Search results for: single cell

Authors: Leo Nnamdi Ozurumba-Dwight

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

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

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Authors: Awais Naeem, Osama Shakeel, Faizan Ullah, Abdul Wahid Anwer

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Introduction: To study the clinic-pathological factors, diagnostic factors and survival of extra-pulmonary small cell carcinoma. Methodology: From 1995 to 2017 all patients with a diagnosis of extra-pulmonary small cell carcinoma were included in the study. Demographic variables and clinic-pathological factors were collected. Management of disease was recorded. Short and long term oncological outcomes were recorded. All data was entered and analyzed in SPSS version 21. Results: A total of 14 patients were included in the study. Median age was 53.42 +/- 16.1 years. There were 5 male and 9 female patients. Most common presentation was dysphagia in 16 patient among esophageal small cell carcinoma and while other patient had pain in abdomen. Mean duration of symptoms was 4.23+/-2.91 months .Most common site is esophagus (n=6) followed by gall bladder(n=3). Almost all of the patients received chemo-radiotherapy. Majority of the patient presented with extensive disease. Five patients (35.7%) died during the follow up period, two (14.3%) were alive and rest of the patients were lost to follow up. Mean follow up period was 22.92 months and median follow up was 15 months. Conclusion: Extra-pulmonary small cell carcinoma is rare and needs to be managed aggressively. All patients should be treated with both systemic and local therapies.

Keywords: small cell carcinoma of esophagus, extrapulmonary small cell carcinoma, small cell carcinoma of gall bladder, small cell carcinoma of rectum, small cell carcinoma of stomach

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Authors: Hossein Pourbashash

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Recent experimental studies have shown that HIV can be transmitted directly from cell to cell when structures called virological synapses form during interactions between T cells. In this article, we describe a new within-host model of HIV infection that incorporates two mechanisms: infection by free virions and the direct cell-to-cell transmission. We conduct the local and global stability analysis of the model. We show that if the basic reproduction number R0 1, the virus is cleared and the disease dies out; if R0 > 1, the virus persists in the host. We also prove that the unique positive equilibrium attracts all positive solutions under additional assumptions on the parameters.

Keywords: HIV virus model, cell-to-cell transmission, global stability, Lyapunov function, second compound matrices

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Authors: Elif Gencturk, Ekin Yurdakul, Ahmet Y. Celik, Senol Mutlu, Kutlu O. Ulgen

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Yeast cells are generally used as a model system of eukaryotes due to their complex genetic structure, rapid growth ability in optimum conditions, easy replication and well-defined genetic system properties. Thus, yeast cells increased the knowledge of the principal pathways in humans. During fermentation, carbohydrates (hexoses and pentoses) degrade into some toxic by-products such as 5-hydroxymethylfurfural (5-HMF or HMF) and furfural. HMF influences the ethanol yield, and ethanol productivity; it interferes with microbial growth and is considered as a potent inhibitor of bioethanol production. In this study, yeast single cell behavior under HMF application was monitored by using a continuous flow single phase microfluidic platform. Microfluidic device in operation is fabricated by hot embossing and thermo-compression techniques from cyclo-olefin polymer (COP). COP is biocompatible, transparent and rigid material and it is suitable for observing fluorescence of cells considering its low auto-fluorescence characteristic. The response of yeast cells was recorded through Red Fluorescent Protein (RFP) tagged Nop56 gene product, which is an essential evolutionary-conserved nucleolar protein, and also a member of the box C/D snoRNP complexes. With the application of HMF, yeast cell proliferation continued but HMF slowed down the cell growth, and after HMF treatment the cell proliferation stopped. By the addition of fresh nutrient medium, the yeast cells recovered after 6 hours of HMF exposure. Thus, HMF application suppresses normal functioning of cell cycle but it does not cause cells to die. The monitoring of Nop56 expression phases of the individual cells shed light on the protein and ribosome synthesis cycles along with their link to growth. Further computational study revealed that the mechanisms underlying the inhibitory or inductive effects of HMF on growth are enriched in functional categories of protein degradation, protein processing, DNA repair and multidrug resistance. The present microfluidic device can successfully be used for studying the effects of inhibitory agents on growth by single cell tracking, thus capturing cell to cell variations. By metabolic engineering techniques, engineered strains can be developed, and the metabolic network of the microorganism can thus be manipulated such that chemical overproduction of target metabolite is achieved along with the maximum growth/biomass yield.  

Keywords: COP, HMF, ribosome biogenesis, thermoplastic microbioreactor, yeast

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Authors: Yingjeng James Li, Yun Jyun Ou, Chih Chi Hsu, Chiao-Chih Hu

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A fuel cell is a device that produces electric power by reacting fuel and oxidant electrochemically. There is no pollution produced from a fuel cell if hydrogen is employed as the fuel. Therefore, a fuel cell is considered as a zero emission device and is a source of green power. A membrane electrode assembly (MEA) is the key component of a fuel cell. It is, therefore, beneficial to develop MEAs with high performance. In this study, an MEA for proton exchange membrane fuel cell (PEMFC) was prepared from a 15-micron thick reinforced PEM. The active area of such MEA is 25 cm2. Carbon supported platinum (Pt/C) was employed as the catalyst for both anode and cathode. The platinum loading is 0.6 mg/cm2 based on the sum of anode and cathode. Commercially available carbon papers coated with a micro porous layer (MPL) serve as gas diffusion layers (GDLs). The original thickness of the GDL is 250 μm. It was compressed down to 163 μm when assembled into the single cell test fixture. Polarization curves were taken by using eight different test conditions. At our standard test condition (cell: 70 °C; anode: pure hydrogen, 100%RH, 1.2 stoic, ambient pressure; cathode: air, 100%RH, 3.0 stoic, ambient pressure), the cell current density is 1250 mA/cm2 at 0.6 V, and 2400 mA/cm2 at 0.4 V. At self-humidified condition and cell temperature of 55 °C, the cell current density is 1050 mA/cm2 at 0.6 V, and 2250 mA/cm2 at 0.4 V. Hydrogen crossover rate of the MEA is 0.0108 mL/min*cm2 according to linear sweep voltammetry experiments. According to the MEA’s Pt loading and the cyclic voltammetry experiments, the Pt electrochemical surface area is 60 m2/g. The ohmic part of the impedance spectroscopy results shows that the membrane resistance is about 60 mΩ*cm2 when the MEA is operated at 0.6 V.

Keywords: fuel cell, membrane electrode assembly, proton exchange membrane, reinforced

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Authors: Fouad Berrabeh, Sabir Messalti

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The performances of the photovoltaic systems are very dependent on different conditions, such as solar irradiation, temperature, etc. Therefore, it is very important to provide detailed studies for different cases in order to provide continuously power, so the photovoltaic system must be properly sized. This paper presents the modelling and simulation of the photovoltaic cell using single diode model. I-V characteristics and P-V characteristics are presented and it verified at different conditions (irradiance effect, temperature effect, series resistance effect).

Keywords: photovoltaic cell, BP SX 150 BP solar photovoltaic module, irradiance effect, temperature effect, series resistance effect, I–V characteristics, P–V characteristics

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Authors: H. Oudira, A. Saifi

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

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

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Authors: Jun Sung Go, Jong Kang Park, Jong Tae Kim

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As semi-conductor manufacturing technology evolves; the single event transient problem becomes more significant issue. Single event transient has a critical impact on both combinational and sequential logic circuits, so it is important to evaluate the soft error tolerance of the circuits at the design stage. In this paper, we present a soft error detecting simulation using scan chain. The simulation model generates a single event transient randomly in the circuit, and detects the soft error during the execution of the test patterns. We verified this model by inserting a scan chain in an 8051 microprocessor using 65 nm CMOS technology. While the test patterns generated by ATPG program are passing through the scan chain, we insert a single event transient and detect the number of soft errors per sub-module. The experiments show that the soft error rates per cell area of the SFR module is 277% larger than other modules.

Keywords: scan chain, single event transient, soft error, 8051 processor

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Authors: Isaac Quiros-Fernandez, Angel Cid-Arregui

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Tumor-reactive T cell receptors (TCRs) are being subject of intense investigation since they offer great potential in adoptive cell therapies against cancer. However, the identification of tumor-specific TCRs has proven challenging, for instance, due to the limited expansion capacity of tumor-infiltrating T cells (TILs) and the extremely low frequencies of tumor-reactive T cells in the repertoire of patients and healthy donors. We have developed an approach for rapid identification and characterization of neoepitope-reactive TCRs from the T cell repertoire of healthy donors. CD8 T cells isolated from multiple donors are subjected to a first sorting step after staining with HLA multimers carrying the peptide of interest. The isolated cells are expanded for two weeks, after which a second sorting is performed using the same peptide-HLA multimers. The cells isolated in this way are then processed for single-cell sequencing of their TCR alpha and beta chains. Newly identified TCRs are cloned in appropriate expression vectors for functional analysis on Jurkat, NK92, and primary CD8 T cells and tumor cells expressing the appropriate antigen. We have identified TCRs specifically binding HLA-A2 presenting epitopes of tumor antigens, which are capable of inducing TCR-mediated cell activation and cytotoxicity in target cancer cell lines. This method allows the identification of tumor-reactive TCRs in about two to three weeks, starting from peripheral blood samples of readily available healthy donors.

Keywords: cancer, TCR, tumor antigens, immunotherapy

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Authors: S. Limpattayanate, M. Hunsom

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A comparison of activity and stability of the as-formed Pt/C, Pt-Co, and Pt-Pd/C electrocatalysts, prepared by a combined approach of impregnation and seeding, was performed. According to the activity test in a single proton exchange membrane (PEM) fuel cell, the oxygen reduction reaction (ORR) activity of the Pt-M/C electro catalyst was slightly lower than that of Pt/C. The j0.9 V and E10 mA/cm2 of the as-prepared electrocatalysts increased in the order of Pt/C>Pt-Co/C>Pt-Pd/C. However, in the medium-to-high current density region, Pt-Pd/C exhibited the best performance. With regard to their stability in a 0.5 M H2SO4 electrolyte solution, the electro chemical surface area decreased as the number of rounds of repetitive potential cycling increased due to the dissolution of the metals within the catalyst structure. For long-term measurement, Pt-Pd/C was the most stable than the other three electrocatalysts.

Keywords: ORR activity, stability, Pt-based electrocatalysts, PEM fuel cell

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Authors: Yuxi Zhu, Zhenqian Chen

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It is of great interest to investigate the thermal properties of non-precious metal catalysts for Proton exchange membrane fuel cell (PEMFC) based on the thermal management requirements. Due to the low symmetry of materials, to accurately obtain the thermal conductivity of materials, it is necessary to obtain the second and third order force constants by combining density functional theory and machine learning interatomic potential. To be specific, the interatomic force constants are obtained by moment tensor potential (MTP), which is trained by the computational trajectory of Ab initio molecular dynamics (AIMD) at 50, 300, 600, and 900 K for 1 ps each, with a time step of 1 fs in the AIMD computation. And then the thermal conductivity can be obtained by solving the Boltzmann transport equation. In this paper, the thermal transport properties of single metal atom catalysts are studied for the first time to our best knowledge by machine-learning interatomic potential (MLIP). Results show that the single metal atom catalysts exhibit anisotropic thermal conductivities and partially exhibit good thermal conductivity. The average lattice thermal conductivities of G-FeN₄, G-CoN₄ and G-NiN₄ at 300 K are 88.61 W/mK, 205.32 W/mK and 210.57 W/mK, respectively. While other single metal atom catalysts show low thermal conductivity due to their low phonon lifetime. The results also show that low-frequency phonons (0-10 THz) dominate thermal transport properties. The results provide theoretical insights into the application of single metal atom catalysts in thermal management.

Keywords: proton exchange membrane fuel cell, single metal atom catalysts, density functional theory, thermal conductivity, machine-learning interatomic potential

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Authors: M. S. Kilic, S. Korkut, B. Hazer

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Newly synthesized Polypropylene-g-Polyethylene glycol polymer was first time used for a compartment-less enzymatic fuel cell. Working electrodes based on Polypropylene-g-Polyethylene glycol were operated as unmediated and mediated system (with ferrocene and gold/cobalt oxide nanoparticles). Glucose oxidase and bilirubin oxidase was selected as anodic and cathodic enzyme, respectively. Glucose was used as fuel in a single-compartment and membrane-less cell. Maximum power density was obtained as 0.65 nW cm-2, 65 nW cm-2, and 23500 nW cm-2 from the unmediated, ferrocene and gold/cobalt oxide modified polymeric film, respectively. Power density was calculated to be ~16000 nW cm-2 for undiluted wastewater sample with gold/cobalt oxide nanoparticles including system.

Keywords: bilirubin oxidase, enzymatic fuel cell, glucose oxidase, nanoparticles

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Authors: Almudena Espin Perez, Tyler Risom, Carl Pelz, Isabel English, Robert M. Angelo, Rosalie Sears, Andrew J. Gentles

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Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly malignancies. The role of the tumor microenvironment (TME) is gaining significant attention in cancer research. Despite ongoing efforts, the nature of the interactions between tumors, immune cells, and stromal cells remains poorly understood. The cell-intrinsic properties that govern cell lineage plasticity in PDAC and extrinsic influences of immune populations require technically challenging approaches due to the inherently heterogeneous nature of PDAC. Understanding the cell lineage plasticity of PDAC will improve the development of novel strategies that could be translated to the clinic. Members of the team have demonstrated that the acquisition of ductal to neuroendocrine lineage plasticity in PDAC confers therapeutic resistance and is a biomarker of poor outcomes in patients. Our approach combines computational methods for deconvolving bulk transcriptomic cancer data using CIBERSORTx and high-throughput single-cell imaging using Multiplexed Ion Beam Imaging (MIBI) to study lineage plasticity in PDAC and its relationship to the infiltrating immune system. The CIBERSORTx algorithm uses signature matrices from immune cells and stroma from sorted and single-cell data in order to 1) infer the fractions of different immune cell types and stromal cells in bulked gene expression data and 2) impute a representative transcriptome profile for each cell type. We studied a unique set of 300 genomically well-characterized primary PDAC samples with rich clinical annotation. We deconvolved the PDAC transcriptome profiles using CIBERSORTx, leveraging publicly available single-cell RNA-seq data from normal pancreatic tissue and PDAC to estimate cell type proportions in PDAC, and digitally reconstruct cell-specific transcriptional profiles from our study dataset. We built signature matrices and optimized by simulations and comparison to ground truth data. We identified cell-type-specific transcriptional programs that contribute to cancer cell lineage plasticity, especially in the ductal compartment. We also studied cell differentiation hierarchies using CytoTRACE and predict cell lineage trajectories for acinar and ductal cells that we believe are pinpointing relevant information on PDAC progression. Collaborators (Angelo lab, Stanford University) has led the development of the Multiplexed Ion Beam Imaging (MIBI) platform for spatial proteomics. We will use in the very near future MIBI from tissue microarray of 40 PDAC samples to understand the spatial relationship between cancer cell lineage plasticity and stromal cells focused on infiltrating immune cells, using the relevant markers of PDAC plasticity identified from the RNA-seq analysis.

Keywords: deconvolution, imaging, microenvironment, PDAC

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Authors: Li Yang, Yang Song, Martin Y. M. Chiang

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Objective: To experimentally substantiate the micromechanical compatibility between cell and scaffold, in the regenerative medicine approach for restoring bone volume, is essential for phenotypic transitions Methods: Through nanotechnology and electrospinning process, nanofibrous scaffolds were fabricated to host dental follicle stem cells (DFSCs). Blends (50:50) of polycaprolactone (PCL) and silk fibroin (SF), mixed with various content of cellulose nanocrystals (CNC, up to 5% in weight), were electrospun to prepare nanofibrous scaffolds with heterogeneous microstructure in terms of fiber size. Colloidal probe atomic force microscopy (AFM) and conventional uniaxial tensile tests measured the scaffold stiffness at the micro-and macro-scale, respectively. The cell elastic modulus and cell-scaffold adhesive interaction (i.e., a chemical function) were examined through single-cell force spectroscopy using AFM. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to determine if the mechanotransduction signal (i.e., Yap1, Wwr2, Rac1, MAPK8, Ptk2 and Wnt5a) is upregulated by the scaffold stiffness at the micro-scale (cellular scale). Results: The presence of CNC produces fibrous scaffolds with a bimodal distribution of fiber diameter. This structural heterogeneity, which is CNC-composition dependent, remarkably modulates the mechanical functionality of scaffolds at microscale and macroscale simultaneously, but not the chemical functionality (i.e., only a single material property is varied). In in vitro tests, the osteogenic differentiation and gene expression associated with mechano-sensitive cell markers correlate to the degree of micromechanical compatibility between DFSCs and the scaffold. Conclusion: Cells require compliant scaffolds to encourage energetically favorable interactions for mechanotransduction, which are converted into changes in cellular biochemistry to direct the phenotypic evolution. The micromechanical compatibility is indeed important to the efficacy of regenerative medicine.

Keywords: phenotype transition, scaffold stiffness, electrospinning, cellulose nanocrystals, single-cell force spectroscopy

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Authors: Chia-Jung Li, Li-Te Lin, Kuan-Hao Tsui

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The study identifies SPP1 as a potential gene associated with ovarian aging, revealing a significant decline in its expression in aged ovaries. SPP1, also known as osteopontin (OPN), is a multifunctional glycoprotein involved with regulatory proteins and pro-inflammatory immune chemokines. However, its genetic links to ovarian aging have not been extensively explored. Spatial transcriptomic analyses were conducted on ovaries from young and aged female mice, along with a sample from a 73-year-old individual. Additionally, single-cell RNA sequencing analysis was performed to identify associations between SPP1 and key genes. The study focused on crucial genes, including ITGAV, ITGB1, CD44, MMP3, and FN1, with a particular emphasis on the correlation between SPP1 and ITGB1. The findings indicate a significant decline in SPP1 expression in aged ovaries, which was consistent in the 73-year-old sample. Single-cell RNA sequencing unveiled associations between SPP1 and key genes, emphasizing a strong co-expression correlation between SPP1 and ITGB1. While the study provides valuable insights, further research is necessary to understand the broader implications and potential applications of SPP1 in ovarian aging. Translating these findings to clinical settings requires careful consideration. The identification of SPP1 as a gene implicated in ovarian aging opens new avenues for advancing precision medicine and refining treatment strategies for conditions related to ovarian aging.

Keywords: SPP1, ovarian aging, spatial transcriptomic, single-cell RNA sequencing

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Authors: Hediyeh Talebi, Shokoofeh Ghiam, Changiz Eslahchi

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Traditionally, Alzheimer’s disease research has focused on genes with significant fold changes, potentially neglecting subtle but biologically important alterations. Our study introduces an integrative approach that highlights genes crucial to underlying biological processes, regardless of their fold change magnitude. Alzheimer's Single-cell RNA-seq data related to the peripheral blood mononuclear cells (PBMC) was extracted from the Gene Expression Omnibus (GEO). After quality control, normalization, scaling, batch effect correction, and clustering, differentially expressed genes (DEGs) were identified with adjusted p-values less than 0.05. These DEGs were categorized based on cell-type, resulting in four datasets, each corresponding to a distinct cell type. To distinguish between cells from healthy individuals and those with Alzheimer's, an adversarial autoencoder with a classifier was employed. This allowed for the separation of healthy and diseased samples. To identify the most influential genes in this classification, the weight matrices in the network, which includes the encoder and classifier components, were multiplied, and focused on the top 20 genes. The analysis revealed that while some of these genes exhibit a high fold change, others do not. These genes, which may be overlooked by previous methods due to their low fold change, were shown to be significant in our study. The findings highlight the critical role of genes with subtle alterations in diagnosing Alzheimer's disease, a facet frequently overlooked by conventional methods. These genes demonstrate remarkable discriminatory power, underscoring the need to integrate biological relevance with statistical measures in gene prioritization. This integrative approach enhances our understanding of the molecular mechanisms in Alzheimer’s disease and provides a promising direction for identifying potential therapeutic targets.

Keywords: alzheimer's disease, single-cell RNA-seq, neural networks, blood biomarkers

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Authors: An Chengrui, Yin Zi, Wu Bingbing, Ma Yuanzhu, Jin Kaixiu, Chen Xiao, Ouyang Hongwei

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Single cell RNA sequence (scRNA-seq) is one of the effective tools to study transcriptomics of biological processes. Recently, similarity measurement of cells is Euclidian distance or its derivatives. However, the process of scRNA-seq is a multi-variate Bernoulli event model, thus we hypothesize that it would be more efficient when the divergence between cells is valued with relative entropy than Euclidian distance. In this study, we compared the performances of Euclidian distance, Spearman correlation distance and Relative Entropy using scRNA-seq data of the early, medial and late stage of limb development generated in our lab. Relative Entropy is better than other methods according to cluster potential test. Furthermore, we developed KL-SNE, an algorithm modifying t-SNE whose definition of divergence between cells Euclidian distance to Kullback–Leibler divergence. Results showed that KL-SNE was more effective to dissect cell heterogeneity than t-SNE, indicating the better performance of relative entropy than Euclidian distance. Specifically, the chondrocyte expressing Comp was clustered together with KL-SNE but not with t-SNE. Surprisingly, cells in early stage were surrounded by cells in medial stage in the processing of KL-SNE while medial cells neighbored to late stage with the process of t-SNE. This results parallel to Heatmap which showed cells in medial stage were more heterogenic than cells in other stages. In addition, we also found that results of KL-SNE tend to follow Gaussian distribution compared with those of the t-SNE, which could also be verified with the analysis of scRNA-seq data from another study on human embryo development. Therefore, it is also an effective way to convert non-Gaussian distribution to Gaussian distribution and facilitate the subsequent statistic possesses. Thus, relative entropy is potentially a better way to determine the divergence of cells in scRNA-seq data analysis.

Keywords: Single cell RNA sequence, Similarity measurement, Relative Entropy, KL-SNE, t-SNE

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Authors: Azza T. Tahera, Eman M. Ahmeda, Nadia A. Khalila, Yassin M. Nissanb

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New 3-(pyridin-4-yl)-[1,2,4] triazolo [4,3-b] pyridazine derivatives 2a-i, 4a,b and 6a,b were designed, synthesized and evaluated as cytotoxic agents. All compounds were investigated for their in vitro cytotoxicity at a single dose 10-5M concentration towards 60 cancer cell lines according to USA NCI protocol. The preliminary screening results showed that the majority of tested compounds exhibited remarkable activity against SR (leukemia) cell panel. Molecular docking for all synthesized compounds was performed on the active site of c-Met kinase. The most active compounds, 2f and 4a were further evaluated at a seven dose level screening and their IC50 as a c-Met kinase inhibitors were determined in vitro.

Keywords: triazolopyridazines, pyridazines, cytotoxic activity, cell panel

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Authors: Hirowati Ali, Aisyah Ellyanti, Dewi Rusnita, Septelia Inawati Wanandi

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Stem cell has been known for its potency to be differentiated in many cells. Recently stem cell has been used for many treatment of degenerative medicine. It is still controversy whether stem cell can be used for therapy or these cells can activate cancer stem cell. SCUBE2 is a novel secreted and membrane-anchored protein which has been reported to its role in better prognosis and inhibition of cancer cell proliferation. Our study aims to observe whether stem cell can up-regulate SCUBE2 gene in MCF7 breast cancer cell line. We used in vitro study using MCF-7 cell treated with stem cell derived from placenta Wharton's jelly which has been known for its stemness and widely used. Our results showed that MCF-7 cell line grows up rapidly in 6-well culture dish. Stem cell was cultured in 6-well dish. After 50%-60% MCF-7 confluence, we co-cultured these cells with stem cells for 24 hours and 48 hours. We hypothesize SCUBE2 gene which is previously known for its higher expression in better prognosis of breast cancer, is up-regulated after stem cells addition in MCF7 culture dishes.

Keywords: breast cancer cells, inhibition of cancer cells, mesenchymal stem cells, SCUBE2

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Authors: Sarim Ahmad

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The Single Cell Gel Electrophoresis Assay (SCGE, known as comet assay) is a potential, uncomplicated, sensitive and state-of-the-art technique for quantitating DNA damage at individual cell level and repair from in vivo and in vitro samples of eukaryotic cells and some prokaryotic cells, being popular in its widespread use in various areas including human biomonitoring, genotoxicology, ecological monitoring and as a tool for research into DNA damage or repair in different cell types in response to a range of DNA damaging agents, cancer risk and therapy. The method involves the encapsulation of cells in a low-melting-point agarose suspension, lysis of the cells in neutral or alkaline (pH > 13) conditions, and electrophoresis of the suspended lysed cells, resulting in structures resembling comets as observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend towards the anode. This is followed by visual analysis with staining of DNA and calculating fluorescence to determine the extent of DNA damage. This can be performed by manual scoring or automatically by imaging software. The assay can, therefore, predict an individual’s tumor sensitivity to radiation and various chemotherapeutic drugs and further assess the oxidative stress within tumors and to detect the extent of DNA damage in various cancerous and precancerous lesions of oral cavity.

Keywords: comet assay, single cell gel electrophoresis, DNA damage, early detection test

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Authors: Dalwinder Singh, Amandeep Verma, Manpreet Kaur, Birmohan Singh

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The detection of pre-cancerous changes using a Pap smear test of cervical cell is the important step for the early diagnosis of cervical cancer. The Pap smear test consists of a sample of human cells taken from the cervix which are analysed to detect cancerous and pre-cancerous stage of the given subject. The manual analysis of these cells is labor intensive and time consuming process which relies on expert cytotechnologist. In this paper, a computer assisted system for the automated analysis of the cervical cells has been proposed. We propose a morphology based approach to the nucleus detection and segmentation of the cytoplasmic region of the given single or multiple overlapped cell. Further, various texture and region based features are calculated from these cells to classify these into normal and abnormal cell. Experimental results on public available dataset show that our system has achieved satisfactory success rate.

Keywords: cervical cancer, cervical tissue, mathematical morphology, texture features

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Authors: Bourenane Bouhafs Naziha

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ARTEA330EC is a fungicide used to inhibit the growth of many types of fungi on and cereals and rice, it is the single largest selling agrochemical that has been widely detected in surface waters in our area (Northeast Algerian). The studies on long-term genotoxic effects of fugicides in different tissues of fish using genotoxic biomarkers are limited. Therefore, in the present study DNA damage by propiconazole in freshwater fish Gambusia affinis by comet assays was investigated. The LC(50)- 96 h of the fungicide was estimated for the fish in a semi-static system. On this basis of LC(50) value sublethal and nonlethal concentrations were determined (25; 50; 75; and 100 ppm). The DNA damage was measured in erythrocytes as the percentage of DNA in comet tails of fishes exposed to above concentrations the fungicide. In general,non significant effects for both the concentrations and time of exposure were observed in treated fish compared with the controls. However It was found that the highest DNA damage was observed at the highest concentration and the longest time of exposure (day 12). The study indicated comet assay to be sensitive and rapid method to detect genotoxicity of propiconasol and other pesticides in fishes.

Keywords: genotoxicity, fungicide, propiconazole, freshwater, Gambusia affinis, alkaline single-cell gel electrophoresis

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Authors: Mitesh Rathod, Jungho Ahn, Noo Li Jeon, Junghoon Lee

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Endothelial cells respond to cues from both biochemical as well as micro mechanical environment. Significant effort has been directed to understand the effects of biochemical signaling, however, relatively little is known about regulation of endothelial cell biology by the micro mechanical environment. Numerous studies have been performed to understand how physical forces regulate endothelial cell behavior. In this regard, past studies have majorly focused on exploring how fluid shear stress governs endothelial cell behavior. Parallel plate flow chambers and rectangular microchannels are routinely employed for applying fluid shear force on endothelial cells. However, these studies fall short in mimicking the in vivo like micro environment from topological aspects. Few studies have only used circular microchannels to replicate in vivo like condition. Seldom efforts have been directed to elucidate the combined effect of topology, substrate rigidity and fluid shear stress on endothelial cell response. In this regard, we demonstrate a facile fabrication process to develop a hybrid polydimethylsiloxane microfluidic platform to study endothelial cell biology. On a single chip microchannels with different cross sections i.e., circular, rectangular and square have been fabricated. In addition, our fabrication approach allows variation in the substrate rigidity along the channel length. Two different variants of polydimethylsiloxane, namely Sylgard 184 and Sylgard 527, were utilized to achieve the variation in rigidity. Moreover, our approach also enables in creating Y bifurcation circular microchannels. Our microfluidic platform thus facilitates for conducting studies pertaining to endothelial cell morphology with respect to change in topology, substrate rigidity and fluid flow on a single chip. The hybrid platform was tested by culturing Human Umbilical Vein Endothelial Cells in circular microchannels with varying substrate rigidity, and exposed to fluid shear stress of 12 dynes/cm² and static conditions. Results indicate the cell area response to flow induced shear stress was governed by the underlying substrate mechanics.

Keywords: hybrid, microfluidic platform, PDMS, shear flow, substrate rigidity

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Authors: Hilal Turkoglu Sasmazel, Ozan Ozkan, Seda Surucu

Abstract:

Tissue engineering is the field of treating defects caused by injuries, trauma or acute/chronic diseases by using artificial scaffolds that mimic the extracellular matrix (ECM), the natural biological support for the tissues and cells within the body. The main aspects of a successful artificial scaffold are (i) large surface area in order to provide multiple anchorage points for cells to attach, (ii) suitable porosity in order to achieve 3 dimensional growth of the cells within the scaffold as well as proper transport of nutrition, biosignals and waste and (iii) physical, chemical and biological compatibility of the material in order to obtain viability throughout the healing process. By hybrid scaffolds where two or more different materials were combined with advanced fabrication techniques into complex structures, it is possible to combine the advantages of individual materials into one single structure while eliminating the disadvantages of each. Adding this to the complex structure provided by advanced fabrication techniques enables obtaining the desired aspects of a successful artificial tissue scaffold. In this study, fibroblast compatibility of poly(ε-caprolactone) (PCL)/chitosan core-shell electrospun hybrid scaffolds with proper mechanical, chemical and physical properties successfully developed in our previous study was investigated. Standard 7-day cell culture was carried out with L929 fibroblast cell line. The viability of the cells cultured with the scaffolds was monitored with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay for every 48 h starting with 24 h after the initial seeding. In this assay, blank commercial tissue culture polystyrene (TCPS) Petri dishes, single electrospun PCL and single electrospun chitosan mats were used as control in order to compare and contrast the performance of the hybrid scaffolds. The adhesion, proliferation, spread and growth of the cells on/within the scaffolds were observed visually on the 3rd and the 7th days of the culture period with confocal laser scanning microscopy (CSLM) and scanning electron microscopy (SEM). The viability assay showed that the hybrid scaffolds caused no toxicity for fibroblast cells and provided a steady increase in cell viability, effectively doubling the cell density for every 48 h for the course of 7 days, as compared to TCPS, single electrospun PCL or chitosan mats. The cell viability on the hybrid scaffold was ~2 fold better compared to TCPS because of its 3D ECM-like structure compared to 2D flat surface of commercially cell compatible TCPS, and the performance was ~2 fold and ~10 fold better compared to single PCL and single chitosan mats, respectively, even though both fabricated similarly with electrospinning as non-woven fibrous structures, because single PCL and chitosan mats were either too hydrophobic or too hydrophilic to maintain cell attachment points. The viability results were verified with visual images obtained with CSLM and SEM, in which cells found to achieve characteristic spindle-like fibroblast shape and spread on the surface as well within the pores successfully at high densities.

Keywords: chitosan, core-shell, fibroblast, electrospinning, PCL

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Authors: Prabhavathi Sundaram, Heidi Abrahamse

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In recent years, nanotechnology coupled with photodynamic therapy (PDT) has received considerable attention in terms of improving the effectiveness of drug delivery in cancer therapeutics. The development of functionalized single-walled carbon nanotubes (SWCNTs) has become revolutionary in targeted photosensitizers delivery since it improves the therapeutic index of drugs. The objective of this study was to prepare, characterize and evaluate the potential of functionalized SWCNTs using hyaluronic acid and loading it with photosensitizer and to effectively target colon cancer cells. The single-walled carbon nanotubes were covalently functionalized with hyaluronic acid and the loaded photosensitizer by non-covalent interaction. The photodynamic effect of SWCNTs is detected under laser irradiation in vitro. The hyaluronic acid-functionalized nanocomposites had a good affinity with CD44 receptors, and it avidly binds on to the surface of CACO-2 cells. The cellular uptake of nanocomposites was studied using fluorescence microscopy using lyso tracker. The anticancer activity of nanocomposites was analyzed in CACO-2 cells using different studies such as cell morphology, cell apoptosis, and nuclear morphology. The combined effect of nanocomposites and PDT improved the therapeutic effect of cancer treatment. The study suggested that the nanocomposites and PDT have great potential in the treatment of colon cancer.

Keywords: colon cancer, hyaluronic acid, single walled carbon nanotubes, photosensitizers, photodynamic therapy

Procedia PDF Downloads 117

Authors: Dong-An Wang

Abstract:

All kinds of microspheres have been extensively employed as carriers for drug, gene and therapeutic cell delivery. Most therapeutic cell delivery microspheres rely on a two-step methodology: fabrication of microspheres and subsequent seeding of cells onto them. In this study, we have developed a novel one-step cell encapsulation technique using a convenient and instant water-in-oil single emulsion approach to form cell-encapsulated gelatin microspheres. This technology is adopted for hyaline cartilage tissue engineering, in which autologous chondrocytes are used as therapeutic cells. Cell viability was maintained throughout and after the microsphere formation (75-100 µm diameters) process that avoids involvement of any covalent bonding reactions or exposure to any further chemicals. Further encapsulation of cell-laden microspheres in alginate gels were performed under 4°C via a prompt process. Upon the formation of alginate constructs, they were immediately relocated into CO2 incubator where the temperature was maintained at 37°C; under this temperature, the cell-laden gelatin microspheres dissolved within hours to yield similarly sized cavities and the chondrocytes were therefore suspended within the cavities inside the alginate gel bulk. Hence, the gelatin cell-laden microspheres served two roles: as cell delivery vehicles which can be removable through temperature curing, and as porogens within an alginate hydrogel construct to provide living space for cell growth and tissue development as well as better permeability for mutual diffusions. These cell-laden microspheres, namely “temperature-cured dissolvable gelatin microsphere based cell carriers” (tDGMCs), were further encapsulated in a chondrocyte-laden alginate scaffold system and analyzed by WST-1, gene expression analyses, biochemical assays, histology and immunochemistry stains. The positive results consistently demonstrated the promise of tDGMC technology in delivering these non-anchorage dependent cells (chondrocytes). It can be further conveniently translated into delivery of other non-anchorage dependent cell species, including stem cells, progenitors or iPS cells, for regeneration of tissues in internal organs, such as engineered hepatogenesis or pancreatic regeneration.

Keywords: biomaterials, tissue engineering, microsphere, hydrogel, porogen, anchorage dependence

Procedia PDF Downloads 396

Authors: Lucretia Ramnath

Abstract:

Fishmeal is extensively used for fish farming but is an expensive fish feed ingredient. A cheaper alternate to fishmeal is single cell protein (SCP) which can be cultivated on fermentable sugars recovered from organic waste streams such as pulp and paper mill sludge (PPMS). PPMS has a high cellulose content, thus is suitable for glucose recovery through enzymatic hydrolysis but is hampered by lignin and ash. To render PPMS amenable for enzymatic hydrolysis, the PPMS waspre-treated to produce a glucose-rich hydrolysate which served as a feed stock for the production of fungal SCP. The PPMS used in this study had the following composition: 72.77% carbohydrates, 8.6% lignin, and 18.63% ash. The pre-treatments had no significant effect on lignin composition but had a substantial effect on carbohydrate and ash content. Enzymatic hydrolysis of screened PPMS was previously optimized through response surface methodology (RSM) and 2-factorial design. The optimized protocol resulted in a hydrolysate containing 46.1 g/L of glucose, of which 86% was recovered after downstream processing by passing through a 100-mesh sieve (38 µm pore size). Vogel’s medium supplemented with 10 g/L hydrolysate successfully supported the growth of Fusarium venenatum, conducted using standard growth conditions; pH 6, 200 rpm, 2.88 g/L ammonium phosphate, 25°C. A maximum F. venenatum biomass of 45 g/L was produced with a yield coefficient of 4.67. Pulp and paper mill sludge hydrolysate contained approximately five times more glucose than what was needed for SCP production and served as a suitable carbon source. We have shown that PPMS can be successfully beneficiated for SCP production.

Keywords: pulp and paper waste, fungi, single cell protein, hydrolysate

Procedia PDF Downloads 207

Authors: Rinat Arbel-Goren, Joel Stavans

Abstract:

Cell-to-cell variations in transcript or protein abundance, called noise, may give rise to phenotypic variability between isogenic cells, enhancing the probability of survival under stress conditions. These variations may be introduced by post-transcriptional regulatory processes such as non-coding, small RNAs stoichiometric degradation of target transcripts in bacteria. We study the iron homeostasis network in Escherichia coli, in which the RyhB small RNA regulates the expression of various targets as a model system. Using fluorescence reporter genes to detect protein levels and single-molecule fluorescence in situ hybridization to monitor transcripts levels in individual cells, allows us to compare noise at both transcript and protein levels. The experimental results and computer simulations show that extrinsic noise buffers through a feed-forward loop configuration the increase in variability introduced at the transcript level by iron deprivation, illuminating the important role that extrinsic noise plays during stress. Surprisingly, extrinsic noise also decouples of fluctuations of two different targets, in spite of RyhB being a common upstream factor degrading both. Thus, phenotypic variability increases under stress conditions by the decoupling of target fluctuations in the same cell rather than by increasing the noise of each. We also present preliminary results on the adaptation of cells to prolonged iron deprivation in order to shed light on the evolutionary role of post-transcriptional downregulation by small RNAs.

Keywords: cell-to-cell variability, Escherichia coli, noise, single-molecule fluorescence in situ hybridization (smFISH), transcript

Procedia PDF Downloads 164

Authors: K. Suresh, R. Arunkumar

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The objective of this study is to investigate the preparation of gramine nano suspension and protective effect of Gramine on the apoptosis of laryngeal cancer cells cell line (HEp-2 and KB). The growth inhibition rate of Hep-2 and KB cells in vitro were measured by MTT assay and apoptosis by, levels of reactive oxygen species, mitochondrial membrane potential, morphological changes and flowcytometry. Based on the results, we determined the effective doses of gramine as 127.23µm/ml for 24 hr and 119.81 µm/ml for 48hr in hep-2 cell line and 147.58 µm ml for 24 hr and 123.74µm µm/ml for 48hr in KB cell line. cytotoxicity effects of gramine were confirmed by treatment of HEp-2 cell and KB cell with IC50 concentration of gramine resulted in sequences of events marked by the enhance the apoptosis accompanied by loss of cell viability, modulation of reactive oxygen species and cell cycle arrest through the induction of G0/G1 phase arrest on HEp-2 cells. Our study suggests that the nanosuspension of gramine possesses the more cytotoxic effect of cancer cells and a novel candidate for cancer chemoprevention.

Keywords: apoptosis, HEp-2 cell line, KB cell line mitochondria, gramine, nanosuspension

Procedia PDF Downloads 454

Authors: Sangwoo Han, Saeed Khaleghi Rahimian, Ying Liu

Abstract:

Vehicle electrification is gaining momentum, and many car manufacturers promise to deliver more electric vehicle (EV) models to consumers in the coming years. In controlling the battery pack, the battery management system (BMS) must maintain optimal battery performance while ensuring the safety of a battery pack. Tasks related to battery performance include determining state-of-charge (SOC), state-of-power (SOP), state-of-health (SOH), cell balancing, and battery charging. Safety related functions include making sure cells operate within specified, static and dynamic voltage window and temperature range, derating power, detecting faulty cells, and warning the user if necessary. The BMS often utilizes an RC circuit model to model a Li-ion cell because of its robustness and low computation cost among other benefits. Because an equivalent circuit model such as the RC model is not a physics-based model, it can never be a prognostic model to predict battery state-of-health and avoid any safety risk even before it occurs. A physics-based Li-ion cell model, on the other hand, is more capable at the expense of computation cost. To avoid the high computation cost associated with a full-order model, many researchers have demonstrated the use of a single particle model (SPM) for BMS applications. One drawback associated with the single particle modeling approach is that it forces to use the average current density in the calculation. The SPM would be appropriate for simulating drive cycles where there is insufficient time to develop a significant current distribution within an electrode. However, under a continuous or high-pulse electrical load, the model may fail to predict cell voltage or Li⁺ plating potential. To overcome this issue, a multi-particle reduced-order model is proposed here. The use of multiple particles combined with either linear or nonlinear charge-transfer reaction kinetics enables to capture current density distribution within an electrode under any type of electrical load. To maintain computational complexity like that of an SPM, governing equations are solved sequentially to minimize iterative solving processes. Furthermore, the model is validated against a full-order model implemented in COMSOL Multiphysics.

Keywords: battery management system, physics-based li-ion cell model, reduced-order model, single-particle and multi-particle model

Procedia PDF Downloads 112