert’s knowledge in the form of human understandable rules and then inserting these rules into a dynamic cell structure (DCS) neural network. The DCS is a form of self-organizing map that can be used for many purposes, including classification and prediction. This particular neural network is considered to be a topology preserving network that starts with no pre-structure, but assumes a structure once trained. The DCS has been used in mission and safety-critical applications, including adaptive flight control and health-monitoring in aerial vehicles. The approach is to insert expert knowledge into the DCS before training. Rules are translated into a pre-structure and then training data are presented. This idea has been demonstrated using the well-known Iris data set and it has been shown that inserting the pre-structure results in better accuracy with the same training.

Keywords: neural network, self-organizing map, rule extraction, rule insertion

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Authors: V. A. Adzika, D. Ayim-Aboagye, T. Gordh

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Background and aims: Prevalence of Sickle Cell Disease (SCD) is high in Ghana but not much is known in terms of research into non-medical strategies for managing and coping with the pain associated with SCD. This study was carried out to examine effective non-medical related strategies patients use to cope and manage their SCD condition. Methods: SCD patients (387) consisting of 180 males and 204 females between 18-65 years old years participated in the study. A cross-sectional research design was used in which participants completed questionnaires on pain, non-medical coping and management strategies, anxiety, and depression. Results of multiple regression analysis showed that socio-demographic characteristics contributed to the variance in the pain associated with SCD. Results: Over 90% of participants reported that pains associated with SCD were the main reason for seeking treatment in SCD crisis. In terms of non-medical related coping strategies, attending a place of worship and praying were the main coping strategies used in SCD crises, suggesting that patients’ beliefs, particularly in a supernatural being, served as a mitigating factor in the process of coping with the pain associated with SCD crisis. Also, avoidance and withdrawal from people and social activities were reported to be strategies used to cope effectively with the pain associated with SCD crisis. Conclusion: This indicates that it is imperative to incorporate non-medical related coping and management strategies, especially religious beliefs and psychosocial factors, to coping and management of the pain associated with SCD.

Keywords: anxiety, depression, sickle cell disease, quality of life, socio-demographic characteristics, Ghana

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Authors: Khairi El Battawy, Monika Skalicki

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The present investigation has been done on pure Egyptian Arabian mares that reared in private horse studs. Fifty non-pregnant mares were selected and examined to classify them as either being reproductively healthy or subfertile mares including clinical endometritis, early embryonic death, granulosa cell tumor, repeat breeder (post-breeding endometritis), and anoestrus mares. The purpose of the study was to assess oxidative/antioxidant biochemical metabolites, lipogram, trace elements and reproductive hormones throughout reproductive conditions in mares during regular estrous, anestrum, early pregnancy, granulose cell tumor, ovulation failure, and endometritis. Results showed intensification of the free radical-dependent process in the blood of infertile mare, especially mares with endometritis. Ultrasonography as a diagnostic tool diagnosis of endometritis in mares was an important step as it revealed much information concerning infertility problem.

Keywords: endometritis, ovulation, oxidative, mare

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Authors: Lian Wu, Mervyn Merrilees

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Chronic Obstructive Pulmonary Disease (COPD) is a worldwide problem, characterized by irreversible and progressive airflow obstruction. In New Zealand, it is currently the 4th commonest cause of death and exacerbations of COPD are a frequent cause of admission to hospital. Serum levels of Clara cell secretory protein-16 (CC-16) are believed to represent Clara cell toxicity. More recently, CC-16 has been found to be associated with smoker COPD. It is produced almost exclusively by non-ciliated Clara cells in the airways, and its primary function is to protect the lungs against oxidative stress and carcinogenesis. After acute exposure to cigarette smoke, serum levels of CC-16 become elevated. CC16 is a potent natural immune-suppressor and anti-inflammatory agent. In vitro, CC16 inhibits both monocyte and polymorphonuclear neutrophils chemotaxis and phagocytosis. CC16 also inhibits fibroblast chemotaxis. However, the role of CC-16 in non-smoking related COPD is still not clear. In this study, we investigated serum CC-16 levels in non-smoking related COPD. Methods: We compared non-smoker patients with COPD (FEV1<60% of predicted, FEV1/FVC <0.7, n=100) and individuals with normal lung function FEV1≥ 80% of predicted and FEV1/FVC≥ 0.7, n=80). All subjects had no smoking history. CC-16 was measured by ELISA. Results and conclusion: Serum CC-16 levels are reduced in individuals with non-smoking related COPD, and there is a weak correlation with disease severity in non-smoking related COPD group compared to non-smoker controls.

Keywords: COPD, CC-16, ELISA, non-smoking-related COPD

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Authors: Mohammadjavad Sotoudeheian, Soroush Nematollahi

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Crohn’s disease (CD) is a chronic gastrointestinal segment inflammation encompassing immune dysregulation in a genetically susceptible individual in response to the environmental triggers and interaction between the microbiome and immune system. Uncontrolled inflammation leads to long-term complications, including fibrotic strictures and enteric fistulae. Increased production of Th1 and Th17-cell cytokines and defects in T-regulatory cells have been associated with CD. Th17-cells are essential for protection against extracellular pathogens, but their atypical activity can cause autoimmunity. Intrinsic defects in the control of programmed cell death in the mucosal T-cell compartment are strongly implicated in the pathogenesis of CD. The apoptosis defect in mucosal T-cells in CD has been endorsed as an imbalance of the Bcl-2 and the Bax. The immune system encounters foreign antigens through microbial colonization of mucosal surfaces or infections. In addition, FOSL downregulated IL-26 expression, a cytokine that marks inflammatory Th17-populations in patients suffering from CD. Furthermore, the expression of IL-23 is associated with the transcription factor primary leucine zipper transcription factor ATF-like (Batf). Batf-deficiency demonstrated the crucial role of Batf in colitis development. Batf and IL-23 mediate their effects by inducing IL-6 production. Strong association of IL-23R, Stat3, and Stat4 with IBD susceptibility point to a critical involvement of T-cells. IL-23R levels in transfer fistula were dependent on the AP-1 transcription factor JunB that additionally controlled levels of RORγt by facilitating DNA binding of Batf. T lymphocytes lacking JunB failed to induce IL-23- and Th17-mediated experimental colitis highlighting the relevance of JunB for the IL-23/ Th17 pathway. The absence of T-bet causes unrestrained Th17-cell differentiation. T-cells are central parts of immune-mediated colon fistula. Especially Th17-cells were highly prevalent in inflamed IBD tissues, as RORγt is effective in preventing colitis. Intraepithelial lymphocytes (IEL) contain unique T-cell subsets, including cells expressing RORγt. Increased activated Th17 and decreased T-regulatory cells in inflamed intestinal tissues had been seen. T-cells differentiate in response to many cytokines, including IL-1β, IL-6, IL-23, and TGF-β, into Th17-cells, a process which is critically dependent on the Batf. IL-23 promotes Th17-cell in the colon. Batf manages the generation of IL-23 induced IL-23R+ Th17-cells. Batf is necessary for TGF-β/IL-6-induced Th17-polarization. Batf-expressing T-cells are the core of T-cell-mediated colitis. The human-specific parts of three AP-1 transcription factors, FOSL1, FOSL2, and BATF, are essential during the early stages of Th17 differentiation. BATF supports the Th17 lineage. FOSL1, FOSL2, and BATF make possession of regulatory loci of genes in the Th17 lineage cascade. The AP1 transcription factor Batf is identified to control intestinal inflammation and seems to regulate pathways within lymphocytes, which could theoretically control the expression of several genes. It shows central regulatory properties over Th17-cell development and is intensely upregulated within IBD-affected tissues. Here, we demonstrated that targeting Batf in IBD appears as a therapeutic approach that reduces colitogenic T-cell activities during fistula formation while aiming to affect inflammation in the gut epithelial cells.

Keywords: immune system, Crohn’s Disease, BATF, T helper cells, Bcl, interleukin, FOSL

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Authors: Hanan Khojah, RuAngelie Edrada-Ebel

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Neurodegenerative disease including Alzheimer’s disease is a major cause of long-term disability. Oxidative stress is frequently implicated as one of the key contributing factors to neurodegenerative diseases. Protection against neuronal damage remains a great challenge for researchers. Ficus carica (commonly known as fig) is a species of great antioxidant nutritional value comprising a protective mechanism against innumerable health disorders related to oxidative stress as well as Alzheimer’s disease. The purpose of this work was to characterize the non-polar active metabolites in Ficus carica endocarp, mesocarp, and exocarp. Crude extracts were prepared using several extraction solvents, which included 1:1 water: ethylacetate, acetone and methanol. The dried extracts were then solvent partitioned between equivalent amounts of water and ethylacetate. Purification and fractionation were accomplished by high-throughput chromatography. The isolated metabolites were tested on their effect on human neuroblastoma cell line by cell viability test and cell cytotoxicity assay with acrolein. Molecular weights of the active metabolites were determined via LC–HRESIMS and GC-EIMS. Metabolomic profiling was performed to identify the active metabolites by using differential expression analysis software (Mzmine) and SIMCA for multivariate analysis. Structural elucidation and identification of the interested active metabolites were studied by 1-D and 2-D NMR. Significant differences in bioactivity against a concentration-dependent assay on acrolein radicals were observed between the three fruit parts. However, metabolites obtained from mesocarp and the endocarp demonstrated bioactivity to scavenge ROS radical. NMR profiling demonstrated that aliphatic compounds such as γ-sitosterol tend to induce neuronal bioactivity and exhibited bioactivity on the cell viability assay. γ-Sitosterol was found in higher concentrations in the mesocarp and was considered as one of the major phytosterol in Ficus carica.

Keywords: alzheimer, Ficus carica, γ-Sitosterol, metabolomics

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Authors: Mehdi Salari

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This work introduces a new experimental method of martensite treatment contains accumulative roll-bonding used for producing the nano/ultrafine grained structure in low carbon steel. The ARB process up to 4 cycles was performed under unlubricated conditions, while the annealing process was carried out in the temperature range of 450–550°C for 30–100 min. The microstructures of the deformed and annealed specimens were investigated. The results showed that in the annealed specimen at 450°C for 30 or 60 min, recrystallization couldn’t be completed. Decrease in time and temperature intensified the volume fraction of the martensite cell blocks. Fully equiaxed nano/ultrafine grained ferrite was developed from the martensite cell blocks during the annealing at temperature around 500°C for 100 min.

Keywords: martensite process, accumulative roll bonding, recrystallization, nanostructure, plain carbon steel

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Authors: Małgorzata Tomczyńska, Joanna Saluk-Bijak

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Background: Graves’ disease is a frequent organ-specific autoimmune thyroid disease, which characterized by the presence of different kind autoantibodies, that, in most cases, act as agonists of the thyrotropin receptor, leading to hyperthyroidism. Role of platelets and lymphocytes can be modulated in the pathophysiology of thyroid autoimmune diseases. Interference in the physiology of platelets can lead to enhanced activity of these cells. Activated platelets can bind to circulating lymphocytes and to affect lymphocyte adhesion. Platelets and lymphocytes can regulate mutual functions. Therefore, the activation of T lymphocytes, as well as blood platelets, is associated with the development of inflammation and oxidative stress within the target tissue. The present study was performed to investigate a platelet-lymphocyte relation by assessing the degree of their mutual aggregation in whole blood of patients with Graves’ disease. Also, the purpose of this study was to examine the impact of platelet interaction on lymphocyte migration capacity. Methods: 30 patients with Graves’ disease were recruited in the study. The matched 30 healthy subjects were served as the control group. Immunophenotyping of lymphocytes was carried out by flow cytometry method. A CytoSelect™ Cell Migration Assay Kit was used to evaluate lymphocyte migration and adhesion to blood platelets. Visual assessment of lymphocyte-platelet aggregate morphology was done using confocal microscope after magnetic cell isolation by Miltenyi Biotec. Results: The migration and functional responses of lymphocytes to blood platelets were greater in the group of Graves’ disease patients compared with healthy controls. The group of Graves’ disease patients exhibited a reduced T lymphocyte and a higher B cell count compared with controls. Based on microscopic analysis, more platelet-lymphocyte aggregates were found in patients than in control. Conclusions: Studies have shown that in Graves' disease, lymphocytes show increased platelet affinity, more strongly migrating toward them, and forming mutual cellular conglomerates. This may be due to the increased activation of blood platelets in this disease.

Keywords: blood platelets, cell migration, Graves’ disease, lymphocytes, lymphocyte-platelet aggregates

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Authors: S. Reshma Reghu, Shiburaj Sugathan, T. G. Nandu, K. B. Ramesh Kumar, Mathew Dan

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Bacterial diseases are considered to be one of the most prevalent health hazards in the developing world and many bacteria are becoming resistant to existing antibiotics making the treatment ineffective. Thus, it is necessary to find novel targets and develop new antibacterial drugs with a novel mechanism of action. The process of bacterial cell division is a novel and attractive target for new antibacterial drug discovery. FtsZ, a homolog of eukaryotic tubulin, is the major protein of the bacterial cell division machinery and is considered as an important antibacterial drug target. Zingiberaceae, the Ginger family consists of aromatic herbs with creeping rhizomes. Many of these plants have antimicrobial properties.This study aimed to determine the anti-bacterial activity of selected Zingiberaceous plants by targeting bacterial cell division protein, FtsZ. Essential oils and methanol extracts of Amomum ghaticum, Alpinia galanga, Kaempferia galanga, K. rotunda, and Zingiber officinale were tested to find its antibacterial efficiency using disc diffusion method against authentic bacterial strains obtained from MTCC (India). Essential oil isolated from A.galanga and Z.officinale were further assayed for FtsZ inhibition assay following non-radioactive malachite green-phosphomolybdate assay using E. coli FtsZ protein obtained from Cytoskelton Inc., USA. Z.officinale essential oil possess FtsZ inhibitory property. A molecular docking study was conducted with the known bioactive compounds of Z. officinale as ligands with the E. coli FtsZ protein homology model. Some of the major constituents of this plant like catechin, epicatechin, and gingerol possess agreeable docking scores. The results of this study revealed that several chemical constituents in Ginger plants can be utilised as potential source of antibacterial activity and it can warrant further investigation through drug discovery studies.

Keywords: antibacterial, FtsZ, zingiberaceae, docking

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Authors: Parul Grover, K. A. Suri, Raj Kumar, Gulshan Bansal

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Background: Nyctanthes arbortristis Linn is traditionally used as anticancer herb in Indian system of medicine, but its introduction into modern system of medicine is still awaited due to lack of systematic scientific studies. Objective: The objective of the present study was to isolate and characterize anticancer phytoconstituents from N. arbortristis L. leaves based on bioactivity guided fractionation. Method: Different extracts of the leaves of the plant were prepared by Soxhlet extractor. Each extract was evaluated for anticancer activity against HL-60 cell lines. Chloroform and HA extract showed potent anticancer activity and hence were selected for fractionation. Fraction C1 from chloroform extract was found to be most potent amongst all when tested against three cell lines (HL-60, A-549, and HCT-116) and thus was selected for further fractionation and a pure compound CP-01 was isolated. RP-HPLC method has been developed for quantification of isolated compound by using Kinetex C-18 column with gradient elution at 0.7 mL/min using mobile phase containing potassium dihydrogen phosphate (0.01 M, pH 3.0) with acetonitrile. The wavelength of maximum absorption (λₘₐₓ) selected was 210 nm. Results: The structure of potent anticancer CP-01 was determined on the basis spectroscopic methods like IR, 1H-NMR, ¹³C-NMR and Mass Spectrometry and it was characterized as 1,1,2-tris(2’,4’-di-tert-butylbenzene)-4,4-dimethyl-pent-1-ene. The content of CP-01 was found to be 0.88 %w/w of chloroform extract and 0.08 %w/w of N.arbortristis leaves. Conclusion: The study supports the traditional use of N. arbortristis as anticancer herb & the identified compound CP-01 can serve as an excellent lead to develop potent and safe anticancer drugs.

Keywords: anticancer, HL-60 cell lines, Nyctanthes arbor-tristis, RP-HPLC

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Authors: Huseyin Apdik, Aysegul Dogan, Selami Demirci, Ezgi Avsar Apdik, Fikrettin Sahin

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Mesenchymal stem cells (MSCs) are crucial cell types for bone maintenance and growth along with resident bone progenitor cells providing bone tissue integrity during osteogenesis and skeletal growth. Any deficiency in this regulation would result in vital bone diseases. Of those, osteoporosis, characterized by a reduction in bone mass and mineral density, is a critical skeletal disease for especially elderly people. The commonly used drugs for the osteoporosis treatment are bisphosphonates (BPs). The most prominent role of BPs is to prevent bone resorption arisen from high osteoclast activity. However, administrations of bisphosphonates may also cause bisphosphonate-induced osteonecrosis of the jaw (BIONJ). Up to the present, the researchers have proposed several circumstances for BIONJ. However, effects of long-term and/or high dose usage of BPs on stem cell’s proliferation, survival, differentiation or maintenance capacity have not been evaluated yet. The present study will be held to; figure out BPs’ effects on MSCs in vitro in the aspect of cell proliferation and toxicity, migration, angiogenic activity, lineage specific gene and protein expression levels, mesenchymal stem cell properties and potential signaling pathways affected by BP treatment. Firstly, mesenchymal stem cell characteristics of Dental Pulp Stem Cells (DPSCs) and Periodontal Ligament Stem Cells (PDLSCs) were proved using flow cytometry analysis. Cell viability analysis was completed to determine the cytotoxic effects of BPs (Zoledronate (Zol), Alendronate (Ale) and Risedronate (Ris)) on DPSCs and PDLSCs by the 3-(4,5-di-methyl-thiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS) assay. Non-toxic concentrations of BPs were determined at 24 h under growth condition, and at 21 days under osteogenic differentiation condition for both cells. The scratch assay was performed to evaluate their migration capacity under the usage of determined of BPs concentrations at 24 h. The results revealed that while the scratch closure is 70% in the control group for DPSCs, it was 57%, 66% and 66% in Zol, Ale and Ris groups, respectively. For PDLSs, while wound closure is 71% in control group, it was 65%, 66% and 66% in Zol, Ale and Ris groups, respectively. As future experiments, tube formation assay and aortic ring assay will be done to determinate angiogenesis abilities of DPSCs and PDLSCs treated with BPs. Expression levels of osteogenic differentiation marker genes involved in bone development will be determined using real time-polymerase change reaction (RT-PCR) assay and expression profiles of important proteins involved in osteogenesis will be evaluated using western blotting assay for osteogenically differentiated MSCs treated with or without BPs. In addition to these, von Kossa staining will be performed to measure calcium mineralization status of MSCs.

Keywords: bisphosphonates, bisphosphonate-induced osteonecrosis of the jaw, mesenchymal stem cells, osteogenesis

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Authors: Karthik Chamari, Vasudha Rudraraju, Gaurav Chaudhari

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Background: Mantle cell lymphoma (MCL) is one of the mature B cell non-Hodgkin lymphomas (NHL). The course of MCL is moderately aggressive and variable, and it has median overall survival of 8 to 10 years. Ibrutinib, a Bruton’s tyrosine kinase inhibitor, was approved by the United States (US) Food and Drug Administration in November of 2013 for the treatment of MCL patients who have received at least one prior therapy. In this study, we aimed to evaluate whether there has been a change in survival and patterns of second primary malignancies (SPMs) among the MCL population in the US after ibrutinib approval. Methods: Using the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER)-18, we conducted a retrospective study with patients diagnosed with MCL (ICD-0-3 code 9673/3) between 2007 and 2018. We divided patients into two six-year cohorts, pre-ibrutinib approval (2007-2012) and post-ibrutinib approval (2013-2018), and compared relative survival rates (RSRs) and standardized incidence ratios (SIRs) of SPMs between cohorts. Results: We included 9,257 patients diagnosed with MCL between 2007 and 2018 in the SEER-18 survival and SIR registries. Of these, 4,205 (45%) patients were included in the pre-ibrutinib cohort, and 5052 (55%) patients were included in the post-ibrutinib cohort. The median follow-up duration for the pre-ibrutinib cohort was 54 months (range 0 to 143 months), and the post-ibrutinib cohort was 20 months (range 0 to 71 months). There was a significant difference in the five-year RSRs between pre-ibrutinib and post-ibrutinib cohorts (57.5% vs. 62.6%, p < 0.005). Out of the 9,257 patients diagnosed with MCL, 920 developed SPMs. A higher proportion of SPMs occurred in the post-ibrutinib cohort (63%) when compared with the pre-ibrutinib cohort (37%). Non-hematological malignancies comprised most of all SPMs. A higher incidence of non-hematological malignancies occurred in the post-ibrutinib cohort (SIR 1.42, 95% CI 1.29 to 1.56) when compared with the pre-ibrutinib cohort (SIR 1.14, 95% CI 1 to 1.3). There was a statistically significant increase in the incidence of cancers of the respiratory tract (SIR 1.77, 95% CI 1.43 to 2.18), urinary tract (SIR 1.61, 95% CI 1.23 to 2.06) when compared with other non-hematological malignancies in post-ibrutinib cohort. Conclusions: Our study results suggest the relative survival rates have increased since the approval of ibrutinib for mantle cell lymphoma patients. Additionally, for some unclear reasons, the incidence of SPM’s (non-hematological malignancies), mainly cancers of the respiratory tract, urinary tract, have increased in the six years following the approval of ibrutinib. Further studies should be conducted to determine the cause of these findings.

Keywords: mantle cell lymphoma, Ibrutinib, relative survival analysis, secondary primary cancers

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Authors: Uma Krishnamoorthy, Vivienne Beavers, Janet Marshall

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Introduction: Cervical cytology showing features raising the suspicion of non cervical glandular neoplasia are reported as code 0 under the United Kingdom National Health Service Cervical screening programme ( NHSCSP). As the suspicion is regarding non cervical neoplasia, smear is reported as normal and patient informed that cervical screening result is normal. GP receives copy of results where it states further referral is indicated in small font within text of report. Background: There were several incidents of delayed diagnosis of endometrial cancer in Lancashire which prompted this Northwest Regional review to enable an understanding of underlying pathology outcome of code zero smears to raise awareness and also to review whether further action on wording of smear results was indicated to prevent such delay. Methodology: All Smears reported at the Manchester cytology centre who process cytology for Lancashire population from March 2013 to March 2014 were reviewed and histological diagnosis outcome of women in whom smear was reported as code zero was reviewed retrospectively . Results: Total smears reported by the cytology centre during this period was approximately 109400. Reports issued with result code 0 among this during this time period was 49.Results revealed that among three fourth (37) of women with code zero smear (N=49), evidence of underlying pathology of non cervical origin was confirmed. Of this, 73 % (36) were due to endometrial pathology with 49 % (24) endometrial carcinoma, 12 % (6)polyp, 4 % atypical endometrial hyperplasia (2), 6 % endometrial hyperplasia without atypia (3), and 2 % adenomyosis (1 case) and 2 % ( 1 case) due to ovarian adenocarcinoma. Conclusion: This review demonstrated that more than half (51 %) of women with a code 0 smear report were diagnosed with underlying carcinoma and 75 % had a confirmed underlying pathology contributory to code 0 smear findings. Recommendations and Action Plan: A local rapid access referral and management pathway for this group of women was implemented as a result of this in our unit. The findings and Pathway were shared with other regional units served by the cytology centre through the Pan Lancashire cervical screening board and through the Cytology centre. Locally, the smear report wording was updated to include a rubber stamp/ print in "Red Bold letters" stating that " URGENT REFERRAL TO GYNAECOLOGY IS INDICATED". Findings were also shared through the Pan Lancashire board with National cervical screening programme board, and revisions to wording of code zero smear reports to highlight the need for Urgent referral has now been agreed at National level to be implemented.

Keywords: code zero smears, endometrial cancer, non cervical glandular neoplasia, ovarian cancer

Procedia PDF Downloads 299

Authors: Seonaid Hume, Gordon Dobie, Graeme West

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Detecting and localizing radioactive sources is a necessity for safe and secure decommissioning of nuclear facilities. An important aspect for the management of the sort-and-segregation process is establishing the spatial distributions and quantities of the waste radionuclides, their type, corresponding activity, and ultimately classification for disposal. The data received from surveys directly informs decommissioning plans, on-site incident management strategies, the approach needed for a new cell, as well as protecting the workforce and the public. Manual classification of nuclear waste from a nuclear cell is time-consuming, expensive, and requires significant expertise to make the classification judgment call. Also, in-cell decommissioning is still in its relative infancy, and few techniques are well-developed. As with any repetitive and routine tasks, there is the opportunity to improve the task of classifying nuclear waste using autonomous systems. Hence, this paper proposes a new framework for the automatic classification of nuclear waste. This framework consists of five main stages; 3D spatial mapping and object detection, object classification, radiological mapping, source localisation based on gathered evidence and finally, waste classification. The first stage of the framework, 3D visual mapping, involves object detection from point cloud data. A review of related applications in other industries is provided, and recommendations for approaches for waste classification are made. Object detection focusses initially on cylindrical objects since pipework is significant in nuclear cells and indeed any industrial site. The approach can be extended to other commonly occurring primitives such as spheres and cubes. This is in preparation of stage two, characterizing the point cloud data and estimating the dimensions, material, degradation, and mass of the objects detected in order to feature match them to an inventory of possible items found in that nuclear cell. Many items in nuclear cells are one-offs, have limited or poor drawings available, or have been modified since installation, and have complex interiors, which often and inadvertently pose difficulties when accessing certain zones and identifying waste remotely. Hence, this may require expert input to feature match objects. The third stage, radiological mapping, is similar in order to facilitate the characterization of the nuclear cell in terms of radiation fields, including the type of radiation, activity, and location within the nuclear cell. The fourth stage of the framework takes the visual map for stage 1, the object characterization from stage 2, and radiation map from stage 3 and fuses them together, providing a more detailed scene of the nuclear cell by identifying the location of radioactive materials in three dimensions. The last stage involves combining the evidence from the fused data sets to reveal the classification of the waste in Bq/kg, thus enabling better decision making and monitoring for in-cell decommissioning. The presentation of the framework is supported by representative case study data drawn from an application in decommissioning from a UK nuclear facility. This framework utilises recent advancements of the detection and mapping capabilities of complex radiation fields in three dimensions to make the process of classifying nuclear waste faster, more reliable, cost-effective and safer.

Keywords: nuclear decommissioning, radiation detection, object detection, waste classification

Procedia PDF Downloads 201

Authors: Norma Binti Alias, Che Rahim Che The, Norfarizan Mohd Said, Sakinah Abdul Hanan, Akhtar Ali

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This paper discusses on the transportation of magnetic drug targeting through blood within vessels, tissues and cells. There are three integrated mathematical models to be discussed and analyze the concentration of drug and blood flow through magnetic nanoparticles. The cell therapy brought advancement in the field of nanotechnology to fight against the tumors. The systematic therapeutic effect of Single Cells can reduce the growth of cancer tissue. The process of this nanoscale phenomena system is able to measure and to model, by identifying some parameters and applying fundamental principles of mathematical modeling and simulation. The mathematical modeling of single cell growth depends on three types of cell densities such as proliferative, quiescent and necrotic cells. The aim of this paper is to enhance the simulation of three types of models. The first model represents the transport of drugs by coupled partial differential equations (PDEs) with 3D parabolic type in a cylindrical coordinate system. This model is integrated by Non-Newtonian flow equations, leading to blood liquid flow as the medium for transportation system and the magnetic force on the magnetic nanoparticles. The interaction between the magnetic force on drug with magnetic properties produces induced currents and the applied magnetic field yields forces with tend to move slowly the movement of blood and bring the drug to the cancer cells. The devices of nanoscale allow the drug to discharge the blood vessels and even spread out through the tissue and access to the cancer cells. The second model is the transport of drug nanoparticles from the vascular system to a single cell. The treatment of the vascular system encounters some parameter identification such as magnetic nanoparticle targeted delivery, blood flow, momentum transport, density and viscosity for drug and blood medium, intensity of magnetic fields and the radius of the capillary. Based on two discretization techniques, finite difference method (FDM) and finite element method (FEM), the set of integrated models are transformed into a series of grid points to get a large system of equations. The third model is a single cell density model involving the three sets of first order PDEs equations for proliferating, quiescent and necrotic cells change over time and space in Cartesian coordinate which regulates under different rates of nutrients consumptions. The model presents the proliferative and quiescent cell growth depends on some parameter changes and the necrotic cells emerged as the tumor core. Some numerical schemes for solving the system of equations are compared and analyzed. Simulation and computation of the discretized model are supported by Matlab and C programming languages on a single processing unit. Some numerical results and analysis of the algorithms are presented in terms of informative presentation of tables, multiple graph and multidimensional visualization. As a conclusion, the integrated of three types mathematical modeling and the comparison of numerical performance indicates that the superior tool and analysis for solving the complete set of magnetic drug delivery system which give significant effects on the growth of the targeted cancer cell.

Keywords: mathematical modeling, visualization, PDE models, magnetic nanoparticle drug delivery model, drug release model, single cell effects, avascular tumor growth, numerical analysis

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Authors: Rohin Sharma, Sumantu Chaulagain

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This paper presents calculations of total energy of InGaN alloy carried out in a disordered quasirandom structure for a triclinic super cell. This structure replicates the disorder and composition effect in the alloy. First principle calculations within the density functional theory with the local density approximation approach is employed to accurately determine total energy of the system. Lattice constants and band gaps associated with the ground states are then estimated for different concentration ratios of the alloy. We provide precise results of quasirandom structures of the alloy and their lattice constants with the total energy and band gap energy of the system for the range of seven different composition ratios and their respective lattice parameters.

Keywords: DFT, ground state, LDA, quasicrystal, triclinic super cell

Procedia PDF Downloads 190

Authors: N. Hasbullah, K. A. Sekak

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The sulfonated poly (ether ether ketone) SPEEK nanofiber membrane were successfully electrospun for Polymer Electrolyte Membrane (PEM) in Proton Exchange Membrane Fuel Cell (PEMFC) and their nanosized properties were investigated. The poly (ether ether ketone) PEEK victrex® grade 90p was sulfonated with concentrated sulfuric acid (95-98% w/w) at room temperature for 60 hours sulfonation times. The degree sulfonation of SPEEK are 70% was determined by H1 NMR and the functional groups of the SPEEK were characterize using FTIR. Then, the SPEEK nanofiber membrane were prepared via electrospinning method using DMAC as a solvent. The SPEEK sample were successfully electrospun using predetermine set up. FESEM show the electrospun fiber mat surface and confirmed the nanostructure membrane cell.

Keywords: polymer electrolyte membrane (PEM), sulfonated poly (ether ether ketone) (SPEEK), degree sulfonation, Electrospinning, Nanofibers

Procedia PDF Downloads 311

Authors: Leila Rashki Ghaleno, Mohamad Amin Hajari, Leila Montazeri, Abdolhossein Shahverdi, Mojtaba Rezazadeh Valojerdi

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Background: Spermatogonia stem cells (SSCs) exhibit pluripotency, enabling them to undergo differentiation into many cell lineages, including neurons, glia, endothelial cells, and hepatocytes when cultured in vitro. Although the specific mechanisms are not yet fully understood, it has been observed that biopolymer agents, such as hyaluronic acid (HA) and alginate (Alg), have the potential to induce transdifferentiation of SSCs. The current work aimed to examine the process of in vitro spermatogenesis and the conversion of mouse testicular cells into hepatocytes and erythrocyte-like cells utilizing the HA-Alg hydrogel. Method: After being extracted from the testes of a 5-day postpartum mouse (5 DPP), the testicular cells were separated into two enzymatic stages and then put into a composite hydrogel containing 0.5% HA and 1% alginate. On days 14 and 28 of culture, the colonies' growth, the cells' viability, and their histology were assessed. Result: Despite observing significant cell proliferation on day 14 and the development of circular-shaped organoids on day 28, it was noted that the organoids generated in the HA-Alg medium tended to maintain their circular morphology on day 28. Notably, the testicular cells underwent transdifferentiation into cell types resembling erythrocytes and hepatocytes. The hepatocyte-like cells exhibited the presence of glycogen and lipid deposits, indicating their hepatocyte-like characteristics. Interestingly, immunostaining analysis revealed the secretion of albumin and the presence of VEGFR on day 14. However, on day 28, albumin expression was not detected, while the expression of Sox9 (a marker for hepatocytes), Vegf, CD34, and C-kit (markers for erythrocytes) showed increased levels in the gene expression evaluation. Conclusion: The present findings indicated that HA-Alg could be a potent and effective agent for the transdifferentiation of testis cells into erythrocyte and hepatocyte-like cells, as recent studies have confirmed the transformation of SSCs into hepatocyte cells during in vitro culture.

Keywords: 3D culture, mouse testicular cell, hyaluronic acid, liver organoids

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Authors: E. M. Hassan, A. L. Kalamkarov

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Two micromechanical models for 3D smart composite with embedded periodic or nearly periodic network of generally orthotropic reinforcements and actuators are developed and applied to cubic structures with unidirectional orientation of constituents. Analytical formulas for the effective piezothermoelastic coefficients are derived using the Asymptotic Homogenization Method (AHM). Finite Element Analysis (FEA) is subsequently developed and used to examine the aforementioned periodic 3D network reinforced smart structures. The deformation responses from the FE simulations are used to extract effective coefficients. The results from both techniques are compared. This work considers piezoelectric materials that respond linearly to changes in electric field, electric displacement, mechanical stress and strain and thermal effects. This combination of electric fields and thermo-mechanical response in smart composite structures is characterized by piezoelectric and thermal expansion coefficients. The problem is represented by unit-cell and the models are developed using the AHM and the FEA to determine the effective piezoelectric and thermal expansion coefficients. Each unit cell contains a number of orthotropic inclusions in the form of structural reinforcements and actuators. Using matrix representation of the coupled response of the unit cell, the effective piezoelectric and thermal expansion coefficients are calculated and compared with results of the asymptotic homogenization method. A very good agreement is shown between these two approaches.

Keywords: asymptotic homogenization method, finite element analysis, effective piezothermoelastic coefficients, 3D smart network composite structures

Procedia PDF Downloads 400

Authors: Mosima M. Mabitsela

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Groundnut is preferably grown on light textured soils. Most of these light textured soils tend to be highly weathered and characterized by high soil acidity and low nutrient status. One major soil factor associated with infertility of acidic soils that can negatively depress groundnut yield is aluminium (Al) toxicity. In plants Al toxicity damages root cells, leading to inhibition of root growth as a result of the suppression of cell division, cell elongation and cell expansion in the apical meristem cells of the root. The end result is that roots become stunted and brittle, root hair development is poor, and the root apices become swollen. This study was conducted to determine the effects of aluminium (Al) toxicity on a range of groundnut varieties. Fifteen cultivars were tested in incremental aluminum (Al) supply in an ebb and flow solution culture laid out in a randomized complete block design. There were six aluminium (Al) treatments viz. 0 µM, 1 µM, 5.7 µM, 14.14 µM, 53.18 µM, and 200 µM. At 1 µM there was no inhibitory effect on the growth of groundnut. The inhibition of groundnut growth was noticeable from 5.7 µM to 200 µM, where the severe effect of aluminium (Al) stress was observed at 200 µM. The cultivars varied in their response to aluminium (Al) supply in solution culture. Groundnuts are one of the most important food crops in the world, and its supply is on a decline due to the light-textured soils that they thrive under as these soils are acidic and can easily solubilize aluminium (Al) to its toxic form. Consequently, there is a need to develop groundnut cultivars with high tolerance to soil acidity.

Keywords: aluminium toxicity, cultivars, reduction, root growth

Procedia PDF Downloads 152

Authors: Leander Van Neste, Kirk Wojno

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The innate immune system reacts to foreign insult in several unique ways, one of which is phagocytosis of perceived threats such as cancer, bacteria, and viruses. The goal of this study was to look for evidence of phagocytosed RNA from tumor cells in circulating monocytes. While all monocytes possess phagocytic capabilities, the non-classical CD14+/FCGR3A+ monocytes and the intermediate CD14++/FCGR3A+ monocytes most actively remove threatening ‘external’ cellular materials. Purified CD14-positive monocyte samples from fourteen patients recently diagnosed with clinically localized prostate cancer (PCa) were investigated by single-cell RNA sequencing using the 10X Genomics protocol followed by paired-end sequencing on Illumina’s NovaSeq. Similarly, samples were processed and used as controls, i.e., one patient underwent biopsy but was found not to harbor prostate cancer (benign), three young, healthy men, and three men previously diagnosed with prostate cancer that recently underwent (curative) radical prostatectomy (post-RP). Sequencing data were mapped using 10X Genomics’ CellRanger software and viable cells were subsequently identified using CellBender, removing technical artifacts such as doublets and non-cellular RNA. Next, data analysis was performed in R, using the Seurat package. Because the main goal was to identify differences between PCa patients and ‘control’ patients, rather than exploring differences between individual subjects, the individual Seurat objects of all 21 patients were merged into one Seurat object per Seurat’s recommendation. Finally, the single-cell dataset was normalized as a whole prior to further analysis. Cell identity was assessed using the SingleR and cell dex packages. The Monaco Immune Data was selected as the reference dataset, consisting of bulk RNA-seq data of sorted human immune cells. The Monaco classification was supplemented with normalized PCa data obtained from The Cancer Genome Atlas (TCGA), which consists of bulk RNA sequencing data from 499 prostate tumor tissues (including 1 metastatic) and 52 (adjacent) normal prostate tissues. SingleR was subsequently run on the combined immune cell and PCa datasets. As expected, the vast majority of cells were labeled as having a monocytic origin (~90%), with the most noticeable difference being the larger number of intermediate monocytes in the PCa patients (13.6% versus 7.1%; p<.001). In men harboring PCa, 0.60% of all purified monocytes were classified as harboring PCa signals when the TCGA data were included. This was 3-fold, 7.5-fold, and 4-fold higher compared to post-RP, benign, and young men, respectively (all p<.001). In addition, with 7.91%, the number of unclassified cells, i.e., cells with pruned labels due to high uncertainty of the assigned label, was also highest in men with PCa, compared to 3.51%, 2.67%, and 5.51% of cells in post-RP, benign, and young men, respectively (all p<.001). It can be postulated that actively phagocytosing cells are hardest to classify due to their dual immune cell and foreign cell nature. Hence, the higher number of unclassified cells and intermediate monocytes in PCa patients might reflect higher phagocytic activity due to tumor burden. This also illustrates that small numbers (~1%) of circulating peripheral blood monocytes that have interacted with tumor cells might still possess detectable phagocytosed tumor RNA.

Keywords: circulating monocytes, phagocytic cells, prostate cancer, tumor immune response

Procedia PDF Downloads 162

Authors: Marinna Madrid, Nabiha Saklayen, Marinus Huber, Nicolas Vogel, Christos Boutopoulos, Michel Meunier, Eric Mazur

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Delivering material into cells is important for a diverse range of biological applications, including gene therapy, cellular engineering and imaging. We present a plasmonic substrate for delivering membrane-impermeable material into cells at high throughput and high efficiency while maintaining cell viability. The substrate fabrication is based on an affordable and fast colloidal self-assembly process. When illuminated with a femtosecond laser, the light interacts with the electrons at the surface of the metal substrate, creating localized surface plasmons that form bubbles via energy dissipation in the surrounding medium. These bubbles come into close contact with the cell membrane to form transient pores and enable entry of membrane-impermeable material via diffusion. We use fluorescence microscopy and flow cytometry to verify delivery of membrane-impermeable material into HeLa CCL-2 cells. We show delivery efficiency and cell viability data for a range of membrane-impermeable cargo, including dyes and biologically relevant material such as siRNA. We estimate the effective pore size by determining delivery efficiency for hard fluorescent spheres with diameters ranging from 20 nm to 2 um. To provide insight to the cell poration mechanism, we relate the poration data to pump-probe measurements of micro- and nano-bubble formation on the plasmonic substrate. Finally, we investigate substrate stability and reusability by using scanning electron microscopy (SEM) to inspect for damage on the substrate after laser treatment. SEM images show no visible damage. Our findings indicate that self-assembled plasmonic substrates are an affordable tool for high-throughput, high-efficiency delivery of material into mammalian cells.

Keywords: femtosecond laser, intracellular delivery, plasmonic, self-assembly

Procedia PDF Downloads 531

Authors: Anil Koklu, Amin Mansoorifar, Ali Beskok

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Dielectric spectroscopy (DS) is a noninvasive, label free technique for a long term real-time measurements of the impedance spectra of biological cells. DS enables characterization of cellular dielectric properties such as membrane capacitance and cytoplasmic conductivity. We have developed a lab-on-a-chip device that uses an electro-activated microwells array for loading, DS measurements, and unloading of biological cells. We utilized from dielectrophoresis (DEP) to capture target cells inside the wells and release them after DS measurement. DEP is a label-free technique that exploits differences among dielectric properties of the particles. In detail, DEP is the motion of polarizable particles suspended in an ionic solution and subjected to a spatially non-uniform external electric field. To the best of our knowledge, this is the first microfluidic chip that combines DEP and DS to analyze biological cells using electro-activated wells. Device performance is tested using two different cell lines of prostate cancer cells (RV122, PC-3). Impedance measurements were conducted at 0.2 V in the 10 kHz to 40 MHz range with 6 s time resolution. An equivalent circuit model was developed to extract the cell membrane capacitance and cell cytoplasmic conductivity from the impedance spectra. We report the time course of the variations in dielectric properties of PC-3 and RV122 cells suspended in low conductivity medium (LCB), which enhances dielectrophoretic and impedance responses, and their response to sudden pH change from a pH of 7.3 to a pH of 5.8. It is shown that microfluidic chip allowed online measurements of dielectric properties of prostate cancer cells and the assessment of the cellular level variations under external stimuli such as different buffer conductivity and pH. Based on these data, we intend to deploy the current device for single cell measurements by fabricating separately addressable N × N electrode platforms. Such a device will allow time-dependent dielectric response measurements for individual cells with the ability of selectively releasing them using negative-DEP and pressure driven flow.

Keywords: microfluidic, microfabrication, lab on a chip, AC electrokinetics, dielectric spectroscopy

Procedia PDF Downloads 151

Authors: Samineh Barmaki, Ville Jokinen, Esko Kankuri

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We report a microfluidic chip that can be used to modify the gaseous microenvironment of a cell-culture in ambient atmospheric conditions. The aim of the study is to show the cellular response to nitric oxide (NO) under hypoxic (oxygen < 5%) condition. Simultaneously targeting to hypoxic and nitric oxide will provide an opportunity for NO‑based therapeutics. Studies on cellular responses to lowered oxygen concentration or to gaseous mediators are usually carried out under a specific macro environment, such as hypoxia chambers, or with specific NO donor molecules that may have additional toxic effects. In our study, the chip consists of a microfluidic layer and a cell culture well, separated by a thin gas permeable polydimethylsiloxane (PDMS) membrane. The main design goal is to separate the gas oxygen scavenger and NO donor solutions, which are often toxic, from the cell media. Two different types of gas exchangers, titled 'pool' and 'meander' were tested. We find that the pool design allows us to reach a higher level of oxygen depletion than meander (24.32 ± 19.82 %vs -3.21 ± 8.81). Our microchip design can make the cells culture more simple and makes it easy to adapt existing cell culture protocols. Our first application is utilizing the chip to create hypoxic conditions on targeted areas of cell culture. In this study, oxygen scavenger sodium sulfite generates hypoxia and its effect on human embryonic kidney cells (HEK-293). The PDMS membrane was coated with fibronectin before initiating cell cultures, and the cells were grown for 48h on the chips before initiating the gas control experiments. The hypoxia experiments were performed by pumping of O₂-depleted H₂O into the microfluidic channel with a flow-rate of 0.5 ml/h. Image-iT® reagent as an oxygen level responser was mixed with HEK-293 cells. The fluorescent signal appears on cells stained with Image-iT® hypoxia reagent (after 6h of pumping oxygen-depleted H₂O through the microfluidic channel in pool area). The exposure to different levels of O₂ can be controlled by varying the thickness of the PDMS membrane. Recently, we improved the design of the microfluidic chip, which can control the microenvironment of two different gases at the same time. The hypoxic response was also improved from the new design of microchip. The cells were grown on the thin PDMS membrane for 30 hours, and with a flowrate of 0.1 ml/h; the oxygen scavenger was pumped into the microfluidic channel. We also show that by pumping sodium nitroprusside (SNP) as a nitric oxide donor activated under light and can generate nitric oxide on top of PDMS membrane. We are aiming to show cellular microenvironment response of HEK-293 cells to both nitric oxide (by pumping SNP) and hypoxia (by pumping oxygen scavenger solution) in separated channels in one microfluidic chip.

Keywords: hypoxia, nitric oxide, microenvironment, microfluidic chip, sodium nitroprusside, SNP

Procedia PDF Downloads 134

Authors: Denis Mustafov, Emmanouil Karteris, Maria Braoudaki

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Glioblastoma multiform (GBM) is a heterogenous primary brain tumour that kills most affected patients. To the authors best knowledge, despite all research efforts there is no early diagnostic biomarker for GBM. MicroRNAs (miRNAs) are short non-coding RNA molecules which are deregulated in many cancers. The aim of this research was to determine miRNAs with a diagnostic impact and to potentially identify promising therapeutic targets for glioblastoma multiform. In silico analysis was performed to identify deregulated miRNAs with diagnostic relevance for glioblastoma. The expression profiles of the chosen miRNAs were then validated in vitro in the human glioblastoma cell lines A172 and U-87MG. Briefly, RNA extraction was carried out using the Trizol method, whilst miRNA extraction was performed using the mirVANA miRNA isolation kit. Quantitative Real-Time Polymerase Chain Reaction was performed to verify their expression. The presence of five target proteins within the A172 cell line was evaluated by Western blotting. The expression of the CORO1C protein within 32 GBM cases was examined via immunohistochemistry. The miRNAs identified in silico included miR-21-5p, miR-34a and miR-128a. These miRNAs were shown to target deregulated GBM genes, such as CDK6, E2F3, BMI1, JAG1, and CORO1C. miR-34a and miR-128a showed low expression profiles in comparison to a control miR-RNU-44 in both GBM cell lines suggesting tumour suppressor properties. Opposing, miR-21-5p demonstrated greater expression indicating that it could potentially function as an oncomiR. Western blotting revealed expression of all five proteins within the A172 cell line. In silico analysis also suggested that CORO1C is a target of miR-128a and miR-34a. Immunohistochemistry demonstrated that 75% of the GBM cases showed moderate to high expression of CORO1C protein. Greater understanding of the deregulated expression of miR-128a and the upregulation of CORO1C in GBM could potentially lead to the identification of a promising diagnostic biomarker signature for glioblastomas.

Keywords: non-coding RNAs, gene expression, brain tumours, immunohistochemistry

Procedia PDF Downloads 91