Validation of in vitro assays in three-dimensional human dermal constructs.

PubMed

Idrees, Ayesha; Chiono, Valeria; Ciardelli, Gianluca; Shah, Siegfried; Viebahn, Richard; Zhang, Xiang; Salber, Jochen

2018-05-01

Three-dimensional cell culture systems are urgently needed for cytocompatibility testing of biomaterials. This work aimed at the development of three-dimensional in vitro dermal skin models and their optimization for cytocompatibility evaluation. Initially "murine in vitro dermal construct" based on L929 cells was generated, leading to the development of "human in vitro dermal construct" consisting of normal human dermal fibroblasts in rat tail tendon collagen type I. To assess the viability of the cells, different assays CellTiter-Blue ® , RealTime-Glo ™ MT, and CellTiter-Glo ® (Promega) were evaluated to optimize the best-suited assay to the respective cell type and three-dimensional system. Z-stack imaging (Live/Dead and Phalloidin/DAPI-Promokine) was performed to visualize normal human dermal fibroblasts inside matrix revealing filopodia-like morphology and a uniform distribution of normal human dermal fibroblasts in matrix. CellTiter-Glo was found to be the optimal cell viability assay among those analyzed. CellTiter-Blue reagent affected the cell morphology of normal human dermal fibroblasts (unlike L929), suggesting an interference with cell biological activity, resulting in less reliable viability data. On the other hand, RealTime-Glo provided a linear signal only with a very low cell density, which made this assay unsuitable for this system. CellTiter-Glo adapted to three-dimensional dermal construct by optimizing the "shaking time" to enhance the reagent penetration and maximum adenosine triphosphate release, indicating 2.4 times higher viability value by shaking for 60 min than for 5 min. In addition, viability results showed that cells were viable inside the matrix. This model would be further advanced with more layers of skin to make a full thickness model.

Visual-servoing optical microscopy

DOEpatents

Callahan, Daniel E.; Parvin, Bahram

2009-06-09

The present invention provides methods and devices for the knowledge-based discovery and optimization of differences between cell types. In particular, the present invention provides visual servoing optical microscopy, as well as analysis methods. The present invention provides means for the close monitoring of hundreds of individual, living cells over time: quantification of dynamic physiological responses in multiple channels; real-time digital image segmentation and analysis; intelligent, repetitive computer-applied cell stress and cell stimulation; and the ability to return to the same field of cells for long-term studies and observation. The present invention further provides means to optimize culture conditions for specific subpopulations of cells.

Visual-servoing optical microscopy

DOEpatents

Callahan, Daniel E [Martinez, CA; Parvin, Bahram [Mill Valley, CA

2011-05-24

The present invention provides methods and devices for the knowledge-based discovery and optimization of differences between cell types. In particular, the present invention provides visual servoing optical microscopy, as well as analysis methods. The present invention provides means for the close monitoring of hundreds of individual, living cells over time; quantification of dynamic physiological responses in multiple channels; real-time digital image segmentation and analysis; intelligent, repetitive computer-applied cell stress and cell stimulation; and the ability to return to the same field of cells for long-term studies and observation. The present invention further provides means to optimize culture conditions for specific subpopulations of cells.

Visual-servoing optical microscopy

DOEpatents

Callahan, Daniel E; Parvin, Bahram

2013-10-01

The present invention provides methods and devices for the knowledge-based discovery and optimization of differences between cell types. In particular, the present invention provides visual servoing optical microscopy, as well as analysis methods. The present invention provides means for the close monitoring of hundreds of individual, living cells over time; quantification of dynamic physiological responses in multiple channels; real-time digital image segmentation and analysis; intelligent, repetitive computer-applied cell stress and cell stimulation; and the ability to return to the same field of cells for long-term studies and observation. The present invention further provides means to optimize culture conditions for specific subpopulations of cells.

Multiple Lytic Origins of Replication Are Required for Optimal Gammaherpesvirus Fitness In Vitro and In Vivo.

PubMed

Sattler, Christine; Steer, Beatrix; Adler, Heiko

2016-03-01

An unresolved question in herpesvirus biology is why some herpesviruses contain more than one lytic origin of replication (oriLyt). Using murine gammaherpesvirus 68 (MHV-68) as model virus containing two oriLyts, we demonstrate that loss of either of the two oriLyts was well tolerated in some situations but not in others both in vitro and in vivo. This was related to the cell type, the organ or the route of inoculation. Depending on the cell type, different cellular proteins, for example Hexim1 and Rbbp4, were found to be associated with oriLyt DNA. Overexpression or downregulation of these proteins differentially affected the growth of mutants lacking either the left or the right oriLyt. Thus, multiple oriLyts are required to ensure optimal fitness in different cell types and tissues.

Liquid-phase-deposited siloxane-based capping layers for silicon solar cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Veith-Wolf, Boris; Wang, Jianhui; Hannu-Kuure, Milja

2015-02-02

We apply non-vacuum processing to deposit dielectric capping layers on top of ultrathin atomic-layer-deposited aluminum oxide (AlO{sub x}) films, used for the rear surface passivation of high-efficiency crystalline silicon solar cells. We examine various siloxane-based liquid-phase-deposited (LPD) materials. Our optimized AlO{sub x}/LPD stacks show an excellent thermal and chemical stability against aluminum metal paste, as demonstrated by measured surface recombination velocities below 10 cm/s on 1.3 Ωcm p-type silicon wafers after firing in a belt-line furnace with screen-printed aluminum paste on top. Implementation of the optimized LPD layers into an industrial-type screen-printing solar cell process results in energy conversion efficiencies ofmore » up to 19.8% on p-type Czochralski silicon.« less

Cell-specific Labeling Enzymes for Analysis of Cell–Cell Communication in Continuous Co-culture*

PubMed Central

Tape, Christopher J.; Norrie, Ida C.; Worboys, Jonathan D.; Lim, Lindsay; Lauffenburger, Douglas A.; Jørgensen, Claus

2014-01-01

We report the orthologous screening, engineering, and optimization of amino acid conversion enzymes for cell-specific proteomic labeling. Intracellular endoplasmic-reticulum-anchored Mycobacterium tuberculosis diaminopimelate decarboxylase (DDCM.tub-KDEL) confers cell-specific meso-2,6-diaminopimelate-dependent proliferation to multiple eukaryotic cell types. Optimized lysine racemase (LyrM37-KDEL) supports D-lysine specific proliferation and efficient cell-specific isotopic labeling. When ectopically expressed in discrete cell types, these enzymes confer 90% cell-specific isotopic labeling efficiency after 10 days of co-culture. Moreover, DDCM.tub-KDEL and LyrM37-KDEL facilitate equally high cell-specific labeling fidelity without daily media exchange. Consequently, the reported novel enzyme pairing can be used to study cell-specific signaling in uninterrupted, continuous co-cultures. Demonstrating the importance of increased labeling stability for addressing novel biological questions, we compare the cell-specific phosphoproteome of fibroblasts in direct co-culture with epithelial tumor cells in both interrupted (daily media exchange) and continuous (no media exchange) co-cultures. This analysis identified multiple cell-specific phosphorylation sites specifically regulated in the continuous co-culture. Given their applicability to multiple cell types, continuous co-culture labeling fidelity, and suitability for long-term cell–cell phospho-signaling experiments, we propose DDCM.tub-KDEL and LyrM37-KDEL as excellent enzymes for cell-specific labeling with amino acid precursors. PMID:24820872

Baculovirus-mediated expression of GPCRs in insect cells.

PubMed

Saarenpää, Tuulia; Jaakola, Veli-Pekka; Goldman, Adrian

2015-01-01

G-protein-coupled receptors (GPCRs) are a large family of seven transmembrane proteins that influence a considerable number of cellular events. For this reason, they are one of the most studied receptor types for their pharmacological and structural properties. Solving the structure of several GPCR receptor types has been possible using almost all expression systems, including Escherichia coli, yeast, mammalian, and insect cells. So far, however, most of the GPCR structures solved have been done using the baculovirus insect cell expression system. The reason for this is mainly due to cost-effectiveness, posttranslational modification efficiency, and overall effortless maintenance. The system has evolved so much that variables starting from vector type, purification tags, cell line, and growth conditions can be varied and optimized countless ways to suit the needs of new constructs. Here, we present the array of techniques that enable the rapid and efficient optimization of expression steps for maximal protein quality and quantity, including our emendations. © 2015 Elsevier Inc. All rights reserved.

Mathematical modeling of a thermovoltaic cell

NASA Technical Reports Server (NTRS)

White, Ralph E.; Kawanami, Makoto

1992-01-01

A new type of battery named 'Vaporvolt' cell is in the early stage of its development. A mathematical model of a CuO/Cu 'Vaporvolt' cell is presented that can be used to predict the potential and the transport behavior of the cell during discharge. A sensitivity analysis of the various transport and electrokinetic parameters indicates which parameters have the most influence on the predicted energy and power density of the 'Vaporvolt' cell. This information can be used to decide which parameters should be optimized or determined more accurately through further modeling or experimental studies. The optimal thicknesses of electrodes and separator, the concentration of the electrolyte, and the current density are determined by maximizing the power density. These parameter sensitivities and optimal design parameter values will help in the development of a better CuO/Cu 'Vaporvolt' cell.

Multiple Lytic Origins of Replication Are Required for Optimal Gammaherpesvirus Fitness In Vitro and In Vivo

PubMed Central

Sattler, Christine; Steer, Beatrix; Adler, Heiko

2016-01-01

An unresolved question in herpesvirus biology is why some herpesviruses contain more than one lytic origin of replication (oriLyt). Using murine gammaherpesvirus 68 (MHV-68) as model virus containing two oriLyts, we demonstrate that loss of either of the two oriLyts was well tolerated in some situations but not in others both in vitro and in vivo. This was related to the cell type, the organ or the route of inoculation. Depending on the cell type, different cellular proteins, for example Hexim1 and Rbbp4, were found to be associated with oriLyt DNA. Overexpression or downregulation of these proteins differentially affected the growth of mutants lacking either the left or the right oriLyt. Thus, multiple oriLyts are required to ensure optimal fitness in different cell types and tissues. PMID:27007137

An intracellular analysis of the visual responses of neurones in cat visual cortex.

PubMed Central

Douglas, R J; Martin, K A; Whitteridge, D

1991-01-01

1. Extracellular and intracellular recordings were made from neurones in the visual cortex of the cat in order to compare the subthreshold membrane potentials, reflecting the input to the neurone, with the output from the neurone seen as action potentials. 2. Moving bars and edges, generated under computer control, were used to stimulate the neurones. The membrane potential was digitized and averaged for a number of trials after stripping the action potentials. Comparison of extracellular and intracellular discharge patterns indicated that the intracellular impalement did not alter the neurones' properties. Input resistance of the neurone altered little during stable intracellular recordings (30 min-2 h 50 min). 3. Intracellular recordings showed two distinct patterns of membrane potential changes during optimal visual stimulation. The patterns corresponded closely to the division of S-type (simple) and C-type (complex) receptive fields. Simple cells had a complex pattern of membrane potential fluctuations, involving depolarizations alternating with hyperpolarizations. Complex cells had a simple single sustained plateau of depolarization that was often followed but not preceded by a hyperpolarization. In both simple and complex cells the depolarizations led to action potential discharges. The hyperpolarizations were associated with inhibition of action potential discharge. 4. Stimulating simple cells with non-optimal directions of motion produced little or no hyperpolarization of the membrane in most cases, despite a lack of action potential output. Directional complex cells always produced a single plateau of depolarization leading to action potential discharge in both the optimal and non-optimal directions of motion. The directionality could not be predicted on the basis of the position of the hyperpolarizing inhibitory potentials found in the optimal direction. 5. Stimulation of simple cells with non-optimal orientations occasionally produced slight hyperpolarizations and inhibition of action potential discharge. Complex cells, which had broader orientation tuning than simple cells, could show marked hyperpolarization for non-optimal orientations, but this was not generally the case. 6. The data do not support models of directionality and orientation that rely solely on strong inhibitory mechanisms to produce stimulus selectivity. PMID:1804981

Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity.

PubMed

Chen, Mei-ling; Guo, Qin; Wang, Rui-zhi; Xu, Juan; Zhou, Chen-wei; Ruan, Hui; He, Guo-qing

2011-07-01

Surface display is effectively utilized to construct a whole-cell biocatalyst. Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast. Here, the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor, recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed. Compared with the wild-type ROL-displaying yeast, the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate. To our knowledge, this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction. Consequently, the yeast whole-cell ROL biocatalyst was constructed with high activity. The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C. Furthermore, this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.

Optimal control for Malaria disease through vaccination

NASA Astrophysics Data System (ADS)

Munzir, Said; Nasir, Muhammad; Ramli, Marwan

2018-01-01

Malaria is a disease caused by an amoeba (single-celled animal) type of plasmodium where anopheles mosquito serves as the carrier. This study examines the optimal control problem of malaria disease spread based on Aron and May (1982) SIR type models and seeks the optimal solution by minimizing the prevention of the spreading of malaria by vaccine. The aim is to investigate optimal control strategies on preventing the spread of malaria by vaccination. The problem in this research is solved using analytical approach. The analytical method uses the Pontryagin Minimum Principle with the symbolic help of MATLAB software to obtain optimal control result and to analyse the spread of malaria with vaccination control.

Will stem cell therapies be safe and effective for treating spinal cord injuries?

PubMed Central

Thomas, Katharine E.; Moon, Lawrence D. F.

2017-01-01

Introduction A large number of different cells including embryonic and adult stem cells have been transplanted into animal models of spinal cord injury, and in many cases these procedures have resulted in modest sensorimotor benefits. In October 2010 the world’s first clinical trial using human embryonic stem cells began, using stem cells converted into oligodendrocyte precursor cells. Sources of data In this review we examine some of the publically-available pre-clinical evidence that some of these cell types improve outcome in animal models of spinal cord injury. Much evidence is not available for public scrutiny, however, being private commercial property of various stem cell companies. Areas of agreement Transplantation of many different types of stem and progenitor cell enhances spontaneous recovery of function when transplanted acutely after spinal cord injury in animal models. Areas of disagreement The common mechanism(s) whereby the generic procedure of cellular transplantation enhances recovery of function are not well understood, although a range of possibilities are usually cited (including preservation of tissue, remyelination, axon sprouting, glial cell replacement). Only in exceptional cases has it been shown that functional recovery depends causally on the survival and differentiation of the transplanted cells. There is no agreement about the optimal cell type for transplantation: candidate stem cells have not yet been compared with each other or with other cell types (e.g., autologous Schwann cells) in a single study. Areas timely for developing research Transplantation of cells into animals with a long lifespan is important to determine whether or not tumours will eventually form. It will also be important to determine whether long-term survival of cells is required for functional recovery, and if so, how many are optimal. PMID:21586446

Improved specific energy Ni-H2 cell

NASA Astrophysics Data System (ADS)

Miller, L.

1985-07-01

Design optimization activities which have evolved and validated the necessary technology to produce Ni-H2 battery cells exhibiting a specific energy of 75-80 Whr/Kg (energy density approximately 73 Whr/L are summarized. Final design validation is currently underway with the production of battery cells for qualification and life testing. The INTELSAT type Ni-H2 battery cell design has been chosen for expository purposes. However, it should be recognized portions of the improved technology could be applied to the Air Force type Ni-H2 battery cell design with equal benefit.

Improved Specific Energy Ni-h2 Cell

NASA Technical Reports Server (NTRS)

Miller, L.

1985-01-01

Design optimization activities which have evolved and validated the necessary technology to produce Ni-H2 battery cells exhibiting a specific energy of 75-80 Whr/Kg (energy density approximately 73 Whr/L are summarized. Final design validation is currently underway with the production of battery cells for qualification and life testing. The INTELSAT type Ni-H2 battery cell design has been chosen for expository purposes. However, it should be recognized portions of the improved technology could be applied to the Air Force type Ni-H2 battery cell design with equal benefit.

Optimization of active cell area on the dye-sensitized solar cell efficiency

NASA Astrophysics Data System (ADS)

Putri, A. W.; Nurosyid, F.; Supriyanto, Agus

2017-11-01

This study is aimed to obtain optimal active area producing high efficiency of DSSC module. The DSSC structure is constructed of TiO2 as working electrode, dye as photosensitizer, platinum as counter electrode, and electrolyte as electron transfer media. TiO2 paste was deposited on Fluorine-doped Tin Oxide (FTO) by screen printing method. Meanwhile, platinum was also coated on FTO via brush painting method. Keithley I-V meter was performed to characterize DSSC electrical property. The active area of each cell was varied of 4.5 cm2, 9 cm2, and 13.5 cm2. Each cell was assembled into a module using an external series connection of Z type. The module was consisted of 12 cells, 6 cells, and 4 cells with module active area of 54 cm2. The optimal active area of DSSC cell is 4.5 cm2 resulting 0.4149% efficiency. In addition, the highest efficiency of DSSC module is 0.2234% acquired by 6 cells assembling.

Optimal fold symmetry of LH2 rings on a photosynthetic membrane

PubMed Central

Cleary, Liam; Chen, Hang; Chuang, Chern; Silbey, Robert J.; Cao, Jianshu

2013-01-01

An intriguing observation of photosynthetic light-harvesting systems is the N-fold symmetry of light-harvesting complex 2 (LH2) of purple bacteria. We calculate the optimal rotational configuration of N-fold rings on a hexagonal lattice and establish two related mechanisms for the promotion of maximum excitation energy transfer (EET). (i) For certain fold numbers, there exist optimal basis cells with rotational symmetry, extendable to the entire lattice for the global optimization of the EET network. (ii) The type of basis cell can reduce or remove the frustration of EET rates across the photosynthetic network. We find that the existence of a basis cell and its type are directly related to the number of matching points S between the fold symmetry and the hexagonal lattice. The two complementary mechanisms provide selection criteria for the fold number and identify groups of consecutive numbers. Remarkably, one such group consists of the naturally occurring 8-, 9-, and 10-fold rings. By considering the inter-ring distance and EET rate, we demonstrate that this group can achieve minimal rotational sensitivity in addition to an optimal packing density, achieving robust and efficient EET. This corroborates our findings i and ii and, through their direct relation to S, suggests the design principle of matching the internal symmetry with the lattice order. PMID:23650366

Optimal fold symmetry of LH2 rings on a photosynthetic membrane.

PubMed

Cleary, Liam; Chen, Hang; Chuang, Chern; Silbey, Robert J; Cao, Jianshu

2013-05-21

An intriguing observation of photosynthetic light-harvesting systems is the N-fold symmetry of light-harvesting complex 2 (LH2) of purple bacteria. We calculate the optimal rotational configuration of N-fold rings on a hexagonal lattice and establish two related mechanisms for the promotion of maximum excitation energy transfer (EET). (i) For certain fold numbers, there exist optimal basis cells with rotational symmetry, extendable to the entire lattice for the global optimization of the EET network. (ii) The type of basis cell can reduce or remove the frustration of EET rates across the photosynthetic network. We find that the existence of a basis cell and its type are directly related to the number of matching points S between the fold symmetry and the hexagonal lattice. The two complementary mechanisms provide selection criteria for the fold number and identify groups of consecutive numbers. Remarkably, one such group consists of the naturally occurring 8-, 9-, and 10-fold rings. By considering the inter-ring distance and EET rate, we demonstrate that this group can achieve minimal rotational sensitivity in addition to an optimal packing density, achieving robust and efficient EET. This corroborates our findings i and ii and, through their direct relation to S, suggests the design principle of matching the internal symmetry with the lattice order.

Baseline and annual repeat rounds of screening: implications for optimal regimens of screening.

PubMed

Henschke, Claudia I; Salvatore, Mary; Cham, Matthew; Powell, Charles A; DiFabrizio, Larry; Flores, Raja; Kaufman, Andrew; Eber, Corey; Yip, Rowena; Yankelevitz, David F

2018-03-01

Differences in results of baseline and subsequent annual repeat rounds provide important information for optimising the regimen of screening. A prospective cohort study of 65,374 was reviewed to examine the frequency/percentages of the largest noncalcified nodule (NCN), lung cancer cell types and Kaplan-Meier (K-M) survival rates, separately for baseline and annual rounds. Of 65,374 baseline screenings, NCNs were identified in 28,279 (43.3%); lung cancer in 737 (1.1%). Of 74,482 annual repeat screenings, new NCNs were identified in 4959 (7%); lung cancer in 179 (0.24%). Only adenocarcinoma was diagnosed in subsolid NCNs. Percentages of lung cancers by cell type were significantly different (p < 0.0001) in the baseline round compared with annual rounds, reflecting length bias, as were the ratios, reflecting lead times. Long-term K-M survival rate was 100% for typical carcinoids and for adenocarcinomas manifesting as subsolid NCNs; 85% (95% CI 81-89%) for adenocarcinoma, 74% (95% CI 63-85%) for squamous cell, 48% (95% CI 34-62%) for small cell. The rank ordering by lead time was the same as the rank ordering by survival rates. The significant differences in the frequency of NCNs and frequency and aggressiveness of diagnosed cancers in baseline and annual repeat need to be recognised for an optimal regimen of screening. • Lung cancer aggressiveness varies considerably by cell type and nodule consistency. • Kaplan-Meier survival rates varied by cell type between 100% and 48%. • The percentages of lung cancers by cell type in screening rounds reflect screening biases. • Rank ordering by cell type survival is consistent with that by lead times. • Empirical evidence provides critical information for the regimen of screening.

Development of defined microbial population standards using fluorescence activated cell sorting for the absolute quantification of S. aureus using real-time PCR.

PubMed

Martinon, Alice; Cronin, Ultan P; Wilkinson, Martin G

2012-01-01

In this article, four types of standards were assessed in a SYBR Green-based real-time PCR procedure for the quantification of Staphylococcus aureus (S. aureus) in DNA samples. The standards were purified S. aureus genomic DNA (type A), circular plasmid DNA containing a thermonuclease (nuc) gene fragment (type B), DNA extracted from defined populations of S. aureus cells generated by Fluorescence Activated Cell Sorting (FACS) technology with (type C) or without purification of DNA by boiling (type D). The optimal efficiency of 2.016 was obtained on Roche LightCycler(®) 4.1. software for type C standards, whereas the lowest efficiency (1.682) corresponded to type D standards. Type C standards appeared to be more suitable for quantitative real-time PCR because of the use of defined populations for construction of standard curves. Overall, Fieller Confidence Interval algorithm may be improved for replicates having a low standard deviation in Cycle Threshold values such as found for type B and C standards. Stabilities of diluted PCR standards stored at -20°C were compared after 0, 7, 14 and 30 days and were lower for type A or C standards compared with type B standards. However, FACS generated standards may be useful for bacterial quantification in real-time PCR assays once optimal storage and temperature conditions are defined.

Magnetic manipulation device for the optimization of cell processing conditions.

PubMed

Ito, Hiroshi; Kato, Ryuji; Ino, Kosuke; Honda, Hiroyuki

2010-02-01

Variability in human cell phenotypes make it's advancements in optimized cell processing necessary for personalized cell therapy. Here we propose a strategy of palm-top sized device to assist physically manipulating cells for optimizing cell preparations. For the design of such a device, we combined two conventional approaches: multi-well plate formatting and magnetic cell handling using magnetite cationic liposomes (MCLs). From our previous works, we showed the labeling applications of MCL on adhesive cells for various tissue engineering approaches. To feasibly transfer cells in multi-well plate, we here evaluated the magnetic response of MCL-labeled suspension type cells. The cell handling performance of Jurkat cells proved to be faster and more robust compared to MACS (Magnetic Cell Sorting) bead methods. To further confirm our strategy, prototype palm-top sized device "magnetic manipulation device (MMD)" was designed. In the device, the actual cell transportation efficacy of Jurkat cells was satisfying. Moreover, as a model of the most distributed clinical cell processing, primary peripheral blood mononuclear cells (PBMCs) from different volunteers were evaluated. By MMD, individual PBMCs indicated to have optimum Interleukin-2 (IL-2) concentrations for the expansion. Such huge differences of individual cells indicated that MMD, our proposing efficient and self-contained support tool, could assist the feasible and cost-effective optimization of cell processing in clinical facilities. Copyright (c) 2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Fast Filtration of Bacterial or Mammalian Suspension Cell Cultures for Optimal Metabolomics Results

PubMed Central

Bordag, Natalie; Janakiraman, Vijay; Nachtigall, Jonny; González Maldonado, Sandra; Bethan, Bianca; Laine, Jean-Philippe; Fux, Elie

2016-01-01

The metabolome offers real time detection of the adaptive, multi-parametric response of the organisms to environmental changes, pathophysiological stimuli or genetic modifications and thus rationalizes the optimization of cell cultures in bioprocessing. In bioprocessing the measurement of physiological intracellular metabolite levels is imperative for successful applications. However, a sampling method applicable to all cell types with little to no validation effort which simultaneously offers high recovery rates, high metabolite coverage and sufficient removal of extracellular contaminations is still missing. Here, quenching, centrifugation and fast filtration were compared and fast filtration in combination with a stabilizing washing solution was identified as the most promising sampling method. Different influencing factors such as filter type, vacuum pressure, washing solutions were comprehensively tested. The improved fast filtration method (MxP® FastQuench) followed by routine lipid/polar extraction delivers a broad metabolite coverage and recovery reflecting well physiological intracellular metabolite levels for different cell types, such as bacteria (Escherichia coli) as well as mammalian cells chinese hamster ovary (CHO) and mouse myeloma cells (NS0).The proposed MxP® FastQuench allows sampling, i.e. separation of cells from medium with washing and quenching, in less than 30 seconds and is robustly designed to be applicable to all cell types. The washing solution contains the carbon source respectively the 13C-labeled carbon source to avoid nutritional stress during sampling. This method is also compatible with automation which would further reduce sampling times and the variability of metabolite profiling data. PMID:27438065

Paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

PubMed

Yeow, Natasha; McLiesh, Heather; Guan, Liyun; Shen, Wei; Garnier, Gil

2016-07-01

A rapid and simple paper-based elution assay for red blood cell antigen typing by the indirect antiglobulin test (IAT) was established. This allows to type blood using IgG antibodies for the important blood groups in which IgM antibodies do not exist. Red blood cells incubated with IgG anti-D were washed with saline and spotted onto the paper assay pre-treated with anti-IgG. The blood spot was eluted with an elution buffer solution in a chromatography tank. Positive samples were identified by the agglutinated and fixed red blood cells on the original spotting area, while red blood cells from negative samples completely eluted away from the spot of origin. Optimum concentrations for both anti-IgG and anti-D were identified to eliminate the washing step after the incubation phase. Based on the no-washing procedure, the critical variables were investigated to establish the optimal conditions for the paper-based assay. Two hundred ten donor blood samples were tested in optimal conditions for the paper test with anti-D and anti-Kell. Positive and negative samples were clearly distinguished. This assay opens up new applications of the IAT on paper including antibody detection and blood donor-recipient crossmatching and extends its uses into non-blood typing applications with IgG antibody-based diagnostics. Graphical abstract A rapid and simple paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

Porous silicon-copper phthalocyanine heterostructure based photoelectrochemical cell

NASA Astrophysics Data System (ADS)

A. Betty, C.; N, Padma; Arora, Shalav; Survaiya, Parth; Bhattacharya, Debarati; Choudhury, Sipra; Roy, Mainak

2018-01-01

A hybrid solar cell consisting of nanostructured p-type porous silicon (PS) deposited with visible light absorbing dye, Copper Phthalocyanine (CuPc) has been prepared in the photoelectrochemical cell configuration. P-type PS with (100) and (111) orientations which have different porous structures were used for studying the effects of the substrate morphology on the cell efficiency. Heterostructures were prepared by depositing three different thicknesses of CuPc for optimizing the cell efficiency. Structural and surface characterizations were studied using XRD, Raman, SEM and AFM on the PS-CuPc heterostructure. XRD spectrum on both plane silicon and porous silicon indicates the π-π stacking of CuPc with increased disorder for CuPc film on porous silicon. Electrochemical characterizations under sun light type radiation have been carried out to evaluate the photosensitivity of the heterostructure. Between the two different substrates, (100) PS gives better photocurrent, possibly due to the higher surface area and lower series resistance of the structure. Among the (100) PS substrates, (100) PS with 15 nm CuPc film gives Voc more than 1 V resulting in higher efficiency for the cell. The study suggests the scope for optimization of solar cell efficiency using various combinations of the substrate structure and thickness of the sensitizing layer.

Silicon solar cell development and radiation effects study for low temperature and low illumination intensity operation, volume 2

NASA Technical Reports Server (NTRS)

Kirkpatrick, A. R.

1972-01-01

The results are presented of a study to determine the effect of in-situ proton irradiation upon low temperature, low intensity performance of several cell types. The cell types were selected in an attempt to distinguish variations in temperature-dependent radiation resistance which could be attributed to the n-p or p-n structure, diffused or implanted junctions, crucible grown or float-zone type base material, and high or low base resistivity. The results indicate that while expected variations of performance occur at room temperature, all cell types degrade more or less similarly at lower temperatures with normalized degradation becoming increasingly rapid as temperature is reduced. Recommendations for an optimized cell for Jupiter probe use are included along with a definition of the testing required on these cells to insure good performance characteristics.

Optimization of Controllable Factors in the Aluminum Silicon Eutectic Paste and Rear Silicon Nitride Mono-Passivation Layer of PERC Solar Cells

NASA Astrophysics Data System (ADS)

Park, Sungeun; Park, Hyomin; Kim, Dongseop; Yang, JungYup; Lee, Dongho; Kim, Young-Su; Kim, Hyun-Jong; Suh, Dongchul; Min, Byoung Koun; Kim, Kyung Nam; Park, Se Jin; Kim, Donghwan; Lee, Hae-Seok; Nam, Junggyu; Kang, Yoonmook

2018-05-01

Passivated emitter and rear contact (PERC) is a promising technology owing to high efficiency can be achieved with p-type wafer and their easily applicable to existing lines. In case of using p-type mono wafer, 0.5-1% efficiency increase is expected with PERC technologies compared to existing Al BSF solar cells, while for multi-wafer solar cells it is 0.5-0.8%. We addressed the optimization of PERC solar cells using the Al paste. The paste was prepared from the aluminum-silicon alloy with eutectic composition to avoid the formation of voids that degrade the open-circuit voltage. The glass frit of the paste was changed to improve adhesion. Scanning electron microscopy revealed voids and local back surface field between the aluminum electrode and silicon base. We confirmed the conditions on the SiNx passivation layer for achieving higher efficiency and better adhesion for long-term stability. The cell characteristics were compared across cells containing different pastes. PERC solar cells with the Al/Si eutectic paste exhibited the efficiency of 19.6%.

Establishment and culture optimization of a new type of pituitary immortalized cell line.

PubMed

Kokubu, Yuko; Asashima, Makoto; Kurisaki, Akira

2015-08-07

The pituitary gland is a center of the endocrine system that controls homeostasis in an organism by secreting various hormones. The glandular anterior pituitary consists of five different cell types, each expressing specific hormones. However, their regulation and the appropriate conditions for their in vitro culture are not well defined. Here, we report the immortalization of mouse pituitary cells by introducing TERT, E6, and E7 transgenes. The immortalized cell lines mainly expressed a thyrotroph-specific thyroid stimulating hormone beta (Tshb). After optimization of the culture conditions, these immortalized cells proliferated and maintained morphological characteristics similar to those of primary pituitary cells under sphere culture conditions in DMEM/F12 medium supplemented with N2, B27, basic FGF, and EGF. These cell lines responded to PKA or PKC pathway activators and induced the expression of Tshb mRNA. Moreover, transplantation of the immortalized cell line into subcutaneous regions and kidney capsules of mice further increased Tshb expression. These results suggest that immortalization of pituitary cells with TERT, E6, and E7 transgenes is a useful method for generating proliferating cells for the in vitro analysis of pituitary regulatory mechanisms. Copyright © 2015 Elsevier Inc. All rights reserved.

Cell-Free Protein Synthesis Enhancement from Real-Time NMR Metabolite Kinetics: Redirecting Energy Fluxes in Hybrid RRL Systems.

PubMed

Panthu, Baptiste; Ohlmann, Théophile; Perrier, Johan; Schlattner, Uwe; Jalinot, Pierre; Elena-Herrmann, Bénédicte; Rautureau, Gilles J P

2018-01-19

A counterintuitive cell-free protein synthesis (CFPS) strategy, based on reducing the ribosomal fraction in rabbit reticulocyte lysate (RRL), triggers the development of hybrid systems composed of RRL ribosome-free supernatant complemented with ribosomes from different mammalian cell-types. Hybrid RRL systems maintain translational properties of the original ribosome cell types, and deliver protein expression levels similar to RRL. Here, we show that persistent ribosome-associated metabolic activity consuming ATP is a major obstacle for maximal protein yield. We provide a detailed picture of hybrid CFPS systems energetic metabolism based on real-time nuclear magnetic resonance (NMR) investigation of metabolites kinetics. We demonstrate that protein synthesis capacity has an upper limit at native ribosome concentration and that lower amounts of the ribosomal fraction optimize energy fluxes toward protein translation, consequently increasing CFPS yield. These results provide a rationalized strategy for further mammalian CFPS developments and reveal the potential of real-time NMR metabolism phenotyping for optimization of cell-free protein expression systems.

IFNγ Signaling Endows DCs with the Capacity to Control Type I Inflammation during Parasitic Infection through Promoting T-bet+ Regulatory T Cells

PubMed Central

Lee, Hyang-Mi; Fleige, Anne; Forman, Ruth; Cho, Sunglim; Khan, Aly Azeem; Lin, Ling-Li; Nguyen, Duc T.; O'Hara-Hall, Aisling; Yin, Zhinan; Hunter, Christopher A.; Muller, Werner; Lu, Li-Fan

2015-01-01

IFNγ signaling drives dendritic cells (DCs) to promote type I T cell (Th1) immunity. Here, we show that activation of DCs by IFNγ is equally crucial for the differentiation of a population of T-bet+ regulatory T (Treg) cells specialized to inhibit Th1 immune responses. Conditional deletion of IFNγ receptor in DCs but not in Treg cells resulted in a severe defect in this specific Treg cell subset, leading to exacerbated immune pathology during parasitic infections. Mechanistically, IFNγ-unresponsive DCs failed to produce sufficient amount of IL-27, a cytokine required for optimal T-bet induction in Treg cells. Thus, IFNγ signalling endows DCs with the ability to efficiently control a specific type of T cell immunity through promoting a corresponding Treg cell population. PMID:25658840

The biogenesis protein PEX14 is an optimal marker for the identification and localization of peroxisomes in different cell types, tissues, and species in morphological studies.

PubMed

Grant, Phillip; Ahlemeyer, Barbara; Karnati, Srikanth; Berg, Timm; Stelzig, Ingra; Nenicu, Anca; Kuchelmeister, Klaus; Crane, Denis I; Baumgart-Vogt, Eveline

2013-10-01

Catalase and ABCD3 are frequently used as markers for the localization of peroxisomes in morphological experiments. Their abundance, however, is highly dependent on metabolic demands, reducing the validity of analyses of peroxisomal abundance and distribution based solely on these proteins. We therefore attempted to find a protein which can be used as an optimal marker for peroxisomes in a variety of species, tissues, cell types and also experimental designs, independently of peroxisomal metabolism. We found that the biogenesis protein peroxin 14 (PEX14) is present in comparable amounts in the membranes of every peroxisome and is optimally suited for immunoblotting, immunohistochemistry, immunofluorescence, and immunoelectron microscopy. Using antibodies against PEX14, we could visualize peroxisomes with almost undetectable catalase content in various mammalian tissue sections (submandibular and adrenal gland, kidney, testis, ovary, brain, and pancreas from mouse, cat, baboon, and human) and cell cultures (primary cells and cell lines). Peroxisome labeling with catalase often showed a similar tissue distribution to the mitochondrial enzyme mitochondrial superoxide dismutase (both responsible for the degradation of reactive oxygen species), whereas ABCD3 exhibited a distinct labeling only in cells involved in lipid metabolism. We increased the sensitivity of our methods by using QuantumDots™, which have higher emission yields compared to classic fluorochromes and are unsusceptible to photobleaching, thereby allowing more exact quantification without artificial mistakes due to heterogeneity of individual peroxisomes. We conclude that PEX14 is indeed the best marker for labeling of peroxisomes in a variety of tissues and cell types in a consistent fashion for comparative morphometry.

Screening somatic cell nuclear transfer parameters for generation of transgenic cloned cattle with intragenomic integration of additional gene copies that encode bovine adipocyte-type fatty acid-binding protein (A-FABP).

PubMed

Guo, Yong; Li, Hejuan; Wang, Ying; Yan, Xingrong; Sheng, Xihui; Chang, Di; Qi, Xiaolong; Wang, Xiangguo; Liu, Yunhai; Li, Junya; Ni, Hemin

2017-02-01

Somatic cell nuclear transfer (SCNT) is frequently used to produce transgenic cloned livestock, but it is still associated with low success rates. To our knowledge, we are the first to report successful production of transgenic cattle that overexpress bovine adipocyte-type fatty acid binding proteins (A-FABPs) with the aid of SCNT. Intragenomic integration of additional A-FABP gene copies has been found to be positively correlated with the intramuscular fat content in different farm livestock species. First, we optimized the cloning parameters to produce bovine embryos integrated with A-FABP by SCNT, such as applied voltage field strength and pulse duration for electrofusion, morphology and size of donor cells, and number of donor cells passages. Then, bovine fibroblast cells from Qinchuan cattle were transfected with A-FABP and used as donor cells for SCNT. Hybrids of Simmental and Luxi local cattle were selected as the recipient females for A-FABP transgenic SCNT-derived embryos. The results showed that a field strength of 2.5 kV/cm with two 10-μs duration electrical pulses was ideal for electrofusion, and 4-6th generation circular smooth type donor cells with diameters of 15-25 μm were optimal for producing transgenic bovine embryos by SCNT, and resulted in higher fusion (80%), cleavage (73%), and blastocyst (27%) rates. In addition, we obtained two transgenic cloned calves that expressed additional bovine A-FABP gene copies, as detected by PCR-amplified cDNA sequencing. We proposed a set of optimal protocols to produce transgenic SCNT-derived cattle with intragenomic integration of ectopic A-FABP-inherited exon sequences.

Microelectrical Impedance Spectroscopy for the Differentiation between Normal and Cancerous Human Urothelial Cell Lines: Real-Time Electrical Impedance Measurement at an Optimal Frequency

PubMed Central

Park, Yangkyu; Kim, Hyeon Woo; Yun, Joho; Seo, Seungwan; Park, Chang-Ju; Lee, Jeong Zoo; Lee, Jong-Hyun

2016-01-01

Purpose. To distinguish between normal (SV-HUC-1) and cancerous (TCCSUP) human urothelial cell lines using microelectrical impedance spectroscopy (μEIS). Materials and Methods. Two types of μEIS devices were designed and used in combination to measure the impedance of SV-HUC-1 and TCCSUP cells flowing through the channels of the devices. The first device (μEIS-OF) was designed to determine the optimal frequency at which the impedance of two cell lines is most distinguishable. The μEIS-OF trapped the flowing cells and measured their impedance at a frequency ranging from 5 kHz to 1 MHz. The second device (μEIS-RT) was designed for real-time impedance measurement of the cells at the optimal frequency. The impedance was measured instantaneously as the cells passed the sensing electrodes of μEIS-RT. Results. The optimal frequency, which maximized the average difference of the amplitude and phase angle between the two cell lines (p < 0.001), was determined to be 119 kHz. The real-time impedance of the cell lines was measured at 119 kHz; the two cell lines differed significantly in terms of amplitude and phase angle (p < 0.001). Conclusion. The μEIS-RT can discriminate SV-HUC-1 and TCCSUP cells by measuring the impedance at the optimal frequency determined by the μEIS-OF. PMID:26998490

Analytical optimal controls for the state constrained addition and removal of cryoprotective agents

PubMed Central

Chicone, Carmen C.; Critser, John K.

2014-01-01

Cryobiology is a field with enormous scientific, financial and even cultural impact. Successful cryopreservation of cells and tissues depends on the equilibration of these materials with high concentrations of permeating chemicals (CPAs) such as glycerol or 1,2 propylene glycol. Because cells and tissues are exposed to highly anisosmotic conditions, the resulting gradients cause large volume fluctuations that have been shown to damage cells and tissues. On the other hand, there is evidence that toxicity to these high levels of chemicals is time dependent, and therefore it is ideal to minimize exposure time as well. Because solute and solvent flux is governed by a system of ordinary differential equations, CPA addition and removal from cells is an ideal context for the application of optimal control theory. Recently, we presented a mathematical synthesis of the optimal controls for the ODE system commonly used in cryobiology in the absence of state constraints and showed that controls defined by this synthesis were optimal. Here we define the appropriate model, analytically extend the previous theory to one encompassing state constraints, and as an example apply this to the critical and clinically important cell type of human oocytes, where current methodologies are either difficult to implement or have very limited success rates. We show that an enormous increase in equilibration efficiency can be achieved under the new protocols when compared to classic protocols, potentially allowing a greatly increased survival rate for human oocytes, and pointing to a direction for the cryopreservation of many other cell types. PMID:22527943

Simulation of a photo-solar generator for an optimal output by a parabolic photovoltaic concentrator of Stirling engine type

NASA Astrophysics Data System (ADS)

Kaddour, A.; Benyoucef, B.

Solar energy is the source of the most promising energy and the powerful one among renewable energies. Photovoltaic electricity (statement) is obtained by direct transformation of the sunlight into electricity, by means of cells statement. Then, we study the operation of cells statement by the digital simulation with an aim of optimizing the output of the parabolic concentrator of Stirling engine type. The Greenius software makes it possible to carry out the digital simulation in 2D and 3D and to study the influence of the various parameters on the characteristic voltage under illumination of the cell. The results obtained enabled us to determine the extrinsic factors which depend on the environment and the intrinsic factors which result from the properties of materials used.

Primary midgut, salivary gland, and ovary cultures from Boophilus microplus.

PubMed

Mosqueda, Juan; Cossío-Bayugar, Raquel; Rodríguez, Elba; Falcón, Alfonso; Ramos, Alberto; Figueroa, Julio V; Alvarez, Antonio

2008-12-01

Primary cell cultures from different tick organs are a valuable tool for host parasite research in the study of the protozoan Babesia sp., which infects different organs of the tick. In this work we describe the generation of midgut, salivary gland, and ovary primary cell cultures from dissections of Boophilus microplus. Midguts, salivary glands, and ovaries were dissected from B. microplus ticks on different days after bovine infestation; different enzymatic disaggregating protocols were tested in the presence of proteolytic enzymes, such as trypsin and collagenase type I and II, for tissue disaggregation and primary cell culture generation. The dissected tick organs obtained 18-20 days after bovine infestation showed a major cellular differentiation and were easier to identify by cellular morphology. The enzymatic disaggregation results showed that each tissue required a different proteolytic enzyme for optimal disaggregation; collagenase type I produced the most complete disaggregation for ovaries but not for midgut or salivary glands. Collagenase type II was effective for salivary glands but performed poorly on ovaries and midgets, and typsin was effective for midguts only. The midgut and ovary primary cell cultures were maintained for 4 weeks in optimal conditions after the cells were no longer viable. The salivary gland cell cultures were viable for 8 months.

Optimizing cryopreservation of human spermatogonial stem cells: comparing the effectiveness of testicular tissue and single cell suspension cryopreservation

PubMed Central

Yango, Pamela; Altman, Eran; Smith, James F.; Klatsky, Peter C.; Tran, Nam D.

2015-01-01

Objective To determine whether optimal human spermatogonial stem cell (SSC) cryopreservation is best achieved with testicular tissue or single cell suspension cryopreservation. This study compares the effectiveness between these two approaches by using testicular SSEA-4+ cells, a known population containing SSCs. Design In vitro human testicular tissues. Setting Academic research unit. Patients Adult testicular tissues (n = 4) collected from subjects with normal spermatogenesis and normal fetal testicular tissues (n = 3). Intervention(s) Testicular tissue vs. single cell suspension cryopreservation. Main Outcome Measures Cell viability, total cell recovery per milligram of tissue, as well as, viable and SSEA-4+ cell recovery. Results Single cell suspension cryopreservation yielded higher recovery of SSEA-4+ cells enriched in adult SSCs whereas fetal SSEA-4+ cell recovery was similar between testicular tissue and single cell suspension cryopreservation. Conclusions Adult and fetal human SSEA-4+ populations exhibited differential sensitivity to cryopreservation based on whether they were cryopreserved in situ as testicular tissues or as single cells. Thus, optimal preservation of human SSCs depends on the patient age, type of samples cryopreserved, and end points of therapeutic applications. PMID:25241367

Neural Stem Cell Differentiation Using Microfluidic Device-Generated Growth Factor Gradient.

PubMed

Kim, Ji Hyeon; Sim, Jiyeon; Kim, Hyun-Jung

2018-04-11

Neural stem cells (NSCs) have the ability to self-renew and differentiate into multiple nervous system cell types. During embryonic development, the concentrations of soluble biological molecules have a critical role in controlling cell proliferation, migration, differentiation and apoptosis. In an effort to find optimal culture conditions for the generation of desired cell types in vitro , we used a microfluidic chip-generated growth factor gradient system. In the current study, NSCs in the microfluidic device remained healthy during the entire period of cell culture, and proliferated and differentiated in response to the concentration gradient of growth factors (epithermal growth factor and basic fibroblast growth factor). We also showed that overexpression of ASCL1 in NSCs increased neuronal differentiation depending on the concentration gradient of growth factors generated in the microfluidic gradient chip. The microfluidic system allowed us to study concentration-dependent effects of growth factors within a single device, while a traditional system requires multiple independent cultures using fixed growth factor concentrations. Our study suggests that the microfluidic gradient-generating chip is a powerful tool for determining the optimal culture conditions.

Single-nucleus analysis of accessible chromatin in developing mouse forebrain reveals cell-type-specific transcriptional regulation.

PubMed

Preissl, Sebastian; Fang, Rongxin; Huang, Hui; Zhao, Yuan; Raviram, Ramya; Gorkin, David U; Zhang, Yanxiao; Sos, Brandon C; Afzal, Veena; Dickel, Diane E; Kuan, Samantha; Visel, Axel; Pennacchio, Len A; Zhang, Kun; Ren, Bing

2018-03-01

Analysis of chromatin accessibility can reveal transcriptional regulatory sequences, but heterogeneity of primary tissues poses a significant challenge in mapping the precise chromatin landscape in specific cell types. Here we report single-nucleus ATAC-seq, a combinatorial barcoding-assisted single-cell assay for transposase-accessible chromatin that is optimized for use on flash-frozen primary tissue samples. We apply this technique to the mouse forebrain through eight developmental stages. Through analysis of more than 15,000 nuclei, we identify 20 distinct cell populations corresponding to major neuronal and non-neuronal cell types. We further define cell-type-specific transcriptional regulatory sequences, infer potential master transcriptional regulators and delineate developmental changes in forebrain cellular composition. Our results provide insight into the molecular and cellular dynamics that underlie forebrain development in the mouse and establish technical and analytical frameworks that are broadly applicable to other heterogeneous tissues.

Intracellular trehalose via transporter TRET1 as a method to cryoprotect CHO-K1 cells.

PubMed

Uchida, Tsutomu; Furukawa, Maho; Kikawada, Takahiro; Yamazaki, Kenji; Gohara, Kazutoshi

2017-08-01

Trehalose is a promising natural cryoprotectant, but its cryoprotective effect is limited due to difficulties in transmembrane transport. Thus, expressing the trehalose transporter TRET1 on various mammalian cells may yield more trehalose applications. In this study, we ran comparative cryopreservation experiments between the TRET1-expressing CHO-K1 cells (CHO-TRET1) and the CHO-K1 cells transfected with an empty vector (CHO-vector). The experiments involve freezing under various trehalose concentrations in an extracellular medium. The freeze-thawing viabilities of CHO-TRET1 cells are higher than those of CHO-vector cells for most freezing conditions. This result differs from control experiments with a transmembrane type cryoprotectant, dimethyl sulfoxide (Me 2 SO), which had similar viabilities in each condition for both cell types. We conclude that the trehalose loaded into the cells with TRET1 significantly improves the cryoprotective effect. The higher viabilities occurred when the extracellular trehalose concentration exceeded 200 mM, with 250-500 mM being optimal, and a cooling rate below 30 K/min, with 5-20 K/min being optimal. Copyright © 2017 Elsevier Inc. All rights reserved.

Layout optimization of DRAM cells using rigorous simulation model for NTD

NASA Astrophysics Data System (ADS)

Jeon, Jinhyuck; Kim, Shinyoung; Park, Chanha; Yang, Hyunjo; Yim, Donggyu; Kuechler, Bernd; Zimmermann, Rainer; Muelders, Thomas; Klostermann, Ulrich; Schmoeller, Thomas; Do, Mun-hoe; Choi, Jung-Hoe

2014-03-01

DRAM chip space is mainly determined by the size of the memory cell array patterns which consist of periodic memory cell features and edges of the periodic array. Resolution Enhancement Techniques (RET) are used to optimize the periodic pattern process performance. Computational Lithography such as source mask optimization (SMO) to find the optimal off axis illumination and optical proximity correction (OPC) combined with model based SRAF placement are applied to print patterns on target. For 20nm Memory Cell optimization we see challenges that demand additional tool competence for layout optimization. The first challenge is a memory core pattern of brick-wall type with a k1 of 0.28, so it allows only two spectral beams to interfere. We will show how to analytically derive the only valid geometrically limited source. Another consequence of two-beam interference limitation is a "super stable" core pattern, with the advantage of high depth of focus (DoF) but also low sensitivity to proximity corrections or changes of contact aspect ratio. This makes an array edge correction very difficult. The edge can be the most critical pattern since it forms the transition from the very stable regime of periodic patterns to non-periodic periphery, so it combines the most critical pitch and highest susceptibility to defocus. Above challenge makes the layout correction to a complex optimization task demanding a layout optimization that finds a solution with optimal process stability taking into account DoF, exposure dose latitude (EL), mask error enhancement factor (MEEF) and mask manufacturability constraints. This can only be achieved by simultaneously considering all criteria while placing and sizing SRAFs and main mask features. The second challenge is the use of a negative tone development (NTD) type resist, which has a strong resist effect and is difficult to characterize experimentally due to negative resist profile taper angles that perturb CD at bottom characterization by scanning electron microscope (SEM) measurements. High resist impact and difficult model data acquisition demand for a simulation model that hat is capable of extrapolating reliably beyond its calibration dataset. We use rigorous simulation models to provide that predictive performance. We have discussed the need of a rigorous mask optimization process for DRAM contact cell layout yielding mask layouts that are optimal in process performance, mask manufacturability and accuracy. In this paper, we have shown the step by step process from analytical illumination source derivation, a NTD and application tailored model calibration to layout optimization such as OPC and SRAF placement. Finally the work has been verified with simulation and experimental results on wafer.

Directed differentiation of embryonic stem cells using a bead-based combinatorial screening method.

PubMed

Tarunina, Marina; Hernandez, Diana; Johnson, Christopher J; Rybtsov, Stanislav; Ramathas, Vidya; Jeyakumar, Mylvaganam; Watson, Thomas; Hook, Lilian; Medvinsky, Alexander; Mason, Chris; Choo, Yen

2014-01-01

We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported.

Multi-mission Ni-H2 battery cell for the 1990's

NASA Technical Reports Server (NTRS)

Miller, Lee; Brill, Jack; Dodson, Gary

1989-01-01

A sufficient production, test and operational database is now available to permit design technology optimization for the next decade. The evolved battery cell design features standardized technology intended to support multiple type missions (e.g., both GEO and LEO). Design analyses and validation test cells demonstrate improved performance plus attractive specific-energy characteristics will be achieved.

Tissue engineering of ligaments: a comparison of bone marrow stromal cells, anterior cruciate ligament, and skin fibroblasts as cell source.

PubMed

Van Eijk, F; Saris, D B F; Riesle, J; Willems, W J; Van Blitterswijk, C A; Verbout, A J; Dhert, W J A

2004-01-01

Anterior cruciate ligament (ACL) reconstruction surgery still has important problems to overcome, such as "donor site morbidity" and the limited choice of grafts in revision surgery. Tissue engineering of ligaments may provide a solution for these problems. Little is known about the optimal cell source for tissue engineering of ligaments. The aim of this study is to determine the optimal cell source for tissue engineering of the anterior cruciate ligament. Bone marrow stromal cells (BMSCs), ACL, and skin fibroblasts were seeded onto a resorbable suture material [poly(L-lactide/glycolide) multifilaments] at five different seeding densities, and cultured for up to 12 days. All cell types tested attached to the suture material, proliferated, and synthesized extracellular matrix rich in collagen type I. On day 12 the scaffolds seeded with BMSCs showed the highest DNA content (p < 0.01) and the highest collagen production (p < 0.05 for the two highest seeding densities). Scaffolds seeded with ACL fibroblasts showed the lowest DNA content and collagen production. Accordingly, BMSCs appear to be the most suitable cells for further study and development of tissue-engineered ligament.

[Effect of cryopreservation on umbilical blood cells and its mechanism].

PubMed

Li, Xin; Chen, Fangping; Jiang, Tiebin; Wang, Erhua; Liu, Jing

2013-07-01

To evaluate the effect of cryopreservation on clonogenic ability and apoptosis rate of mono-nuclear cells and CD34+ cells in umbilical blood (UB), and to choose the index to present the freezing injury and optimize the cryopreservation of UB. The mono-nuclear cells (MNC) and CD34+ cells were separated from UB and frozen.After 30 days, they were thawed in warm water. Clonogenic capacity and clonogenic recovery before and after the cryopreservation was compared. We also used Annexin V-FITC-PI to investigate the apoptosis rate of the cells before and after the cryopreservation of these 2 types of cells. The number of colony forming unit-granulocyte/monocyte (CFU-GMs) was not changed after freezing and thawing in both MNCs and CD34+ cells, while the number of colony forming unit-granulocyte, erythrocyte, monocyte and megakaryocyte (CFU-GEMM) was obviously reduced after freezing in CD34+ cells. The 2 types of cryopreserved cells had certain degree of apoptosis before the cryopreservation. MNC-type cryopreservation increased the cells apoptosis a little, while CD34+-type cryopreservation increased more. The cells have certain degree of apoptosis before the cryopreservation. The freezing and thawing procedure does affect the early stage progenitor cells-CFU-GEMM in the CD34+- type cryopreserved cells in UB. The damage may be induced by the cell apoptosis.

Non Linear Programming (NLP) Formulation for Quantitative Modeling of Protein Signal Transduction Pathways

PubMed Central

Morris, Melody K.; Saez-Rodriguez, Julio; Lauffenburger, Douglas A.; Alexopoulos, Leonidas G.

2012-01-01

Modeling of signal transduction pathways plays a major role in understanding cells' function and predicting cellular response. Mathematical formalisms based on a logic formalism are relatively simple but can describe how signals propagate from one protein to the next and have led to the construction of models that simulate the cells response to environmental or other perturbations. Constrained fuzzy logic was recently introduced to train models to cell specific data to result in quantitative pathway models of the specific cellular behavior. There are two major issues in this pathway optimization: i) excessive CPU time requirements and ii) loosely constrained optimization problem due to lack of data with respect to large signaling pathways. Herein, we address both issues: the former by reformulating the pathway optimization as a regular nonlinear optimization problem; and the latter by enhanced algorithms to pre/post-process the signaling network to remove parts that cannot be identified given the experimental conditions. As a case study, we tackle the construction of cell type specific pathways in normal and transformed hepatocytes using medium and large-scale functional phosphoproteomic datasets. The proposed Non Linear Programming (NLP) formulation allows for fast optimization of signaling topologies by combining the versatile nature of logic modeling with state of the art optimization algorithms. PMID:23226239

Non Linear Programming (NLP) formulation for quantitative modeling of protein signal transduction pathways.

PubMed

Mitsos, Alexander; Melas, Ioannis N; Morris, Melody K; Saez-Rodriguez, Julio; Lauffenburger, Douglas A; Alexopoulos, Leonidas G

2012-01-01

Modeling of signal transduction pathways plays a major role in understanding cells' function and predicting cellular response. Mathematical formalisms based on a logic formalism are relatively simple but can describe how signals propagate from one protein to the next and have led to the construction of models that simulate the cells response to environmental or other perturbations. Constrained fuzzy logic was recently introduced to train models to cell specific data to result in quantitative pathway models of the specific cellular behavior. There are two major issues in this pathway optimization: i) excessive CPU time requirements and ii) loosely constrained optimization problem due to lack of data with respect to large signaling pathways. Herein, we address both issues: the former by reformulating the pathway optimization as a regular nonlinear optimization problem; and the latter by enhanced algorithms to pre/post-process the signaling network to remove parts that cannot be identified given the experimental conditions. As a case study, we tackle the construction of cell type specific pathways in normal and transformed hepatocytes using medium and large-scale functional phosphoproteomic datasets. The proposed Non Linear Programming (NLP) formulation allows for fast optimization of signaling topologies by combining the versatile nature of logic modeling with state of the art optimization algorithms.

Protein expression of preferred human codon-optimized Gaussia luciferase genes with an artificial open-reading frame in mammalian and bacterial cells.

PubMed

Inouye, Satoshi; Suzuki, Takahiro

2016-12-01

The protein expressions of three preferred human codon-optimized Gaussia luciferase genes (pGLuc, EpGLuc, and KpGLuc) were characterized in mammalian and bacterial cells by comparing them with those of wild-type Gaussia luciferase gene (wGLuc) and human codon-optimized Gaussia luciferase gene (hGLuc). Two synthetic genes of EpGLuc and KpGLuc containing the complete preferred human codons have an artificial open-reading frame; however, they had the similar protein expression levels to those of pGLuc and hGLuc in mammalian cells. In bacterial cells, the protein expressions of pGLuc, EpGLuc, and KpGLuc with approximately 65% GC content were the same and showed approximately 60% activities of wGLuc and hGLuc. The artificial open-reading frame in EpGLuc and KpGLuc did not affect the protein expression in mammalian and bacterial cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Modeling and predictions of biphasic mechanosensitive cell migration altered by cell-intrinsic properties and matrix confinement.

PubMed

Pathak, Amit

2018-04-12

Motile cells sense the stiffness of their extracellular matrix (ECM) through adhesions and respond by modulating the generated forces, which in turn lead to varying mechanosensitive migration phenotypes. Through modeling and experiments, cell migration speed is known to vary with matrix stiffness in a biphasic manner, with optimal motility at an intermediate stiffness. Here, we present a two-dimensional cell model defined by nodes and elements, integrated with subcellular modeling components corresponding to mechanotransductive adhesion formation, force generation, protrusions and node displacement. On 2D matrices, our calculations reproduce the classic biphasic dependence of migration speed on matrix stiffness and predict that cell types with higher force-generating ability do not slow down on very stiff matrices, thus disabling the biphasic response. We also predict that cell types defined by lower number of total receptors require stiffer matrices for optimal motility, which also limits the biphasic response. For a cell type with robust biphasic migration on 2D surface, simulations in channel-like confined environments of varying width and height predict faster migration in more confined matrices. Simulations performed in shallower channels predict that the biphasic mechanosensitive cell migration response is more robust on 2D micro-patterns as compared to the channel-like 3D confinement. Thus, variations in the dimensionality of matrix confinement alters the way migratory cells sense and respond to the matrix stiffness. Our calculations reveal new phenotypes of stiffness- and topography-sensitive cell migration that critically depend on both cell-intrinsic and matrix properties. These predictions may inform our understanding of various mechanosensitive modes of cell motility that could enable tumor invasion through topographically heterogeneous microenvironments. © 2018 IOP Publishing Ltd.

Enrichment of cardiac differentiation of mouse embryonic stem cells by optimizing the hanging drop method.

PubMed

Chen, Ming; Lin, Yong-Qing; Xie, Shuang-Lun; Wu, Hong-Fu; Wang, Jing-Feng

2011-04-01

Hanging drop (HD) culture is used to induce differentiation of embryonic stem cells (ESCs) into other cell types including cardiomyocytes. However, the factors affecting cardiac differentiation of ESCs with this method remain incompletely understood. We have investigated the effects of the starting number of ESCs in embryoid bodies (EBs) and the time of EB adherence to gelatin-coated plates on cardiac differentiation: cardiac differentiation was increased in the EBs by a larger number of ESCs and was decreased by plating EBs at day 4 or earlier. These two factors can thus be optimized to enrich the cardiac differentiation in ESCs using the HD method.

The simulation of CZTS solar cell for performance improvement

NASA Astrophysics Data System (ADS)

Kumar, Atul; Thakur, Ajay D.

2018-05-01

A Copper-Zinc-Tin-Sulphide (CZTS) based solar cell of Mo/CZTS/CdS/ZnO is simulated using SCAPS. Quantum efficiency and IV curve of the simulated output of CZTS solar cell is mapped with highest efficiency reported in literature for CZTS solar cell. A modification in back contact thus shottky barrier, spike type band alignment at the CZTS-n type layer junction and higher electron mobility (owing to alkali doping in CZT)S are implement in simulation of CZTS solar cell. An improvement in the solar cell efficiency compared to the standard cell configuration of Mo/CZTS/CdS/ZnO is found. CZTS is plagued with low Voc and low FF which can be increased by optimization as suggested in paper.

Human MAIT-cell responses to Escherichia coli: activation, cytokine production, proliferation, and cytotoxicity.

PubMed

Dias, Joana; Sobkowiak, Michał J; Sandberg, Johan K; Leeansyah, Edwin

2016-07-01

Mucosa-associated invariant T cells are a large and relatively recently described innate-like antimicrobial T-cell subset in humans. These cells recognize riboflavin metabolites from a range of microbes presented by evolutionarily conserved major histocompatibility complex, class I-related molecules. Given the innate-like characteristics of mucosa-associated invariant T cells and the novel type of antigens they recognize, new methodology must be developed and existing methods refined to allow comprehensive studies of their role in human immune defense against microbial infection. In this study, we established protocols to examine a range of mucosa-associated invariant T-cell functions as they respond to antigen produced by Escherichia coli These improved and dose- and time-optimized experimental protocols allow detailed studies of MR1-dependent mucosa-associated invariant T-cell responses to Escherichia coli pulsed antigen-presenting cells, as assessed by expression of activation markers and cytokines, by proliferation, and by induction of apoptosis and death in major histocompatibility complex, class I-related-expressing target cells. The novel and optimized protocols establish a framework of methods and open new possibilities to study mucosa-associated invariant T-cell immunobiology, using Escherichia coli as a model antigen. Furthermore, we propose that these robust experimental systems can also be adapted to study mucosa-associated invariant T-cell responses to other microbes and types of antigen-presenting cells. © The Author(s).

Multi-mission Ni-H2 battery cells for the 1990's

NASA Technical Reports Server (NTRS)

Miller, Lee; Brill, Jack; Dodson, Gary

1989-01-01

A sufficient production, test and operational database is now available to permit design technology optimization for the next decade. The evolved battery cell design features standardized technology intended to support multiple type missions (e.g., both GEO and LEO). Design analysis and validation test cells demonstrate that improved performance plus attractive specific-energy characteristics will be achieved.

Formulation Changes Affect Material Properties and Cell Behavior in HA-Based Hydrogels.

PubMed

Lawyer, Thomas; McIntosh, Kristen; Clavijo, Cristian; Potekhina, Lydia; Mann, Brenda K

2012-01-01

To develop and optimize new scaffold materials for tissue engineering applications, it is important to understand how changes to the scaffold affect the cells that will interact with that scaffold. In this study, we used a hyaluronic acid- (HA-) based hydrogel as a synthetic extracellular matrix, containing modified HA (CMHA-S), modified gelatin (Gtn-S), and a crosslinker (PEGda). By varying the concentrations of these components, we were able to change the gelation time, enzymatic degradation, and compressive modulus of the hydrogel. These changes also affected fibroblast spreading within the hydrogels and differentially affected the proliferation and metabolic activity of fibroblasts and mesenchymal stem cells (MSCs). In particular, PEGda concentration had the greatest influence on gelation time, compressive modulus, and cell spreading. MSCs appeared to require a longer period of adjustment to the new microenvironment of the hydrogels than fibroblasts. Fibroblasts were able to proliferate in all formulations over the course of two weeks, but MSCs did not. Metabolic activity changed for each cell type during the two weeks depending on the formulation. These results highlight the importance of determining the effect of matrix composition changes on a particular cell type of interest in order to optimize the formulation for a given application.

Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells.

PubMed

Sidney, Laura E; Branch, Matthew J; Dua, Harminder S; Hopkinson, Andrew

2015-12-01

The limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. Primary human corneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. All media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Directed Differentiation of Embryonic Stem Cells Using a Bead-Based Combinatorial Screening Method

PubMed Central

Tarunina, Marina; Hernandez, Diana; Johnson, Christopher J.; Rybtsov, Stanislav; Ramathas, Vidya; Jeyakumar, Mylvaganam; Watson, Thomas; Hook, Lilian; Medvinsky, Alexander; Mason, Chris; Choo, Yen

2014-01-01

We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported. PMID:25251366

Stem cell gene therapy for fanconi anemia: report from the 1st international Fanconi anemia gene therapy working group meeting.

PubMed

Tolar, Jakub; Adair, Jennifer E; Antoniou, Michael; Bartholomae, Cynthia C; Becker, Pamela S; Blazar, Bruce R; Bueren, Juan; Carroll, Thomas; Cavazzana-Calvo, Marina; Clapp, D Wade; Dalgleish, Robert; Galy, Anne; Gaspar, H Bobby; Hanenberg, Helmut; Von Kalle, Christof; Kiem, Hans-Peter; Lindeman, Dirk; Naldini, Luigi; Navarro, Susana; Renella, Raffaele; Rio, Paula; Sevilla, Julián; Schmidt, Manfred; Verhoeyen, Els; Wagner, John E; Williams, David A; Thrasher, Adrian J

2011-07-01

Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA.

Chir99021 and Valproic acid reduce the proliferative advantage of Apc mutant cells.

PubMed

Langlands, Alistair J; Carroll, Thomas D; Chen, Yu; Näthke, Inke

2018-02-15

More than 90% of colorectal cancers carry mutations in Apc that drive tumourigenesis. A 'just-right' signalling model proposes that Apc mutations stimulate optimal, but not excessive Wnt signalling, resulting in a growth advantage of Apc mutant over wild-type cells. Reversal of this growth advantage constitutes a potential therapeutic approach. We utilised intestinal organoids to compare the growth of Apc mutant and wild-type cells. Organoids derived from Apc Min/+ mice recapitulate stages of intestinal polyposis in culture. They eventually form spherical cysts that reflect the competitive growth advantage of cells that have undergone loss of heterozygosity (LOH). We discovered that this emergence of cysts was inhibited by Chiron99021 and Valproic acid, which potentiates Wnt signalling. Chiron99021 and Valproic acid restrict the growth advantage of Apc mutant cells while stimulating that of wild-type cells, suggesting that excessive Wnt signalling reduces the relative fitness of Apc mutant cells. As a proof of concept, we demonstrated that Chiron99021-treated Apc mutant organoids were rendered susceptible to TSA-induced apoptosis, while wild-type cells were protected.

Human leucocyte antigen class I-redirected anti-tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells.

PubMed

Tan, M P; Dolton, G M; Gerry, A B; Brewer, J E; Bennett, A D; Pumphrey, N J; Jakobsen, B K; Sewell, A K

2017-01-01

CD4 + T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4 + T cells occur in low frequency, express relatively low-affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4 + T cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the co-receptor CD8 glycoprotein in CD4 + cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4 + and CD8 + T cells expressing wild-type and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4 + T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4 + T cells than CD8 + T cells. These results indicate that the CD4 + T cell component of current adoptive therapies using TCRs optimized for CD8 + T cells is below par and that there is room for substantial improvement. © 2016 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.

Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment.

PubMed

Bender, Ruben R; Muth, Anke; Schneider, Irene C; Friedel, Thorsten; Hartmann, Jessica; Plückthun, Andreas; Maisner, Andrea; Buchholz, Christian J

2016-06-01

Receptor-targeted lentiviral vectors (LVs) can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV) glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance). Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV) glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4) was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.

Steam Methane Reformation Testing for Air-Independent Solid Oxide Fuel Cell Systems

NASA Technical Reports Server (NTRS)

Mwara, Kamwana N.

2015-01-01

Recently, NASA has been looking into utilizing landers that can be propelled by LOX-CH (sub 4), to be used for long duration missions. Using landers that utilize such propellants, also provides the opportunity to use solid oxide fuel cells as a power option, especially since they are able to process methane into a reactant through fuel reformation. One type of reformation, called steam methane reformation, is a process to reform methane into a hydrogen-rich product by reacting methane and steam (fuel cell exhaust) over a catalyst. A steam methane reformation system could potentially use the fuel cell's own exhaust to create a reactant stream that is hydrogen-rich, and requires less internal reforming of the incoming methane. Also, steam reformation may hold some advantages over other types of reforming, such as partial oxidation (PROX) reformation. Steam reformation does not require oxygen, while up to 25 percent can be lost in PROX reformation due to unusable CO (sub 2) reformation. NASA's Johnson Space Center has conducted various phases of steam methane reformation testing, as a viable solution for in-space reformation. This has included using two different types of catalysts, developing a custom reformer, and optimizing the test system to find the optimal performance parameters and operating conditions.

A classification scheme for risk assessment methods.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stamp, Jason Edwin; Campbell, Philip LaRoche

2004-08-01

This report presents a classification scheme for risk assessment methods. This scheme, like all classification schemes, provides meaning by imposing a structure that identifies relationships. Our scheme is based on two orthogonal aspects--level of detail, and approach. The resulting structure is shown in Table 1 and is explained in the body of the report. Each cell in the Table represent a different arrangement of strengths and weaknesses. Those arrangements shift gradually as one moves through the table, each cell optimal for a particular situation. The intention of this report is to enable informed use of the methods so that amore » method chosen is optimal for a situation given. This report imposes structure on the set of risk assessment methods in order to reveal their relationships and thus optimize their usage.We present a two-dimensional structure in the form of a matrix, using three abstraction levels for the rows and three approaches for the columns. For each of the nine cells in the matrix we identify the method type by name and example. The matrix helps the user understand: (1) what to expect from a given method, (2) how it relates to other methods, and (3) how best to use it. Each cell in the matrix represent a different arrangement of strengths and weaknesses. Those arrangements shift gradually as one moves through the table, each cell optimal for a particular situation. The intention of this report is to enable informed use of the methods so that a method chosen is optimal for a situation given. The matrix, with type names in the cells, is introduced in Table 2 on page 13 below. Unless otherwise stated we use the word 'method' in this report to refer to a 'risk assessment method', though often times we use the full phrase. The use of the terms 'risk assessment' and 'risk management' are close enough that we do not attempt to distinguish them in this report. The remainder of this report is organized as follows. In Section 2 we provide context for this report--what a 'method' is and where it fits. In Section 3 we present background for our classification scheme--what other schemes we have found, the fundamental nature of methods and their necessary incompleteness. In Section 4 we present our classification scheme in the form of a matrix, then we present an analogy that should provide an understanding of the scheme, concluding with an explanation of the two dimensions and the nine types in our scheme. In Section 5 we present examples of each of our classification types. In Section 6 we present conclusions.« less

Establishment and culture optimization of a new type of pituitary immortalized cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kokubu, Yuko; Asashima, Makoto; Life Science Center of TARA, The University of Tsukuba, Ibaraki-ken 305-8577

The pituitary gland is a center of the endocrine system that controls homeostasis in an organism by secreting various hormones. The glandular anterior pituitary consists of five different cell types, each expressing specific hormones. However, their regulation and the appropriate conditions for their in vitro culture are not well defined. Here, we report the immortalization of mouse pituitary cells by introducing TERT, E6, and E7 transgenes. The immortalized cell lines mainly expressed a thyrotroph-specific thyroid stimulating hormone beta (Tshb). After optimization of the culture conditions, these immortalized cells proliferated and maintained morphological characteristics similar to those of primary pituitary cells undermore » sphere culture conditions in DMEM/F12 medium supplemented with N2, B27, basic FGF, and EGF. These cell lines responded to PKA or PKC pathway activators and induced the expression of Tshb mRNA. Moreover, transplantation of the immortalized cell line into subcutaneous regions and kidney capsules of mice further increased Tshb expression. These results suggest that immortalization of pituitary cells with TERT, E6, and E7 transgenes is a useful method for generating proliferating cells for the in vitro analysis of pituitary regulatory mechanisms. - Highlights: • Mouse pituitary cell lines were immortalized by introducing TERT, E6, and E7. • The immortalized cell lines mainly expressed thyroid stimulating hormone beta. • The cell lines responded to PKA or PKC pathway activators, and induced Tshb.« less

Stem cell therapy. Use of differentiated pluripotent stem cells as replacement therapy for treating disease.

PubMed

Fox, Ira J; Daley, George Q; Goldman, Steven A; Huard, Johnny; Kamp, Timothy J; Trucco, Massimo

2014-08-22

Pluripotent stem cells (PSCs) directed to various cell fates holds promise as source material for treating numerous disorders. The availability of precisely differentiated PSC-derived cells will dramatically affect blood component and hematopoietic stem cell therapies and should facilitate treatment of diabetes, some forms of liver disease and neurologic disorders, retinal diseases, and possibly heart disease. Although an unlimited supply of specific cell types is needed, other barriers must be overcome. This review of the state of cell therapies highlights important challenges. Successful cell transplantation will require optimizing the best cell type and site for engraftment, overcoming limitations to cell migration and tissue integration, and occasionally needing to control immunologic reactivity, as well as a number of other challenges. Collaboration among scientists, clinicians, and industry is critical for generating new stem cell-based therapies. Copyright © 2014, American Association for the Advancement of Science.

Sample types applied for molecular diagnosis of therapeutic management of advanced non-small cell lung cancer in the precision medicine.

PubMed

Han, Yanxi; Li, Jinming

2017-10-26

In this era of precision medicine, molecular biology is becoming increasingly significant for the diagnosis and therapeutic management of non-small cell lung cancer. The specimen as the primary element of the whole testing flow is particularly important for maintaining the accuracy of gene alteration testing. Presently, the main sample types applied in routine diagnosis are tissue and cytology biopsies. Liquid biopsies are considered as the most promising alternatives when tissue and cytology samples are not available. Each sample type possesses its own strengths and weaknesses, pertaining to the disparity of sampling, preparation and preservation procedures, the heterogeneity of inter- or intratumors, the tumor cellularity (percentage and number of tumor cells) of specimens, etc., and none of them can individually be a "one size to fit all". Therefore, in this review, we summarized the strengths and weaknesses of different sample types that are widely used in clinical practice, offered solutions to reduce the negative impact of the samples and proposed an optimized strategy for choice of samples during the entire diagnostic course. We hope to provide valuable information to laboratories for choosing optimal clinical specimens to achieve comprehensive functional genomic landscapes and formulate individually tailored treatment plans for NSCLC patients that are in advanced stages.

Characterization of an in vitro system for the synthesis of mRNA from human parainfluenza virus type 3.

PubMed

De, B P; Galinski, M S; Banerjee, A K

1990-03-01

A cell extract derived from human parainfluenza virus type 3-infected human lung carcinoma (HLC) cells synthesized mRNA in vitro. Under optimal conditions, the extract was able to support transcription of all virus-encoded genes as determined by hybridization analyses. The RNA products contained full-length poly(A)-containing mRNA species similar to those observed in acutely infected cells. Further purification of the viral nucleocapsids from the infected HLC cell extract resulted in total loss of the capacity of the extract to synthesize mRNA in vitro. However, the addition of cytoplasmic extracts from uninfected HLC cells to the nucleocapsid preparations restored transcription to levels observed in the infected cell lysates, indicating requirement of a host factor(s) in the human parainfluenza virus type 3 transcription process. In distinction to the abundant transcription observed in the cell extract from HLC cells, cell extract prepared from CV-1 cells failed to support transcription in vitro. High levels of RNase activity in the cell extract from CV-1 cells appears to be the principal reason for this difference.

Multivalent glycopeptide dendrimers for the targeted delivery of antigens to dendritic cells.

PubMed

García-Vallejo, Juan J; Ambrosini, Martino; Overbeek, Annemieke; van Riel, Wilhelmina E; Bloem, Karien; Unger, Wendy W J; Chiodo, Fabrizio; Bolscher, Jan G; Nazmi, Kamran; Kalay, Hakan; van Kooyk, Yvette

2013-04-01

Dendritic cells are the most powerful type of antigen presenting cells. Current immunotherapies targeting dendritic cells have shown a relative degree of success but still require further improvement. One of the most important issues to solve is the efficiency of antigen delivery to dendritic cells in order to achieve an appropriate uptake, processing, and presentation to Ag-specific T cells. C-type lectins have shown to be ideal receptors for the targeting of antigens to dendritic cells and allow the use of their natural ligands - glycans - instead of antibodies. Amongst them, dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) is an interesting candidate due to its biological properties and the availability of its natural carbohydrate ligands. Using Le(b)-conjugated poly(amido amine) (PAMAM) dendrimers we aimed to characterize the optimal level of multivalency necessary to achieve the desired internalization, lysosomal delivery, Ag-specific T cell proliferation, and cytokine response. Increasing DC-SIGN ligand multivalency directly translated in an enhanced binding, which might also be interesting for blocking purposes. Internalization, routing to lysosomal compartments, antigen presentation and cytokine response could be optimally achieved with glycopeptide dendrimers carrying 16-32 glycan units. This report provides the basis for the design of efficient targeting of peptide antigens for the immunotherapy of cancer, autoimmunity and infectious diseases. Copyright © 2012 Elsevier Ltd. All rights reserved.

Targeting the kinesin Eg5 to monitor siRNA transfection in mammalian cells.

PubMed

Weil, D; Garçon, L; Harper, M; Duménil, D; Dautry, F; Kress, M

2002-12-01

RNA interference, the inhibition of gene expression by double-stranded RNA, provides a powerful tool for functional studies once the sequence of a gene is known. In most mammalian cells, only short molecules can be used because long ones induce the interferon pathway. With the identification of a proper target sequence, the penetration of the oligonucleotides constitutes the most serious limitation in the application of this technique. Here we show that a small interfering RNA (siRNA) targeting the mRNA of the kinesin Eg5 induces a rapid mitotic arrest and provides a convenient assay for the optimization of siRNA transfection. Thus, dose responses can be established for different transfection techniques, highlighting the great differences in response to transfection techniques of various cell types. We report that the calcium phosphate precipitation technique can be an efficient and cost-effective alternative to Oligofectamine in some adherent cells, while electroporation can be efficient for some cells growing in suspension such as hematopoietic cells and some adherent cells. Significantly, the optimal parameters for the electroporation of siRNA differ from those for plasmids, allowing the use of milder conditions that induce less cell toxicity. In summary, a single siRNA leading to an easily assayed phenotype can be used to monitor the transfection of siRNA into any type of proliferating cells of both human and murine origin.

Optimization and comparative analysis of plant organellar DNA enrichment methods suitable for next generation sequencing

USDA-ARS?s Scientific Manuscript database

Plant organellar genomes contain large repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy) and abundance of sub-types may change through de...

Progress and Challenges in Macroencapsulation Approaches for Type 1 Diabetes (T1D) Treatment: Cells, Biomaterials, and Devices

PubMed Central

Song, Shang; Roy, Shuvo

2018-01-01

Macroencapsulation technology has been an attractive topic in the field of treatment for Type 1 diabetes due to mechanical stability, versatility, and retrievability of the macrocapsule design. Macro-capsules can be categorized into extravascular and intravascular devices, in which solute transport relies either on diffusion or convection, respectively. Failure of macroencapsulation strategies can be due to limited regenerative capacity of the encased insulin-producing cells, sub-optimal performance of encapsulation biomaterials, insufficient immunoisolation, excessive blood thrombosis for vascular perfusion devices, and inadequate modes of mass transfer to support cell viability and function. However, significant technical advancements have been achieved in macroencapsulation technology, namely reducing diffusion distance for oxygen and nutrients, using pro-angiogenic factors to increase vascularization for islet engraftment, and optimizing membrane permeability and selectivity to prevent immune attacks from host’s body. This review presents an overview of existing macroencapsulation devices and discusses the advances based on tissue-engineering approaches that will stimulate future research and development of macroencapsulation technology. PMID:26615050

Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs.

PubMed

Bertero, Alessandro; Pawlowski, Matthias; Ortmann, Daniel; Snijders, Kirsten; Yiangou, Loukia; Cardoso de Brito, Miguel; Brown, Stephanie; Bernard, William G; Cooper, James D; Giacomelli, Elisa; Gambardella, Laure; Hannan, Nicholas R F; Iyer, Dharini; Sampaziotis, Fotios; Serrano, Felipe; Zonneveld, Mariëlle C F; Sinha, Sanjay; Kotter, Mark; Vallier, Ludovic

2016-12-01

Inducible loss of gene function experiments are necessary to uncover mechanisms underlying development, physiology and disease. However, current methods are complex, lack robustness and do not work in multiple cell types. Here we address these limitations by developing single-step optimized inducible gene knockdown or knockout (sOPTiKD or sOPTiKO) platforms. These are based on genetic engineering of human genomic safe harbors combined with an improved tetracycline-inducible system and CRISPR/Cas9 technology. We exemplify the efficacy of these methods in human pluripotent stem cells (hPSCs), and show that generation of sOPTiKD/KO hPSCs is simple, rapid and allows tightly controlled individual or multiplexed gene knockdown or knockout in hPSCs and in a wide variety of differentiated cells. Finally, we illustrate the general applicability of this approach by investigating the function of transcription factors (OCT4 and T), cell cycle regulators (cyclin D family members) and epigenetic modifiers (DPY30). Overall, sOPTiKD and sOPTiKO provide a unique opportunity for functional analyses in multiple cell types relevant for the study of human development. © 2016. Published by The Company of Biologists Ltd.

Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis.

PubMed

Cherubin, Patrick; Quiñones, Beatriz; Teter, Ken

2018-02-06

Ricin, Shiga toxin, exotoxin A, and diphtheria toxin are AB-type protein toxins that act within the host cytosol and kill the host cell through pathways involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. Intoxication is therefore viewed as an irreversible process. Using flow cytometry and a fluorescent reporter system to monitor protein synthesis, we show a single molecule of cytosolic toxin is not sufficient for complete inhibition of protein synthesis or cell death. Furthermore, cells can recover from intoxication: cells with a partial loss of protein synthesis will, upon removal of the toxin, increase the level of protein production and survive the toxin challenge. Thus, in contrast to the prevailing model, ongoing toxin delivery to the cytosol appears to be required for the death of cells exposed to sub-optimal toxin concentrations.

Optimization of FNAC findings as a preoperative diagnostic aid for odontogenic cysts.

PubMed

Jain, Garima; Shetty, Pushparaja

2015-01-01

Fine-needle aspiration cytology (FNAC) is not a definitive preoperative diagnostic procedure done for all cases of odontogenic cysts. This is because of the inconsistent results obtained with it. This study was done to optimize FNAC findings and help in preoperative characterization of odontogenic cysts. Cystic fluid was collected and centrifuged from 50 odontogenic cysts that were planned for excision. Three smears were prepared from the cell sediment obtained after centrifugation and stained. The stained sections were examined for presence and type of epithelial cells, to formulate a preopererative diagnosis. Epithelial cells were detected in 46% cases in smear 1, 48% cases in smear 2, and 52% cases in smear 3. When all three smears from one case were studied, 86% cases showed epithelial cells for evaluation. Cystic aspirate should be centrifuged and the entire cell sediment should be examined by making multiple smears for evaluation of cystic epithelial lining cells.

A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype.

PubMed

Ellis, Brian L; Hirsch, Matthew L; Barker, Jenny C; Connelly, Jon P; Steininger, Robert J; Porteus, Matthew H

2013-03-06

The ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Viruses can be a useful tool for this delivery, particularly in difficult to transfect cell types. Adeno-associated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity. There are now a number of natural for that designed AAV serotypes that each has a differential ability to infect a variety of cell types. Although transduction studies have been completed, the bulk of the studies have been done in vivo, and there has never been a comprehensive study of transduction ex vivo/in vitro. Each cell type was infected with each serotype at a multiplicity of infection of 100,000 viral genomes/cell and transduction was analyzed by flow cytometry + . We found that AAV1 and AAV6 have the greatest ability to transduce a wide range of cell types, however, for particular cell types, there are specific serotypes that provide optimal transduction. In this work, we describe the transduction efficiency of ten different AAV serotypes in thirty-four different mammalian cell lines and primary cell types. Although these results may not be universal due to numerous factors such as, culture conditions and/ or cell growth rates and cell heterogeneity, these results provide an important and unique resource for investigators who use AAV as an ex vivo gene delivery vector or who work with cells that are difficult to transfect.

Study of p-type and intrinsic materials for amorphous silicon based solar cells

NASA Astrophysics Data System (ADS)

Du, Wenhui

This dissertation summarizes the research work on the investigation and optimization of high efficiency hydrogenated amorphous silicon (a-Si:H) based thin film n-i-p single-junction and multi-junction solar cells, deposited using radio frequency (RF) and very high frequency (VHF) plasma enhanced chemical vapor deposition (PECVD) techniques. The fabrication and characterization of high quality p-type and intrinsic materials for a-Si:H based solar cells have been systematically and intensively studied. Hydrogen dilution, substrate temperature, gas flow rate, RF- or VHF-power density, and films deposition time have been optimized to obtain "on-the-edge" materials. To understand the material structure of the silicon p-layer providing a high Voc a-Si:H solar cell, hydrogenated amorphous, protocrystalline, and nanocrystalline silicon p-layers have been prepared using RF-PECVD and characterized by Raman spectroscopy and high resolution transmission electronic microscopy (HRTEM). It was found that the optimum Si:H p-layer for n-i-p a-Si:H solar cells is composed of fine-grained nanocrystals with crystallite sizes in the range of 3-5 nm embedded in an amorphous network. Using the optimized p-layer, an a-Si:H single-junction solar cell with a very high Voc value of 1.042 V and a FF value of 0.74 has been obtained. a-Si:H, a-SiGe:H and nc-Si:H i-layers have been prepared using RF- and VHF-PECVD techniques and monitored by different optical and electrical characterizations. Single-junction a-Si:H, a-SiGe and nc-Si:H cells have been developed and optimized. Intermediate bandgap a-SiGe:H solar cells achieved efficiencies over 12.5%. On the basis of optimized component cells, we achieved a-Si:Hla-SiGe:H tandem solar cells with efficiencies of ˜12.9% and a-Si:H/a-SiGe:H/a-SiGe:H triple-junction cells with efficiencies of ˜12.03%. VHF-PECVD technique was used to increase the deposition rates of the narrow bandgap materials. The deposition rate for a-SiGe:H i-layer attained 9 A/sec and the solar cell had a V oc of 0.588 V, Jsc of 20.4 mA/cm2, FF of 0.63, and efficiency of 7.6%. Preliminary research on the preparation of a-Si:Hlnc-Si:H tandem solar cells and a-Si:Hla-SiGe:Hlnc-Si:H triple-junction cells has also been undertaken using VHF nc-Si:H bottom cells with deposition rates of 6 A/sec. All I-V measurements were carried out under AM1.5G (100 MW/cm2) and the cell area was 0.25 cm2.

Record high efficiency of screen-printed silicon aluminum back surface field solar cell: 20.29%

NASA Astrophysics Data System (ADS)

Kim, Ki Hyung; Park, Chang Sub; Doo Lee, Jae; Youb Lim, Jong; Yeon, Je Min; Kim, Il Hwan; Lee, Eun Joo; Cho, Young Hyun

2017-08-01

We have achieved a record high cell efficiency of 20.29% for an industrial 6-in. p-type monocrystalline silicon solar cell with a full-area aluminum back surface field (Al-BSF) by simply modifying the cell structure and optimizing the process with the existing cell production line. The cell efficiency was independently confirmed by the Solar Energy Research Institute of Singapore (SERIS). To increase the cell efficiency, for example, in four busbars, double printing, a lightly doped emitter with a sheet resistance of 90 to 100 Ω/□, and front surface passivation by using silicon oxynitride (SiON) on top of a silicon nitride (SiN x ) antireflection layer were adopted. To optimize front side processing, PC1D simulation was carried out prior to cell fabrication. The resulting efficiency gain is 0.64% compared with that in the reference cells with three busbars, a single antireflection coating layer, and a low-sheet-resistance emitter.

Optimization and control of perfusion cultures using a viable cell probe and cell specific perfusion rates.

PubMed

Dowd, Jason E; Jubb, Anthea; Kwok, K Ezra; Piret, James M

2003-05-01

Consistent perfusion culture production requires reliable cell retention and control of feed rates. An on-line cell probe based on capacitance was used to assay viable biomass concentrations. A constant cell specific perfusion rate controlled medium feed rates with a bioreactor cell concentration of approximately 5 x 10(6) cells mL(-1). Perfusion feeding was automatically adjusted based on the cell concentration signal from the on-line biomass sensor. Cell specific perfusion rates were varied over a range of 0.05 to 0.4 nL cell(-1) day(-1). Pseudo-steady-state bioreactor indices (concentrations, cellular rates and yields) were correlated to cell specific perfusion rates investigated to maximize recombinant protein production from a Chinese hamster ovary cell line. The tissue-type plasminogen activator concentration was maximized ( approximately 40 mg L(-1)) at 0.2 nL cell(-1) day(-1). The volumetric protein productivity ( approximately 60 mg L(-1) day(-1) was maximized above 0.3 nL cell(-1) day(-1). The use of cell specific perfusion rates provided a straightforward basis for controlling, modeling and optimizing perfusion cultures.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components.

PubMed

Qing, Chang; Wei-ding, Cui; Wei-min, Fan

2011-04-01

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

High throughput RNAi assay optimization using adherent cell cytometry.

PubMed

Nabzdyk, Christoph S; Chun, Maggie; Pradhan, Leena; Logerfo, Frank W

2011-04-25

siRNA technology is a promising tool for gene therapy of vascular disease. Due to the multitude of reagents and cell types, RNAi experiment optimization can be time-consuming. In this study adherent cell cytometry was used to rapidly optimize siRNA transfection in human aortic vascular smooth muscle cells (AoSMC). AoSMC were seeded at a density of 3000-8000 cells/well of a 96 well plate. 24 hours later AoSMC were transfected with either non-targeting unlabeled siRNA (50 nM), or non-targeting labeled siRNA, siGLO Red (5 or 50 nM) using no transfection reagent, HiPerfect or Lipofectamine RNAiMax. For counting cells, Hoechst nuclei stain or Cell Tracker green were used. For data analysis an adherent cell cytometer, Celigo® was used. Data was normalized to the transfection reagent alone group and expressed as red pixel count/cell. After 24 hours, none of the transfection conditions led to cell loss. Red fluorescence counts were normalized to the AoSMC count. RNAiMax was more potent compared to HiPerfect or no transfection reagent at 5 nM siGLO Red (4.12 +/-1.04 vs. 0.70 +/-0.26 vs. 0.15 +/-0.13 red pixel/cell) and 50 nM siGLO Red (6.49 +/-1.81 vs. 2.52 +/-0.67 vs. 0.34 +/-0.19). Fluorescence expression results supported gene knockdown achieved by using MARCKS targeting siRNA in AoSMCs. This study underscores that RNAi delivery depends heavily on the choice of delivery method. Adherent cell cytometry can be used as a high throughput-screening tool for the optimization of RNAi assays. This technology can accelerate in vitro cell assays and thus save costs.

High throughput RNAi assay optimization using adherent cell cytometry

PubMed Central

2011-01-01

Background siRNA technology is a promising tool for gene therapy of vascular disease. Due to the multitude of reagents and cell types, RNAi experiment optimization can be time-consuming. In this study adherent cell cytometry was used to rapidly optimize siRNA transfection in human aortic vascular smooth muscle cells (AoSMC). Methods AoSMC were seeded at a density of 3000-8000 cells/well of a 96well plate. 24 hours later AoSMC were transfected with either non-targeting unlabeled siRNA (50 nM), or non-targeting labeled siRNA, siGLO Red (5 or 50 nM) using no transfection reagent, HiPerfect or Lipofectamine RNAiMax. For counting cells, Hoechst nuclei stain or Cell Tracker green were used. For data analysis an adherent cell cytometer, Celigo® was used. Data was normalized to the transfection reagent alone group and expressed as red pixel count/cell. Results After 24 hours, none of the transfection conditions led to cell loss. Red fluorescence counts were normalized to the AoSMC count. RNAiMax was more potent compared to HiPerfect or no transfection reagent at 5 nM siGLO Red (4.12 +/-1.04 vs. 0.70 +/-0.26 vs. 0.15 +/-0.13 red pixel/cell) and 50 nM siGLO Red (6.49 +/-1.81 vs. 2.52 +/-0.67 vs. 0.34 +/-0.19). Fluorescence expression results supported gene knockdown achieved by using MARCKS targeting siRNA in AoSMCs. Conclusion This study underscores that RNAi delivery depends heavily on the choice of delivery method. Adherent cell cytometry can be used as a high throughput-screening tool for the optimization of RNAi assays. This technology can accelerate in vitro cell assays and thus save costs. PMID:21518450

Reliability evaluation of high-performance, low-power FinFET standard cells based on mixed RBB/FBB technique

NASA Astrophysics Data System (ADS)

Wang, Tian; Cui, Xiaoxin; Ni, Yewen; Liao, Kai; Liao, Nan; Yu, Dunshan; Cui, Xiaole

2017-04-01

With shrinking transistor feature size, the fin-type field-effect transistor (FinFET) has become the most promising option in low-power circuit design due to its superior capability to suppress leakage. To support the VLSI digital system flow based on logic synthesis, we have designed an optimized high-performance low-power FinFET standard cell library based on employing the mixed FBB/RBB technique in the existing stacked structure of each cell. This paper presents the reliability evaluation of the optimized cells under process and operating environment variations based on Monte Carlo analysis. The variations are modelled with Gaussian distribution of the device parameters and 10000 sweeps are conducted in the simulation to obtain the statistical properties of the worst-case delay and input-dependent leakage for each cell. For comparison, a set of non-optimal cells that adopt the same topology without employing the mixed biasing technique is also generated. Experimental results show that the optimized cells achieve standard deviation reduction of 39.1% and 30.7% at most in worst-case delay and input-dependent leakage respectively while the normalized deviation shrinking in worst-case delay and input-dependent leakage can be up to 98.37% and 24.13%, respectively, which demonstrates that our optimized cells are less sensitive to variability and exhibit more reliability. Project supported by the National Natural Science Foundation of China (No. 61306040), the State Key Development Program for Basic Research of China (No. 2015CB057201), the Beijing Natural Science Foundation (No. 4152020), and Natural Science Foundation of Guangdong Province, China (No. 2015A030313147).

Modeling and Optimization of Commercial Buildings and Stationary Fuel Cell Systems (Presentation)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ainscough, C.; McLarty, D.; Sullivan, R.

2013-10-01

This presentation describes the Distributed Generation Building Energy Assessment Tool (DG-BEAT) developed by the National Renewable Energy Laboratory and the University of California Irvine. DG-BEAT is designed to allow stakeholders to assess the economics of installing stationary fuel cell systems in a variety of building types in the United States.

Stem Cell Gene Therapy for Fanconi Anemia: Report from the 1st International Fanconi Anemia Gene Therapy Working Group Meeting

PubMed Central

Tolar, Jakub; Adair, Jennifer E; Antoniou, Michael; Bartholomae, Cynthia C; Becker, Pamela S; Blazar, Bruce R; Bueren, Juan; Carroll, Thomas; Cavazzana-Calvo, Marina; Clapp, D Wade; Dalgleish, Robert; Galy, Anne; Gaspar, H Bobby; Hanenberg, Helmut; Von Kalle, Christof; Kiem, Hans-Peter; Lindeman, Dirk; Naldini, Luigi; Navarro, Susana; Renella, Raffaele; Rio, Paula; Sevilla, Julián; Schmidt, Manfred; Verhoeyen, Els; Wagner, John E; Williams, David A; Thrasher, Adrian J

2011-01-01

Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA. PMID:21540837

Optimization of the Production of Inactivated Clostridium novyi Type B Vaccine Using Computational Intelligence Techniques.

PubMed

Aquino, P L M; Fonseca, F S; Mozzer, O D; Giordano, R C; Sousa, R

2016-07-01

Clostridium novyi causes necrotic hepatitis in sheep and cattle, as well as gas gangrene. The microorganism is strictly anaerobic, fastidious, and difficult to cultivate in industrial scale. C. novyi type B produces alpha and beta toxins, with the alpha toxin being linked to the presence of specific bacteriophages. The main strategy to combat diseases caused by C. novyi is vaccination, employing vaccines produced with toxoids or with toxoids and bacterins. In order to identify culture medium components and concentrations that maximized cell density and alpha toxin production, a neuro-fuzzy algorithm was applied to predict the yields of the fermentation process for production of C. novyi type B, within a global search procedure using the simulated annealing technique. Maximizing cell density and toxin production is a multi-objective optimization problem and could be treated by a Pareto approach. Nevertheless, the approach chosen here was a step-by-step one. The optimum values obtained with this approach were validated in laboratory scale, and the results were used to reload the data matrix for re-parameterization of the neuro-fuzzy model, which was implemented for a final optimization step with regards to the alpha toxin productivity. With this methodology, a threefold increase of alpha toxin could be achieved.

Optimized Methods for the Isolation of Arabidopsis Female Central Cells and Their Nuclei

PubMed Central

Park, Kyunghyuk; Frost, Jennifer M.; Adair, Adam James; Kim, Dong Min; Yun, Hyein; Brooks, Janie S.; Fischer, Robert L.; Choi, Yeonhee

2016-01-01

The Arabidopsis female gametophyte contains seven cells with eight haploid nuclei buried within layers of sporophytic tissue. Following double fertilization, the egg and central cells of the gametophyte develop into the embryo and endosperm of the seed, respectively. The epigenetic status of the central cell has long presented an enigma due both to its inaccessibility, and the fascinating epigenome of the endosperm, thought to have been inherited from the central cell following activity of the DEMETER demethylase enzyme, prior to fertilization. Here, we present for the first time, a method to isolate pure populations of Arabidopsis central cell nuclei. Utilizing a protocol designed to isolate leaf mesophyll protoplasts, we systematically optimized each step in order to efficiently separate central cells from the female gametophyte. We use initial manual pistil dissection followed by the derivation of central cell protoplasts, during which process the central cell emerges from the micropylar pole of the embryo sac. Then, we use a modified version of the Isolation of Nuclei TAgged in specific Cell Types (INTACT) protocol to purify central cell nuclei, resulting in a purity of 75–90% and a yield sufficient to undertake downstream molecular analyses. We find that the process is highly dependent on the health of the original plant tissue used, and the efficiency of protoplasting solution infiltration into the gametophyte. By isolating pure central cell populations, we have enabled elucidation of the physiology of this rare cell type, which in the future will provide novel insights into Arabidopsis reproduction. PMID:27788573

Fatty acid production in genetically modified cyanobacteria

PubMed Central

Liu, Xinyao; Sheng, Jie; Curtiss III, Roy

2011-01-01

To avoid costly biomass recovery in photosynthetic microbial biofuel production, we genetically modified cyanobacteria to produce and secrete fatty acids. Starting with introducing an acyl–acyl carrier protein thioesterase gene, we made six successive generations of genetic modifications of cyanobacterium Synechocystis sp. PCC6803 wild type (SD100). The fatty acid secretion yield was increased to 197 ± 14 mg/L of culture in one improved strain at a cell density of 1.0 × 109 cells/mL by adding codon-optimized thioesterase genes and weakening polar cell wall layers. Although these strains exhibited damaged cell membranes at low cell densities, they grew more rapidly at high cell densities in late exponential and stationary phase and exhibited less cell damage than cells in wild-type cultures. Our results suggest that fatty acid secreting cyanobacteria are a promising technology for renewable biofuel production. PMID:21482809

A Minimally Invasive Method for Retrieving Single Adherent Cells of Different Types from Cultures

PubMed Central

Zeng, Jia; Mohammadreza, Aida; Gao, Weimin; Merza, Saeed; Smith, Dean; Kelbauskas, Laimonas; Meldrum, Deirdre R.

2014-01-01

The field of single-cell analysis has gained a significant momentum over the last decade. Separation and isolation of individual cells is an indispensable step in almost all currently available single-cell analysis technologies. However, stress levels introduced by such manipulations remain largely unstudied. We present a method for minimally invasive retrieval of selected individual adherent cells of different types from cell cultures. The method is based on a combination of mechanical (shear flow) force and biochemical (trypsin digestion) treatment. We quantified alterations in the transcription levels of stress response genes in individual cells exposed to varying levels of shear flow and trypsinization. We report optimal temperature, RNA preservation reagents, shear force and trypsinization conditions necessary to minimize changes in the stress-related gene expression levels. The method and experimental findings are broadly applicable and can be used by a broad research community working in the field of single cell analysis. PMID:24957932

Positioning Vascularized Composite Allotransplantation in the Spectrum of Transplantation

DTIC Science & Technology

2017-10-01

have now shown that the efficacy of both protocols is dependent upon a radiation-sensitive donor bone marrow (BM) cell type that is of T or B cell... dependent VCA survival. IL-2C Therapy Increases the Number but Not Function of Foxp3 CD4+ Treg Cells To test the effects of JES6-1 mAb-based IL-2C...which Treg cell- dependent immunoregulation has considerable potential. These IL-2C studies are now “in press” (4). TASK 5: OPTIMAL COMBINATION

Optimal voltage stimulation parameters for network-mediated responses in wild type and rd10 mouse retinal ganglion cells

NASA Astrophysics Data System (ADS)

Jalligampala, Archana; Sekhar, Sudarshan; Zrenner, Eberhart; Rathbun, Daniel L.

2017-04-01

To further improve the quality of visual percepts elicited by microelectronic retinal prosthetics, substantial efforts have been made to understand how retinal neurons respond to electrical stimulation. It is generally assumed that a sufficiently strong stimulus will recruit most retinal neurons. However, recent evidence has shown that the responses of some retinal neurons decrease with excessively strong stimuli (a non-monotonic response function). Therefore, it is necessary to identify stimuli that can be used to activate the majority of retinal neurons even when such non-monotonic cells are part of the neuronal population. Taking these non-monotonic responses into consideration, we establish the optimal voltage stimulation parameters (amplitude, duration, and polarity) for epiretinal stimulation of network-mediated (indirect) ganglion cell responses. We recorded responses from 3958 mouse retinal ganglion cells (RGCs) in both healthy (wild type, WT) and a degenerating (rd10) mouse model of retinitis pigmentosa—using flat-mounted retina on a microelectrode array. Rectangular monophasic voltage-controlled pulses were presented with varying voltage, duration, and polarity. We found that in 4-5 weeks old rd10 mice the RGC thresholds were comparable to those of WT. There was a marked response variability among mouse RGCs. To account for this variability, we interpolated the percentage of RGCs activated at each point in the voltage-polarity-duration stimulus space, thus identifying the optimal voltage-controlled pulse (-2.4 V, 0.88 ms). The identified optimal voltage pulse can activate at least 65% of potentially responsive RGCs in both mouse strains. Furthermore, this pulse is well within the range of stimuli demonstrated to be safe and effective for retinal implant patients. Such optimized stimuli and the underlying method used to identify them support a high yield of responsive RGCs and will serve as an effective guideline for future in vitro investigations of retinal electrostimulation by establishing standard stimuli for each unique experimental condition.

High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells.

PubMed

Steyer, Benjamin; Carlson-Stevermer, Jared; Angenent-Mari, Nicolas; Khalil, Andrew; Harkness, Ty; Saha, Krishanu

2016-04-01

Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of multiple components. Both the Cas9 endonuclease and a single guide RNA, that defines the genomic target, need to be present and co-localized within the nucleus for efficient gene-editing to occur. This work describes a new high-throughput screening platform for the optimization of CRISPR-Cas9 delivery strategies. By exploiting high content image analysis and microcontact printed plates, multi-parametric gene-editing outcome data from hundreds to thousands of isolated cell populations can be screened simultaneously. Employing this platform, we systematically screened four commercially available cationic lipid transfection materials with a range of RNAs encoding the CRISPR-Cas9 system. Analysis of Cas9 expression and editing of a fluorescent mCherry reporter transgene within human embryonic kidney cells was monitored over several days after transfection. Design of experiments analysis enabled rigorous evaluation of delivery materials and RNA concentration conditions. The results of this analysis indicated that the concentration and identity of transfection material have significantly greater effect on gene-editing than ratio or total amount of RNA. Cell subpopulation analysis on microcontact printed plates, further revealed that low cell number and high Cas9 expression, 24h after CRISPR-Cas9 delivery, were strong predictors of gene-editing outcomes. These results suggest design principles for the development of materials and transfection strategies with lipid-based materials. This platform could be applied to rapidly optimize materials for gene-editing in a variety of cell/tissue types in order to advance genomic medicine, regenerative biology and drug discovery. CRISPR-Cas9 is a new gene-editing technology for "genome surgery" that is anticipated to treat genetic diseases. This technology uses multiple components of the Cas9 system to cut out disease-causing mutations in the human genome and precisely suture in therapeutic sequences. Biomaterials based delivery strategies could help transition these technologies to the clinic. The design space for materials based delivery strategies is vast and optimization is essential to ensuring the safety and efficacy of these treatments. Therefore, new methods are required to rapidly and systematically screen gene-editing efficacy in human cells. This work utilizes an innovative platform to generate and screen many formulations of synthetic biomaterials and components of the CRISPR-Cas9 system in parallel. On this platform, we watch genome surgery in action using high content image analysis. These capabilities enabled us to identify formulation parameters for Cas9-material complexes that can optimize gene-editing in a specific human cell type. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Histological quantification of brain tissue inflammatory cell infiltration after focal cerebral infarction: a systematic review.

PubMed

Russek, Natanya S; Jensen, Matthew B

2014-03-01

Ischemic stroke is a leading cause of death and disability, and current treatments to limit tissue injury and improve recovery are limited. Cerebral infarction is accompanied by intense brain tissue inflammation involving many inflammatory cell types that may cause both negative and positive effects on outcomes. Many potential neuroprotective and neurorestorative treatments may affect, and be affected by, this inflammatory cell infiltration, so that accurate quantification of this tissue response is needed. We performed a systematic review of histological methods to quantify brain tissue inflammatory cell infiltration after cerebral infarction. We found reports of multiple techniques to quantify different inflammatory cell types. We found no direct comparison studies and conclude that more research is needed to optimize the assessment of this important stroke outcome.

Adnectin-Based Design of Chimeric Antigen Receptor for T Cell Engineering.

PubMed

Han, Xiaolu; Cinay, Gunce E; Zhao, Yifan; Guo, Yunfei; Zhang, Xiaoyang; Wang, Pin

2017-11-01

Although chimeric antigen receptor (CAR)-engineered T cell therapy has achieved encouraging clinical trial results for treating hematological cancers, further optimization can likely expand this therapeutic success to more patients and other cancer types. Most CAR constructs used in clinical trials incorporate single chain variable fragment (scFv) as the extracellular antigen recognition domain. The immunogenicity of nonhuman scFv could cause host rejection against CAR T cells and compromise their persistence and efficacy. The limited availability of scFvs and slow discovery of new monoclonal antibodies also limit the development of novel CAR constructs. Adnectin, a class of affinity molecules derived from the tenth type III domain of human fibronectin, can be an alternative to scFv as an antigen-binding moiety in the design of CAR molecules. We constructed adnectin-based CARs targeting epithelial growth factor receptor (EGFR) and found that compared to scFv-based CAR, T cells engineered with adnectin-based CARs exhibited equivalent cell-killing activity against target H292 lung cancer cells in vitro and had comparable antitumor efficacy in xenograft tumor-bearing mice in vivo. In addition, with optimal affinity tuning, adnectin-based CAR showed higher selectivity on target cells with high EGFR expression than on those with low expression. This new design of adnectin CARs can potentially facilitate the development of T cell immunotherapy for cancer and other diseases. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Development of an encapsulated stem cell-based therapy for diabetes.

PubMed

Tomei, Alice Anna; Villa, Chiara; Ricordi, Camillo

2015-01-01

Islet transplantation can treat the most severe cases of type 1 diabetes but it currently requires deceased donor pancreata as an islet source and chronic immunosuppression to prevent rejection and recurrence of autoimmunity. Stem cell-derived insulin-producing cells may address the shortage of organ donors, whereas cell encapsulation may reduce or eliminate the requirement for immunosuppression, minimizing the risks associated with the islet transplantation procedure, and potentially prolonging graft survival. This review focuses on the design principles for immunoisolation devices and on stem cell differentiation into insulin-producing cell products. The reader will gain understanding of the different types of immunoisolation devices and the key parameters that affect the outcome of the encapsulated graft. Progresses in stem cell differentiation towards mature endocrine islet cells, including the most recent clinical trials and the challenges associated with the application of immunoisolation devices designed for primary islets to stem-cell products, are also discussed. Recent advancements in the field of stem cell-derived islet cell products and immunoisolation strategies hold great promise for type 1 diabetes. However, a combination product including both cells and an immunoisolation strategy still needs to be optimized and tested for safety and efficacy.

Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity.

PubMed

Salvat, Regina S; Verma, Deeptak; Parker, Andrew S; Kirsch, Jack R; Brooks, Seth A; Bailey-Kellogg, Chris; Griswold, Karl E

2017-06-27

Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 β-lactamase (P99βL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide-MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 10 9 variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99βL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening.

High performance hybrid silicon micropillar solar cell based on light trapping characteristics of Cu nanoparticles

NASA Astrophysics Data System (ADS)

Zhang, Yulong; Fan, Zhiqiang; Zhang, Weijia; Ma, Qiang; Jiang, Zhaoyi; Ma, Denghao

2018-05-01

High performance silicon combined structure (micropillar with Cu nanoparticles) solar cell has been synthesized from N-type silicon substrates based on the micropillar array. The combined structure solar cell exhibited higher short circuit current rather than the silicon miropillar solar cell, which the parameters of micropillar array are the same. Due to the Cu nanoparticles were decorated on the surface of silicon micropillar array, the photovoltaic properties of cells have been improved. In addition, the optimal efficiency of 11.5% was measured for the combined structure solar cell, which is better than the silicon micropillar cell.

Tumor cell-derived microparticles: a new form of cancer vaccine.

PubMed

Zhang, Huafeng; Huang, Bo

2015-08-01

For cancer vaccines, tumor antigen availability is currently not an issue due to technical advances. However, the generation of optimal immune stimulation during vaccination is challenging. We have recently demonstrated that tumor cell-derived microparticles (MP) can function as a new form of potent cancer vaccine by efficiently activating type I interferon pathway in a cGAS/STING dependent manner.

Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis

USDA-ARS?s Scientific Manuscript database

Shiga toxin 1, exotoxin A, diphtheria toxin and ricin are all AB-type protein toxins that act within the host cytosol to kill the host cell through a pathway involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. In...

A Novel Cell Type Enables B. subtilis to Escape from Unsuccessful Sporulation in Minimal Medium.

PubMed

Defeu Soufo, Hervé Joël

2016-01-01

Sporulation is the most enduring survival strategy developed by several bacterial species. However, spore development of the model organism Bacillus subtilis has mainly been studied by means of media or conditions optimized for the induction of sporogenesis. Here, I show that during prolonged growth during stationary phase in minimal medium, B. subtilis undergoes an asymmetric cell division that produces small and round-shaped, DNA containing cells. In contrast to wild-type cells, mutants harboring spo0A or spoIIIE / sftA double mutations neither sporulate nor produce this special cell type, providing evidence that the small round cells emerge from the abortion of endospore formation. In most cases observed, the small round cells arise in the presence of sigma H but absence of sigma F activity, different from cases of abortive sporulation described for rich media. These data suggest that in minimal media, many cells are able to initiate but fail to complete spore development, and therefore return to normal growth as rods. This work reveals that the continuation of asymmetric cell division, which results in the formation of the small round cells, is a way for cells to delay or escape from-unsuccessful-sporulation. Based on these findings, I suggest to name the here described cell type as "dwarf cells" to distinguish them from the well-known minicells observed in mutants defective in septum placement or proper chromosome partitioning.

Potential of Immobilized Whole-Cell Methylocella tundrae as a Biocatalyst for Methanol Production from Methane.

PubMed

Mardina, Primata; Li, Jinglin; Patel, Sanjay K S; Kim, In-Won; Lee, Jung-Kul; Selvaraj, Chandrabose

2016-07-28

Methanol is a versatile compound that can be biologically synthesized from methane (CH4) by methanotrophs using a low energy-consuming and environment-friendly process. Methylocella tundrae is a type II methanotroph that can utilize CH4 as a carbon and energy source. Methanol is produced in the first step of the metabolic pathway of methanotrophs and is further oxidized into formaldehyde. Several parameters must be optimized to achieve high methanol production. In this study, we optimized the production conditions and process parameters for methanol production. The optimum incubation time, substrate, pH, agitation rate, temperature, phosphate buffer and sodium formate concentration, and cell concentration were determined to be 24 h, 50% CH4, pH 7, 150 rpm, 30°C, 100 mM and 50 mM, and 18 mg/ml, respectively. The optimization of these parameters significantly improved methanol production from 0.66 to 5.18 mM. The use of alginate-encapsulated cells resulted in enhanced methanol production stability and reusability of cells after five cycles of reuse under batch culture conditions.

Optimizing the design and in vitro evaluation of bioreactive glucose oxidase-microspheres for enhanced cytotoxicity against multidrug resistant breast cancer cells.

PubMed

Cheng, Ji; Liu, Qun; Shuhendler, Adam J; Rauth, Andrew M; Wu, Xiao Yu

2015-06-01

Glucose oxidase (GOX) encapsulated in alginate-chitosan microspheres (GOX-MS) was shown in our previous work to produce reactive oxygen species (ROS) in situ and exhibit anticancer effects in vitro and in vivo. The purpose of present work was to optimize the design and thus enhance the efficacy of GOX-MS against multidrug resistant (MDR) cancer cells. GOX-MS with different mean diameters of 4, 20 or 140 μm were prepared using an emulsification-internal gelation-adsorption-chitosan coating method with varying compositions and conditions. The GOX loading efficiency, loading level, relative bioactivity of GOX-MS, and GOX leakage were determined and optimal chitosan concentrations in the coating solution were identified. The influence of particle size on cellular uptake, ROS generation, cytotoxicity and their underlying mechanisms was investigated. At the same GOX dose and incubation time, smaller sized GOX-MS produced larger amounts of H2O2 in cell culture medium and greater cytotoxicity toward murine breast cancer MDR (EMT6/AR1.0) and wild type (EMT6/WT) cells. Fluorescence and confocal laser scanning microscopy revealed significant uptake of small sized (4 μm) GOX-MS by both MDR and WT cells, but no cellular uptake of large (140 μm) GOX-MS. The GOX-MS were equally effective in killing both MDR cells and WT cells. The cytotoxicity of the GOX formulations was positively correlated with membrane damage and lipid peroxidation. GOX-MS induced greater membrane damage and lipid peroxidation in MDR cells than the WT cells. These results suggest that the optimized, small micron-sized GOX-MS are highly effective against MDR breast cancer cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Genomic tissue typing and optimal antithymocyte globuline dose using unrelated donors results in similar survival and relapse as HLA-identical siblings in haematopoietic stem-cell transplantation for leukaemia.

PubMed

Remberger, Mats; Mattsson, Jonas; Hausenberger, Dan; Schaffer, Marie; Svahn, Britt-Marie; Ringdén, Olle

2008-05-01

Sixty-one leukaemia patients treated with haematopoietic stem cell transplantation (HSCT) from a genomic human leucocyte antigen (HLA)-A, -B and -DRbeta1 matched unrelated donor (MUD) were compared with 121 patients with an HLA-identical sibling donor. All patients received conventional conditioning. We selected all patients with unrelated donors who received optimal antithymocyte globuline (ATG) dose, 6 mg/kg. One hundred and seven patients received stem cells from peripheral blood and 75 patients received bone marrow (BM) cells. The incidences of acute graft-versus-host disease (GVHD) grades II-IV were 33.4% and 34.7% in the MUD and sibling group, respectively. After year 2001, the incidence of chronic GVHD was similar in the two groups (27.8% vs. 25.8%). There was no difference in overall survival (60% vs. 60%), transplant-related mortality (18.6% vs. 16.6%) and relapse (23% vs. 26.4%) between the two groups. Haematopoietic stem cell transplantation with unrelated donors results in similar GVHD, relapse and survival as compared to using sibling donors. Reasons for this may be improved tissue-typing techniques and supportive care and optimisation of the ATG dose.

Optimized shielding for space radiation protection

NASA Technical Reports Server (NTRS)

Wilson, J. W.; Cucinotta, F. A.; Kim, M. H.; Schimmerling, W.

2001-01-01

Future deep space mission and International Space Station exposures will be dominated by the high-charge and -energy (HZE) ions of the Galactic Cosmic Rays (GCR). A few mammalian systems have been extensively tested over a broad range of ion types and energies. For example, C3H10T1/2 cells, V79 cells, and Harderian gland tumors have been described by various track-structure dependent response models. The attenuation of GCR induced biological effects depends strongly on the biological endpoint, response model used, and material composition. Optimization of space shielding is then driven by the nature of the response model and the transmission characteristics of the given material.

Optimized Shielding for Space Radiation Protection

NASA Technical Reports Server (NTRS)

Wilson, J. W.; Cucinotta, F. A.; Kim, M.-H. Y.; Schimmerling, W.

2000-01-01

Abstract. Future deep space mission and International Space Station exposures will be dominated by the high-charge and -energy (HZE) ions of the Galactic Cosmic Rays (GCR). A few mammalian systems have been extensively tested over a broad range of ion types and energies. For example, C3H10T1/2 cells, V79 cells, and Harderian gland tumors have been described by various track-structure dependent response models. The attenuation of GCR induced biological effects depends strongly on the biological endpoint, response model used, and material composition. Optimization of space shielding is then driven by the nature of the response model and the transmission characteristics of the given material.

Optimal fault-tolerant control strategy of a solid oxide fuel cell system

NASA Astrophysics Data System (ADS)

Wu, Xiaojuan; Gao, Danhui

2017-10-01

For solid oxide fuel cell (SOFC) development, load tracking, heat management, air excess ratio constraint, high efficiency, low cost and fault diagnosis are six key issues. However, no literature studies the control techniques combining optimization and fault diagnosis for the SOFC system. An optimal fault-tolerant control strategy is presented in this paper, which involves four parts: a fault diagnosis module, a switching module, two backup optimizers and a controller loop. The fault diagnosis part is presented to identify the SOFC current fault type, and the switching module is used to select the appropriate backup optimizer based on the diagnosis result. NSGA-II and TOPSIS are employed to design the two backup optimizers under normal and air compressor fault states. PID algorithm is proposed to design the control loop, which includes a power tracking controller, an anode inlet temperature controller, a cathode inlet temperature controller and an air excess ratio controller. The simulation results show the proposed optimal fault-tolerant control method can track the power, temperature and air excess ratio at the desired values, simultaneously achieving the maximum efficiency and the minimum unit cost in the case of SOFC normal and even in the air compressor fault.

Application of response surface methodology to maximize the productivity of scalable automated human embryonic stem cell manufacture.

PubMed

Ratcliffe, Elizabeth; Hourd, Paul; Guijarro-Leach, Juan; Rayment, Erin; Williams, David J; Thomas, Robert J

2013-01-01

Commercial regenerative medicine will require large quantities of clinical-specification human cells. The cost and quality of manufacture is notoriously difficult to control due to highly complex processes with poorly defined tolerances. As a step to overcome this, we aimed to demonstrate the use of 'quality-by-design' tools to define the operating space for economic passage of a scalable human embryonic stem cell production method with minimal cell loss. Design of experiments response surface methodology was applied to generate empirical models to predict optimal operating conditions for a unit of manufacture of a previously developed automatable and scalable human embryonic stem cell production method. Two models were defined to predict cell yield and cell recovery rate postpassage, in terms of the predictor variables of media volume, cell seeding density, media exchange and length of passage. Predicted operating conditions for maximized productivity were successfully validated. Such 'quality-by-design' type approaches to process design and optimization will be essential to reduce the risk of product failure and patient harm, and to build regulatory confidence in cell therapy manufacturing processes.

Sensing in tissue bioreactors

NASA Astrophysics Data System (ADS)

Rolfe, P.

2006-03-01

Specialized sensing and measurement instruments are under development to aid the controlled culture of cells in bioreactors for the fabrication of biological tissues. Precisely defined physical and chemical conditions are needed for the correct culture of the many cell-tissue types now being studied, including chondrocytes (cartilage), vascular endothelial cells and smooth muscle cells (blood vessels), fibroblasts, hepatocytes (liver) and receptor neurones. Cell and tissue culture processes are dynamic and therefore, optimal control requires monitoring of the key process variables. Chemical and physical sensing is approached in this paper with the aim of enabling automatic optimal control, based on classical cell growth models, to be achieved. Non-invasive sensing is performed via the bioreactor wall, invasive sensing with probes placed inside the cell culture chamber and indirect monitoring using analysis within a shunt or a sampling chamber. Electroanalytical and photonics-based systems are described. Chemical sensing for gases, ions, metabolites, certain hormones and proteins, is under development. Spectroscopic analysis of the culture medium is used for measurement of glucose and for proteins that are markers of cell biosynthetic behaviour. Optical interrogation of cells and tissues is also investigated for structural analysis based on scatter.

Three-dimensional epithelial tissues generated from human embryonic stem cells.

PubMed

Hewitt, Kyle J; Shamis, Yulia; Carlson, Mark W; Aberdam, Edith; Aberdam, Daniel; Garlick, Jonathan A

2009-11-01

The use of pluripotent human embryonic stem (hES) cells for tissue engineering may provide advantages over traditional sources of progenitor cells because of their ability to give rise to multiple cell types and their unlimited expansion potential. We derived cell populations with properties of ectodermal and mesenchymal cells in two-dimensional culture and incorporated these divergent cell populations into three-dimensional (3D) epithelial tissues. When grown in specific media and substrate conditions, two-dimensional cultures were enriched in cells (EDK1) with mesenchymal morphology and surface markers. Cells with a distinct epithelial morphology (HDE1) that expressed cytokeratin 12 and beta-catenin at cell junctions became the predominant cell type when EDK1 were grown on surfaces enriched in keratinocyte-derived extracellular matrix proteins. When these cells were incorporated into the stromal and epithelial tissue compartments of 3D tissues, they generated multilayer epithelia similar to those generated with foreskin-derived epithelium and fibroblasts. Three-dimensional tissues demonstrated stromal cells with morphologic features of mature fibroblasts, type IV collagen deposition in the basement membrane, and a stratified epithelium that expressed cytokeratin 12. By deriving two distinct cell lineages from a common hES cell source to fabricate complex tissues, it is possible to explore environmental cues that will direct hES-derived cells toward optimal tissue form and function.

Biological shielding test of hot cells with high active source 60Co (300 TBq)

NASA Astrophysics Data System (ADS)

Švrčula, P.; Zoul, D.; Zimina, M.; Petříčková, A.; Adamíková, T.; Schulc, M.; Srba, O.

2017-11-01

This article describes a method for testing of the efficiency of the biological shielding of the hot cell facility, which were constructed as a part of the project SUSEN. Ten hot cells and one semi-hot cell are present in the facility Radiochemistry II. The shielding is made from steel plates. In order to demonstrate sufficient efficiency of the biological shielding of the hot cells and a correspondence between measured and contractual values at selected points. The test was done using sealed high activity 60Co sources. The results are also used as a proof of the optimization of radiation protection for the workplace of this type. The results confirm significant optimization of radiation protection at the workplace. The dose received by a staff do not exceed one tens of annual limit during active service. Obtained results fulfill general requirements of radiation protection and will be used for further active service of hot cells facility.

Bioengineered anterior cruciate ligament

NASA Technical Reports Server (NTRS)

Martin, Ivan (Inventor); Altman, Gregory (Inventor); Kaplan, David (Inventor); Vunjak-Novakovic, Gordana (Inventor)

2001-01-01

The present invention provides a method for producing an anterior cruciate ligament ex vivo. The method comprises seeding pluripotent stem cells in a three dimensional matrix, anchoring the seeded matrix by attachment to two anchors, and culturing the cells within the matrix under conditions appropriate for cell growth and regeneration, while subjecting the matrix to one or more mechanical forces via movement of one or both of the attached anchors. Bone marrow stromal cells are preferably used as the pluripotent cells in the method. Suitable matrix materials are materials to which cells can adhere, such as a gel made from collagen type I. Suitable anchor materials are materials to which the matrix can attach, such as Goinopra coral and also demineralized bone. Optimally, the mechanical forces to which the matrix is subjected mimic mechanical stimuli experienced by an anterior cruciate ligament in vivo. This is accomplished by delivering the appropriate combination of tension, compression, torsion, and shear, to the matrix. The bioengineered ligament which is produced by this method is characterized by a cellular orientation and/or matrix crimp pattern in the direction of the applied mechanical forces, and also by the production of collagen type I, collagen type III, and fibronectin proteins along the axis of mechanical load produced by the mechanical forces. Optimally, the ligament produced has fiber bundles which are arranged into a helical organization. The method for producing an anterior cruciate ligament can be adapted to produce a wide range of tissue types ex vivo by adapting the anchor size and attachment sites to reflect the size of the specific type of tissue to be produced, and also adapting the specific combination of forces applied, to mimic the mechanical stimuli experienced in vivo by the specific type of tissue to be produced. The methods of the present invention can be further modified to incorporate other stimuli experienced in vivo by the particular developing tissue, some examples of the stimuli being chemical stimuli, and electro-magnetic stimuli. Some examples of tissue which can be produced include other ligaments in the body (hand, wrist, elbow, knee), tendon, cartilage, bone, muscle, and blood vessels.

Generation of Oligodendrogenic Spinal Neural Progenitor Cells From Human Induced Pluripotent Stem Cells.

PubMed

Khazaei, Mohamad; Ahuja, Christopher S; Fehlings, Michael G

2017-08-14

This unit describes protocols for the efficient generation of oligodendrogenic neural progenitor cells (o-NPCs) from human induced pluripotent stem cells (hiPSCs). Specifically, detailed methods are provided for the maintenance and differentiation of hiPSCs, human induced pluripotent stem cell-derived neural progenitor cells (hiPS-NPCs), and human induced pluripotent stem cell-oligodendrogenic neural progenitor cells (hiPSC-o-NPCs) with the final products being suitable for in vitro experimentation or in vivo transplantation. Throughout, cell exposure to growth factors and patterning morphogens has been optimized for both concentration and timing, based on the literature and empirical experience, resulting in a robust and highly efficient protocol. Using this derivation procedure, it is possible to obtain millions of oligodendrogenic-NPCs within 40 days of initial cell plating which is substantially shorter than other protocols for similar cell types. This protocol has also been optimized to use translationally relevant human iPSCs as the parent cell line. The resultant cells have been extensively characterized both in vitro and in vivo and express key markers of an oligodendrogenic lineage. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

Wiring Economy of Pyramidal Cells in the Juvenile Rat Somatosensory Cortex

PubMed Central

Bielza, Concha; Larrañaga, Pedro; DeFelipe, Javier

2016-01-01

Ever since Cajal hypothesized that the structure of neurons is designed in such a way as to save space, time and matter, numerous researchers have analyzed wiring properties at different scales of brain organization. Here we test the hypothesis that individual pyramidal cells, the most abundant type of neuron in the cerebral cortex, optimize brain connectivity in terms of wiring length. In this study, we analyze the neuronal wiring of complete basal arborizations of pyramidal neurons in layer II, III, IV, Va, Vb and VI of the hindlimb somatosensory cortical region of postnatal day 14 rats. For each cell, we search for the optimal basal arborization and compare its length with the length of the real dendritic structure. Here the optimal arborization is defined as the arborization that has the shortest total wiring length provided that all neuron bifurcations are respected and the extent of the dendritic arborizations remain unchanged. We use graph theory and evolutionary computation techniques to search for the minimal wiring arborizations. Despite morphological differences between pyramidal neurons located in different cortical layers, we found that the neuronal wiring is near-optimal in all cases (the biggest difference between the shortest synthetic wiring found for a dendritic arborization and the length of its real wiring was less than 5%). We found, however, that the real neuronal wiring was significantly closer to the best solution found in layers II, III and IV. Our studies show that the wiring economy of cortical neurons is related not to the type of neurons or their morphological complexities but to general wiring economy principles. PMID:27832100

Wiring Economy of Pyramidal Cells in the Juvenile Rat Somatosensory Cortex.

PubMed

Anton-Sanchez, Laura; Bielza, Concha; Larrañaga, Pedro; DeFelipe, Javier

2016-01-01

Ever since Cajal hypothesized that the structure of neurons is designed in such a way as to save space, time and matter, numerous researchers have analyzed wiring properties at different scales of brain organization. Here we test the hypothesis that individual pyramidal cells, the most abundant type of neuron in the cerebral cortex, optimize brain connectivity in terms of wiring length. In this study, we analyze the neuronal wiring of complete basal arborizations of pyramidal neurons in layer II, III, IV, Va, Vb and VI of the hindlimb somatosensory cortical region of postnatal day 14 rats. For each cell, we search for the optimal basal arborization and compare its length with the length of the real dendritic structure. Here the optimal arborization is defined as the arborization that has the shortest total wiring length provided that all neuron bifurcations are respected and the extent of the dendritic arborizations remain unchanged. We use graph theory and evolutionary computation techniques to search for the minimal wiring arborizations. Despite morphological differences between pyramidal neurons located in different cortical layers, we found that the neuronal wiring is near-optimal in all cases (the biggest difference between the shortest synthetic wiring found for a dendritic arborization and the length of its real wiring was less than 5%). We found, however, that the real neuronal wiring was significantly closer to the best solution found in layers II, III and IV. Our studies show that the wiring economy of cortical neurons is related not to the type of neurons or their morphological complexities but to general wiring economy principles.

Correction of murine Rag2 severe combined immunodeficiency by lentiviral gene therapy using a codon-optimized RAG2 therapeutic transgene.

PubMed

van Til, Niek P; de Boer, Helen; Mashamba, Nomusa; Wabik, Agnieszka; Huston, Marshall; Visser, Trudi P; Fontana, Elena; Poliani, Pietro Luigi; Cassani, Barbara; Zhang, Fang; Thrasher, Adrian J; Villa, Anna; Wagemaker, Gerard

2012-10-01

Recombination activating gene 2 (RAG2) deficiency results in severe combined immunodeficiency (SCID) with complete lack of T and B lymphocytes. Initial gammaretroviral gene therapy trials for other types of SCID proved effective, but also revealed the necessity of safe vector design. We report the development of lentiviral vectors with the spleen focus forming virus (SF) promoter driving codon-optimized human RAG2 (RAG2co), which improved phenotype amelioration compared to native RAG2 in Rag2(-/-) mice. With the RAG2co therapeutic transgene, T-cell receptor (TCR) and immunoglobulin repertoire, T-cell mitogen responses, plasma immunoglobulin levels and T-cell dependent and independent specific antibody responses were restored. However, the thymus double positive T-cell population remained subnormal, possibly due to the SF virus derived element being sensitive to methylation/silencing in the thymus, which was prevented by replacing the SF promoter by the previously reported silencing resistant element (ubiquitous chromatin opening element (UCOE)), and also improved B-cell reconstitution to eventually near normal levels. Weak cellular promoters were effective in T-cell reconstitution, but deficient in B-cell reconstitution. We conclude that immune functions are corrected in Rag2(-/-) mice by genetic modification of stem cells using the UCOE driven codon-optimized RAG2, providing a valid optional vector for clinical implementation.

A reliable protocol for the isolation of viable, chondrogenically differentiated human mesenchymal stem cells from high-density pellet cultures.

PubMed

Ullah, Mujib; Hamouda, Houda; Stich, Stefan; Sittinger, Michael; Ringe, Jochen

2012-12-01

Administration of chondrogenically differentiated mesenchymal stem cells (MSC) is discussed as a promising approach for the regenerative treatment of injured or diseased cartilage. The high-density pellet culture is the standard culture for chondrogenic differentiation, but cells in pellets secrete extracellular matrix (ECM) that they become entrapped in. Protocols for cell isolation from pellets often result in cell damage and dedifferentiation towards less differentiated MSC. Therefore, our aim was to develop a reliable protocol for the isolation of viable, chondrogenically differentiated MSC from high-density pellet cultures. Human bone marrow MSC were chondrogenically stimulated with transforming growth factor-β3, and the cartilaginous structure of the pellets was verified by alcian blue staining of cartilage proteoglycans, antibody staining of cartilage collagen type II, and quantitative real-time reverse-transcription polymerase chain reaction of the marker genes COL2A1 and SOX9. Trypsin and collagenases II and P were tested alone or in combination, and for different concentrations and times, to find a protocol for optimized pellet digestion. Whereas trypsin was not able to release viable cells, 90-min digestion with 300 U of collagenase II, 20 U of collagenase P, and 2 mM CaCl2 worked quite well and resulted in about 2.5×10(5) cells/pellet. The protocol was further optimized for the separation of released cells and ECM from each other. Cells were alcian blue and collagen type II positive and expressed COL2A1 and SOX9, verifying a chondrogenic character. However, they had different morphological shapes. The ECM was also uniformly alcian blue and collagen type II positive but showed different organizational and structural forms. To conclude, our protocol allows the reliable isolation of a defined number of viable, chondrogenically differentiated MSC from high-density pellet cultures. Such cells, as well as the ECM components, are of interest as research tools and for cartilage tissue engineering.

Microfluidic differential immunocapture biochip for specific leukocyte counting

PubMed Central

Hassan, Umer; Watkins, Nicholas N; Reddy, Bobby; Damhorst, Gregory; Bashir, Rashid

2016-01-01

Enumerating specific cell types from whole blood can be very useful for research and diagnostic purposes—e.g., for counting of cD4 and cD8 t cells in HIV/aIDs diagnostics. We have developed a biosensor based on a differential immunocapture technology to enumerate specific cells in 30 min using 10 µl of blood. this paper provides a comprehensive stepwise protocol to replicate our biosensor for cD4 and cD8 cell counts. the biochip can also be adapted to enumerate other specific cell types such as somatic cells or cells from tissue or liquid biopsies. capture of other specific cells requires immobilization of their corresponding antibodies within the capture chamber. therefore, this protocol is useful for research into areas surrounding immunocapture-based biosensor development. the biosensor production requires 24 h, a one-time cell capture optimization takes 6–9 h, and the final cell counting experiment in a laboratory environment requires 30 min to complete. PMID:26963632

Role of CD4+ T cells in a protective immune response against Cryptococcus neoformans in the central nervous system.

PubMed

Uicker, William C; McCracken, James P; Buchanan, Kent L

2006-02-01

Cryptococcosis is a life-threatening disease caused by the encapsulated yeast, Cryptococcus neoformans. Although infection with C. neoformans is initiated in the lungs, morbidity and mortality is mostly associated with infections of the central nervous system (CNS). Individuals with deficiencies in cell-mediated immunity, such as patients with AIDS, are more susceptible to disseminated cryptococcosis, highlighting the importance of cell-mediated immunity and CD4+ T cells in host resistance against C. neoformans. Using a mouse model of cryptococcal meningoencephalitis, we have shown that immunization of mice with a cryptococcal antigen induced a protective immune response that crossed the blood-brain barrier and initiated an immune response directly in the CNS if C. neoformans was present. The regional protective response was characteristic of a Type-1 (Th1) response in the types of cells present at the site of infection and in the cytokines and chemokines expressed. Here, we extend those findings and report that CD4+ T cells are required for survival of immune mice infected directly in the brain with C. neoformans and sensitized CD4 + T cells can transfer partial protection to naive mice infected intracerebrally with C. neoformans. Furthermore, CD4 + T cells were also important for optimal infiltration of inflammatory cells at the site of infection and in the expression of cytokines and chemokines associated with protection in the brain. Lastly, CD4+ T cells were required for optimal regional production and secretion of IFNgamma and in the significantly increased expression of iNOS in C. neoformans-infected brains of immune mice.

Tetherin Suppresses Type I Interferon Signaling by Targeting MAVS for NDP52-Mediated Selective Autophagic Degradation in Human Cells.

PubMed

Jin, Shouheng; Tian, Shuo; Luo, Man; Xie, Weihong; Liu, Tao; Duan, Tianhao; Wu, Yaoxing; Cui, Jun

2017-10-19

Tetherin (BST2/CD317) is an interferon-inducible antiviral factor known for its ability to block the release of enveloped viruses from infected cells. Yet its role in type I interferon (IFN) signaling remains poorly defined. Here, we demonstrate that Tetherin is a negative regulator of RIG-I like receptor (RLR)-mediated type I IFN signaling by targeting MAVS. The induction of Tetherin by type I IFN accelerates MAVS degradation via ubiquitin-dependent selective autophagy in human cells. Moreover, Tetherin recruits E3 ubiquitin ligase MARCH8 to catalyze K27-linked ubiquitin chains on MAVS at lysine 7, which serves as a recognition signal for NDP52-dependent autophagic degradation. Taken together, our findings reveal a negative feedback loop of RLR signaling generated by Tetherin-MARCH8-MAVS-NDP52 axis and provide insights into a better understanding of the crosstalk between selective autophagy and optimal deactivation of type I IFN signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation of a Passive Heat Exchanger Based Cooling System for Fuel Cell Applications

NASA Technical Reports Server (NTRS)

Colozza, Anthony J.; Burke, Kenneth A.

2011-01-01

Fuel cell cooling is conventionally performed with an actively controlled, dedicated coolant loop that exchanges heat with a separate external cooling loop. To simplify this system the concept of directly cooling a fuel cell utilizing a coolant loop with a regenerative heat exchanger to preheat the coolant entering the fuel cell with the coolant exiting the fuel cell was analyzed. The preheating is necessary to minimize the temperature difference across the fuel cell stack. This type of coolant system would minimize the controls needed on the coolant loop and provide a mostly passive means of cooling the fuel cell. The results indicate that an operating temperature of near or greater than 70 C is achievable with a heat exchanger effectiveness of around 90 percent. Of the heat exchanger types evaluated with the same type of fluid on the hot and cold side, a counter flow type heat exchanger would be required which has the possibility of achieving the required effectiveness. The number of heat transfer units required by the heat exchanger would be around 9 or greater. Although the analysis indicates the concept is feasible, the heat exchanger design would need to be developed and optimized for a specific fuel cell operation in order to achieve the high effectiveness value required.

Methods of improving the efficiency of photovoltaic cells. [including X ray analysis

NASA Technical Reports Server (NTRS)

Loferski, J. J.; Roessler, B.; Crisman, E. E.; Chen, L. Y.; Kaul, R.

1974-01-01

Work on aluminum-alloyed silicon grating cells is continued. Optimization of the geometry (grating line width and spacing) confirms the analysis of such cells. A 1 sq cm grating cell was fabricated and its i-V characteristic was measured under an AMO solar simulator. It is found that the efficiency of this cell would be about 7.9%, if it were covered by the usual antireflection coating. The surface of the cell is not covered by a diffused junction. The response is blue shifted; the current is somewhat higher than that produced by a commercial Si cell. However, the open circuit voltage is low, and attempts to optimize the open circuit voltage of the aluminum-alloy junctions are described. A preliminary X-ray topographic examination of GaAs specimens of the type commonly used to make solar cells is studied. The X-ray study shows that the wafers are filled with regions having strain gradients, possibly caused by precipitates. It is possible that a correlation exists between the presence of low mechanical perfection and minority carrier diffusion lengths of GaAs crystals.

Probing Photocurrent Nonuniformities in the Subcells of Monolithic Perovskite/Silicon Tandem Solar Cells.

PubMed

Song, Zhaoning; Werner, Jérémie; Shrestha, Niraj; Sahli, Florent; De Wolf, Stefaan; Niesen, Björn; Watthage, Suneth C; Phillips, Adam B; Ballif, Christophe; Ellingson, Randy J; Heben, Michael J

2016-12-15

Perovskite/silicon tandem solar cells with high power conversion efficiencies have the potential to become a commercially viable photovoltaic option in the near future. However, device design and optimization is challenging because conventional characterization methods do not give clear feedback on the localized chemical and physical factors that limit performance within individual subcells, especially when stability and degradation is a concern. In this study, we use light beam induced current (LBIC) to probe photocurrent collection nonuniformities in the individual subcells of perovskite/silicon tandems. The choices of lasers and light biasing conditions allow efficiency-limiting effects relating to processing defects, optical interference within the individual cells, and the evolution of water-induced device degradation to be spatially resolved. The results reveal several types of microscopic defects and demonstrate that eliminating these and managing the optical properties within the multilayer structures will be important for future optimization of perovskite/silicon tandem solar cells.

Photothermal monitoring of interaction of carcinoma cells with cytostatic drugs in vitro

NASA Astrophysics Data System (ADS)

Lapotko, Dmitri; Hanna, Ehab; Cannon, Martin

2003-06-01

Background/problem. Monitoring of tumor response to cancer chemotherapy and dose optimization for specific patients are the key factors for successful application of anti-tumor drugs. Using patient's tumor cells for preliminary in vitro drug screening may allow optimal selection of drug type and dose. Method. Single cell state was studied with photothermal microscope. Carcinoma cells were irradiated at 427 nm with 8 ns laser pulse with energy 30 - 40 μJ. Cell photothermal (PT) response amplitude and shape from each cell were analyzed and amount of cells that produced specific PT response was used as PT parameter. Parallel experiment included cell viability control. Results were obtained for two cytotoxic chemotherapy agents -- Platinol-aq and Adrucil. Incubation of cell suspensions for 90 min at 20 and 37°C caused changes in cell PT parameters. Reaction of carcinoma cells to the drug was very similar to reaction of hepatocytes to respiratory chain inhibition and reaction of RBC to osmotic pressure decrease. PT effect was found to be dose-dependent. PT method allows detecting drug-induced changes before cell death or morphological changes and therefore can be fast and sensitive modality for control of chemotherapy.

Optimization of trypsins for influenza A/H1N1 virus replication in MDCK SI-6 cells, a novel MDCK cell line.

PubMed

Iskandar, Viska I; Sasaki, Yutaka; Yoshino, Naoto; Abubakar, Raden Z R; Sato, Shigehiro; Muraki, Yasushi

2018-02-01

A cell-based vaccine production method for influenza virus may be an effective and more rapid alternative to egg-based systems. For high-yield virus production, the effect of bovine, porcine, fungal, and recombinant trypsins on influenza A/H1N1 virus replication in MDCK SI-6 cells (SI-6 cells), a novel MDCK cell line developed by our research group, was examined. SI-6 cells infected with influenza A/H1N1 virus were incubated in the presence of four trypsin types at various concentrations, and virus yields in the culture medium were evaluated by a hemagglutination (HA) assay. Virus growth was most efficient in the presence of bovine and porcine trypsins. An analysis of the optimized concentration and definitive HA titer of each trypsin by Gaussian distribution revealed that comparable high virus yields (166.1 and 164.2 HAU/50μl) were obtained at the optimized concentrations of bovine (0.4μg/ml) and porcine (2.1μg/ml) trypsins, respectively, the yields of which were significantly higher than that of fungal and recombinant trypsins. We conclude that bovine and porcine trypsins are suitable for influenza A/H1N1 virus replication in SI-6 cells. This result complements our previous study and suggests the possible application of SI-6 cells to the development of cell-based influenza vaccines. Copyright © 2017 Elsevier B.V. All rights reserved.

Impact of dopant concentrations on emitter formation with spin on dopant source in n-type crystalline silicon solar cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singha, Bandana; Solanki, Chetan Singh

Use of a suitable dopant source for emitter formation is an essential requirement in n-type crystalline silicon solar cells. Boron spin on dopant source, used as alternative to mostly used BBr{sub 3} liquid source, can yield an emitter with less diffusion induced defects under controlled conditions. Different concentrations of commercially available spin on dopant source is used and optimized in this work for sheet resistance values of the emitter ranging from 30 Ω/□ to 70 Ω/□ with emitter doping concentrations suitable for ohmic contacts. The dopant concentrations diluted with different ratios improves the carrier lifetime and thus improves the emittermore » performance. Hence use of suitable dopant source is essential in forming emitters in n-type crystalline silicon solar cells.« less

An optimized library for reference-based deconvolution of whole-blood biospecimens assayed using the Illumina HumanMethylationEPIC BeadArray.

PubMed

Salas, Lucas A; Koestler, Devin C; Butler, Rondi A; Hansen, Helen M; Wiencke, John K; Kelsey, Karl T; Christensen, Brock C

2018-05-29

Genome-wide methylation arrays are powerful tools for assessing cell composition of complex mixtures. We compare three approaches to select reference libraries for deconvoluting neutrophil, monocyte, B-lymphocyte, natural killer, and CD4+ and CD8+ T-cell fractions based on blood-derived DNA methylation signatures assayed using the Illumina HumanMethylationEPIC array. The IDOL algorithm identifies a library of 450 CpGs, resulting in an average R 2  = 99.2 across cell types when applied to EPIC methylation data collected on artificial mixtures constructed from the above cell types. Of the 450 CpGs, 69% are unique to EPIC. This library has the potential to reduce unintended technical differences across array platforms.

Novel Multiplex Fluorescent PCR-Based Method for HLA Typing and Preimplantational Genetic Diagnosis of β-Thalassemia.

PubMed

Khosravi, Sharifeh; Salehi, Mansour; Ramezanzadeh, Mahboobeh; Mirzaei, Hamed; Salehi, Rasoul

2016-05-01

Thalassemia is curable by bone marrow transplantation; however, finding suitable donors with defined HLA combination remains a major challenge. Cord blood stem cells with preselected HLA system through preimplantation genetic diagnosis (PGD) proved very useful for resolving scarce HLA-matched bone marrow donors. A thalassemia trait couple with an affected child was included in this study. We used informative STR markers at the HLA and beta globin loci to develop a single cell multiplex fluorescent PCR protocol. The protocol was extensively optimized on single lymphocytes isolated from the couple's peripheral blood. The optimized protocol was applied on single blastomeres biopsied from day 3 cleavage stage IVF embryos of the couple. Four IVF embryos biopsied on day 3 and a single blastomere of each were provided for genetic diagnosis of combined β-thalassemia mutations and HLA typing. Of these, one embryo was diagnosed as homozygous normal for the thalassemia mutation and HLA matched with the existing affected sibling. The optimized protocol worked well in PGD clinical cycle for selection of thalassemia-unaffected embryos with the desired HLA system. Copyright © 2016 IMSS. Published by Elsevier Inc. All rights reserved.

Screening a strain of Aspergillus niger and optimization of fermentation conditions for degradation of aflatoxin B₁.

PubMed

Zhang, Wei; Xue, Beibei; Li, Mengmeng; Mu, Yang; Chen, Zhihui; Li, Jianping; Shan, Anshan

2014-11-13

Aflatoxin B₁, a type of highly toxic mycotoxin produced by some species belonging to the Aspergillus genus, such as Aspergillus flavus and Aspergillus parasiticus, is widely distributed in feed matrices. Here, coumarin was used as the sole carbon source to screen microorganism strains that were isolated from types of feed ingredients. Only one isolate (ND-1) was able to degrade aflatoxin B₁ after screening. ND-1 isolate, identified as a strain of Aspergillus niger using phylogenetic analysis on the basis of 18S rDNA, could remove 26.3% of aflatoxin B₁ after 48 h of fermentation in nutrient broth (NB). Optimization of fermentation conditions for aflatoxin B₁ degradation by selected Aspergillus niger was also performed. These results showed that 58.2% of aflatoxin B₁ was degraded after 24 h of culture under the optimal fermentation conditions. The aflatoxin B₁ degradation activity of Aspergillus niger supernatant was significantly stronger than cells and cell extracts. Furthermore, effects of temperature, heat treatment, pH, and metal ions on aflatoxin B₁ degradation by the supernatant were examined. Results indicated that aflatoxin B₁ degradation of Aspergillus niger is enzymatic and this process occurs in the extracellular environment.

Initial blood storage experiment

NASA Technical Reports Server (NTRS)

Surgenor, Douglas MACN.

1988-01-01

The design of the Initial Blood Storage Experiment (IBSE) was based upon a carefully controlled comparison between identical sets of human blood cell suspensions - red cells, white cell, and platelets - one set of which was transported aboard the Columbia on a 6 day 11 hour mission, and the other held on the ground. Both sets were carried inside stainless steel dewars within specially fabricated flight hardware. Individual bags of cell suspensions were randomly assigned with respect to ground vs orbit status, dewar chamber, and specific location within the dewar. To foster optimal preservation, each cell type was held under specific optimal conditions of pH, ionic strength, solute concentration, gas tension, and temperature. An added variable in this initial experiment was provided by the use of three different polymer/plasticizer formulations for the sealed bags which held the blood cells. At termination of the experiment, aliquots of the suspensions, identified only by code, were distributed to be assayed. Assays were selected to constitute a broad survey of cellular properties and thereby maximize the chances of detection of gravitational effects. A total of 74 different outcome measurements were reported for statistical analysis. When the measurements were completed, the results were entered into the IBSE data base, at which time the data were matched with the original blood bag numbers to determine their status with respect to polymer/plasticizer type, orbit status (orbit or ground), and storage position within the experimental hardware. The data were studied by analysis of variance. Initially, type of bag and orbital status were main factors; later more detailed analyses were made on specific issues such as position in the hardware and specific plastic. If the analysis of variance indicated a statistical significance at the 5 percent level the corresponding p-value was reported.

Neural differentiation of novel multipotent progenitor cells from cryopreserved human umbilical cord blood

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Myoung Woo; Moon, Young Joon; Yang, Mal Sook

2007-06-29

Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells, with practical and ethical advantages. To date, the presence of other stem cells in UCB remains to be established. We investigated whether other stem cells are present in cryopreserved UCB. Seeded mononuclear cells formed adherent colonized cells in optimized culture conditions. Over a 4- to 6-week culture period, colonized cells gradually developed into adherent mono-layer cells, which exhibited homogeneous fibroblast-like morphology and immunophenotypes, and were highly proliferative. Isolated cells were designated 'multipotent progenitor cells (MPCs)'. Under appropriate conditions for 2 weeks, MPCs differentiated into neural tissue-specific cell types,more » including neuron, astrocyte, and oligodendrocyte. Differentiated cells presented their respective markers, specifically, NF-L and NSE for neurons, GFAP for astrocytes, and myelin/oligodendrocyte for oligodendrocytes. In this study, we successfully isolated MPCs from cryopreserved UCB, which differentiated into the neural tissue-specific cell types. These findings suggest that cryopreserved human UCB is a useful alternative source of neural progenitor cells, such as MPCs, for experimental and therapeutic applications.« less

Immuno Nanoparticles Integrated Electrical Control of Targeted Cancer Cell Development Using Whole Cell Bioelectronic Device

PubMed Central

Hondroulis, Evangelia; Zhang, Rui; Zhang, Chengxiao; Chen, Chunying; Ino, Kosuke; Matsue, Tomokazu; Li, Chen-Zhong

2014-01-01

Electrical properties of cells determine most of the cellular functions, particularly ones which occur in the cell's membrane. Manipulation of these electrical properties may provide a powerful electrotherapy option for the treatment of cancer as cancerous cells have been shown to be more electronegative than normal proliferating cells. Previously, we used an electrical impedance sensing system (EIS) to explore the responses of cancerous SKOV3 cells and normal HUVEC cells to low intensity (<2 V/cm) AC electric fields, determining that the optimal frequency for SKOV3 proliferation arrest was 200 kHz, without harming the non-cancerous HUVECs. In this study, to determine if these effects are cell type dependant, human breast adenocarcinoma cells (MCF7) were subjected to a range of frequencies (50 kHz-2 MHz) similar to the previously tested SKOV3. For the MCF7, an optimal frequency of 100 kHz was determined using the EIS, indicating a higher sensitivity towards the applied field. Further experiments specifically targeting the two types of cancer cells using HER2 antibody functionalized gold nanoparticles (HER2-AuNPs) were performed to determine if enhanced electric field strength can be induced via the application of nanoparticles, consequently leading to the killing of the cancerous cells without affecting non cancerous HUVECs and MCF10a providing a platform for the development of a non-invasive cancer treatment without any harmful side effects. The EIS was used to monitor the real-time consequences on cellular viability and a noticeable decrease in the growth profile of the MCF7 was observed with the application of the HER2-AuNPs and the electric fields indicating specific inhibitory effects on dividing cells in culture. To further understand the effects of the externally applied field to the cells, an Annexin V/EthD-III assay was performed to determine the cell death mechanism indicating apoptosis. The zeta potential of the SKOV3 and the MCF7 before and after incorporation of the HER2-AuNPs was also obtained indicating a decrease in zeta potential with the incorporation of the nanoparticles. The outcome of this research will improve our fundamental understanding of the behavior of cancer cells and define optimal parameters of electrotherapy for clinical and drug delivery applications. PMID:25057316

Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio.

PubMed

Yang, Tianzhi; Martin, Paige; Fogarty, Brittany; Brown, Alison; Schurman, Kayla; Phipps, Roger; Yin, Viravuth P; Lockman, Paul; Bai, Shuhua

2015-06-01

The blood-brain barrier (BBB) essentially restricts therapeutic drugs from entering into the brain. This study tests the hypothesis that brain endothelial cell derived exosomes can deliver anticancer drug across the BBB for the treatment of brain cancer in a zebrafish (Danio rerio) model. Four types of exosomes were isolated from brain cell culture media and characterized by particle size, morphology, total protein, and transmembrane protein markers. Transport mechanism, cell uptake, and cytotoxicity of optimized exosome delivery system were tested. Brain distribution of exosome delivered anticancer drugs was evaluated using transgenic zebrafish TG (fli1: GFP) embryos and efficacies of optimized formations were examined in a xenotransplanted zebrafish model of brain cancer model. Four exosomes in 30-100 diameters showed different morphologies and exosomes derived from brain endothelial cells expressed more CD63 tetraspanins transmembrane proteins. Optimized exosomes increased the uptake of fluorescent marker via receptor mediated endocytosis and cytotoxicity of anticancer drugs in cancer cells. Images of the zebrafish showed exosome delivered anticancer drugs crossed the BBB and entered into the brain. In the brain cancer model, exosome delivered anticancer drugs significantly decreased fluorescent intensity of xenotransplanted cancer cells and tumor growth marker. Brain endothelial cell derived exosomes could be potentially used as a carrier for brain delivery of anticancer drug for the treatment of brain cancer.

A toxicity cost function approach to optimal CPA equilibration in tissues.

PubMed

Benson, James D; Higgins, Adam Z; Desai, Kunjan; Eroglu, Ali

2018-02-01

There is growing need for cryopreserved tissue samples that can be used in transplantation and regenerative medicine. While a number of specific tissue types have been successfully cryopreserved, this success is not general, and there is not a uniform approach to cryopreservation of arbitrary tissues. Additionally, while there are a number of long-established approaches towards optimizing cryoprotocols in single cell suspensions, and even plated cell monolayers, computational approaches in tissue cryopreservation have classically been limited to explanatory models. Here we develop a numerical approach to adapt cell-based CPA equilibration damage models for use in a classical tissue mass transport model. To implement this with real-world parameters, we measured CPA diffusivity in three human-sourced tissue types, skin, fibroid and myometrium, yielding propylene glycol diffusivities of 0.6 × 10 -6  cm 2 /s, 1.2 × 10 -6  cm 2 /s and 1.3 × 10 -6  cm 2 /s, respectively. Based on these results, we numerically predict and compare optimal multistep equilibration protocols that minimize the cell-based cumulative toxicity cost function and the damage due to excessive osmotic gradients at the tissue boundary. Our numerical results show that there are fundamental differences between protocols designed to minimize total CPA exposure time in tissues and protocols designed to minimize accumulated CPA toxicity, and that "one size fits all" stepwise approaches are predicted to be more toxic and take considerably longer than needed. Copyright © 2017 Elsevier Inc. All rights reserved.

Alpha-fetoprotein-producing ovarian clear cell adenocarcinoma with fetal gut differentiation: a rare case report and literature review.

PubMed

Chao, Wei-Ting; Liu, Chia-Hao; Lai, Chiung-Ru; Chen, Yi-Jen; Chuang, Chi-Mu; Wang, Peng-Hui

2018-06-22

Alpha-fetoprotein (AFP) is a useful tumor marker for ovarian germ cell tumors, particularly yolk sac tumor (YST). It is valuable for both diagnosis and further follow-up. Epithelial ovarian carcinoma (EOC) rarely secretes AFP, especially for clear cell type and in the postmenopausal women. Based on the limited knowledge about AFP-producing clear cell type EOC, a case and literature review on this topic is extensively reviewed. We report a 55-year-old postmenopausal woman experienced vaginal spotting for one month, and serum level of AFP was 60,721 ng/ml initially. Histological examination was clear cell type EOC. Tumor cells revealed strong immunoreactivity for glypican-3 (GPC3) and AFP and weak for hepatocyte nuclear factor-1 beta (HNF-1 beta), but negative for CD30, making the diagnosis of AFP-producing clear cell type EOC with fetal gut differentiation in focal areas, FIGO (International Federation of Gynecology and Obstetrics) IIIc. Although the patient underwent an intensive treatment, including optimal debulking surgery and multi-agent chemotherapy, the patient died of disease. To provide a better understanding of clinical and molecular characteristics of the AFP-producing clear cell type EOC, we conducted a systematic literature review. A total of three papers described the AFP-producing clear cell type EOC are available. The overall survival rate of these cases, including the current case is 50%. Although immunohistochemical examination is not always needed in routine for the diagnosis of clear cell type EOC, to distinguish from other tumors, especially germ cell tumors, or to provide the better way to monitor therapeutic response or to evaluate the disease status, immunostaining, including GPC3, HNF-1 beta, CD30, cytokeratin 7 or 20, and AFP is taken into account. Due to rarity, the appropriate chemotherapy regimen and the biological behavior of AFP-producing clear cell type EOC are still unclear.

Muramyl Peptide-Enhanced Sleep: Pharmacological Optimization of Performance

DTIC Science & Technology

1990-06-01

Dower, S . K., S . R. Kronheim, T. P. Hopp, M. Cantrell, M. Deeley, S . Gillis, C. S . Henney, and D. L. Urdal. The cell surface receptors for...sleep-promoting factor isolated from human urine. J. Biol. Chem. 259:12652-12658, 1984. 90. Martin, S . A., R. S . Rosenthal, and K. Biemann. Fast atom...AD REPORT NUMBER0 TITLE Muramyl Peptide-Enhanced Sleep: Pharmacological Optimization of Performance TYPE OF REPORT Annj~Jl AUTHOR ( s ) O I James M

Optimization of Dosing for EGFR-Mutant Non–Small Cell Lung Cancer with Evolutionary Cancer Modeling

PubMed Central

Chmielecki, Juliann; Foo, Jasmine; Oxnard, Geoffrey R.; Hutchinson, Katherine; Ohashi, Kadoaki; Somwar, Romel; Wang, Lu; Amato, Katherine R.; Arcila, Maria; Sos, Martin L.; Socci, Nicholas D.; Viale, Agnes; de Stanchina, Elisa; Ginsberg, Michelle S.; Thomas, Roman K.; Kris, Mark G.; Inoue, Akira; Ladanyi, Marc; Miller, Vincent A.; Michor, Franziska; Pao, William

2012-01-01

Non–small cell lung cancers (NSCLCs) that harbor mutations within the epidermal growth factor receptor (EGFR) gene are sensitive to the tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. Unfortunately, all patients treated with these drugs will acquire resistance, most commonly as a result of a secondary mutation within EGFR (T790M). Because both drugs were developed to target wild-type EGFR, we hypothesized that current dosing schedules were not optimized for mutant EGFR or to prevent resistance. To investigate this further, we developed isogenic TKI-sensitive and TKI-resistant pairs of cell lines that mimic the behavior of human tumors. We determined that the drug-sensitive and drug-resistant EGFR-mutant cells exhibited differential growth kinetics, with the drug-resistant cells showing slower growth. We incorporated these data into evolutionary mathematical cancer models with constraints derived from clinical data sets. This modeling predicted alternative therapeutic strategies that could prolong the clinical benefit of TKIs against EGFR-mutant NSCLCs by delaying the development of resistance. PMID:21734175

Reporter gene imaging of targeted T cell immunotherapy in recurrent glioma.

PubMed

Keu, Khun Visith; Witney, Timothy H; Yaghoubi, Shahriar; Rosenberg, Jarrett; Kurien, Anita; Magnusson, Rachel; Williams, John; Habte, Frezghi; Wagner, Jamie R; Forman, Stephen; Brown, Christine; Allen-Auerbach, Martin; Czernin, Johannes; Tang, Winson; Jensen, Michael C; Badie, Behnam; Gambhir, Sanjiv S

2017-01-18

High-grade gliomas are aggressive cancers that often become rapidly fatal. Immunotherapy using CD8 + cytotoxic T lymphocytes (CTLs), engineered to express both herpes simplex virus type 1 thymidine kinase (HSV1-TK) and interleukin-13 (IL-13) zetakine chimeric antigen receptor (CAR), is a treatment strategy with considerable potential. To optimize this and related immunotherapies, it would be helpful to monitor CTL viability and trafficking to glioma cells. We show that noninvasive positron emission tomography (PET) imaging with 9-[4-[ 18 F]fluoro-3-(hydroxymethyl)butyl]guanine ([ 18 F]FHBG) can track HSV1-tk reporter gene expression present in CAR-engineered CTLs. [ 18 F]FHBG imaging was safe and enabled the longitudinal imaging of T cells stably transfected with a PET reporter gene in patients. Further optimization of this imaging approach for monitoring in vivo cell trafficking should greatly benefit various cell-based therapies for cancer. Copyright © 2017, American Association for the Advancement of Science.

On the MTD paradigm and optimal control for multi-drug cancer chemotherapy.

PubMed

Ledzewicz, Urszula; Schättler, Heinz; Gahrooi, Mostafa Reisi; Dehkordi, Siamak Mahmoudian

2013-06-01

In standard chemotherapy protocols, drugs are given at maximum tolerated doses (MTD) with rest periods in between. In this paper, we briey discuss the rationale behind this therapy approach and, using as example multidrug cancer chemotherapy with a cytotoxic and cytostatic agent, show that these types of protocols are optimal in the sense of minimizing a weighted average of the number of tumor cells (taken both at the end of therapy and at intermediate times) and the total dose given if it is assumed that the tumor consists of a homogeneous population of chemotherapeutically sensitive cells. A 2-compartment linear model is used to model the pharmacokinetic equations for the drugs.

Cardiomyocytes from phorbol myristate acetate-activated mesenchymal stem cells restore electromechanical function in infarcted rat hearts

PubMed Central

Song, Heesang; Hwang, Hye Jin; Chang, Woochul; Song, Byeong-Wook; Cha, Min-Ji; Lim, Soyeon; Choi, Eun Ju; Ham, Onju; Lee, Chang Youn; Park, Jun-Hee; Lee, Se-Yeon; Choi, Eunmi; Lee, Chungkeun; Lee, Myoungho; Lee, Moon-Hyoung; Kim, Sung-Hou; Jang, Yangsoo; Hwang, Ki-Chul

2011-01-01

Despite the safety and feasibility of mesenchymal stem cell (MSC) therapy, an optimal cell type has not yet emerged in terms of electromechanical integration in infarcted myocardium. We found that poor to moderate survival benefits of MSC-implanted rats were caused by incomplete electromechanical integration induced by tissue heterogeneity between myocytes and engrafted MSCs in the infarcted myocardium. Here, we report the development of cardiogenic cells from rat MSCs activated by phorbol myristate acetate, a PKC activator, that exhibited high expressions of cardiac-specific markers and Ca2+ homeostasis-related proteins and showed adrenergic receptor signaling by norepinephrine. Histological analysis showed high connexin 43 coupling, few inflammatory cells, and low fibrotic markers in myocardium implanted with these phorbol myristate acetate-activated MSCs. Infarct hearts implanted with these cells exhibited restoration of conduction velocity through decreased tissue heterogeneity and improved myocardial contractility. These findings have major implications for the development of better cell types for electromechanical integration of cell-based treatment for infarcted myocardium. PMID:21173226

Retinal ganglion cell responses to voltage and current stimulation in wild-type and rd1 mouse retinas

NASA Astrophysics Data System (ADS)

Goo, Yong Sook; Ye, Jang Hee; Lee, Seokyoung; Nam, Yoonkey; Ryu, Sang Baek; Kim, Kyung Hwan

2011-06-01

Retinal prostheses are being developed to restore vision for those with retinal diseases such as retinitis pigmentosa or age-related macular degeneration. Since neural prostheses depend upon electrical stimulation to control neural activity, optimal stimulation parameters for successful encoding of visual information are one of the most important requirements to enable visual perception. In this paper, we focused on retinal ganglion cell (RGC) responses to different stimulation parameters and compared threshold charge densities in wild-type and rd1 mice. For this purpose, we used in vitro retinal preparations of wild-type and rd1 mice. When the neural network was stimulated with voltage- and current-controlled pulses, RGCs from both wild-type and rd1 mice responded; however the temporal pattern of RGC response is very different. In wild-type RGCs, a single peak within 100 ms appears, while multiple peaks (approximately four peaks) with ~10 Hz rhythm within 400 ms appear in RGCs in the degenerated retina of rd1 mice. We find that an anodic phase-first biphasic voltage-controlled pulse is more efficient for stimulation than a biphasic current-controlled pulse based on lower threshold charge density. The threshold charge densities for activation of RGCs both with voltage- and current-controlled pulses are overall more elevated for the rd1 mouse than the wild-type mouse. Here, we propose the stimulus range for wild-type and rd1 retinas when the optimal modulation of a RGC response is possible.

Therapeutic potential of umbilical cord blood cells for type 1 diabetes mellitus.

PubMed

He, Binbin; Li, Xia; Yu, Haibo; Zhou, Zhiguang

2015-11-01

Type 1 diabetes mellitus (T1DM) is a chronic disorder that results from autoimmune-mediated destruction of pancreatic islet β-cells. However, to date, no conventional intervention has successfully treated the disease. The optimal therapeutic method for T1DM should effectively control the autoimmunity, restore immune homeostasis, preserve residual β-cells, reverse β-cell destruction, and protect the regenerated insulin-producing cells against re-attack. Umbilical cord blood is rich in regulatory T (T(reg)) cells and multiple types of stem cells that exhibit immunomodulating potential and hold promise in their ability to restore peripheral tolerance towards pancreatic islet β-cells through remodeling of immune responses and suppression of autoreactive T cells. Recently, reinfusion of autologous umbilical cord blood or immune cells from cord blood has been proposed as a novel therapy for T1DM, with the advantages of no risk to the donors, minimal ethical concerns, a low incidence of graft-versus-host disease and easy accessibility. In this review, we revisit the role of autologous umbilical cord blood or immune cells from cord blood-based applications for the treatment of T1DM. © 2015 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

A Novel Cell Type Enables B. subtilis to Escape from Unsuccessful Sporulation in Minimal Medium

PubMed Central

Defeu Soufo, Hervé Joël

2016-01-01

Sporulation is the most enduring survival strategy developed by several bacterial species. However, spore development of the model organism Bacillus subtilis has mainly been studied by means of media or conditions optimized for the induction of sporogenesis. Here, I show that during prolonged growth during stationary phase in minimal medium, B. subtilis undergoes an asymmetric cell division that produces small and round-shaped, DNA containing cells. In contrast to wild-type cells, mutants harboring spo0A or spoIIIE/sftA double mutations neither sporulate nor produce this special cell type, providing evidence that the small round cells emerge from the abortion of endospore formation. In most cases observed, the small round cells arise in the presence of sigma H but absence of sigma F activity, different from cases of abortive sporulation described for rich media. These data suggest that in minimal media, many cells are able to initiate but fail to complete spore development, and therefore return to normal growth as rods. This work reveals that the continuation of asymmetric cell division, which results in the formation of the small round cells, is a way for cells to delay or escape from—unsuccessful—sporulation. Based on these findings, I suggest to name the here described cell type as “dwarf cells” to distinguish them from the well-known minicells observed in mutants defective in septum placement or proper chromosome partitioning. PMID:27891124

Optimal design and operation of solid oxide fuel cell systems for small-scale stationary applications

NASA Astrophysics Data System (ADS)

Braun, Robert Joseph

The advent of maturing fuel cell technologies presents an opportunity to achieve significant improvements in energy conversion efficiencies at many scales; thereby, simultaneously extending our finite resources and reducing "harmful" energy-related emissions to levels well below that of near-future regulatory standards. However, before realization of the advantages of fuel cells can take place, systems-level design issues regarding their application must be addressed. Using modeling and simulation, the present work offers optimal system design and operation strategies for stationary solid oxide fuel cell systems applied to single-family detached dwellings. A one-dimensional, steady-state finite-difference model of a solid oxide fuel cell (SOFC) is generated and verified against other mathematical SOFC models in the literature. Fuel cell system balance-of-plant components and costs are also modeled and used to provide an estimate of system capital and life cycle costs. The models are used to evaluate optimal cell-stack power output, the impact of cell operating and design parameters, fuel type, thermal energy recovery, system process design, and operating strategy on overall system energetic and economic performance. Optimal cell design voltage, fuel utilization, and operating temperature parameters are found using minimization of the life cycle costs. System design evaluations reveal that hydrogen-fueled SOFC systems demonstrate lower system efficiencies than methane-fueled systems. The use of recycled cell exhaust gases in process design in the stack periphery are found to produce the highest system electric and cogeneration efficiencies while achieving the lowest capital costs. Annual simulations reveal that efficiencies of 45% electric (LHV basis), 85% cogenerative, and simple economic paybacks of 5--8 years are feasible for 1--2 kW SOFC systems in residential-scale applications. Design guidelines that offer additional suggestions related to fuel cell-stack sizing and operating strategy (base-load or load-following and cogeneration or electric-only) are also presented.

Comparative transfection of DNA into primary and transformed mammalian cells from different lineages

PubMed Central

2010-01-01

Background The delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Numerous chemical and physical approaches have been used to deliver the DNA, but their efficacy has been variable and is highly dependent on the cell type to be transfected. Results Studies were undertaken to evaluate and compare the transfection efficacy of several chemical reagents to that of the electroporation/nucleofection system using both adherent cells (primary and transformed airway epithelial cells and primary fibroblasts as well as embryonic stem cells) and cells in suspension (primary hematopoietic stem/progenitor cells and lymphoblasts). With the exception of HEK 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering DNA into the cells as determined by cell proliferation and GFP expression, respectively. Lipofectamine and nucleofection of HEK 293 were essentially equivalent in terms of toxicity and efficiency. Transient transfection efficiency in all the cell systems ranged from 40%-90%, with minimal toxicity and no apparent species specificity. Differences in efficiency and toxicity were cell type/system specific. Conclusions In general, the Amaxa electroporation/nucleofection system appears superior to other chemical systems. However, there are cell-type and species specific differences that need to be evaluated empirically to optimize the conditions for transfection efficiency and cell survival. PMID:20144189

A reliable method for spectrophotometric determination of glycine betaine in cell suspension and other systems.

PubMed

Valadez-Bustos, Ma Guadalupe; Aguado-Santacruz, Gerardo Armando; Tiessen-Favier, Axel; Robledo-Paz, Alejandrina; Muñoz-Orozco, Abel; Rascón-Cruz, Quintin; Santacruz-Varela, Amalio

2016-04-01

Glycine betaine is a quaternary ammonium compound that accumulates in a large variety of species in response to different types of stress. Glycine betaine counteracts adverse effects caused by abiotic factors, preventing the denaturation and inactivation of proteins. Thus, its determination is important, particularly for scientists focused on relating structural, biochemical, physiological, and/or molecular responses to plant water status. In the current work, we optimized the periodide technique for the determination of glycine betaine levels. This modification permitted large numbers of samples taken from a chlorophyllic cell line of the grass Bouteloua gracilis to be analyzed. Growth kinetics were assessed using the chlorophyllic suspension to determine glycine betaine levels in control (no stress) cells and cells osmotically stressed with 14 or 21% polyethylene glycol 8000. After glycine extraction, different wavelengths and reading times were evaluated in a spectrophotometer to determine the optimal quantification conditions for this osmolyte. Optimal results were obtained when readings were taken at a wavelength of 290 nm at 48 h after dissolving glycine betaine crystals in dichloroethane. We expect this modification to provide a simple, rapid, reliable, and cheap method for glycine betaine determination in plant samples and cell suspension cultures. Copyright © 2016 Elsevier Inc. All rights reserved.

Towards efficient and cost-effective inverted hybrid organic solar cells using inorganic semiconductor in the active layer

NASA Astrophysics Data System (ADS)

Imran, M.; Ikram, M.; Dilpazir, S.; Nafees, M.; Ali, S.; Geng, J.

2017-11-01

The article investigates the effects of NiO (p-type) and TiO2 (n-type) nanoparticles (NPs) on the performance of poly(3-hexylthiophene) (P3HT) and (phenyl-C61-butyric acid methylester) (PCBM) based devices with an inverse geometry. Various weight ratios of these nanoparticles were mixed in the polymer solution using 1,2-dichlorobenzene as solvent. An optimal amount of NPs-doped active layer exhibited higher power conversion efficiency (PCE) of 3.85% as compared to the reference cell, which exhibited an efficiency of 3.40% under white light illumination intensity of 100 mW/cm2. Enhanced PCE originates from increased film roughness and light harvesting due to increased absorption range upon mixing an optimal amount of NPs in the organic-based active layer. Further addition of NiO and TiO2 concentration relative to PCBM resulted in significant agglomeration of nanoparticles leading to degraded device parameters.

Optimization of lipid-assisted nanoparticle for disturbing neutrophils-related inflammation.

PubMed

Liu, Yang; Cao, Zhi-Ting; Xu, Cong-Fei; Lu, Zi-Dong; Luo, Ying-Li; Wang, Jun

2018-07-01

Inflammation is closely related to the development of many diseases and is commonly characterized by abnormal infiltration of immune cells, especially neutrophils. The current therapeutics of inflammatory diseases give little attention to direct modulation of these diseases with respect to immune cells. Nanoparticles are applied for efficient drug delivery into the disease-related immune cells, but their performance is significantly affected by their surface properties. In this study, to optimize the properties of nanoparticles for modulating neutrophils-related inflammation, we prepared a library of poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-b-PLGA)-based cationic lipid-assisted nanoparticles (CLANs) with different surface PEG density and surface charge. Optimized CLANs for neutrophils targeting were screened in high-fat diet (HFD)-induced type 2 diabetes (T2D) mice. Then, a CRISPR-Cas9 plasmid expressing a guide RNA (gRNA) targeting neutrophil elastase (NE) was encapsulated into the optimized CLAN and denoted as CLAN pCas9/gNE . After intravenous injection, CLAN pCas9/gNE successfully disrupted the NE gene of neutrophils and mitigated the insulin resistance of T2D mice via reducing the inflammation in epididymal white adipose tissue (eWAT) and in the liver. This strategy provides an example of abating the inflammatory microenvironment by directly modulating immune cells with nanoparticles carrying genome editing tools. Copyright © 2018 Elsevier Ltd. All rights reserved.

Laser capture microdissection of embryonic cells and preparation of RNA for microarray assays.

PubMed

Redmond, Latasha C; Pang, Christopher J; Dumur, Catherine; Haar, Jack L; Lloyd, Joyce A

2014-01-01

In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice-isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure(®) LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM.

Laser Capture Microdissection of Embryonic Cells and Preparation of RNA for Microarray Assays

PubMed Central

Redmond, Latasha C.; Pang, Christopher J.; Dumur, Catherine; Haar, Jack L.; Lloyd, Joyce A.

2014-01-01

In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice–isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure® LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM. PMID:24318813

Efficiency enhancement using a Zn1- x Ge x -O thin film as an n-type window layer in Cu2O-based heterojunction solar cells

NASA Astrophysics Data System (ADS)

Minami, Tadatsugu; Nishi, Yuki; Miyata, Toshihiro

2016-05-01

Efficiency enhancement was achieved in Cu2O-based heterojunction solar cells fabricated with a zinc-germanium-oxide (Zn1- x Ge x -O) thin film as the n-type window layer and a p-type Na-doped Cu2O (Cu2O:Na) sheet prepared by thermally oxidizing Cu sheets. The Ge content (x) dependence of the obtained photovoltaic properties of the heterojunction solar cells is mainly explained by the conduction band discontinuity that results from the electron affinity difference between Zn1- x Ge x -O and Cu2O:Na. The optimal value of x in Zn1- x Ge x -O thin films prepared by pulsed laser deposition was observed to be 0.62. An efficiency of 8.1% was obtained in a MgF2/Al-doped ZnO/Zn0.38Ge0.62-O/Cu2O:Na heterojunction solar cell.

Peptide-MHC Class I Tetramers Can Fail To Detect Relevant Functional T Cell Clonotypes and Underestimate Antigen-Reactive T Cell Populations.

PubMed

Rius, Cristina; Attaf, Meriem; Tungatt, Katie; Bianchi, Valentina; Legut, Mateusz; Bovay, Amandine; Donia, Marco; Thor Straten, Per; Peakman, Mark; Svane, Inge Marie; Ott, Sascha; Connor, Tom; Szomolay, Barbara; Dolton, Garry; Sewell, Andrew K

2018-04-01

Peptide-MHC (pMHC) multimers, usually used as streptavidin-based tetramers, have transformed the study of Ag-specific T cells by allowing direct detection, phenotyping, and enumeration within polyclonal T cell populations. These reagents are now a standard part of the immunology toolkit and have been used in many thousands of published studies. Unfortunately, the TCR-affinity threshold required for staining with standard pMHC multimer protocols is higher than that required for efficient T cell activation. This discrepancy makes it possible for pMHC multimer staining to miss fully functional T cells, especially where low-affinity TCRs predominate, such as in MHC class II-restricted responses or those directed against self-antigens. Several recent, somewhat alarming, reports indicate that pMHC staining might fail to detect the majority of functional T cells and have prompted suggestions that T cell immunology has become biased toward the type of cells amenable to detection with multimeric pMHC. We use several viral- and tumor-specific pMHC reagents to compare populations of human T cells stained by standard pMHC protocols and optimized protocols that we have developed. Our results confirm that optimized protocols recover greater populations of T cells that include fully functional T cell clonotypes that cannot be stained by regular pMHC-staining protocols. These results highlight the importance of using optimized procedures that include the use of protein kinase inhibitor and Ab cross-linking during staining to maximize the recovery of Ag-specific T cells and serve to further highlight that many previous quantifications of T cell responses with pMHC reagents are likely to have considerably underestimated the size of the relevant populations. Copyright © 2018 The Authors.

Interleukin (IL)-1 Receptor Signaling on Graft Parenchymal Cells Regulates Memory and De Novo Donor-Reactive CD8 T Cell Responses to Cardiac Allografts1

PubMed Central

Iida, Shoichi; Tsuda, Hidetoshi; Tanaka, Toshiaki; Kish, Danielle D.; Abe, Toyofumi; Su, Charles A.; Abe, Ryo; Tanabe, Kazunari; Valujskikh, Anna; Baldwin, William M.; Fairchild, Robert L.

2016-01-01

Reperfusion of organ allografts induces a potent inflammatory response that directs rapid memory T cell, neutrophil and macrophage graft infiltration and their activation to express functions mediating graft tissue injury. The role of cardiac allograft IL-1 receptor signaling in this early inflammation and the downstream primary alloimmune response was investigated. When compared to complete MHC-mismatched wild type cardiac allografts, IL-1R−/− allografts had marked decreases in endogenous memory CD8 T cell and neutrophil infiltration and expression of proinflammatory mediators at early times after transplant whereas endogenous memory CD4 T cell and macrophage infiltration was not decreased. IL-1R−/− allograft recipients also had marked decreases in de novo donor-reactive CD8, but not CD4, T cell development to IFN-γ-producing cells. CD8 T cell-mediated rejection of IL-1R−/− cardiac allografts took 3 weeks longer than wild type allografts. Cardiac allografts from reciprocal bone marrow reconstituted IL-1R−/−/wild type chimeric donors indicated that IL-1R signaling on graft non-hematopoietic-derived, but not bone marrow-derived, cells is required for the potent donor-reactive memory and primary CD8 T cell alloimmune responses observed in response to wild type allografts. These studies implicate IL-1R-mediated signals by allograft parenchymal cells in generating the stimuli provoking development and elicitation of optimal alloimmune responses to the grafts. PMID:26856697

Engineering zonal cartilage through bioprinting collagen type II hydrogel constructs with biomimetic chondrocyte density gradient.

PubMed

Ren, Xiang; Wang, Fuyou; Chen, Cheng; Gong, Xiaoyuan; Yin, Li; Yang, Liu

2016-07-20

Cartilage tissue engineering is a promising approach for repairing and regenerating cartilage tissue. To date, attempts have been made to construct zonal cartilage that mimics the cartilaginous matrix in different zones. However, little attention has been paid to the chondrocyte density gradient within the articular cartilage. We hypothesized that the chondrocyte density gradient plays an important role in forming the zonal distribution of extracellular matrix (ECM). In this study, collagen type II hydrogel/chondrocyte constructs were fabricated using a bioprinter. Three groups were created according to the total cell seeding density in collagen type II pre-gel: Group A, 2 × 10(7) cells/mL; Group B, 1 × 10(7) cells/mL; and Group C, 0.5 × 10(7) cells/mL. Each group included two types of construct: one with a biomimetic chondrocyte density gradient and the other with a single cell density. The constructs were cultured in vitro and harvested at 0, 1, 2, and 3 weeks for cell viability testing, reverse-transcription quantitative PCR (RT-qPCR), biochemical assays, and histological analysis. We found that total ECM production was positively correlated with the total cell density in the early culture stage, that the cell density gradient distribution resulted in a gradient distribution of ECM, and that the chondrocytes' biosynthetic ability was affected by both the total cell density and the cell distribution pattern. Our results suggested that zonal engineered cartilage could be fabricated by bioprinting collagen type II hydrogel constructs with a biomimetic cell density gradient. Both the total cell density and the cell distribution pattern should be optimized to achieve synergistic biological effects.

Stem cell technology for tendon regeneration: current status, challenges, and future research directions

PubMed Central

Lui, Pauline Po Yee

2015-01-01

Tendon injuries are a common cause of physical disability. They present a clinical challenge to orthopedic surgeons because injured tendons respond poorly to current treatments without tissue regeneration and the time required for rehabilitation is long. New treatment options are required. Stem cell-based therapies offer great potential to promote tendon regeneration due to their high proliferative, synthetic, and immunomodulatory activities as well as their potential to differentiate to the target cell types and undergo genetic modification. In this review, I first recapped the challenges of tendon repair by reviewing the anatomy of tendon. Next, I discussed the advantages and limitations of using different types of stem cells compared to terminally differentiated cells for tendon tissue engineering. The safety and efficacy of application of stem cells and their modified counterparts for tendon tissue engineering were then summarized after a systematic literature search in PubMed. The challenges and future research directions to enhance, optimize, and standardize stem cell-based therapies for augmenting tendon repair were then discussed. PMID:26715856

Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum

PubMed Central

Knecht, David A.; Silale, Augustinas; Traynor, David; Williams, Thomas D.; Thomason, Peter A.; Insall, Robert H.; Chubb, Jonathan R.; Kay, Robert R.; Veltman, Douwe M.

2018-01-01

Dictyostelium has a mature technology for molecular-genetic manipulation based around transfection using several different selectable markers, marker re-cycling, homologous recombination and insertional mutagenesis, all supported by a well-annotated genome. However this technology is optimized for mutant, axenic cells that, unlike non-axenic wild type, can grow in liquid medium. There is a pressing need for methods to manipulate wild type cells and ones with defects in macropinocytosis, neither of which can grow in liquid media. Here we present a panel of molecular genetic techniques based on the selection of Dictyostelium transfectants by growth on bacteria rather than liquid media. As well as extending the range of strains that can be manipulated, these techniques are faster than conventional methods, often giving usable numbers of transfected cells within a few days. The methods and plasmids described here allow efficient transfection with extrachromosomal vectors, as well as chromosomal integration at a ‘safe haven’ for relatively uniform cell-to-cell expression, efficient gene knock-in and knock-out and an inducible expression system. We have thus created a complete new system for the genetic manipulation of Dictyostelium cells that no longer requires cell feeding on liquid media. PMID:29847546

β-Globin-Expressing Definitive Erythroid Progenitor Cells Generated from Embryonic and Induced Pluripotent Stem Cell-Derived Sacs.

PubMed

Fujita, Atsushi; Uchida, Naoya; Haro-Mora, Juan J; Winkler, Thomas; Tisdale, John

2016-06-01

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell transfusion. However, when using traditional methods with embryoid bodies, ES cell-derived erythroid cells predominantly express embryonic type ɛ-globin, with lesser fetal type γ-globin and very little adult type β-globin. Furthermore, no β-globin expression is detected in iPS cell-derived erythroid cells. ES cell-derived sacs (ES sacs) have been recently used to generate functional platelets. Due to its unique structure, we hypothesized that ES sacs serve as hemangioblast-like progenitors capable to generate definitive erythroid cells that express β-globin. With our ES sac-derived erythroid differentiation protocol, we obtained ∼120 erythroid cells per single ES cell. Both primitive (ɛ-globin expressing) and definitive (γ- and β-globin expressing) erythroid cells were generated from not only ES cells but also iPS cells. Primitive erythropoiesis is gradually switched to definitive erythropoiesis during prolonged ES sac maturation, concurrent with the emergence of hematopoietic progenitor cells. Primitive and definitive erythroid progenitor cells were selected on the basis of glycophorin A or CD34 expression from cells within the ES sacs before erythroid differentiation. This selection and differentiation strategy represents an important step toward the development of in vitro erythroid cell production systems from pluripotent stem cells. Further optimization to improve expansion should be required for clinical application. Stem Cells 2016;34:1541-1552. © 2016 AlphaMed Press.

Paper-based device for rapid typing of secondary human blood groups.

PubMed

Li, Miaosi; Then, Whui Lyn; Li, Lizi; Shen, Wei

2014-01-01

We report the use of bioactive paper for typing of secondary human blood groups. Our recent work on using bioactive paper for human blood typing has led to the discovery of a new method for identifying haemagglutination of red blood cells. The primary human blood groups, i.e., ABO and RhD groups, have been successfully typed with this method. Clinically, however, many secondary blood groups can also cause fatal blood transfusion accidents, despite the fact that the haemagglutination reactions of secondary blood groups are generally weaker than those of the primary blood groups. We describe the design of a user-friendly sensor for rapid typing of secondary blood groups using bioactive paper. We also present mechanistic insights into interactions between secondary blood group antibodies and red blood cells obtained using confocal microscopy. Haemagglutination patterns under different conditions are revealed for optimization of the assay conditions.

Advances in fuel cell vehicle design

NASA Astrophysics Data System (ADS)

Bauman, Jennifer

Factors such as global warming, dwindling fossil fuel reserves, and energy security concerns combine to indicate that a replacement for the internal combustion engine (ICE) vehicle is needed. Fuel cell vehicles have the potential to address the problems surrounding the ICE vehicle without imposing any significant restrictions on vehicle performance, driving range, or refuelling time. Though there are currently some obstacles to overcome before attaining the widespread commercialization of fuel cell vehicles, such as improvements in fuel cell and battery durability, development of a hydrogen infrastructure, and reduction of high costs, the fundamental concept of the fuel cell vehicle is strong: it is efficient, emits zero harmful emissions, and the hydrogen fuel can be produced from various renewable sources. Therefore, research on fuel cell vehicle design is imperative in order to improve vehicle performance and durability, increase efficiency, and reduce costs. This thesis makes a number of key contributions to the advancement of fuel cell vehicle design within two main research areas: powertrain design and DC/DC converters. With regards to powertrain design, this research first analyzes various powertrain topologies and energy storage system types. Then, a novel fuel cell-battery-ultracapacitor topology is presented which shows reduced mass and cost, and increased efficiency, over other promising topologies found in the literature. A detailed vehicle simulator is created in MATLAB/Simulink in order to simulate and compare the novel topology with other fuel cell vehicle powertrain options. A parametric study is performed to optimize each powertrain and general conclusions for optimal topologies, as well as component types and sizes, for fuel cell vehicles are presented. Next, an analytical method to optimize the novel battery-ultracapacitor energy storage system based on maximizing efficiency, and minimizing cost and mass, is developed. This method can be applied to any system utilizing the novel battery-ultracapacitor energy storage system and is not limited in application to only fuel cell vehicles. With regards to DC/DC converters, it is important to design efficient and light-weight converters for use in fuel cell and other electric vehicles to improve overall vehicle fuel economy. Thus, this research presents a novel soft-switching method, the capacitor-switched regenerative snubber, for the high-power DC/DC boost converters commonly used in fuel cell vehicles. This circuit is shown to increase the efficiency and reduce the overall mass of the DC/DC boost converter.

The Effect of Temperature on the Optimization of Photovoltaic Cells Using Silvaco ATLAS Modeling

DTIC Science & Technology

2010-09-01

the Office of Management and Budget, Paperwork Reduction Project (0704-0188) Washington DC 20503. 1. AGENCY USE ONLY (Leave blank) 2. REPORT DATE ...September 2010 3. REPORT TYPE AND DATES COVERED Master’s Thesis 4. TITLE AND SUBTITLE The Effect of Temperature on the Optimization of...light source used in this thesis. The sun provides an average of 135 mW/cm2 of input power for objects in orbit around the Earth and as much as 100 mW

Preparation of poly-L-lysine functionalized magnetic nanoparticles and their influence on viability of cancer cells

NASA Astrophysics Data System (ADS)

Khmara, I.; Koneracka, M.; Kubovcikova, M.; Zavisova, V.; Antal, I.; Csach, K.; Kopcansky, P.; Vidlickova, I.; Csaderova, L.; Pastorekova, S.; Zatovicova, M.

2017-04-01

This study was aimed at development of biocompatible amino-functionalized magnetic nanoparticles as carriers of specific antibodies able to detect and/or target cancer cells. Poly-L-lysine (PLL)-modified magnetic nanoparticle samples with different PLL/Fe3O4 content were prepared and tested to define the optimal PLL/Fe3O4 weight ratio. The samples were characterized for particle size and morphology (SEM, TEM and DLS), and surface properties (zeta potential measurements). The optimal PLL/Fe3O4 weight ratio of 1.0 based on both zeta potential and DLS measurements was in agreement with the UV/VIS measurements. Magnetic nanoparticles with the optimal PLL content were conjugated with antibody specific for the cancer biomarker carbonic anhydrase IX (CA IX), which is induced by hypoxia, a physiologic stress present in solid tumors and linked with aggressive tumor behavior. CA IX is localized on the cell surface with the antibody-binding epitope facing the extracellular space and is therefore suitable for antibody-based targeting of tumor cells. Here we showed that PLL/Fe3O4 magnetic nanoparticles exhibit cytotoxic activities in a cell type-dependent manner and bind to cells expressing CA IX when conjugated with the CA IX-specific antibody. These data support further investigations of the CA IX antibody-conjugated, magnetic field-guided/activated nanoparticles as tools in anticancer strategies.

[Optimization of labeling and localizing bacterial membrane and nucleus with FM4-64 and Hoechst dyes].

PubMed

Wang, Jing; Han, Yanping; Yang, Ruifu; Zhao, Xingxu

2015-08-04

To observe cell membrane and nucleus in bacteria for subcellular localization. FM4-64 and Hoechst were dyed that can label cell membrane and nucleus, respectively. Both dyes were used to co-stain the membranes and nucleus of eight bacterial strains ( Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Yersinia pestis, Legionella pneumonia, Vibrio cholerae and Bacillus anthracis). E. coli was dyed with different dye concentrations and times and then observed by confocal fluorescence microscopic imaging. Fluorescence intensity of cell membrane and nucleus is affected by dye concentrations and times. The optimal conditions were determined as follows: staining cell membrane with 20 μg/mL FM4-64 for 1 min and cell nucleus with 20 μg/mL Hoechst for 20 min. Gram-negative bacteria were dyed better than gram-positive bacteria with FM4-64dye. FM4-64 and Hoechst can be used to stain membrane and nucleus in different types of bacteria. Co-staining bacterial membrane and nucleus provides the reference to observe cell structure in prokaryotes for studying subcellular localization.

Optimization of GaAs Nanowire Pin Junction Array Solar Cells by Using AlGaAs/GaAs Heterojunctions

NASA Astrophysics Data System (ADS)

Wu, Yao; Yan, Xin; Wei, Wei; Zhang, Jinnan; Zhang, Xia; Ren, Xiaomin

2018-04-01

We optimized the performance of GaAs nanowire pin junction array solar cells by introducing AlGaAs/GaAs heterejunctions. AlGaAs is used for the p type top segment for axial junctions and the p type outer shell for radial junctions. The AlGaAs not only serves as passivation layers for GaAs nanowires but also confines the optical generation in the active regions, reducing the recombination loss in heavily doped regions and the minority carrier recombination at the top contact. The results show that the conversion efficiency of GaAs nanowires can be greatly enhanced by using AlGaAs for the p segment instead of GaAs. A maximum efficiency enhancement of 8.42% has been achieved in this study. And for axial nanowire, by using AlGaAs for the top p segment, a relatively long top segment can be employed without degenerating device performance, which could facilitate the fabrication and contacting of nanowire array solar cells. While for radial nanowires, AlGaAs/GaAs nanowires show better tolerance to p-shell thickness and surface condition.

Optimization of GaAs Nanowire Pin Junction Array Solar Cells by Using AlGaAs/GaAs Heterojunctions.

PubMed

Wu, Yao; Yan, Xin; Wei, Wei; Zhang, Jinnan; Zhang, Xia; Ren, Xiaomin

2018-04-25

We optimized the performance of GaAs nanowire pin junction array solar cells by introducing AlGaAs/GaAs heterejunctions. AlGaAs is used for the p type top segment for axial junctions and the p type outer shell for radial junctions. The AlGaAs not only serves as passivation layers for GaAs nanowires but also confines the optical generation in the active regions, reducing the recombination loss in heavily doped regions and the minority carrier recombination at the top contact. The results show that the conversion efficiency of GaAs nanowires can be greatly enhanced by using AlGaAs for the p segment instead of GaAs. A maximum efficiency enhancement of 8.42% has been achieved in this study. And for axial nanowire, by using AlGaAs for the top p segment, a relatively long top segment can be employed without degenerating device performance, which could facilitate the fabrication and contacting of nanowire array solar cells. While for radial nanowires, AlGaAs/GaAs nanowires show better tolerance to p-shell thickness and surface condition.

High-performance integrated perovskite and organic solar cells with efficient near-infrared harvesting

NASA Astrophysics Data System (ADS)

Kim, Junghwan; Lee, Kwanghee

2016-09-01

The integration of planar-type perovskite (Eg 1.5 eV) solar cells (PSCs) with a bulk-heterojunction (BHJ) composite comprising a near-infrared (NIR) absorbing conjugated polymer (Eg < 1.4 eV) and a fullerene derivative is a promising approach to overcoming the narrow absorption limit of typical PSCs. Nevertheless, integrated solar cells (ISCs) suffer from low fill factors (FFs) and inefficient NIR harvesting, mainly due to poor charge transport in the BHJ films. Here, we successfully demonstrate highly efficient P-I-N perovskite/BHJ ISCs with an enhanced FF and improved NIR harvesting by introducing a novel n-type semiconducting polymer and a new processing additive into the BHJ films. The optimized ISCs exhibit a power conversion efficiency (PCE) of 16.36%, which far surpasses that of the reference PSCs ( 14.70%) due to the increased current density (Jsc 20.04 mA cm-2) resulting from the additional NIR harvesting. Meanwhile, the optimized ISCs maintain a high FF of 77% and an open-circuit voltage (Voc) of 1.06 V. These results indicate that this approach is a versatile means of overcoming the absorption and theoretical efficiency limits of state-ofthe- art PSCs.

Engineering Escherichia coli into a protein delivery system for mammalian cells.

PubMed

Reeves, Analise Z; Spears, William E; Du, Juan; Tan, Kah Yong; Wagers, Amy J; Lesser, Cammie F

2015-05-15

Many Gram-negative pathogens encode type 3 secretion systems, sophisticated nanomachines that deliver proteins directly into the cytoplasm of mammalian cells. These systems present attractive opportunities for therapeutic protein delivery applications; however, their utility has been limited by their inherent pathogenicity. Here, we report the reengineering of a laboratory strain of Escherichia coli with a tunable type 3 secretion system that can efficiently deliver heterologous proteins into mammalian cells, thereby circumventing the need for virulence attenuation. We first introduced a 31 kB region of Shigella flexneri DNA that encodes all of the information needed to form the secretion nanomachine onto a plasmid that can be directly propagated within E. coli or integrated into the E. coli chromosome. To provide flexible control over type 3 secretion and protein delivery, we generated plasmids expressing master regulators of the type 3 system from either constitutive or inducible promoters. We then constructed a Gateway-compatible plasmid library of type 3 secretion sequences to enable rapid screening and identification of sequences that do not perturb function when fused to heterologous protein substrates and optimized their delivery into mammalian cells. Combining these elements, we found that coordinated expression of the type 3 secretion system and modified target protein substrates produces a nonpathogenic strain that expresses, secretes, and delivers heterologous proteins into mammalian cells. This reengineered system thus provides a highly flexible protein delivery platform with potential for future therapeutic applications.

Tobacco BY-2 Media Component Optimization for a Cost-Efficient Recombinant Protein Production

PubMed Central

Häkkinen, Suvi T.; Reuter, Lauri; Nuorti, Ninni; Joensuu, Jussi J.; Rischer, Heiko; Ritala, Anneli

2018-01-01

Plant cells constitute an attractive platform for production of recombinant proteins as more and more animal-free products and processes are desired. One of the challenges in using plant cells as production hosts has been the costs deriving from expensive culture medium components. In this work, the aim was to optimize the levels of most expensive components in the nutrient medium without compromising the accumulation of biomass and recombinant protein yields. Wild-type BY-2 culture and transgenic tobacco BY-2 expressing green fluorescent protein–Hydrophobin I (GFP-HFBI) fusion protein were used to determine the most inexpensive medium composition. One particularly high-accumulating BY-2 clone, named ‘Hulk,’ produced 1.1 ± 0.2 g/l GFP-HFBI in suspension and kept its high performance during prolonged subculturing. In addition, both cultures were successfully cryopreserved enabling truly industrial application of this plant cell host. With the optimized culture medium, 43–55% cost reduction with regard to biomass and up to 69% reduction with regard to recombinant protein production was achieved. PMID:29434617

Tobacco BY-2 Media Component Optimization for a Cost-Efficient Recombinant Protein Production.

PubMed

Häkkinen, Suvi T; Reuter, Lauri; Nuorti, Ninni; Joensuu, Jussi J; Rischer, Heiko; Ritala, Anneli

2018-01-01

Plant cells constitute an attractive platform for production of recombinant proteins as more and more animal-free products and processes are desired. One of the challenges in using plant cells as production hosts has been the costs deriving from expensive culture medium components. In this work, the aim was to optimize the levels of most expensive components in the nutrient medium without compromising the accumulation of biomass and recombinant protein yields. Wild-type BY-2 culture and transgenic tobacco BY-2 expressing green fluorescent protein-Hydrophobin I (GFP-HFBI) fusion protein were used to determine the most inexpensive medium composition. One particularly high-accumulating BY-2 clone, named 'Hulk,' produced 1.1 ± 0.2 g/l GFP-HFBI in suspension and kept its high performance during prolonged subculturing. In addition, both cultures were successfully cryopreserved enabling truly industrial application of this plant cell host. With the optimized culture medium, 43-55% cost reduction with regard to biomass and up to 69% reduction with regard to recombinant protein production was achieved.

Rapid isolation of cancer cells using microfluidic deterministic lateral displacement structure.

PubMed

Liu, Zongbin; Huang, Fei; Du, Jinghui; Shu, Weiliang; Feng, Hongtao; Xu, Xiaoping; Chen, Yan

2013-01-01

This work reports a microfluidic device with deterministic lateral displacement (DLD) arrays allowing rapid and label-free cancer cell separation and enrichment from diluted peripheral whole blood, by exploiting the size-dependent hydrodynamic forces. Experiment data and theoretical simulation are presented to evaluate the isolation efficiency of various types of cancer cells in the microfluidic DLD structure. We also demonstrated the use of both circular and triangular post arrays for cancer cell separation in cell solution and blood samples. The device was able to achieve high cancer cell isolation efficiency and enrichment factor with our optimized design. Therefore, this platform with DLD structure shows great potential on fundamental and clinical studies of circulating tumor cells.

Labeling single cell for in-vivo study of cell fate mapping and lineage tracing

NASA Astrophysics Data System (ADS)

He, Sicong; Xu, Jin; Wu, Yi; Tian, Ye; Sun, Qiqi; Wen, Zilong; Qu, Jianan Y.

2018-02-01

Cell fate mapping and lineage tracing are significant ways to understanding the developmental origins of biological tissues. It requires labeling individual cells and tracing the development of their progeny. We develop an infrared laser-evoked gene operator heat-shock microscope system to achieve single-cell labeling in zebrafish. With a fluorescent thermometry technique, we measure the temperature increase in zebrafish tissues induced by infrared laser and identify the optimal heat shock conditions for single-cell gene induction in different types of zebrafish cells. We use this technique to study the fate mapping of T lymphocytes and discover the distinct waves of lymphopoiesis during the zebrafish development.

(Re)Building a Kidney

PubMed Central

Carroll, Thomas J.; Cleaver, Ondine; Gossett, Daniel R.; Hoshizaki, Deborah K.; Hubbell, Jeffrey A.; Humphreys, Benjamin D.; Jain, Sanjay; Jensen, Jan; Kaplan, David L.; Kesselman, Carl; Ketchum, Christian J.; Little, Melissa H.; McMahon, Andrew P.; Shankland, Stuart J.; Spence, Jason R.; Valerius, M. Todd; Wertheim, Jason A.; Wessely, Oliver; Zheng, Ying; Drummond, Iain A.

2017-01-01

(Re)Building a Kidney is a National Institute of Diabetes and Digestive and Kidney Diseases-led consortium to optimize approaches for the isolation, expansion, and differentiation of appropriate kidney cell types and the integration of these cells into complex structures that replicate human kidney function. The ultimate goals of the consortium are two-fold: to develop and implement strategies for in vitro engineering of replacement kidney tissue, and to devise strategies to stimulate regeneration of nephrons in situ to restore failing kidney function. Projects within the consortium will answer fundamental questions regarding human gene expression in the developing kidney, essential signaling crosstalk between distinct cell types of the developing kidney, how to derive the many cell types of the kidney through directed differentiation of human pluripotent stem cells, which bioengineering or scaffolding strategies have the most potential for kidney tissue formation, and basic parameters of the regenerative response to injury. As these projects progress, the consortium will incorporate systematic investigations in physiologic function of in vitro and in vivo differentiated kidney tissue, strategies for engraftment in experimental animals, and development of therapeutic approaches to activate innate reparative responses. PMID:28096308

Wavelength, beam size and type dependences of cerebral low-level light therapy: A Monte Carlo study on visible Chinese human

NASA Astrophysics Data System (ADS)

Li, Ting; Zhao, Yue; Duan, Meixue; Sun, Yunlong; Li, Kai

2014-02-01

Low level light therapy (LLLT) has been clinically utilized for many indications in medicine requiring protection from cell/tissue death, stimulation of healing and repair of injuries, pain reduction, swelling and inflammation. Presently, use of LLLT to treat stroke, traumatic brain injury, and cognitive dysfunction is attracting growing interest. Near-infrared light can penetrate into the brain tissue, allowing noninvasive treatment to be carried out with few treatment-related adverse events. Optimization of LLLT treatment effect is one key issue of the field; however, only a few experimental tests on mice for wavelength selection have been reported. We addressed this issue by low-cost, straightforward and quantitative comparisons on light dosage distribution in Visible Chinese human head with Monte Carlo modeling of light propagation. Optimized selection in wavelength, beam type and size were given based on comparisons among frequently-used setups (i.e., wavelengths: 660 nm, 810 nm, 980 nm; beam type: Gaussian and flat beam; beam diameter: 2 cm, 4 cm, 6cm).This study provided an efficient way to guide optimization of LLLT setup and selection on wavelength, beam type and size for clinical brain LLLT.

Open source machine-learning algorithms for the prediction of optimal cancer drug therapies.

PubMed

Huang, Cai; Mezencev, Roman; McDonald, John F; Vannberg, Fredrik

2017-01-01

Precision medicine is a rapidly growing area of modern medical science and open source machine-learning codes promise to be a critical component for the successful development of standardized and automated analysis of patient data. One important goal of precision cancer medicine is the accurate prediction of optimal drug therapies from the genomic profiles of individual patient tumors. We introduce here an open source software platform that employs a highly versatile support vector machine (SVM) algorithm combined with a standard recursive feature elimination (RFE) approach to predict personalized drug responses from gene expression profiles. Drug specific models were built using gene expression and drug response data from the National Cancer Institute panel of 60 human cancer cell lines (NCI-60). The models are highly accurate in predicting the drug responsiveness of a variety of cancer cell lines including those comprising the recent NCI-DREAM Challenge. We demonstrate that predictive accuracy is optimized when the learning dataset utilizes all probe-set expression values from a diversity of cancer cell types without pre-filtering for genes generally considered to be "drivers" of cancer onset/progression. Application of our models to publically available ovarian cancer (OC) patient gene expression datasets generated predictions consistent with observed responses previously reported in the literature. By making our algorithm "open source", we hope to facilitate its testing in a variety of cancer types and contexts leading to community-driven improvements and refinements in subsequent applications.

Tumor-Specific Chromosome Mis-Segregation Controls Cancer Plasticity by Maintaining Tumor Heterogeneity

PubMed Central

Hu, Yuanjie; Ru, Ning; Xiao, Huasheng; Chaturbedi, Abhishek; Hoa, Neil T.; Tian, Xiao-Jun; Zhang, Hang; Ke, Chao; Yan, Fengrong; Nelson, Jodi; Li, Zhenzhi; Gramer, Robert; Yu, Liping; Siegel, Eric; Zhang, Xiaona; Jia, Zhenyu; Jadus, Martin R.; Limoli, Charles L.; Linskey, Mark E.; Xing, Jianhua; Zhou, Yi-Hong

2013-01-01

Aneuploidy with chromosome instability is a cancer hallmark. We studied chromosome 7 (Chr7) copy number variation (CNV) in gliomas and in primary cultures derived from them. We found tumor heterogeneity with cells having Chr7-CNV commonly occurs in gliomas, with a higher percentage of cells in high-grade gliomas carrying more than 2 copies of Chr7, as compared to low-grade gliomas. Interestingly, all Chr7-aneuploid cell types in the parental culture of established glioma cell lines reappeared in single-cell-derived subcultures. We then characterized the biology of three syngeneic glioma cultures dominated by different Chr7-aneuploid cell types. We found phenotypic divergence for cells following Chr7 mis-segregation, which benefited overall tumor growth in vitro and in vivo. Mathematical modeling suggested the involvement of chromosome instability and interactions among cell subpopulations in restoring the optimal equilibrium of tumor cell types. Both our experimental data and mathematical modeling demonstrated that the complexity of tumor heterogeneity could be enhanced by the existence of chromosomes with structural abnormality, in addition to their mis-segregations. Overall, our findings show, for the first time, the involvement of chromosome instability in maintaining tumor heterogeneity, which underlies the enhanced growth, persistence and treatment resistance of cancers. PMID:24282558

Advanced Solar Cells for Satellite Power Systems

NASA Technical Reports Server (NTRS)

Flood, Dennis J.; Weinberg, Irving

1994-01-01

The multiple natures of today's space missions with regard to operational lifetime, orbital environment, cost and size of spacecraft, to name just a few, present such a broad range of performance requirements to be met by the solar array that no single design can suffice to meet them all. The result is a demand for development of specialized solar cell types that help to optimize overall satellite performance within a specified cost range for any given space mission. Historically, space solar array performance has been optimized for a given mission by tailoring the features of silicon solar cells to account for the orbital environment and average operating conditions expected during the mission. It has become necessary to turn to entirely new photovoltaic materials and device designs to meet the requirements of future missions, both in the near and far term. This paper will outline some of the mission drivers and resulting performance requirements that must be met by advanced solar cells, and provide an overview of some of the advanced cell technologies under development to meet them. The discussion will include high efficiency, radiation hard single junction cells; monolithic and mechanically stacked multiple bandgap cells; and thin film cells.

Advanced solar cells for satellite power systems

NASA Astrophysics Data System (ADS)

Flood, Dennis J.; Weinberg, Irving

1994-11-01

The multiple natures of today's space missions with regard to operational lifetime, orbital environment, cost and size of spacecraft, to name just a few, present such a broad range of performance requirements to be met by the solar array that no single design can suffice to meet them all. The result is a demand for development of specialized solar cell types that help to optimize overall satellite performance within a specified cost range for any given space mission. Historically, space solar array performance has been optimized for a given mission by tailoring the features of silicon solar cells to account for the orbital environment and average operating conditions expected during the mission. It has become necessary to turn to entirely new photovoltaic materials and device designs to meet the requirements of future missions, both in the near and far term. This paper will outline some of the mission drivers and resulting performance requirements that must be met by advanced solar cells, and provide an overview of some of the advanced cell technologies under development to meet them. The discussion will include high efficiency, radiation hard single junction cells; monolithic and mechanically stacked multiple bandgap cells; and thin film cells.

A Single-Cell View of the BtsSR/YpdAB Pyruvate Sensing Network in Escherichia coli and Its Biological Relevance.

PubMed

Vilhena, Cláudia; Kaganovitch, Eugen; Shin, Jae Yen; Grünberger, Alexander; Behr, Stefan; Kristoficova, Ivica; Brameyer, Sophie; Kohlheyer, Dietrich; Jung, Kirsten

2018-01-01

Fluctuating environments and individual physiological diversity force bacteria to constantly adapt and optimize the uptake of substrates. We focus here on two very similar two-component systems (TCSs) of Escherichia coli belonging to the LytS/LytTR family: BtsS/BtsR (formerly YehU/YehT) and YpdA/YpdB. Both TCSs respond to extracellular pyruvate, albeit with different affinities, typically during postexponential growth, and each system regulates expression of a single transporter gene, yjiY and yhjX , respectively. To obtain insights into the biological significance of these TCSs, we analyzed the activation of the target promoters at the single-cell level. We found unimodal cell-to-cell variability; however, the degree of variance was strongly influenced by the available nutrients and differed between the two TCSs. We hypothesized that activation of either of the TCSs helps individual cells to replenish carbon resources. To test this hypothesis, we compared wild-type cells with the btsSR ypdAB mutant under two metabolically modulated conditions: protein overproduction and persister formation. Although all wild-type cells were able to overproduce green fluorescent protein (GFP), about half of the btsSR ypdAB population was unable to overexpress GFP. Moreover, the percentage of persister cells, which tolerate antibiotic stress, was significantly lower in the wild-type cells than in the btsSR ypdAB population. Hence, we suggest that the BtsS/BtsR and YpdA/YpdB network contributes to a balancing of the physiological state of all cells within a population. IMPORTANCE Histidine kinase/response regulator (HK/RR) systems enable bacteria to respond to environmental and physiological fluctuations. Escherichia coli and other members of the Enterobacteriaceae possess two similar LytS/LytTR-type HK/RRs, BtsS/BtsR (formerly YehU/YehT) and YpdA/YpdB, which form a functional network. Both systems are activated in response to external pyruvate, typically when cells face overflow metabolism during post-exponential growth. Single-cell analysis of the activation of their respective target genes yjiY and yhjX revealed cell-to-cell variability, and the range of variation was strongly influenced by externally available nutrients. Based on the phenotypic characterization of a btsSR ypdAB mutant compared to the parental strain, we suggest that this TCS network supports an optimization of the physiological state of the individuals within the population. Copyright © 2017 American Society for Microbiology.

Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments.

PubMed

Van Valen, David A; Kudo, Takamasa; Lane, Keara M; Macklin, Derek N; Quach, Nicolas T; DeFelice, Mialy M; Maayan, Inbal; Tanouchi, Yu; Ashley, Euan A; Covert, Markus W

2016-11-01

Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domains of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.

Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments

DOE PAGES

Van Valen, David A.; Kudo, Takamasa; Lane, Keara M.; ...

2016-11-04

Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domainsmore » of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.« less

Gene Editing in Human Lymphoid Cells: Role for Donor DNA, Type of Genomic Nuclease and Cell Selection Method.

PubMed

Zotova, Anastasia; Lopatukhina, Elena; Filatov, Alexander; Khaitov, Musa; Mazurov, Dmitriy

2017-11-02

Programmable endonucleases introduce DNA breaks at specific sites, which are repaired by non-homologous end joining (NHEJ) or homology recombination (HDR). Genome editing in human lymphoid cells is challenging as these difficult-to-transfect cells may also inefficiently repair DNA by HDR. Here, we estimated efficiencies and dynamics of knockout (KO) and knockin (KI) generation in human T and B cell lines depending on repair template, target loci and types of genomic endonucleases. Using zinc finger nuclease (ZFN), we have engineered Jurkat and CEM cells with the 8.2 kb human immunodeficiency virus type 1 (HIV-1) ∆Env genome integrated at the adeno-associated virus integration site 1 (AAVS1) locus that stably produce virus particles and mediate infection upon transfection with helper vectors. Knockouts generated by ZFN or clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) double nicking techniques were comparably efficient in lymphoid cells. However, unlike polyclonal sorted cells, gene-edited cells selected by cloning exerted tremendous deviations in functionality as estimated by replication of HIV-1 and human T cell leukemia virus type 1 (HTLV-1) in these cells. Notably, the recently reported high-fidelity eCas9 1.1 when combined to the nickase mutation displayed gene-dependent decrease in on-target activity. Thus, the balance between off-target effects and on-target efficiency of nucleases, as well as choice of the optimal method of edited cell selection should be taken into account for proper gene function validation in lymphoid cells.

Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Valen, David A.; Kudo, Takamasa; Lane, Keara M.

Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domainsmore » of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.« less

Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments

PubMed Central

Van Valen, David A.; Lane, Keara M.; Quach, Nicolas T.; Maayan, Inbal

2016-01-01

Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domains of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems. PMID:27814364

Functional and cytometric examination of different human lung epithelial cell types as drug transport barriers.

PubMed

Min, Kyoung Ah; Rosania, Gus R; Kim, Chong-Kook; Shin, Meong Cheol

2016-03-01

To develop inhaled medications, various cell culture models have been used to examine the transcellular transport or cellular uptake properties of small molecules. For the reproducible high throughput screening of the inhaled drug candidates, a further verification of cell architectures as drug transport barriers can contribute to establishing appropriate in vitro cell models. In the present study, side-by-side experiments were performed to compare the structure and transport function of three lung epithelial cells (Calu-3, normal human bronchial primary cells (NHBE), and NL-20). The cells were cultured on the nucleopore membranes in the air-liquid interface (ALI) culture conditions, with cell culture medium in the basolateral side only, starting from day 1. In transport assays, paracellular transport across all three types of cells appeared to be markedly different with the NHBE or Calu-3 cells, showing low paracellular permeability and high TEER values, while the NL-20 cells showed high paracellular permeability and low TEER. Quantitative image analysis of the confocal microscope sections further confirmed that the Calu-3 cells formed intact cell monolayers in contrast to the NHBE and NL-20 cells with multilayers. Among three lung epithelial cell types, the Calu-3 cell cultures under the ALI condition showed optimal cytometric features for mimicking the biophysical characteristics of in vivo airway epithelium. Therefore, the Calu-3 cell monolayers could be used as functional cell barriers for the lung-targeted drug transport studies.

Functional and cytometric examination of different human lung epithelial cell types as drug transport barriers

PubMed Central

Min, Kyoung Ah; Rosania, Gus R.; Kim, Chong-Kook; Shin, Meong Cheol

2016-01-01

To develop inhaled medications, various cell culture models have been used to examine the transcellular transport or cellular uptake properties of small molecules. For the reproducible high throughput screening of the inhaled drug candidates, a further verification of cell architectures as drug transport barriers can contribute to establishing appropriate in vitro cell models. In the present study, side-by-side experiments were performed to compare the structure and transport function of three lung epithelial cells (Calu-3, normal human bronchial primary cells (NHBE), and NL-20). The cells were cultured on the nucleopore membranes in the air-liquid interface (ALI) culture conditions, with cell culture medium in the basolateral side only, starting from day 1. In transport assays, paracellular transport across all three types of cells appeared to be markedly different with the NHBE or Calu-3 cells, showing low paracellular permeability and high TEER values, while the NL-20 cells showed high paracellular permeability and low TEER. Quantitative image analysis of the confocal microscope sections further confirmed that the Calu-3 cells formed intact cell monolayers in contrast to the NHBE and NL-20 cells with multilayers. Among three lung epithelial cell types, the Calu-3 cell cultures under the ALI condition showed optimal cytometric features for mimicking the biophysical characteristics of in vivo airway epithelium. Therefore, the Calu-3 cell monolayers could be used as functional cell barriers for the lung-targeted drug transport studies. PMID:26746641

An Efficient Electroporation Protocol for the Genetic Modification of Mammalian Cells

PubMed Central

Chicaybam, Leonardo; Barcelos, Camila; Peixoto, Barbara; Carneiro, Mayra; Limia, Cintia Gomez; Redondo, Patrícia; Lira, Carla; Paraguassú-Braga, Flávio; Vasconcelos, Zilton Farias Meira De; Barros, Luciana; Bonamino, Martin Hernán

2017-01-01

Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Electroporation using square-wave generating devices, like Lonza’s Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work, we show that our in-house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology. PMID:28168187

Thin-film module circuit design: Practical and reliability aspects

NASA Technical Reports Server (NTRS)

Daiello, R. V.; Twesme, E. N.

1985-01-01

This paper will address several aspects of the design and construction of submodules based on thin film amorphous silicon (a-Si) p i n solar cells. Starting from presently attainable single cell characteristics, and a realistic set of specifications, practical module designs are discussed from the viewpoints of efficient designs, the fabrication requirements, and reliability concerns. The examples center mostly on series interconnected modules of the superstrate type with detailed discussions of each portion of the structure in relation to its influence on module efficiency. Emphasis is placed on engineering topics such as: area coverage, optimal geometries, and cost and reliability. Practical constraints on achieving optimal designs, along with some examples of potential pitfalls in the manufacture and subsequent performance of a-Si modules are discussed.

Electrospun Collagen/Silk Tissue Engineering Scaffolds: Fiber Fabrication, Post-Treatment Optimization, and Application in Neural Differentiation of Stem Cells

NASA Astrophysics Data System (ADS)

Zhu, Bofan

Biocompatible scaffolds mimicking the locally aligned fibrous structure of native extracellular matrix (ECM) are in high demand in tissue engineering. In this thesis research, unidirectionally aligned fibers were generated via a home-built electrospinning system. Collagen type I, as a major ECM component, was chosen in this study due to its support of cell proliferation and promotion of neuroectodermal commitment in stem cell differentiation. Synthetic dragline silk proteins, as biopolymers with remarkable tensile strength and superior elasticity, were also used as a model material. Good alignment, controllable fiber size and morphology, as well as a desirable deposition density of fibers were achieved via the optimization of solution and electrospinning parameters. The incorporation of silk proteins into collagen was found to significantly enhance mechanical properties and stability of electrospun fibers. Glutaraldehyde (GA) vapor post-treatment was demonstrated as a simple and effective way to tune the properties of collagen/silk fibers without changing their chemical composition. With 6-12 hours GA treatment, electrospun collagen/silk fibers were not only biocompatible, but could also effectively induce the polarization and neural commitment of stem cells, which were optimized on collagen rich fibers due to the unique combination of biochemical and biophysical cues imposed to cells. Taken together, electrospun collagen rich composite fibers are mechanically strong, stable and provide excellent cell adhesion. The unidirectionally aligned fibers can accelerate neural differentiation of stem cells, representing a promising therapy for neural tissue degenerative diseases and nerve injuries.

Genipin-crosslinked microcarriers mediating hepatocellular aggregates formation and functionalities.

PubMed

Lau, Ting Ting; Wang, Chunming; Png, Sze Wei; Su, Kai; Wang, Dong-An

2011-01-01

In engineered regenerative medicine, various types of scaffolds have been customized to pursue the optimal environment for different types of therapeutic cells. In liver therapeutic research, hepatocytes require attachment to solid anchors for survival and proliferation before they could grow into cellular aggregates with enhanced functionalities. Among the various biomaterials scaffolds and vehicles, microspherical cell carriers are suited to these requirements. Individual spheres may provide two-dimensional (2D) cell-affinitive surfaces for cell adhesion and spreading; whereas multiple microcarriers may form three-dimensional (3D) matrices with inter-spherical space for cell expansion and multicellular aggregation. In this study, we culture human liver carcinoma cell line (HepG2) cells on genipin-crosslinked gelatin microspheres of two different sizes. Results suggest that both microcarriers support cell adhesion, proliferation, and spontaneous formation of hepatocellular aggregates, among which the spheres with bigger size (200-300 μm) seem more favorable than the smaller ones in terms of aggregate formation and liver specific functionalities. These findings suggest that the genipin-crosslinked microcarrier is a competent vehicle for liver cell delivery. Copyright © 2010 Wiley Periodicals, Inc.

Role of Chemokines and Trafficking of Immune Cells in Parasitic Infections

PubMed Central

McGovern, Kathryn E.; Wilson, Emma H.

2014-01-01

Parasites are diverse eukaryotic pathogens that can have complex life cycles. Their clearance, or control within a mammalian host requires the coordinated effort of the immune system. The cell types recruited to areas of infection can combat the disease, promote parasite replication and survival, or contribute to disease pathology. Location and timing of cell recruitment can be crucial. In this review, we explore the role chemokines play in orchestrating and balancing the immune response to achieve optimal control of parasite replication without promoting pathology. PMID:25383073

Status of nickel-hydrogen cell technology

NASA Technical Reports Server (NTRS)

Warnock, D. R.

1980-01-01

Nickel hydrogen cell technology has been developed which solves the problems of thermal management, oxygen management, electrolyte management, and electrical and mechanical design peculiar to this new type of battery. This technology was weight optimized for low orbit operation using computer modeling programs but is near optimum for other orbits. Cells ranging in capacity up to about 70 ampere-hours can be made from components of a single standard size and are available from two manufacturers. The knowledge gained is now being applied to the development of two extensions to the basic design: a second set of larger standard components that will cover the capacity range up to 150 ampere-hours; and the development of multicell common pressure vessel modules to reduce volume, cost and weight. A manufacturing technology program is planned to optimize the producibility of the cell design and reduce cost. The most important areas for further improvement are life and reliability which are governed by electrode and separator technology.

An Investigation into III-V Compounds to Reach 20% Efficiency with Minimum Cell Thickness in Ultrathin-Film Solar Cells

NASA Astrophysics Data System (ADS)

Haque, K. A. S. M. Ehteshamul; Galib, Md. Mehedi Hassan

2013-10-01

III-V single-junction solar cells have already achieved very high efficiency levels. However, their use in terrestrial applications is limited by the high fabrication cost. High-efficiency, ultrathin-film solar cells can effectively solve this problem, as their material requirement is minimum. This work presents a comparison among several III-V compounds that have high optical absorption capability as well as optimum bandgap (around 1.4 eV) for use as solar cell absorbers. The aim is to observe and compare the ability of these materials to reach a target efficiency level of 20% with minimum possible cell thickness. The solar cell considered has an n-type ZnSe window layer, an n-type Al0.1Ga0.9As emitter layer, and a p-type Ga0.5In0.5P back surface field (BSF) layer. Ge is used as the substrate. In the initial design, a p-type InP base was sandwiched between the emitter and the BSF layer, and the design parameters for the device were optimized by analyzing the simulation outcomes with ADEPT/F, a one-dimensional (1D) simulation tool. Then, the minimum cell thickness that achieves 20% efficiency was determined by observing the efficiency variation with cell thickness. Afterwards, the base material was changed to a few other selected III-V compounds, and for each case, the minimum cell thickness was determined in a similar manner. Finally, these cell thickness values were compared and analyzed to identify more effective base layer materials for III-V single-junction solar cells.

In vitro generation of renal tubular epithelial cells from fibroblasts: implications for precision and regenerative medicine in nephrology.

PubMed

Wyatt, Christina M; Dubois, Nicole

2017-02-01

Prior efforts to generate renal epithelial cells in vitro have relied on pluripotent or bone marrow-derived mesenchymal stem cells. A recent publication in Nature Cell Biology describes the generation of induced tubular epithelial cells from fibroblasts, potentially offering a novel platform for personalized drug toxicity screening and in vitro disease modeling. This report serves as a promising proof of principle study and opens future research directions, including the optimization of the reprogramming process, efficient translation to adult human fibroblasts, and the generation of highly specific functional renal cell types. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

The neurotrophic effects of different human dental mesenchymal stem cells.

PubMed

Kolar, Mallappa K; Itte, Vinay N; Kingham, Paul J; Novikov, Lev N; Wiberg, Mikael; Kelk, Peyman

2017-10-03

The current gold standard treatment for peripheral nerve injury is nerve grafting but this has disadvantages such as donor site morbidity. New techniques focus on replacing these grafts with nerve conduits enhanced with growth factors and/or various cell types such as mesenchymal stem cells (MSCs). Dental-MSCs (D-MSCs) including stem cells obtained from apical papilla (SCAP), dental pulp stem cells (DPSC), and periodontal ligament stem cells (PDLSC) are potential sources of MSCs for nerve repair. Here we present the characterization of various D-MSCs from the same human donors for peripheral nerve regeneration. SCAP, DPSC and PDLSC expressed BDNF, GDNF, NGF, NTF3, ANGPT1 and VEGFA growth factor transcripts. Conditioned media from D-MSCs enhanced neurite outgrowth in an in vitro assay. Application of neutralizing antibodies showed that brain derived neurotrophic factor plays an important mechanistic role by which the D-MSCs stimulate neurite outgrowth. SCAP, DPSC and PDLSC were used to treat a 10 mm nerve gap defect in a rat sciatic nerve injury model. All the stem cell types significantly enhanced axon regeneration after two weeks and showed neuroprotective effects on the dorsal root ganglia neurons. Overall the results suggested SCAP to be the optimal dental stem cell type for peripheral nerve repair.

A model for genetic and epigenetic regulatory networks identifies rare pathways for transcription factor induced pluripotency

NASA Astrophysics Data System (ADS)

Artyomov, Maxim; Meissner, Alex; Chakraborty, Arup

2010-03-01

Most cells in an organism have the same DNA. Yet, different cell types express different proteins and carry out different functions. This is because of epigenetic differences; i.e., DNA in different cell types is packaged distinctly, making it hard to express certain genes while facilitating the expression of others. During development, upon receipt of appropriate cues, pluripotent embryonic stem cells differentiate into diverse cell types that make up the organism (e.g., a human). There has long been an effort to make this process go backward -- i.e., reprogram a differentiated cell (e.g., a skin cell) to pluripotent status. Recently, this has been achieved by transfecting certain transcription factors into differentiated cells. This method does not use embryonic material and promises the development of patient-specific regenerative medicine, but it is inefficient. The mechanisms that make reprogramming rare, or even possible, are poorly understood. We have developed the first computational model of transcription factor-induced reprogramming. Results obtained from the model are consistent with diverse observations, and identify the rare pathways that allow reprogramming to occur. If validated, our model could be further developed to design optimal strategies for reprogramming and shed light on basic questions in biology.

Multifactorial Experimental Design to Optimize the Anti-Inflammatory and Proangiogenic Potential of Mesenchymal Stem Cell Spheroids.

PubMed

Murphy, Kaitlin C; Whitehead, Jacklyn; Falahee, Patrick C; Zhou, Dejie; Simon, Scott I; Leach, J Kent

2017-06-01

Mesenchymal stem cell therapies promote wound healing by manipulating the local environment to enhance the function of host cells. Aggregation of mesenchymal stem cells (MSCs) into three-dimensional spheroids increases cell survival and augments their anti-inflammatory and proangiogenic potential, yet there is no consensus on the preferred conditions for maximizing spheroid function in this application. The objective of this study was to optimize conditions for forming MSC spheroids that simultaneously enhance their anti-inflammatory and proangiogenic nature. We applied a design of experiments (DOE) approach to determine the interaction between three input variables (number of cells per spheroid, oxygen tension, and inflammatory stimulus) on MSC spheroids by quantifying secretion of prostaglandin E 2 (PGE 2 ) and vascular endothelial growth factor (VEGF), two potent molecules in the MSC secretome. DOE results revealed that MSC spheroids formed with 40,000 cells per spheroid in 1% oxygen with an inflammatory stimulus (Spheroid 1) would exhibit enhanced PGE 2 and VEGF production versus those formed with 10,000 cells per spheroid in 21% oxygen with no inflammatory stimulus (Spheroid 2). Compared to Spheroid 2, Spheroid 1 produced fivefold more PGE 2 and fourfold more VEGF, providing the opportunity to simultaneously upregulate the secretion of these factors from the same spheroid. The spheroids induced macrophage polarization, sprout formation with endothelial cells, and keratinocyte migration in a human skin equivalent model-demonstrating efficacy on three key cell types that are dysfunctional in chronic non-healing wounds. We conclude that DOE-based analysis effectively identifies optimal culture conditions to enhance the anti-inflammatory and proangiogenic potential of MSC spheroids. Stem Cells 2017;35:1493-1504. © 2017 AlphaMed Press.

Model of Exploratory Search for Mating Partners by Fission Yeast

NASA Astrophysics Data System (ADS)

Hurwitz, Daniel; Bendezu, Felipe; Martin, Sophie; Vavylonis, Dimitrios

2014-03-01

During conditions of nitrogen starvation, the model eukaryote S. pombe (fission yeast) undergoes sexual sporulation. Because fission yeast are non-motile, contact between opposite mating types during spore formation is accomplished by polarizing growth, via the Rho GTP-ase Cdc42, in each mating type towards the selected mate, a process known as shmooing. Recent findings showed that cells pick one of their neighboring compatible mates by randomizing the position of the Cdc42 complex about the cell membrane, such that the complex is stabilized near areas of high concentration of the opposite mating type pheromone. We developed Monte Carlo simulations to model partner finding in populations of mating cells and in small cell clusters. We assume that pheromones are secreted at the site of Cdc42 accumulation and that the Cdc42 dwell time increases in response to increasing pheromone concentration. We measured the number of cells that succeed in successful reciprocal pairing, the number of cells that were unable to find a partner, and the number of cells that picked a partner already engaged with another cell. For optimal cell pairing, we find the pheromone concentration decay length is around 1 micron, of order the cell size. We show that non-linear response of Cdc42 dwell time to pheromone concentration improves the number of successful pairs for a given spatial cell distribution. We discuss how these results compare to non-exploratory pairing mechanisms.

Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells.

PubMed

Rostovskaya, Maria; Bredenkamp, Nicholas; Smith, Austin

2015-10-19

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification. © 2015 The Authors.

Are V1 Simple Cells Optimized for Visual Occlusions? A Comparative Study

PubMed Central

Bornschein, Jörg; Henniges, Marc; Lücke, Jörg

2013-01-01

Simple cells in primary visual cortex were famously found to respond to low-level image components such as edges. Sparse coding and independent component analysis (ICA) emerged as the standard computational models for simple cell coding because they linked their receptive fields to the statistics of visual stimuli. However, a salient feature of image statistics, occlusions of image components, is not considered by these models. Here we ask if occlusions have an effect on the predicted shapes of simple cell receptive fields. We use a comparative approach to answer this question and investigate two models for simple cells: a standard linear model and an occlusive model. For both models we simultaneously estimate optimal receptive fields, sparsity and stimulus noise. The two models are identical except for their component superposition assumption. We find the image encoding and receptive fields predicted by the models to differ significantly. While both models predict many Gabor-like fields, the occlusive model predicts a much sparser encoding and high percentages of ‘globular’ receptive fields. This relatively new center-surround type of simple cell response is observed since reverse correlation is used in experimental studies. While high percentages of ‘globular’ fields can be obtained using specific choices of sparsity and overcompleteness in linear sparse coding, no or only low proportions are reported in the vast majority of studies on linear models (including all ICA models). Likewise, for the here investigated linear model and optimal sparsity, only low proportions of ‘globular’ fields are observed. In comparison, the occlusive model robustly infers high proportions and can match the experimentally observed high proportions of ‘globular’ fields well. Our computational study, therefore, suggests that ‘globular’ fields may be evidence for an optimal encoding of visual occlusions in primary visual cortex. PMID:23754938

Heterogeneous Human Periodontal Ligament-Committed Progenitor and Stem Cell Populations Exhibit a Unique Cementogenic Property Under In Vitro and In Vivo Conditions.

PubMed

Shinagawa-Ohama, Rei; Mochizuki, Mai; Tamaki, Yuichi; Suda, Naoto; Nakahara, Taka

2017-05-01

An undesirable complication that arises during dental treatments is external apical-root resorption, which causes root-cementum and root-dentin loss. To induce de novo cementogenesis, stem cell therapy is required. Cementum-forming cells (cementoblasts) are known to be differentiated from periodontal-lineage mesenchymal stem cells (MSCs), which are derived from the dental follicle (DF) in developing tissues and the periodontal ligament (PDL) in adult tissues, but the periodontal-lineage MSC type that is optimal for inducing de novo cementogenesis remains unidentified, as does the method to isolate these cells from harvested tissues. Thus, we investigated the cementogenic potential of DF- and PDL-derived MSCs that were isolated by using two widely used cell-isolation methods: enzymatic digestion and outgrowth (OG) methods. DF- and PDL-derived cells isolated by using both methods proliferated actively, and all four isolated cell types showed MSC gene/protein expression phenotype and ability to differentiate into adipogenic and chondrogenic lineages. Furthermore, cementogenic-potential analysis revealed that all cell types produced alizarin red S-positive mineralized materials in in vitro cultures. However, PDL-OG cells presented unique cementogenic features, such as nodular formation of mineralized deposits displaying a cellular intrinsic fiber cementum-like structure, as well as a higher expression of cementoblast-specific genes than in the other cell types. Moreover, in in vivo transplantation experiments, PDL-OG cells formed cellular cementum-like hard tissue containing embedded osteocalcin-positive cells, whereas the other cells formed acellular cementum-like materials. Given that the root-cementum defect is likely regenerated through cellular cementum deposition, PDL-OG cell-based therapies might potentially facilitate the de novo cellular cementogenesis required for regenerating the root defect.

Differentiation and Characterization of Dopaminergic Neurons From Baboon Induced Pluripotent Stem Cells.

PubMed

Grow, Douglas A; Simmons, DeNard V; Gomez, Jorge A; Wanat, Matthew J; McCarrey, John R; Paladini, Carlos A; Navara, Christopher S

2016-09-01

: The progressive death of dopamine producing neurons in the substantia nigra pars compacta is the principal cause of symptoms of Parkinson's disease (PD). Stem cells have potential therapeutic use in replacing these cells and restoring function. To facilitate development of this approach, we sought to establish a preclinical model based on a large nonhuman primate for testing the efficacy and safety of stem cell-based transplantation. To this end, we differentiated baboon fibroblast-derived induced pluripotent stem cells (biPSCs) into dopaminergic neurons with the application of specific morphogens and growth factors. We confirmed that biPSC-derived dopaminergic neurons resemble those found in the human midbrain based on cell type-specific expression of dopamine markers TH and GIRK2. Using the reverse transcriptase quantitative polymerase chain reaction, we also showed that biPSC-derived dopaminergic neurons express PAX6, FOXA2, LMX1A, NURR1, and TH genes characteristic of this cell type in vivo. We used perforated patch-clamp electrophysiology to demonstrate that biPSC-derived dopaminergic neurons fired spontaneous rhythmic action potentials and high-frequency action potentials with spike frequency adaption upon injection of depolarizing current. Finally, we showed that biPSC-derived neurons released catecholamines in response to electrical stimulation. These results demonstrate the utility of the baboon model for testing and optimizing the efficacy and safety of stem cell-based therapeutic approaches for the treatment of PD. Functional dopamine neurons were produced from baboon induced pluripotent stem cells, and their properties were compared to baboon midbrain cells in vivo. The baboon has advantages as a clinically relevant model in which to optimize the efficacy and safety of stem cell-based therapies for neurodegenerative diseases, such as Parkinson's disease. Baboons possess crucial neuroanatomical and immunological similarities to humans, and baboon pluripotent stem cells can be differentiated into functional neurons that mimic those in the human brain, thus laying the foundation for the utility of the baboon model for evaluating stem cell therapies. ©AlphaMed Press.

Epithelial V-Like Antigen Mediates Efficacy of Anti-Alpha4 Integrin Treatment in a Mouse Model of Multiple Sclerosis

PubMed Central

Wright, Erik; Rahgozar, Kusha; Hallworth, Nicholas; Lanker, Stefan; Carrithers, Michael D.

2013-01-01

Natalizumab inhibits the transmigration of activated T lymphocytes into the brain and is highly efficacious in multiple sclerosis (MS). However, from a pharmacogenomic perspective, its efficacy and safety in specific patients remain unclear. Here our goal was to analyze the effects of epithelial V-like antigen (EVA) on anti-alpha4 integrin (VLA4) efficacy in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). EVA has been previously characterized in human CD4 T lymphocytes, mouse thymic development, and choroid plexus epithelial cells. Further analysis here demonstrated expression in B lymphocytes and an increase in EVA+ lymphocytes following immunization. Following active induction of EAE using the MOG35–55 active immunization model, EVA deficient mice developed more severe EAE and white matter tissue injury as compared to wild type controls. This severe EAE phenotype did not respond to anti-VLA4 treatment. In both the control antibody and anti-VLA4 conditions, these mice demonstrated persistent CNS invasion of mature B lymphocyte (CD19+, CD21+, sIgG+), increased serum autoantibody levels, and extensive complement and IgG deposition within lesions containing CD5+IgG+ cells. Wild type mice treated with control antibody also demonstrated the presence of CD19+, CD21+, sIgG+ cells within the CNS during peak EAE disease severity and detectable serum autoantibody. In contrast, wild type mice treated with anti-VLA4 demonstrated reduced serum autoantibody levels as compared to wild type controls and EVA-knockout mice. As expected, anti-VLA4 treatment in wild type mice reduced the total numbers of all CNS mononuclear cells and markedly decreased CD4 T lymphocyte invasion. Treatment also reduced the frequency of CD19+, CD21+, sIgG+ cells in the CNS. These results suggest that anti-VLA4 treatment may reduce B lymphocyte associated autoimmunity in some individuals and that EVA expression is necessary for an optimal therapeutic response. We postulate that these findings could optimize the selection of treatment responders. PMID:23951051

IL-1 Receptor Signaling on Graft Parenchymal Cells Regulates Memory and De Novo Donor-Reactive CD8 T Cell Responses to Cardiac Allografts.

PubMed

Iida, Shoichi; Tsuda, Hidetoshi; Tanaka, Toshiaki; Kish, Danielle D; Abe, Toyofumi; Su, Charles A; Abe, Ryo; Tanabe, Kazunari; Valujskikh, Anna; Baldwin, William M; Fairchild, Robert L

2016-03-15

Reperfusion of organ allografts induces a potent inflammatory response that directs rapid memory T cell, neutrophil, and macrophage graft infiltration and their activation to express functions mediating graft tissue injury. The role of cardiac allograft IL-1 receptor (IL-1R) signaling in this early inflammation and the downstream primary alloimmune response was investigated. When compared with complete MHC-mismatched wild-type cardiac allografts, IL-1R(-/-) allografts had marked decreases in endogenous memory CD8 T cell and neutrophil infiltration and expression of proinflammatory mediators at early times after transplant, whereas endogenous memory CD4 T cell and macrophage infiltration was not decreased. IL-1R(-/-) allograft recipients also had marked decreases in de novo donor-reactive CD8, but not CD4, T cell development to IFN-γ-producing cells. CD8 T cell-mediated rejection of IL-1R(-/-) cardiac allografts took 3 wk longer than wild-type allografts. Cardiac allografts from reciprocal bone marrow reconstituted IL-1R(-/-)/wild-type chimeric donors indicated that IL-1R signaling on graft nonhematopoietic-derived, but not bone marrow-derived, cells is required for the potent donor-reactive memory and primary CD8 T cell alloimmune responses observed in response to wild-type allografts. These studies implicate IL-1R-mediated signals by allograft parenchymal cells in generating the stimuli-provoking development and elicitation of optimal alloimmune responses to the grafts. Copyright © 2016 by The American Association of Immunologists, Inc.

Optimal degrees of synaptic connectivity

PubMed Central

Litwin-Kumar, Ashok; Harris, Kameron Decker; Axel, Richard; Sompolinsky, Haim; Abbott, L. F.

2017-01-01

Summary Synaptic connectivity varies widely across neuronal types. Cerebellar granule cells receive five orders of magnitude fewer inputs than the Purkinje cells they innervate, and cerebellum-like circuits including the insect mushroom body also exhibit large divergences in connectivity. In contrast, the number of inputs per neuron in cerebral cortex is more uniform and large. We investigate how the dimension of a representation formed by a population of neurons depends on how many inputs they each receive and what this implies for learning associations. Our theory predicts that the dimensions of the cerebellar granule-cell and Drosophila Kenyon-cell representations are maximized at degrees of synaptic connectivity that match those observed anatomically, showing that sparse connectivity is sometimes superior to dense connectivity. When input synapses are subject to supervised plasticity, however, dense wiring becomes advantageous, suggesting that the type of plasticity exhibited by a set of synapses is a major determinant of connection density. PMID:28215558

Effect of Cell Aspect Ratio on Swarming Bacteria

NASA Astrophysics Data System (ADS)

Ilkanaiv, Bella; Kearns, Daniel B.; Ariel, Gil; Be'er, Avraham

2017-04-01

Swarming bacteria collectively migrate on surfaces using flagella, forming dynamic whirls and jets that consist of millions of individuals. Because some swarming bacteria elongate prior to actual motion, cell aspect ratio may play a significant role in the collective dynamics. Extensive research on self-propelled rodlike particles confirms that elongation promotes alignment, strongly affecting the dynamics. Here, we study experimentally the collective dynamics of variants of swarming Bacillus subtilis that differ in length. We show that the swarming statistics depends on the aspect ratio in a critical, fundamental fashion not predicted by theory. The fastest motion was obtained for the wild-type and variants that are similar in length. However, shorter and longer cells exhibit anomalous, non-Gaussian statistics and nonexponential decay of the autocorrelation function, indicating lower collective motility. These results suggest that the robust mechanisms to maintain aspect ratios may be important for efficient swarming motility. Wild-type cells are optimal in this sense.

Modeling to Optimize Terminal Stem Cell Differentiation

PubMed Central

Gallicano, G. Ian

2013-01-01

Embryonic stem cell (ESC), iPCs, and adult stem cells (ASCs) all are among the most promising potential treatments for heart failure, spinal cord injury, neurodegenerative diseases, and diabetes. However, considerable uncertainty in the production of ESC-derived terminally differentiated cell types has limited the efficiency of their development. To address this uncertainty, we and other investigators have begun to employ a comprehensive statistical model of ESC differentiation for determining the role of intracellular pathways (e.g., STAT3) in ESC differentiation and determination of germ layer fate. The approach discussed here applies the Baysian statistical model to cell/developmental biology combining traditional flow cytometry methodology and specific morphological observations with advanced statistical and probabilistic modeling and experimental design. The final result of this study is a unique tool and model that enhances the understanding of how and when specific cell fates are determined during differentiation. This model provides a guideline for increasing the production efficiency of therapeutically viable ESCs/iPSCs/ASC derived neurons or any other cell type and will eventually lead to advances in stem cell therapy. PMID:24278782

Determination of synthetic lethal interactions in KRAS oncogene-dependent cancer cells reveals novel therapeutic targeting strategies

PubMed Central

Steckel, Michael; Molina-Arcas, Miriam; Weigelt, Britta; Marani, Michaela; Warne, Patricia H; Kuznetsov, Hanna; Kelly, Gavin; Saunders, Becky; Howell, Michael; Downward, Julian; Hancock, David C

2012-01-01

Oncogenic mutations in RAS genes are very common in human cancer, resulting in cells with well-characterized selective advantages, but also less well-understood vulnerabilities. We have carried out a large-scale loss-of-function screen to identify genes that are required by KRAS-transformed colon cancer cells, but not by derivatives lacking this oncogene. Top-scoring genes were then tested in a larger panel of KRAS mutant and wild-type cancer cells. Cancer cells expressing oncogenic KRAS were found to be highly dependent on the transcription factor GATA2 and the DNA replication initiation regulator CDC6. Extending this analysis using a collection of drugs with known targets, we found that cancer cells with mutant KRAS showed selective addiction to proteasome function, as well as synthetic lethality with topoisomerase inhibition. Combination targeting of these functions caused improved killing of KRAS mutant cells relative to wild-type cells. These observations suggest novel targets and new ways of combining existing therapies for optimal effect in RAS mutant cancers, which are traditionally seen as being highly refractory to therapy. PMID:22613949

Single Stem Cell Imaging and Analysis Reveals Telomere Length Differences in Diseased Human and Mouse Skeletal Muscles.

PubMed

Tichy, Elisia D; Sidibe, David K; Tierney, Matthew T; Stec, Michael J; Sharifi-Sanjani, Maryam; Hosalkar, Harish; Mubarak, Scott; Johnson, F Brad; Sacco, Alessandra; Mourkioti, Foteini

2017-10-10

Muscle stem cells (MuSCs) contribute to muscle regeneration following injury. In many muscle disorders, the repeated cycles of damage and repair lead to stem cell dysfunction. While telomere attrition may contribute to aberrant stem cell functions, methods to accurately measure telomere length in stem cells from skeletal muscles have not been demonstrated. Here, we have optimized and validated such a method, named MuQ-FISH, for analyzing telomere length in MuSCs from either mice or humans. Our analysis showed no differences in telomere length between young and aged MuSCs from uninjured wild-type mice, but MuSCs isolated from young dystrophic mice exhibited significantly shortened telomeres. In corroboration, we demonstrated that telomere attrition is present in human dystrophic MuSCs, which underscores its importance in diseased regenerative failure. The robust technique described herein provides analysis at a single-cell resolution and may be utilized for other cell types, especially rare populations of cells. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

A critical-like collective state leads to long-range cell communication in Dictyostelium discoideum aggregation

PubMed Central

De Palo, Giovanna; Yi, Darvin; Endres, Robert G.

2017-01-01

The transition from single-cell to multicellular behavior is important in early development but rarely studied. The starvation-induced aggregation of the social amoeba Dictyostelium discoideum into a multicellular slug is known to result from single-cell chemotaxis towards emitted pulses of cyclic adenosine monophosphate (cAMP). However, how exactly do transient, short-range chemical gradients lead to coherent collective movement at a macroscopic scale? Here, we developed a multiscale model verified by quantitative microscopy to describe behaviors ranging widely from chemotaxis and excitability of individual cells to aggregation of thousands of cells. To better understand the mechanism of long-range cell—cell communication and hence aggregation, we analyzed cell—cell correlations, showing evidence of self-organization at the onset of aggregation (as opposed to following a leader cell). Surprisingly, cell collectives, despite their finite size, show features of criticality known from phase transitions in physical systems. By comparing wild-type and mutant cells with impaired aggregation, we found the longest cell—cell communication distance in wild-type cells, suggesting that criticality provides an adaptive advantage and optimally sized aggregates for the dispersal of spores. PMID:28422986

Do immunotherapy and beta cell replacement play a synergistic role in the treatment of type 1 diabetes?

PubMed

Li, Dong-Sheng; Warnock, Garth L; Tu, Han-Jun; Ao, Ziliang; He, Zehua; Lu, Hong; Dai, Long-Jun

2009-10-07

Type 1 diabetes (T1D) is the result of the autoimmune response against pancreatic insulin-producing ss-cells. Its ultimate consequence is beta-cell insufficiency-mediated dysregulation of blood glucose control. In terms of T1D treatment, immunotherapy addresses the cause of T1D, mainly through re-setting the balance between autoimmunity and regulatory mechanisms. Regulatory T cells play an important role in this immune intervention. An alternative T1D treatment is beta-cell replacement, which can reverse the consequence of the disease by replacing destroyed beta-cells in the diabetic pancreas. The applicable insulin-producing cells can be directly obtained from islet transplantation or generated from other cell sources such as autologous adult stem cells, embryonic stem cells, and induced pluripotent stem cells. In this review, we summarize the recent research progress and analyze the possible advantages and disadvantages of these two therapeutic options especially focusing on the potential synergistic effect on T1D treatment. Exploring the optimal combination of immunotherapy and beta-cell replacement will pave the way to the most effective cure for this devastating disease.

A stepwise approach for the reproducible optimization of PAMO expression in Escherichia coli for whole-cell biocatalysis

PubMed Central

2012-01-01

Background Baeyer-Villiger monooxygenases (BVMOs) represent a group of enzymes of considerable biotechnological relevance as illustrated by their growing use as biocatalyst in a variety of synthetic applications. However, due to their increased use the reproducible expression of BVMOs and other biotechnologically relevant enzymes has become a pressing matter while knowledge about the factors governing their reproducible expression is scattered. Results Here, we have used phenylacetone monooxygenase (PAMO) from Thermobifida fusca, a prototype Type I BVMO, as a model enzyme to develop a stepwise strategy to optimize the biotransformation performance of recombinant E. coli expressing PAMO in 96-well microtiter plates in a reproducible fashion. Using this system, the best expression conditions of PAMO were investigated first, including different host strains, temperature as well as time and induction period for PAMO expression. This optimized system was used next to improve biotransformation conditions, the PAMO-catalyzed conversion of phenylacetone, by evaluating the best electron donor, substrate concentration, and the temperature and length of biotransformation. Combining all optimized parameters resulted in a more than four-fold enhancement of the biocatalytic performance and, importantly, this was highly reproducible as indicated by the relative standard deviation of 1% for non-washed cells and 3% for washed cells. Furthermore, the optimized procedure was successfully adapted for activity-based mutant screening. Conclusions Our optimized procedure, which provides a comprehensive overview of the key factors influencing the reproducible expression and performance of a biocatalyst, is expected to form a rational basis for the optimization of miniaturized biotransformations and for the design of novel activity-based screening procedures suitable for BVMOs and other NAD(P)H-dependent enzymes as well. PMID:22720747

Porous Silicon Nanoparticle Photosensitizers for Singlet Oxygen and Their Phototoxicity Against Cancer Cells

PubMed Central

Xiao, Ling; Gu, Luo; Howell, Stephen B.; Sailor, Michael J.

2011-01-01

Porous Si nanoparticles, prepared from electrochemically etched single crystal Si wafers, function as photosensitizers to generate 1O2 in ethanol and in aqueous media. The preparation conditions for the porous Si nanoparticles were optimized to maximize (1) the yield of material; (2) its quantum yield of 1O2 production; and (3) its in vitro degradation properties. The optimal formulation was determined to consist of nanoparticles 146 ± 7 nm in diameter, with nominal pore sizes of 12 ± 4 nm. The quantum yield for 1O2 production is 0.10 ± 0.02 in ethanol and 0.17 ± 0.01 in H2O. HeLa or NIH-3T3 cells treated with 100 µg/mL porous Si nanoparticles and exposed to 60 J/cm2 white light (infrared filtered, 100 mW/cm2 for 10 min) exhibit ~ 45% cell death, while controls containing no nanoparticles show 10% or 25% cell death, respectively. The dark control experiment yields < 10% cytotoxicity for either cell type. PMID:21452822

Comparison and optimization of machine learning methods for automated classification of circulating tumor cells.

PubMed

Lannin, Timothy B; Thege, Fredrik I; Kirby, Brian J

2016-10-01

Advances in rare cell capture technology have made possible the interrogation of circulating tumor cells (CTCs) captured from whole patient blood. However, locating captured cells in the device by manual counting bottlenecks data processing by being tedious (hours per sample) and compromises the results by being inconsistent and prone to user bias. Some recent work has been done to automate the cell location and classification process to address these problems, employing image processing and machine learning (ML) algorithms to locate and classify cells in fluorescent microscope images. However, the type of machine learning method used is a part of the design space that has not been thoroughly explored. Thus, we have trained four ML algorithms on three different datasets. The trained ML algorithms locate and classify thousands of possible cells in a few minutes rather than a few hours, representing an order of magnitude increase in processing speed. Furthermore, some algorithms have a significantly (P < 0.05) higher area under the receiver operating characteristic curve than do other algorithms. Additionally, significant (P < 0.05) losses to performance occur when training on cell lines and testing on CTCs (and vice versa), indicating the need to train on a system that is representative of future unlabeled data. Optimal algorithm selection depends on the peculiarities of the individual dataset, indicating the need of a careful comparison and optimization of algorithms for individual image classification tasks. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Diagnosis and Prevention of Infection by Phlebotomus Fever Group Viruses

DTIC Science & Technology

1991-05-03

proficient, but non- optimal expression vectors [e.g., pAcRP6, see below]. The two types of recombinant viruses were used to infect Spodoptera ... frugiperda cells and the expressed PT viral proteins characterised (Overton et al., 1987). Recombinant AcNPV having the S DNA in one orientation expressed...proteins virus were raised in mice using the corresponding S. frugiperda infected cell extracts and were employed to identify N and NSs proteins in PT virus

Plasmid transfection in bovine cells: Optimization using a realtime monitoring of green fluorescent protein and effect on gene reporter assay.

PubMed

Osorio, Johan S; Bionaz, Massimo

2017-08-30

Gene reporter technology (GRT) has opened several new avenues for monitoring biological events including the activation of transcription factors, which are central to the study of nutrigenomics. However, this technology relies heavily on the insertion of foreign plasmid DNA into the nuclei of cells (i.e., transfection), which can be very challenging and highly variable among cell types. The objective of this study was to investigate the optimal conditions to generate reliable GRT assay data on bovine immortalized cell lines, Madin Darby Bovine Kidney (MDBK) and bovine mammary epithelial alveolar (MACT) cells. Results are reported for two experiments. In Experiment 1, using 96 well-plate and a robotic inverted fluorescent microscope, we compared transfection efficiency among commercially available transfection reagents (TR) Lipofectamine® 3000 (Lipo3), Lipofectamine® LTX (LipoLTX), and TransIT-X2® (TransX2), three doses of TR (i.e., 0.15, 0.3, and 0.4μL/well), and three doses of Green Fluorescent Protein plasmid DNA (i.e., 10, 25, and 50ng/well). Transfection efficiency and mortality rate were analyzed using CellProfiler software. Transfection efficiency increased until the end of the experiment (20h post-transfection) at which point MACT had greater transfection than MDBK cells (16.3% vs. 2.2%). It is unclear the reason for the low transfection in MDBK cells. Maximal transfection efficiency was obtained with 0.3μL/well of LipoLTX plus 25ng/well of plasmid DNA (ca. 29.5±1.9%) and 0.15μL/well of LipoLTX plus 25ng/well of plasmid DNA (ca. 4.0±0.4%) for MACT and MDBK cells, respectively. The higher amount of TR and DNA was generally associated with higher cell mortality. Using high, medium, and low transfection efficiency conditions determined in Experiment 1, we performed a GRT assay for peroxisome proliferator-activated response element (PPRE) luciferase in MACT and MDBK cells treated with 10nM or 100nM of synthetic Peroxisome Proliferator-activated Receptor β/σ (PPARβ/σ) agonist. The GRT assay was unaffected by poor transfection in MACT cells although the high transfection hampered the possibility of detecting differences between 10 and 100nM of the PPARβ/δ agonist. In MDBK cells, low transfection efficiency (<2.0%) failed to detect any differences with GRT assay. The level of transfection was positively associated with a lower coefficient of variation of GRT data. Overall, our data indicates that results of GRT assays are affected by transfection efficiency and a minimum transfection of 2% is required. Thus, factors such as TR type, TR amount, and DNA plasmid amount need to be optimized for a specific cell type before performing GRT assays. Copyright © 2017 Elsevier B.V. All rights reserved.

Hydrocarbons Are Essential for Optimal Cell Size, Division, and Growth of Cyanobacteria1[OPEN

PubMed Central

Lea-Smith, David J.; Nürnberg, Dennis J.; Baers, Laura L.; Davey, Matthew P.; Parolini, Lucia; Huber, Roland G.; Cotton, Charles A. R.; Mastroianni, Giulia; Bombelli, Paolo; Ungerer, Petra; Stevens, Tim J.; Howe, Christopher J.

2016-01-01

Cyanobacteria are intricately organized, incorporating an array of internal thylakoid membranes, the site of photosynthesis, into cells no larger than other bacteria. They also synthesize C15-C19 alkanes and alkenes, which results in substantial production of hydrocarbons in the environment. All sequenced cyanobacteria encode hydrocarbon biosynthesis pathways, suggesting an important, undefined physiological role for these compounds. Here, we demonstrate that hydrocarbon-deficient mutants of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 exhibit significant phenotypic differences from wild type, including enlarged cell size, reduced growth, and increased division defects. Photosynthetic rates were similar between strains, although a minor reduction in energy transfer between the soluble light harvesting phycobilisome complex and membrane-bound photosystems was observed. Hydrocarbons were shown to accumulate in thylakoid and cytoplasmic membranes. Modeling of membranes suggests these compounds aggregate in the center of the lipid bilayer, potentially promoting membrane flexibility and facilitating curvature. In vivo measurements confirmed that Synechococcus sp. PCC 7002 mutants lacking hydrocarbons exhibit reduced thylakoid membrane curvature compared to wild type. We propose that hydrocarbons may have a role in inducing the flexibility in membranes required for optimal cell division, size, and growth, and efficient association of soluble and membrane bound proteins. The recent identification of C15-C17 alkanes and alkenes in microalgal species suggests hydrocarbons may serve a similar function in a broad range of photosynthetic organisms. PMID:27707888

Increasing platelet concentration in platelet-rich plasma inhibits anterior cruciate ligament cell function in three-dimensional culture.

PubMed

Yoshida, Ryu; Cheng, Mingyu; Murray, Martha M

2014-02-01

Tissue engineering is one new strategy being developed to treat ACL ruptures. One such approach is bio-enhanced ACL repair, where a suture repair is supplemented with a bio-active scaffold containing platelets. However, the optimal concentration of platelets to stimulate ACL healing is not known. We hypothesized that increasing platelet concentrations in the scaffold would enhance critical cell behaviors. Porcine ACL fibroblasts were obtained from explant culture and suspended in platelet poor plasma (PPP), 1× platelet-rich plasma (PRP), 3× PRP, 5× PRP, or phosphate buffered saline (PBS). The cell suspensions were cultured in a 3D collagen scaffold. Cellular metabolism (MTT assay), apoptosis (TUNEL assay), and gene expression for type I and type III collagen were measured. 1× PRP significantly outperformed 5× PRP in all parameters studied: Type I and III collagen gene expression, apoptosis prevention, and cell metabolism stimulation. ACL fibroblasts cultured with 1× PRP had the highest type I and type III collagen gene expression. 1× PRP and PPP groups had the highest cell metabolism and lowest apoptosis rates. Concentration of platelets had significant effects on the behavior of ACL fibroblasts; thus, it is an important parameter that should be specified in clinical or basic science studies. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Fullerene C{sub 70} as a p-type donor in organic photovoltaic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhuang, Taojun; Wang, Xiao-Feng, E-mail: charles1976110@hotmail.com, E-mail: zrhong@ucla.edu, E-mail: kid@yz.yamagata-u.ac.jp; Sano, Takeshi

2014-09-01

Fullerenes and their derivatives have been widely used as n-type materials in organic transistor and photovoltaic devices. Though it is believed that they shall be ambipolar in nature, there have been few direct experimental proofs for that. In this work, fullerene C{sub 70}, known as an efficient acceptor, has been employed as a p-type electron donor in conjunction with 1,4,5,8,9,11-hexaazatriphenylene hexacarbonitrile as an electron acceptor in planar-heterojunction (PHJ) organic photovoltaic (OPV) cells. High fill factors (FFs) of more than 0.70 were reliably achieved with the C{sub 70} layer even up to 100 nm thick in PHJ cells, suggesting the superior potentialmore » of fullerene C{sub 70} as the p-type donor in comparison to other conventional donor materials. The optimal efficiency of these unconventional PHJ cells was 2.83% with a short-circuit current of 5.33 mA/cm{sup 2}, an open circuit voltage of 0.72 V, and a FF of 0.74. The results in this work unveil the potential of fullerene materials as donors in OPV devices, and provide alternative approaches towards future OPV applications.« less

Additives and salts for dye-sensitized solar cells electrolytes: what is the best choice?

NASA Astrophysics Data System (ADS)

Bella, Federico; Sacco, Adriano; Pugliese, Diego; Laurenti, Marco; Bianco, Stefano

2014-10-01

A multivariate chemometric approach is proposed for the first time for performance optimization of I-/I3- liquid electrolytes for dye-sensitized solar cells (DSSCs). Over the years the system composed by iodide/triiodide redox shuttle dissolved in organic solvent has been enriched with the addition of different specific cations and chemical compounds to improve the photoelectrochemical behavior of the cell. However, usually such additives act favorably with respect to some of the cell parameters and negatively to others. Moreover, the combined action of different compounds often yields contradictory results, and from the literature it is not possible to identify an optimal recipe. We report here a systematic work, based on a multivariate experimental design, to statistically and quantitatively evaluate the effect of different additives on the photovoltaic performances of the device. The effect of cation size in iodine salts, the iodine/iodide ratio in the electrolyte and the effect of type and concentration of additives are mutually evaluated by means of a Design of Experiment (DoE) approach. Through this statistical method, the optimization of the overall parameters is demonstrated with a limited number of experimental trials. A 25% improvement on the photovoltaic conversion efficiency compared with that obtained with a commercial electrolyte is demonstrated.

HEK293 cell culture media study towards bioprocess optimization: Animal derived component free and animal derived component containing platforms.

PubMed

Liste-Calleja, Leticia; Lecina, Martí; Cairó, Jordi Joan

2014-04-01

The increasing demand for biopharmaceuticals produced in mammalian cells has lead industries to enhance bioprocess volumetric productivity through different strategies. Among those strategies, cell culture media development is of major interest. In the present work, several commercially available culture media for Human Embryonic Kidney cells (HEK293) were evaluated in terms of maximal specific growth rate and maximal viable cell concentration supported. The main objective was to provide different cell culture platforms which are suitable for a wide range of applications depending on the type and the final use of the product obtained. Performing simple media supplementations with and without animal derived components, an enhancement of cell concentration from 2 × 10(6) cell/mL to 17 × 10(6) cell/mL was achieved in batch mode operation. Additionally, the media were evaluated for adenovirus production as a specific application case of HEK293 cells. None of the supplements interfered significantly with the adenovirus infection although some differences were encountered in viral productivity. To the best of our knowledge, the high cell density achieved in the work presented has never been reported before in HEK293 batch cell cultures and thus, our results are greatly promising to further study cell culture strategies in bioreactor towards bioprocess optimization. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Surgery on spinal epidural metastases (SEM) in renal cell carcinoma: a plea for a new paradigm.

PubMed

Bakker, Nicolaas A; Coppes, Maarten H; Vergeer, Rob A; Kuijlen, Jos M A; Groen, Rob J M

2014-09-01

Prediction models for outcome of decompressive surgical resection of spinal epidural metastases (SEM) have in common that they have been developed for all types of SEM, irrespective of the type of primary tumor. It is our experience in clinical practice, however, that these models often fail to accurately predict outcome in the individual patient. To investigate whether decision making could be optimized by applying tumor-specific prediction models. For the proof of concept, we analyzed patients with SEM from renal cell carcinoma that we have operated on. Retrospective chart analysis 2006 to 2012. Twenty-one consecutive patients with symptomatic SEM of renal cell carcinoma. Predictive factors for survival. Next to established predictive factors for survival, we analyzed the predictive value of the Motzer criteria in these patients. The Motzer criteria comprise a specific and validated risk model for survival in patients with renal cell carcinoma. After multivariable analysis, only Motzer intermediate (hazard ratio [HR] 17.4, 95% confidence interval [CI] 1.82-166, p=.01) and high risk (HR 39.3, 95% CI 3.10-499, p=.005) turned out to be significantly associated with survival in patients with renal cell carcinoma that we have operated on. In this study, we have demonstrated that decision making could have been optimized by implementing the Motzer criteria next to established prediction models. We, therefore, suggest that in future, in patients with SEM from renal cell carcinoma, the Motzer criteria are also taken into account. Copyright © 2014 Elsevier Inc. All rights reserved.

Optimizing prognosis-related key miRNA-target interactions responsible for cancer metastasis.

PubMed

Zhao, Hongying; Yuan, Huating; Hu, Jing; Xu, Chaohan; Liao, Gaoming; Yin, Wenkang; Xu, Liwen; Wang, Li; Zhang, Xinxin; Shi, Aiai; Li, Jing; Xiao, Yun

2017-12-12

Increasing evidence suggests that the abnormality of microRNAs (miRNAs) and their downstream targets is frequently implicated in the pathogenesis of human cancers, however, the clinical benefit of causal miRNA-target interactions has been seldom studied. Here, we proposed a computational method to optimize prognosis-related key miRNA-target interactions by combining transcriptome and clinical data from thousands of TCGA tumors across 16 cancer types. We obtained a total of 1,956 prognosis-related key miRNA-target interactions between 112 miRNAs and 1,443 their targets. Interestingly, these key target genes are specifically involved in tumor progression-related functions, such as 'cell adhesion' and 'cell migration'. Furthermore, they are most significantly correlated with 'tissue invasion and metastasis', a hallmark of metastasis, in ten distinct types of cancer through the hallmark analysis. These results implicated that the prognosis-related key miRNA-target interactions were highly associated with cancer metastasis. Finally, we observed that the combination of these key miRNA-target interactions allowed to distinguish patients with good prognosis from those with poor prognosis both in most TCGA cancer types and independent validation sets, highlighting their roles in cancer metastasis. We provided a user-friendly database named miRNATarget (freely available at http://biocc.hrbmu.edu.cn/miRNATar/), which provides an overview of the prognosis-related key miRNA-target interactions across 16 cancer types.

An authentic imaging probe to track cell fate from beginning to end.

PubMed

Lee, Seung Koo; Mortensen, Luke J; Lin, Charles P; Tung, Ching-Hsuan

2014-10-17

Accurate tracing of cell viability is critical for optimizing delivery methods and evaluating the efficacy and safety of cell therapeutics. A nanoparticle-based cell tracker is developed to image cell fate from live to dead. The particle is fabricated from two types of optically quenched polyelectrolytes, a life indicator and a death indicator, through electrostatic interactions. On incubation with cells, the fabricated bifunctional nanoprobes are taken up efficiently and the first colour is produced by normal intracellular proteolysis, reflecting the healthy status of the cells. Depending on the number of coated layers, the signal can persist for several replication cycles. However, as the cells begin dying, the second colour appears quickly to reflect the new cell status. Using this chameleon-like cell tracker, live cells can be distinguished from apoptotic and necrotic cells instantly and definitively.

Beyond the frontiers of neuronal types

PubMed Central

Battaglia, Demian; Karagiannis, Anastassios; Gallopin, Thierry; Gutch, Harold W.; Cauli, Bruno

2012-01-01

Cortical neurons and, particularly, inhibitory interneurons display a large diversity of morphological, synaptic, electrophysiological, and molecular properties, as well as diverse embryonic origins. Various authors have proposed alternative classification schemes that rely on the concomitant observation of several multimodal features. However, a broad variability is generally observed even among cells that are grouped into a same class. Furthermore, the attribution of specific neurons to a single defined class is often difficult, because individual properties vary in a highly graded fashion, suggestive of continua of features between types. Going beyond the description of representative traits of distinct classes, we focus here on the analysis of atypical cells. We introduce a novel paradigm for neuronal type classification, assuming explicitly the existence of a structured continuum of diversity. Our approach, grounded on the theory of fuzzy sets, identifies a small optimal number of model archetypes. At the same time, it quantifies the degree of similarity between these archetypes and each considered neuron. This allows highlighting archetypal cells, which bear a clear similarity to a single model archetype, and edge cells, which manifest a convergence of traits from multiple archetypes. PMID:23403725

Fundamentals of pulmonary drug delivery.

PubMed

Groneberg, D A; Witt, C; Wagner, U; Chung, K F; Fischer, A

2003-04-01

Aerosol administration of peptide-based drugs plays an important role in the treatment of pulmonary and systemic diseases and the unique cellular properties of airway epithelium offers a great potential to deliver new compounds. As the relative contributions from the large airways to the alveolar space are important to the local and systemic availability, the sites and mechanism of uptake and transport of different target compounds have to be characterized. Among the different respiratory cells, the ciliated epithelial cells of the larger and smaller airways and the type I and type II pneumocytes are the key players in pulmonary drug transport. With their diverse cellular characteristics, each of these cell types displays a unique uptake possibility. Next to the knowledge of these cellular aspects, the nature of aerosolized drugs, characteristics of delivery systems and the depositional and pulmonary clearance mechanisms display major targets to optimize pulmonary drug delivery. Based on the growing knowledge on pulmonary cell biology and pathophysiology due to modern methods of molecular biology, the future characterization of pulmonary drug transport pathways can lead to new strategies in aerosol drug therapy.

Design study of large area 8 cm x 8 cm wrapthrough cells for space station

NASA Technical Reports Server (NTRS)

Garlick, George F. J.; Lillington, David R.

1987-01-01

The design of large area silicon solar cells for the projected NASA space station is discussed. It is based on the NASA specification for the cells which calls for an 8 cm by 8 cm cell of wrapthrough type with gridded back contacts. The beginning of life (BOL) power must be 1.039 watts per cell or larger and maximum end of life (EOL) after 10 years in the prescribed orbit under an equivalent 1MeV electron radiation damage fluence of 5 times 10 to the 13th power e/square cm. On orbit efficiency is to be optimized by a low thermal absorptance goal (thermal alpha) of .63.

Optimal management of immune-related adverse events resulting from treatment with immune checkpoint inhibitors: a review and update.

PubMed

Nagai, Hiroki; Muto, Manabu

2018-06-01

Over the last two decades, molecular-targeted agents have become mainstream treatment for many types of malignancies and have improved the overall survival of patients. However, most patients eventually develop resistance to these targeted therapies. Recently, immunotherapies such as immune checkpoint inhibitors have revolutionized the treatment paradigm for many types of malignancies. Immune checkpoint inhibitors have been approved for treatment of melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, Hodgkin's lymphoma, bladder cancer and gastric cancer. However, oncologists have been faced with immune-related adverse events caused by immune checkpoint inhibitors; these are generally mild but can be fatal in some cases. Because immune checkpoint inhibitors have distinct toxicity profiles from those of chemotherapy or targeted therapy, many oncologists are not familiar with the principles for optimal management of immune-related adverse events, which require early recognition and appropriate treatment without delay. To achieve this, oncologists must educate patients and health-care workers, develop checklists of appropriate tests for immune-related adverse events and collaborate closely with organ specialists. Clinical questions that remain include whether immune checkpoint inhibitors should be administered to patients with autoimmune disease and whether patients for whom immune-related adverse events lead to delays in immunotherapy should be retreated. In addition, the predicted use of combination immunotherapies in the near future means that oncologists will face a higher incidence and severity of immune-related adverse events. This review provides an overview of the optimal management of immune-related adverse events attributed to immune checkpoint inhibitors.

Need for optimizing catalyst loading for achieving affordable microbial fuel cells.

PubMed

Singh, Inderjeet; Chandra, Amreesh

2013-08-01

Microbial fuel cell (MFC) technology is a promising technology for electricity production together with simultaneous water treatment. Catalysts play an important role in deciding the MFC performance. In most reports, effect of catalyst - both type and quantity is not optimized. In this paper, synthesis of nanorods of MnO2-catalyst particles for application in Pt-free MFCs is reported. The effect of catalyst loading i.e., weight ratio, with respect to conducting element and binder has been optimized by employing large number of combinations. Using simple theoretical model, it is shown that too high (or low) concentration of catalysts result in loss of MFC performance. The operation of MFC has been investigated using domestic wastewater as source of bio-waste for obtaining real world situation. Maximum power density of ∼61 mW/m(2) was obtained when weight ratio of catalyst and conducting species was 1:1. Suitable reasons are given to explain the outcomes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cellular reprogramming dynamics follow a simple 1D reaction coordinate

NASA Astrophysics Data System (ADS)

Teja Pusuluri, Sai; Lang, Alex H.; Mehta, Pankaj; Castillo, Horacio E.

2018-01-01

Cellular reprogramming, the conversion of one cell type to another, induces global changes in gene expression involving thousands of genes, and understanding how cells globally alter their gene expression profile during reprogramming is an ongoing problem. Here we reanalyze time-course data on cellular reprogramming from differentiated cell types to induced pluripotent stem cells (iPSCs) and show that gene expression dynamics during reprogramming follow a simple 1D reaction coordinate. This reaction coordinate is independent of both the time it takes to reach the iPSC state as well as the details of the experimental protocol used. Using Monte-Carlo simulations, we show that such a reaction coordinate emerges from epigenetic landscape models where cellular reprogramming is viewed as a ‘barrier-crossing’ process between cell fates. Overall, our analysis and model suggest that gene expression dynamics during reprogramming follow a canonical trajectory consistent with the idea of an ‘optimal path’ in gene expression space for reprogramming.

Recruited Monocytes and Type 2 Immunity Promote Lung Regeneration following Pneumonectomy.

PubMed

Lechner, Andrew J; Driver, Ian H; Lee, Jinwoo; Conroy, Carmen M; Nagle, Abigail; Locksley, Richard M; Rock, Jason R

2017-07-06

To investigate the role of immune cells in lung regeneration, we used a unilateral pneumonectomy model that promotes the formation of new alveoli in the remaining lobes. Immunofluorescence and single-cell RNA sequencing found CD115+ and CCR2+ monocytes and M2-like macrophages accumulating in the lung during the peak of type 2 alveolar epithelial stem cell (AEC2) proliferation. Genetic loss of function in mice and adoptive transfer studies revealed that bone marrow-derived macrophages (BMDMs) traffic to the lung through a CCL2-CCR2 chemokine axis and are required for optimal lung regeneration, along with Il4ra-expressing leukocytes. Our data suggest that these cells modulate AEC2 proliferation and differentiation. Finally, we provide evidence that group 2 innate lymphoid cells are a source of IL-13, which promotes lung regeneration. Together, our data highlight the potential for immunomodulatory therapies to stimulate alveologenesis in adults. Copyright © 2017 Elsevier Inc. All rights reserved.

The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Claire Y.-H., E-mail: CHuang1@cdc.go; Butrapet, Siritorn; Moss, Kelly J.

The flaviviral envelope (E) protein directs virus-mediated membrane fusion. To investigate membrane fusion as a requirement for virus growth, we introduced 27 unique mutations into the fusion peptide of an infectious cDNA clone of dengue 2 virus and recovered seven stable mutant viruses. The fusion efficiency of the mutants was impaired, demonstrating for the first time the requirement for specific FP AAs in optimal fusion. Mutant viruses exhibited different growth kinetics and/or genetic stabilities in different cell types and adult mosquitoes. Virus particles could be recovered following RNA transfection of cells with four lethal mutants; however, recovered viruses could notmore » re-infect cells. These viruses could enter cells, but internalized virus appeared to be retained in endosomal compartments of infected cells, thus suggesting a fusion blockade. Mutations of the FP also resulted in reduced virus reactivity with flavivirus group-reactive antibodies, confirming earlier reports using virus-like particles.« less

Lithium D-cell study

NASA Technical Reports Server (NTRS)

Size, P.; Takeuchi, Esther S.

1993-01-01

The purpose of this contract is to evaluate parametrically the effects of various factors including the electrolyte type, electrolyte concentration, depolarizer type, and cell configuration on lithium cell electrical performance and safety. This effort shall allow for the selection and optimization of cell design for future NASA applications while maintaining close ties with WGL's continuous improvements in manufacturing processes and lithium cell design. Taguchi experimental design techniques are employed in this task, and allow for a maximum amount of information to be obtained while requiring significantly less cells than if a full factorial design were employed. Acceptance testing for this task is modeled after the NASA Document EP5-83-025, Revision C, for cell weights, OCV's and load voltages. The performance attributes that are studied in this effort are fresh capacity and start-up characteristics evaluated at two rates and two temperatures, shelf-life characteristics including start-up and capacity retention, and iterative microcalorimetry measurements. Abuse testing includes forced over discharge at two rates with and without diode protection, temperature tolerance testing, and shorting tests at three rates with the measurement of heat generated during shorting conditions.

Efficient genome editing of differentiated renal epithelial cells.

PubMed

Hofherr, Alexis; Busch, Tilman; Huber, Nora; Nold, Andreas; Bohn, Albert; Viau, Amandine; Bienaimé, Frank; Kuehn, E Wolfgang; Arnold, Sebastian J; Köttgen, Michael

2017-02-01

Recent advances in genome editing technologies have enabled the rapid and precise manipulation of genomes, including the targeted introduction, alteration, and removal of genomic sequences. However, respective methods have been described mainly in non-differentiated or haploid cell types. Genome editing of well-differentiated renal epithelial cells has been hampered by a range of technological issues, including optimal design, efficient expression of multiple genome editing constructs, attainable mutation rates, and best screening strategies. Here, we present an easily implementable workflow for the rapid generation of targeted heterozygous and homozygous genomic sequence alterations in renal cells using transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR) system. We demonstrate the versatility of established protocols by generating novel cellular models for studying autosomal dominant polycystic kidney disease (ADPKD). Furthermore, we show that cell culture-validated genetic modifications can be readily applied to mouse embryonic stem cells (mESCs) for the generation of corresponding mouse models. The described procedure for efficient genome editing can be applied to any cell type to study physiological and pathophysiological functions in the context of precisely engineered genotypes.

Sniffing out cancer using the JPL electronic nose: a pilot study of a novel approach to detection and differentiation of brain cancer.

PubMed

Kateb, Babak; Ryan, M A; Homer, M L; Lara, L M; Yin, Yufang; Higa, Kerin; Chen, Mike Y

2009-08-01

A proof-of-concept study was done to determine whether an electronic nose developed for air quality monitoring at the Jet Propulsion Laboratory (JPL) could be used to distinguish between the odors of organ and tumor tissues, with an eye to using such a device as one of several modes in multi-modal imaging and tumor differentiation during surgery. We hypothesized that the JPL electronic nose (ENose) would be able to distinguish between the odors of various organ and tumor tissues. The odor signatures, or array response, of two organs, chicken heart and chicken liver, and cultured glioblastoma and melanoma tumor cell lines were recorded using the JPL Electronic Nose. The overall array responses were compared to determine whether they were sufficiently different to allow the organs and cell lines to be identified by their array responses. The ENose was able to distinguish between the two types of organ tissue and between the two types of tumor cell lines. The variation in array response for the organ tissues was 19% and between the two types of cultured cell lines was 22%. This study shows that it is possible to use an electronic nose to distinguish between two types of tumor cells and between two types of organ tissue. As we conducted the experiment with a sensor array built for air quality monitoring rather than for medical purposes, it may be possible to select an array that is optimized to distinguish between different types of cells and organ tissues. Further focused studies are needed to investigate the odor signatures of different cells as well as cellular proliferation, growth, differentiation and infiltration.

MicroRNA-139 suppresses proliferation in luminal type breast cancer cells by targeting Topoisomerase II alpha

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hua, Wei; State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, 710032 Xi'an; Sa, Ke-Di

The classification of molecular subtypes of breast cancer improves the prognostic accuracy and therapeutic benefits in clinic. However, because of the complexity of breast cancer, more biomarkers and functional molecules need to be explored. Here, analyzing the data in a huge cohort of breast cancer patients, we found that Topoisomerase II alpha (TOP2a), an important target of chemotherapy is a biomarker for prognosis in luminal type breast cancer patients, but not in basal like or HER2 positive breast cancer patients. We identified that miR-139, a previous reported anti-metastatic microRNA targets 3’-untranslated region (3′UTR) of TOP2a mRNA. Further more, we revealedmore » that the forced expression of miR-139 reduces the TOP2a expression at both mRNA and protein levels. And our functional experiments showed that the ectopic expression of miR-139 remarkably inhibits proliferation in luminal type breast cancer cells, while exogenous TOP2a expression could rescue inhibition of cell proliferation mediated by miR-139. Collectively, our present study demonstrates the miR-139-TOP2a regulatory axis is important for proliferation in luminal type breast cancer cells. This functional link may help us to further understand the specificity of subtypes of breast cancer and optimize the strategy of cancer treatment. - Highlights: • High levels of TOP2a expression are closely associated with poor prognosis in luminal type breast cancer patients. • TOP2a is a novel target of miR-139. • Overexpression of miR-139 inhibits proliferation in luminal type breast cancer cells. • TOP2a is essential for miR-139-induced growth arrest in luminal type breast cancer cells.« less

Multidimensional biochemical information processing of dynamical patterns

NASA Astrophysics Data System (ADS)

Hasegawa, Yoshihiko

2018-02-01

Cells receive signaling molecules by receptors and relay information via sensory networks so that they can respond properly depending on the type of signal. Recent studies have shown that cells can extract multidimensional information from dynamical concentration patterns of signaling molecules. We herein study how biochemical systems can process multidimensional information embedded in dynamical patterns. We model the decoding networks by linear response functions, and optimize the functions with the calculus of variations to maximize the mutual information between patterns and output. We find that, when the noise intensity is lower, decoders with different linear response functions, i.e., distinct decoders, can extract much information. However, when the noise intensity is higher, distinct decoders do not provide the maximum amount of information. This indicates that, when transmitting information by dynamical patterns, embedding information in multiple patterns is not optimal when the noise intensity is very large. Furthermore, we explore the biochemical implementations of these decoders using control theory and demonstrate that these decoders can be implemented biochemically through the modification of cascade-type networks, which are prevalent in actual signaling pathways.

Multidimensional biochemical information processing of dynamical patterns.

PubMed

Hasegawa, Yoshihiko

2018-02-01

Cells receive signaling molecules by receptors and relay information via sensory networks so that they can respond properly depending on the type of signal. Recent studies have shown that cells can extract multidimensional information from dynamical concentration patterns of signaling molecules. We herein study how biochemical systems can process multidimensional information embedded in dynamical patterns. We model the decoding networks by linear response functions, and optimize the functions with the calculus of variations to maximize the mutual information between patterns and output. We find that, when the noise intensity is lower, decoders with different linear response functions, i.e., distinct decoders, can extract much information. However, when the noise intensity is higher, distinct decoders do not provide the maximum amount of information. This indicates that, when transmitting information by dynamical patterns, embedding information in multiple patterns is not optimal when the noise intensity is very large. Furthermore, we explore the biochemical implementations of these decoders using control theory and demonstrate that these decoders can be implemented biochemically through the modification of cascade-type networks, which are prevalent in actual signaling pathways.

Development path and current status of the NANIVID: a new device for cancer cell studies.

PubMed

Raja, Waseem Khan; Padgen, Michael R; Williams, James K; Gertler, Frank B; Wyckoff, Jeffrey B; Condeelis, John S; Castracane, James

2012-03-29

Cancer cells create a unique microenvironment in vivo that enables migration to distant organs. To better understand the tumor micro-environment, special tools and devices are required to monitor the interactions between different cell types and the effects of particular chemical gradients. Our study presents the design and optimization of a versatile chemotaxis device, the nano-intravital device (NANIVID), which consists of etched and bonded glass substrates that create a soluble factor reservoir. The device contains a customized hydrogel blend that is loaded with epidermal growth factor (EGF), which diffuses from the outlet to create a chemotactic gradient that can be sustained for many hours in order to attract specific cells to the device. A microelectrode array is under development for quantification of cell collection and will be incorporated into future device generations. Additionally, the NANIVID can be modified to generate gradients of other soluble factors in order to initiate controlled changes to the microenvironment including the induction of hypoxia, manipulation of extracellular matrix stiffness, etc. The focus of the article is to present the design and optimization of the device towards wide ranging applications of cancer cell dynamics in vitro and, ultimately, implantation for in vivo investigations.

Remodeling of bovine oviductal epithelium by mitosis of secretory cells.

PubMed

Ito, Sayaka; Kobayashi, Yoshihiko; Yamamoto, Yuki; Kimura, Koji; Okuda, Kiyoshi

2016-11-01

Two types of oviductal epithelial cells, secretory and ciliated, play crucial roles in the first days after fertilization in mammals. Secretory cells produce various molecules promoting embryo development, while ciliated cells facilitate transport of oocytes and zygotes by ciliary beating. The proportions of the two cell types change during the estrous cycle. The proportion of ciliated cells on the oviductal luminal surface is abundant at the follicular phase, whereas the proportion of secretory cells gradually increases with the formation of the corpus luteum. In the present study, we hypothesize that the proportions of ciliated and secretory epithelial cells are regulated by mitosis. The proportion of the cells being positive for FOXJ1 (a ciliated cell marker) or Ki67 (a mitosis marker) in epithelial cells during the estrous cycle were immunohistochemically examined. Ki67 and FOXJ1 or PAX8 (a secretory cell marker), were double-stained to clarify which types of epithelial cells undergo mitosis. In the ampulla, the percentage of FOXJ1-positive cells was highest at the day of ovulation (Day 0) and decreased by about 50 % by Days 8-12, while in the isthmus it did not change during the estrous cycle. The proportion of Ki67-positive cells was highest at around the time of ovulation in both the ampulla and isthmus. All the Ki67-positive cells were PAX8-positive and FOXJ1-negative in both the ampulla and isthmus. These findings suggest that epithelial remodeling, which is regulated by differentiation and/or proliferation of secretory cells of the oviduct, provides the optimal environment for gamete transport, fertilization and embryonic development.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agostoni, Marco; Lucker, Ben F.; Smith, Matthew A. Y.

Phycobilisomes (PBSs) are pigment-rich super-complexes required for efficient harvest and transfer of light energy to photosynthetic reaction centers of cyanobacteria. The model cyanobacterium Fremyella diplosiphon is able to adjust PBS pigmentation and size in response to the prevailing light spectrum through a process called complementary chromatic acclimation to optimize spectral light absorption, concomitantly optimizing photosynthesis and growth. We explored the fitness costs versus advantages of modulating antennae size and composition under sinusoidal continuous and fluctuating light conditions in F. diplosiphon by comparing growth of wild-type (WT) cells with a mutant strain deficient in PBSs in both monoculture and polyculture conditions.more » Comparative analyses of WT and the PBS-deficient FdCh1 strain under continuous vs. fluctuating sinusoidal light suggest a potential fitness advantage for maintaining PBSs in WT cells during continuous light and a fitness cost during transitions to and acclimation under fluctuating light. Here, we explored the physiological changes correlated with the observed differential growth to understand the dynamics and biochemical bases of comparative fitness of distinct strains under defined growth conditions. Wild-type F. diplosiphon appears to accumulate longer PBS rods and exhibits higher oxidative stress under fluctuating light conditions than continuous sinusoidal light, which may impact responses and the fitness of cells that do not adapt to rapid changes in external light.« less

Silica nanoparticles on front glass for efficiency enhancement in superstrate-type amorphous silicon solar cells

NASA Astrophysics Data System (ADS)

Das, Sonali; Banerjee, Chandan; Kundu, Avra; Dey, Prasenjit; Saha, Hiranmay; Datta, Swapan K.

2013-10-01

Antireflective coating on front glass of superstrate-type single junction amorphous silicon solar cells (SCs) has been applied using highly monodispersed and stable silica nanoparticles (NPs). The silica NPs having 300 nm diameter were synthesized by Stober technique where the size of the NPs was controlled by varying the alcohol medium. The synthesized silica NPs were analysed by dynamic light scattering technique and Fourier transform infrared spectroscopy. The NPs were spin coated on glass side of fluorinated tin oxide (SnO2: F) coated glass superstrate and optimization of the concentration of the colloidal solution, spin speed and number of coated layers was done to achieve minimum reflection characteristics. An estimation of the distribution of the NPs for different optimization parameters has been done using field-emission scanning electron microscopy. Subsequently, the transparent conducting oxide coated glass with the layer having the minimum reflectance is used for fabrication of amorphous silicon SC. Electrical analysis of the fabricated cell indicates an improvement of 6.5% in short-circuit current density from a reference of 12.40 mA cm-2 while the open circuit voltage and the fill factor remains unaltered. A realistic optical model has also been proposed to gain an insight into the system.

Role of STAT1 in Chlamydia-Induced Type-1 Interferon Production in Oviduct Epithelial Cells

PubMed Central

Hosey, Kristen Lynette; Hu, Sishun

2015-01-01

We previously reported that Chlamydia muridarum-infected murine oviduct epithelial cells (OE cells) secrete interferon β (IFN-β) in a mostly TLR3-dependent manner. However, C. muridarum-infected TLR3-deficient OE cells were still able to secrete detectable levels of IFN-β into the supernatants, suggesting that other signaling pathways contribute to Chlamydia-induced IFN-β synthesis in these cells. We investigated the role of STAT1 as a possible contributor in the Chlamydia-induced type-1 IFN production in wild-type (WT) and TLR3-deficient OE cells to ascertain its putative role at early- and late-times during Chlamydia infection. Our data show that C. muridarum infection significantly increased STAT1 gene expression and protein activation in WT OE cells; however, TLR3-deficient OE cells showed diminished STAT1 protein activation and gene expression. There was significantly less IFN-β detected in the supernatants of C. muridarum-infected OE cells derived from mice deficient in STAT1 when compared with WT OE cells, which suggest that STAT1 is required for the optimal synthesis of IFN-β during infection. Real-time quantitative polymerase chain reaction analyses of signaling components of the type-1 IFN signaling pathway demonstrated equal upregulation in the expression of STAT2 and IRF7 genes in the WT and TLR3-deficient OE cells, but no upregulation in these genes in the STAT1-deficient OE cells. Finally, experiments in which INFAR1 was blocked with neutralizing antibody revealed that IFNAR1-mediated signaling was critical to the Chlamydia-induced upregulation in IFN-α gene transcription, but had no role in the Chlamydia-induced upregulation in IFN-β gene transcription. PMID:26262558

Toxicity Minimized Cryoprotectant Addition and Removal Procedures for Adherent Endothelial Cells

PubMed Central

Davidson, Allyson Fry; Glasscock, Cameron; McClanahan, Danielle R.; Benson, James D.; Higgins, Adam Z.

2015-01-01

Ice-free cryopreservation, known as vitrification, is an appealing approach for banking of adherent cells and tissues because it prevents dissociation and morphological damage that may result from ice crystal formation. However, current vitrification methods are often limited by the cytotoxicity of the concentrated cryoprotective agent (CPA) solutions that are required to suppress ice formation. Recently, we described a mathematical strategy for identifying minimally toxic CPA equilibration procedures based on the minimization of a toxicity cost function. Here we provide direct experimental support for the feasibility of these methods when applied to adherent endothelial cells. We first developed a concentration- and temperature-dependent toxicity cost function by exposing the cells to a range of glycerol concentrations at 21°C and 37°C, and fitting the resulting viability data to a first order cell death model. This cost function was then numerically minimized in our state constrained optimization routine to determine addition and removal procedures for 17 molal (mol/kg water) glycerol solutions. Using these predicted optimal procedures, we obtained 81% recovery after exposure to vitrification solutions, as well as successful vitrification with the relatively slow cooling and warming rates of 50°C/min and 130°C/min. In comparison, conventional multistep CPA equilibration procedures resulted in much lower cell yields of about 10%. Our results demonstrate the potential for rational design of minimally toxic vitrification procedures and pave the way for extension of our optimization approach to other adherent cell types as well as more complex systems such as tissues and organs. PMID:26605546

Cytotoxicity of cadmium-free quantum dots and their use in cell bioimaging.

PubMed

Soenen, Stefaan J; Manshian, Bella B; Aubert, Tangi; Himmelreich, Uwe; Demeester, Jo; De Smedt, Stefaan C; Hens, Zeger; Braeckmans, Kevin

2014-06-16

The use of quantum dots (QDots) as bright and photostable probes for long-term fluorescence imaging is gaining more interest. Thus far, (pre)clinical use of QDots remains limited, which is primarily caused by the potential toxicity of QDots. Most QDots consist of Cd2+ ions, which are known to cause high levels of toxicity. In order to overcome this problem, several strategies have been tested, such as the generation of cadmium-free QDots. In the present study, two types of cadmium-free QDots, composed of ZnSe/ZnS (QDotZnSe) and InP/ZnS (QDotInP), were studied with respect to their cytotoxicity and cellular uptake in a variety of cell types. A multiparametric cytotoxicity approach is used, where the QDots are studied with respect to cell viability, oxidative stress, cell morphology, stem cell differentiation, and neurite outgrowth. The data reveal slight differences in uptake levels for both types of QDots (maximal for QDotZnSe), but clear differences in cytotoxicity and cell functionality effects exist, with highest toxicity for QDotZnSe. Differences between cell types and between both types of QDots can be explained by the intrinsic sensitivity of certain cell types and chemical composition of the QDots. At concentrations at which no toxic effects can be observed, the functionality of the QDots for fluorescence cell visualization is evaluated, revealing that the higher brightness of QDotZnSe overcomes most of the toxicity issues compared to that of QDotInP. Comparing the results obtained with common Cd2+-containing QDots tested under identical conditions, the importance of particle functionality is demonstrated, revealing that cadmium-free QDots tested in this study are not significantly better than Cd2+-containing QDots for long-term cell imaging and that more work needs to be performed in optimizing the brightness and surface chemistry of cadmium-free QDots for them to replace currently used Cd2+-containing QDots.

Normal Untreated Jurkat Cells

NASA Technical Reports Server (NTRS)

2004-01-01

Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. The objective of the research was to define a way to differentiate between effects due to microgravity and those due to possible stress from non-optimal spaceflight conditions. These Jurkat cells, a human acute T-cell leukemia was obtained to evaluate three types of potential experimental stressors: a) Temperature elevation; b) Serum starvation; and c) Centrifugal force. The data from previous spaceflight experiments showed that actin filaments and cell shape are significantly different for the control. These normal cells serve as the baseline for future spaceflight experiments.

Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells.

PubMed

Ouyang, Liliang; Yao, Rui; Zhao, Yu; Sun, Wei

2016-09-16

3D cell printing is an emerging technology for fabricating complex cell-laden constructs with precise and pre-designed geometry, structure and composition to overcome the limitations of 2D cell culture and conventional tissue engineering scaffold technology. This technology enables spatial manipulation of cells and biomaterials, also referred to as 'bioink', and thus allows study of cellular interactions in a 3D microenvironment and/or in the formation of functional tissues and organs. Recently, many efforts have been made to develop new bioinks and to apply more cell sources for better biocompatibility and biofunctionality. However, the influences of printing parameters on the shape fidelity of 3D constructs as well as on cell viability after the cell printing process have been poorly characterized. Furthermore, parameter optimization based on a specific cell type might not be suitable for other types of cells, especially cells with high sensibility. In this study, we systematically studied the influence of bioink properties and printing parameters on bioink printability and embryonic stem cell (ESC) viability in the process of extrusion-based cell printing, also known as bioplotting. A novel method was established to determine suitable conditions for bioplotting ESCs to achieve both good printability and high cell viability. The rheological properties of gelatin/alginate bioinks were evaluated to determine the gelation properties under different bioink compositions, printing temperatures and holding times. The bioink printability was characterized by a newly developed semi-quantitative method. The results demonstrated that bioinks with longer gelation times would result in poorer printability. The live/dead assay showed that ESC viability increased with higher printing temperatures and lower gelatin concentrations. Furthermore, an exponential relationship was obtained between ESC viability and induced shear stress. By defining the proper printability and acceptable viability ranges, a combined parameters region was obtained. This study provides guidance for parameter optimization and the fine-tuning of 3D cell printing processes regarding both bioink printability and cell viability after bioplotting, especially for easily damaged cells, like ESCs.

Recombination-activating gene 1 (Rag1)-deficient mice with severe combined immunodeficiency treated with lentiviral gene therapy demonstrate autoimmune Omenn-like syndrome.

PubMed

van Til, Niek P; Sarwari, Roya; Visser, Trudi P; Hauer, Julia; Lagresle-Peyrou, Chantal; van der Velden, Guus; Malshetty, Vidyasagar; Cortes, Patricia; Jollet, Arnaud; Danos, Olivier; Cassani, Barbara; Zhang, Fang; Thrasher, Adrian J; Fontana, Elena; Poliani, Pietro L; Cavazzana, Marina; Verstegen, Monique M A; Villa, Anna; Wagemaker, Gerard

2014-04-01

Recombination-activating gene 1 (RAG1) deficiency results in severe combined immunodeficiency (SCID) caused by a complete lack of T and B lymphocytes. If untreated, patients succumb to recurrent infections. We sought to develop lentiviral gene therapy for RAG1-induced SCID and to test its safety. Constructs containing the viral spleen-focus-forming virus (SF), ubiquitous promoters, or cell type-restricted promoters driving sequence-optimized RAG1 were compared for efficacy and safety in sublethally preconditioned Rag1(-/-) mice undergoing transplantation with transduced bone marrow progenitors. Peripheral blood CD3(+) T-cell reconstitution was achieved with SF, ubiquitous promoters, and cell type-restricted promoters but 3- to 18-fold lower than that seen in wild-type mice, and with a compromised CD4(+)/CD8(+) ratio. Mitogen-mediated T-cell responses and T cell-dependent and T cell-independent B-cell responses were not restored, and T-cell receptor patterns were skewed. Reconstitution of mature peripheral blood B cells was approximately 20-fold less for the SF vector than in wild-type mice and often not detectable with the other promoters, and plasma immunoglobulin levels were abnormal. Two months after transplantation, gene therapy-treated mice had rashes with cellular tissue infiltrates, activated peripheral blood CD44(+)CD69(+) T cells, high plasma IgE levels, antibodies against double-stranded DNA, and increased B cell-activating factor levels. Only rather high SF vector copy numbers could boost T- and B-cell reconstitution, but mRNA expression levels during T- and B-cell progenitor stages consistently remained less than wild-type levels. These results underline that further development is required for improved expression to successfully treat patients with RAG1-induced SCID while maintaining low vector copy numbers and minimizing potential risks, including autoimmune reactions resembling Omenn syndrome. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Customizing the targeting of IGF-1 receptor.

PubMed

Baserga, Renato

2009-02-01

The type 1 IGF receptor (IGF-IR) is activated by two ligands, IGF-1 and IGF-2, and by insulin at supraphysiological concentrations. It plays a significant role in the growth of normal and abnormal cells, and antibodies against the IGF-IR are now in clinical trials. Targeting of the IGF-IR in cancer cells (by antibodies or other means) can be improved by the appropriate selection of responsive tumors. This review focuses on the optimization of IGF-IR targeting in human cancer.

Tenogenesis of bone marrow-, adipose-, and tendon-derived stem cells in a dynamic bioreactor.

PubMed

Youngstrom, Daniel W; LaDow, Jade E; Barrett, Jennifer G

2016-11-01

Tendons are frequently damaged and fail to regenerate, leading to pain, loss of function, and reduced quality of life. Mesenchymal stem cells (MSCs) possess clinically useful tissue-regenerative properties and have been exploited for use in tendon tissue engineering and cell therapy. However, MSCs exhibit phenotypic heterogeneity based on the donor tissue used, and the efficacy of cell-based treatment modalities may be improved by optimizing cell source based on relative differentiation capacity. Equine MSCs were isolated from bone marrow (BM), adipose (AD), and tendon (TN), expanded in monolayer prior to seeding on decellularized tendon scaffolds (DTS), and cell-laden constructs were placed in a bioreactor designed to mimic the biophysical environment of the tendon. It was hypothesized that TN MSCs would differentiate toward a tendon cell phenotype better than BM and AD MSCs in response to a conditioning period involving cyclic mechanical stimulation for 1 hour per day at 3% strain and 0.33 Hz. All cell types integrated into DTS adopted an elongated morphology similar to tenocytes, expressed tendon marker genes, and improved tissue mechanical properties after 11 days. TN MSCs expressed the greatest levels of scleraxis, collagen type-I, and cartilage oligomeric matrix protein. Major histocompatibility class-II protein mRNA expression was not detected in any of the MSC types, suggesting low immunogenicity for allogeneic transplantation. The results suggest that TN MSCs are the ideal cell type for regenerative medicine therapies for tendinopathies, exhibiting the most mature tendon-like phenotype in vitro. When TN MSCs are unavailable, BM or AD MSCs may serve as robust alternatives.

Optimization of Organic Solar Cells: Materials, Devices and Interfaces

NASA Astrophysics Data System (ADS)

Zhou, Nanjia

Due to the increasing demand for sustainable clean energy, photovoltaic cells have received intensified attention in the past decade in both academia and industry. Among the types of cells, organic photovoltaic (OPV) cells offer promise as alternatives to conventional inorganic-type solar cells owning to several unique advantages such as low material and fabrication cost. To maximize power conversion efficiencies (PCEs), extensive research efforts focus on frontier molecular orbital (FMO) energy engineering of photoactive materials. Towards this objective, a series of novel donor polymers incorporating a new building block, bithiophene imide (BTI) group are developed, with narrow bandgap and low-lying highest occupied molecular orbital (HOMO) energies to increase short circuit current density, Jsc, and open circuit voltage, Voc.. Compared to other PV technologies, OPVs often suffer from large internal recombination loss and relatively low fill factors (FFs) <70%. Through a combination of materials design and device architecture optimization strategies to improve both microscopic and macroscopic thin film morphology, OPVs with PCEs up to 8.7% and unprecedented FF approaching 80% are obtained. Such high FF are close to those typically achieved in amorphous Si solar cells. Systematic variations of polymer chemical structures lead to understanding of structure-property relationships between polymer geometry and the resulting blend film morphology characteristics which are crucial for achieving high local mobilities and long carrier lifetimes. Instead of using fullerene as the acceptors, an alternative type of OPV is developed employing a high electron mobility polymer, P(NDI2OD-T2), as the acceptor. To improve the all-polymer blend film morphology, the influence of basic solvent properties such as solvent boiling point and solubility on polymer phase separation and charge transport properties is investigated, yielding to a high PCE of 2.7% for all-polymer solar cells. To take advantages of the inherent mechanical flexibility associated with organic materials, the development of transparent, flexible substrates to replace the conventionally used polycrystalline ITO electrodes is highly desirable. Employing an ultraflexible amorphous zinc indium tin oxide (a-ZITO) transparent conducting oxide (TCO), highly efficient OPVs with similar PCEs to rigid ones are obtained. Furthermore, these cells show no significant PCE reduction under controlled bending test.

Cell structure and function in the visual cortex of the cat

PubMed Central

Kelly, J. P.; Van Essen, D. C.

1974-01-01

1. The organization of the visual cortex was studied with a technique that allows one to determine the physiology and morphology of individual cells. Micro-electrodes filled with the fluorescent dye Procion yellow were used to record intracellularly from cells in area 17 of the cat. The visual receptive field of each neurone was classified as simple, complex, or hypercomplex, and the cell was then stained by the iontophoretic injection of dye. 2. Fifty neurones were successfully examined in this way, and their structural features were compared to the varieties of cell types seen in Golgi preparations of area 17. The majority of simple units were stellate cells, whereas the majority of complex and hypercomplex units were pyramidal cells. Several neurones belonged to less common morphological types, such as double bouquet cells. Simple cells were concentrated in layer IV, hypercomplex cells in layer II + III, and complex cells in layers II + III, V and VI. 3. Electrically inexcitable cells that had high resting potentials but no impulse activity were stained and identified as glial cells. Glial cells responded to visual stimuli with slow graded depolarizations, and many of them showed a preference for a stimulus orientation similar to the optimal orientation for adjacent neurones. 4. The results show that there is a clear, but not absolute correlation between the major structural and functional classes of cells in the visual cortex. This approach, linking the physiological properties of a single cell to a given morphological type, will help in furthering our understanding of the cerebral cortex. ImagesPlate 4Plate 1Plate 2Plate 3 PMID:4136579

Scaffold architecture and fibrin gels promote meniscal cell proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pawelec, K. M., E-mail: pawelec.km@gmail.com, E-mail: jw626@cam.ac.uk; Best, S. M.; Cameron, R. E.

2015-01-01

Stability of the knee relies on the meniscus, a complex connective tissue with poor healing ability. Current meniscal tissue engineering is inadequate, as the signals for increasing meniscal cell proliferation have not been established. In this study, collagen scaffold structure, isotropic or aligned, and fibrin gel addition were tested. Metabolic activity was promoted by fibrin addition. Cellular proliferation, however, was significantly increased by both aligned architectures and fibrin addition. None of the constructs impaired collagen type I production or triggered adverse inflammatory responses. It was demonstrated that both fibrin gel addition and optimized scaffold architecture effectively promote meniscal cell proliferation.

Nanometer-Scale Electrical Potential Profiling Across Perovskite Solar Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiao, Chuanxiao; Jiang, Chun-Sheng; Ke, Weijun

2016-11-21

We used Kelvin probe force microscopy to study the potential distribution on cross-section of perovskite solar cells with different types of electron-transporting layers (ETLs). Our results explain the low open-circuit voltage and fill factor in ETL-free cells, and support the fact that intrinsic SnO2 as an alternative ETL material can make high-performance devices. Furthermore, the potential-profiling results indicate a reduction in junction-interface recombination by the optimized SnO2 layer and adding a fullerene layer, which is consistent with the improved device performance and current-voltage hysteresis.

Design of high-efficiency, radiation-hard, GaInP/GaAs solar cells

NASA Technical Reports Server (NTRS)

Kurtz, Sarah R.; Bertness, K. A.; Kibbler, A. E.; Kramer, C.; Olson, J. M.

1994-01-01

In recently years, Ga(0.5)In((0.5)P/GaAs cells have drawn increased attention both because of their high efficiencies and because they are well suited for space applications. They can be grown and processed as two-junction devices with roughly twice the voltage and half the current of GaAs cells. They have low temperature coefficients, and have good potential for radiation hardness. We have previously reported the effects of electron irradiation on test cells which were not optimally designed for space. From those results we estimated that an optimally designed cell could achieve 20 percent after irradiation with 10(exp 15) cm(exp -2) 1 MeV electrons. Modeling studies predicted that slightly higher efficiencies may be achievable. Record efficiencies for EOL performance of other types of cells are significantly lower. Even the best Si and InP cells have BOL efficiencies lower than the EOL efficiency we report here. Good GaAs cells have an EOL efficiency of 16 percent. The InP/Ga(0.5)In(0.5)As two-junction, two-terminal device has a BOL efficiency as high as 22.2 percent, but radiation results for these cells were limited. In this study we use the previous modeling and irradiation results to design a set of Ga(0.5)In(0.5)P/GaAs cells that will demonstrate the importance of the design parameters and result in high-efficiency devices. We report record AMO efficiencies: a BOL efficiency of 25.7 percent for a device optimized for BOL performance and two of different designs with EOL efficiencies of 19.6 percent (at 10(exp 15) cm(exp -2) 1MeV electrons). We vary the bottom-cell base doping and the top-cell thickness to show the effects of these two important design parameters. We get an unexpected result indicating that the dopant added to the bottom-cell base also increases the degradation of the top cell.

[Stem cells in adults].

PubMed

Borge, O J; Funderud, S

2001-08-30

We present a literature review of the plasticity observed by adult stem cells. We have reviewed the literature regarding stem cells from adults in order to summarise their ability to generate cells of other types than those of the tissue/organ from which they were isolated. Adult stem cells have recently been demonstrated to terminally differentiate into cells of other tissues than those from which they were originally isolated. For example, bone marrow cells have been shown to generate liver, nerve, heart and skeletal muscle cells in addition to their well-known ability to produce blood and mesenchymal cells. Most studies demonstrate a proof-of-principle in animal models; much more research is needed before adult stem cells can be utilised in human medicine. However, the published reports are encouraging and give reasons for a cautious optimism with regard to future clinical use.

Avian photoreceptor patterns represent a disordered hyperuniform solution to a multiscale packing problem

NASA Astrophysics Data System (ADS)

Jiao, Yang; Lau, Timothy; Hatzikirou, Haralampos; Meyer-Hermann, Michael; Corbo, Joseph C.; Torquato, Salvatore

2014-02-01

Optimal spatial sampling of light rigorously requires that identical photoreceptors be arranged in perfectly regular arrays in two dimensions. Examples of such perfect arrays in nature include the compound eyes of insects and the nearly crystalline photoreceptor patterns of some fish and reptiles. Birds are highly visual animals with five different cone photoreceptor subtypes, yet their photoreceptor patterns are not perfectly regular. By analyzing the chicken cone photoreceptor system consisting of five different cell types using a variety of sensitive microstructural descriptors, we find that the disordered photoreceptor patterns are "hyperuniform" (exhibiting vanishing infinite-wavelength density fluctuations), a property that had heretofore been identified in a unique subset of physical systems, but had never been observed in any living organism. Remarkably, the patterns of both the total population and the individual cell types are simultaneously hyperuniform. We term such patterns "multihyperuniform" because multiple distinct subsets of the overall point pattern are themselves hyperuniform. We have devised a unique multiscale cell packing model in two dimensions that suggests that photoreceptor types interact with both short- and long-ranged repulsive forces and that the resultant competition between the types gives rise to the aforementioned singular spatial features characterizing the system, including multihyperuniformity. These findings suggest that a disordered hyperuniform pattern may represent the most uniform sampling arrangement attainable in the avian system, given intrinsic packing constraints within the photoreceptor epithelium. In addition, they show how fundamental physical constraints can change the course of a biological optimization process. Our results suggest that multihyperuniform disordered structures have implications for the design of materials with novel physical properties and therefore may represent a fruitful area for future research.

CD34+ Testicular Stromal Cells Support Long-Term Expansion of Embryonic and Adult Stem and Progenitor Cells

PubMed Central

Kim, Jiyeon; Seandel, Marco; Falciatori, Ilaria; Wen, Duancheng; Rafii, Shahin

2010-01-01

Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multi-potent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. PMID:18669907

Local Inflammatory Cues Regulate Differentiation and Persistence of CD8+ Tissue-Resident Memory T Cells.

PubMed

Bergsbaken, Tessa; Bevan, Michael J; Fink, Pamela J

2017-04-04

Many pathogens initiate infection at mucosal surfaces, and tissue-resident memory T (Trm) cells play an important role in protective immunity, yet the tissue-specific signals that regulate Trm differentiation are poorly defined. During Yersinia infection, CD8 + T cell recruitment to areas of inflammation within the intestine is required for differentiation of the CD103 - CD69 + Trm subset. Intestinal proinflammatory microenvironments have elevated interferon (IFN)-β and interleukin-12 (IL-12), which regulated Trm markers, including CD103. Type I interferon-receptor- or IL-12-receptor-deficient T cells functioned similarly to wild-type (WT) cells during infection; however, the inability of T cells to respond to inflammation resulted in defective differentiation of CD103 - CD69 + Trm cells and reduced Trm persistence. Intestinal macrophages were the main producers of IFN-β and IL-12 during infection, and deletion of CCR2 + IL-12-producing cells reduced the size of the CD103 - Trm population. These data indicate that intestinal inflammation drives phenotypic diversity and abundance of Trm cells for optimal tissue-specific immunity. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Developmental insights from early mammalian embryos and core signaling pathways that influence human pluripotent cell growth and differentiation.

PubMed

Chen, Kevin G; Mallon, Barbara S; Johnson, Kory R; Hamilton, Rebecca S; McKay, Ronald D G; Robey, Pamela G

2014-05-01

Human pluripotent stem cells (hPSCs) have two potentially attractive applications: cell replacement-based therapies and drug discovery. Both require the efficient generation of large quantities of clinical-grade stem cells that are free from harmful genomic alterations. The currently employed colony-type culture methods often result in low cell yields, unavoidably heterogeneous cell populations, and substantial chromosomal abnormalities. Here, we shed light on the structural relationship between hPSC colonies/embryoid bodies and early-stage embryos in order to optimize current culture methods based on the insights from developmental biology. We further highlight core signaling pathways that underlie multiple epithelial-to-mesenchymal transitions (EMTs), cellular heterogeneity, and chromosomal instability in hPSCs. We also analyze emerging methods such as non-colony type monolayer (NCM) and suspension culture, which provide alternative growth models for hPSC expansion and differentiation. Furthermore, based on the influence of cell-cell interactions and signaling pathways, we propose concepts, strategies, and solutions for production of clinical-grade hPSCs, stem cell precursors, and miniorganoids, which are pivotal steps needed for future clinical applications. Published by Elsevier B.V.

Optimization of Streptomyces bacteriophage phi C31 integrase system to prevent post integrative gene silencing in pulmonary type II cells.

PubMed

Aneja, Manish Kumar; Geiger, Johannes; Imker, Rabea; Uzgun, Senta; Kormann, Michael; Hasenpusch, Guenther; Maucksch, Christof; Rudolph, Carsten

2009-12-31

phi C31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study aimed at optimizing elements of phi C31 integrase system for alveolar type II cells. Luciferase and beta-galactosidase activities were measured at different time points post transfection. 5-Aza-2'deoxycytidine (AZA) and trichostatin A (TSA) were used to inhibit DNA methyltransferase and histone deacetylase complex (HDAC) respectively. In A549 cells, expression of the integrase using a CMV promoter resulted in highest integrase activity, whereas in MLE12 cells, both CAG and CMV promoter were equally effective. Effect of polyA site was observed only in A549 cells, where replacement of SV40 polyA by bovine growth hormone (BGH) polyA site resulted in an enhancement of integrase activity. Addition of a C-terminal SV40 nuclear localization signal (NLS) did not result in any significant increase in integrase activity. Long-term expression studies with AZA and TSA, provided evidence for post-integrative gene silencing. In MLE12 cells, both DNA methylases and HDACs played a significant role in silencing, whereas in A549 cells, it could be attributed majorly to HDAC activity. Donor plasmids comprising cellular promoters ubiquitin B (UBB), ubiquitin C (UCC) and elongation factor 1 alpha (EF1 alpha) in an improved backbone prevented post-integrative gene silencing. In contrast to A549 and MLE12 cells, no silencing could be observed in human bronchial epithelial cells, BEAS-2B. Donor plasmid coding for murine erythropoietin under the EF1 alpha promoter when combined with phi C31 integrase resulted in higher long-term erythropoietin expression and subsequently higher hematocrit levels in mice after intravenous delivery to the lungs. These results provide evidence for cell specific post integrative gene silencing with C31 integrase and demonstrate the pivotal role of donor plasmid in long-term expression attained with this system.

Insulin-Like Growth Factor-Type 1 Receptor Inhibitor NVP-AEW541 Enhances Radiosensitivity of PTEN Wild-Type but Not PTEN-Deficient Human Prostate Cancer Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Isebaert, Sofie F., E-mail: sofie.isebaert@med.kuleuven.be; Swinnen, Johannes V.; McBride, William H.

2011-09-01

Purpose: During the past decade, many clinical trials with both monoclonal antibodies and small molecules that target the insulin-like growth factor-type 1 receptor (IGF-1R) have been launched. Despite the important role of IGF-1R signaling in radioresistance, studies of such agents in combination with radiotherapy are lagging behind. Therefore, the aim of this study was to investigate the effect of the small molecule IGF-1R kinase inhibitor NVP-AEW541 on the intrinsic radioresistance of prostate cancer cells. Methods and Materials: The effect of NVP-AEW541 on cell proliferation, cell viability, IGF-1R signaling, radiosensitivity, cell cycle distribution, and double strand break repair was determined inmore » three human prostate cancer cell lines (PC3, DU145, 22Rv1). Moreover, the importance of the PTEN pathway status was explored by means of transfection experiments with constitutively active Akt or inactive kinase-dead Akt. Results: NVP-AEW541 inhibited cell proliferation and decreased cell viability in a time-and dose-dependent manner in all three cell lines. Radiosensitization was observed in the PTEN wild-type cell lines DU145 and 22Rv1 but not in the PTEN-deficient PC3 cell line. NVP-AEW541-induced radiosensitization coincided with downregulation of phospho-Akt levels and high levels of residual double strand breaks. The importance of PTEN status in the radiosensitization effect was confirmed by transfection experiments with constitutively active Akt or inactive kinase-dead Akt. Conclusions: NVP-AEW541 enhances the effect of ionizing radiation in PTEN wild-type, but not in PTEN-deficient, prostate cancer cells. Proper patient selection based on the PTEN status of the tumor will be critical to the achievement of optimal results in clinical trials in which the combination of radiotherapy and this IGF-1R inhibitor is being explored.« less

Continuous in vivo infusion of interferon-gamma (IFN-γ) enhances engraftment of syngeneic wild-type cells in Fanca–/– and Fancg–/– mice

PubMed Central

Si, Yue; Ciccone, Samantha; Yang, Feng-Chun; Yuan, Jin; Zeng, Daisy; Chen, Shi; van de Vrugt, Henri J.; Critser, John; Arwert, Fre; Haneline, Laura S.; Clapp, D. Wade

2006-01-01

Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc–/– cells to interferon-gamma (IFN-γ), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc–/– mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca–/– and Fancg–/– mice are hypersensitive to IFN-γ and that in vivo infusion of IFN-γ at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-γ conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients. PMID:16946306

Continuous in vivo infusion of interferon-gamma (IFN-gamma) enhances engraftment of syngeneic wild-type cells in Fanca-/- and Fancg-/- mice.

PubMed

Si, Yue; Ciccone, Samantha; Yang, Feng-Chun; Yuan, Jin; Zeng, Daisy; Chen, Shi; van de Vrugt, Henri J; Critser, John; Arwert, Fre; Haneline, Laura S; Clapp, D Wade

2006-12-15

Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc-/- cells to interferon-gamma (IFN-gamma), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc-/- mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca-/- and Fancg-/- mice are hypersensitive to IFN-gamma and that in vivo infusion of IFN-gamma at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-gamma conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients.

Assessment of cell culture and polymerase chain reaction procedures for the detection of polioviruses in wastewater.

PubMed Central

Grabow, W. O.; Botma, K. L.; de Villiers, J. C.; Clay, C. G.; Erasmus, B.

1999-01-01

WHO considers that environmental surveillance for wild-type polioviruses is potentially important for surveillance for acute flaccid paralysis as a means of confirming eradication of poliomyelitis. The present study investigated methods for detecting polioviruses in a variety of water environments in South Africa. Most polioviruses were isolated on L20B mouse cells, which, however, were not selective: 16 reoviruses and 8 enteroviruses, apparently animal strains, were also isolated on these cells. Vaccine strains of polioviruses were isolated from surface waters during and shortly after two rounds of mass vaccination of children in an informal settlement where there was no sewerage. The results demonstrated the feasibility of poliovirus surveillance in such settlements. It was also evident that neither poliovirus vaccine strains nor other viruses were likely to interfere significantly with the detection of wild-type polioviruses. Optimal isolation of polioviruses was accomplished by parallel inoculation of L20B mouse cells and at least the PLC/PRF/5 human liver and buffalo green monkey (BGM) kidney cell lines. Analysis of cell cultures using the polymerase chain reaction revealed that 319 test samples contained at least 263 human enteroviruses that failed to produce a cytopathogenic effect. This type of analysis thus significantly increased the sensitivity of enterovirus detection. PMID:10680244

[Stem Cells in the Brain of Mammals and Human: Fundamental and Applied Aspects].

PubMed

Aleksandrova, M A; Marey, M V

2015-01-01

Brain stem cells represent an extremely intriguing phenomenon. The aim of our review is to present an integrity vision of their role in the brain of mammals and humans, and their clinical perspectives. Over last two decades, investigations of biology of the neural stem cells produced significant changes in general knowledge about the processes of development and functioning of the brain. Researches on the cellular and molecular mechanisms of NSC differentiation and behavior led to new understanding of their involvement in learning and memory. In the regenerative medicine, original therapeutic approaches to neurodegenerative brain diseases have been elaborated due to fundamental achievements in this field. They are based on specific regenerative potential of neural stem cells and progenitor cells, which possess the ability to replace dead cells and express crucially significant biologically active factors that are missing in the pathological brain. For the needs of cell substitution therapy in the neural diseases, adequate methods of maintaining stem cells in culture and their differentiation into different types of neurons and glial cells, have been developed currently. The success of modern cellular technologies has significantly expanded the range of cells used for cell therapy. The near future may bring new perspective and distinct progress in brain cell therapy due to optimizing the cells types most promising for medical needs.

Cytoplasmic Flow Enhances Organelle Dispersion in Eukaryotic Cells

NASA Astrophysics Data System (ADS)

Koslover, Elena; Mogre, Saurabh; Chan, Caleb; Theriot, Julie

The cytoplasm of a living cell is an active environment through which intracellular components move and mix. We explore, using theoretical modeling coupled with microrheological measurements, the efficiency of particle dispersion via different modes of transport within this active environment. In particular, we focus on the role of cytoplasmic flow over different scales in contributing to organelle transport within two different cell types. In motile neutrophil cells, we show that bulk fluid flow associated with rapid cell deformation enhances particle transport to and from the cell periphery. In narrow fungal hyphae, localized flows due to hydrodynamic entrainment are shown to contribute to optimally efficient organelle dispersion. Our results highlight the importance of non-traditional modes of transport associated with flow of the cytoplasmic fluid in the distribution of organelles throughout eukaryotic cells.

Stem Cell Therapy for Incontinence: Where Are We Now? What is the Realistic Potential?

PubMed Central

Dissaranan, Charuspong; Cruz, Michelle A.; Couri, Bruna M.; Goldman, Howard B.

2011-01-01

A significant number of women experience stress urinary incontinence (SUI), which greatly affects their quality of life. Recent research investigating utilization of stem cells and their derivatives for the prevention and treatment of SUI has been performed to test the effect of cell source and method of administration in several animal models of SUI. The type of stem cell, timing of optimal dose or doses after injury, mechanism of action of stem cells, and route of administration must be investigated both preclinically and clinically before stem cell therapy becomes a possible treatment for SUI, although the future of this therapy looks promising. This article reviews the progress in stem cell research for incontinence and describes areas of future work as suggested by research in other fields. PMID:21842258

Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium.

PubMed

Peng, Lin; Yu, Xiao; Li, Chengyuan; Cai, Yanfei; Chen, Yun; He, Yang; Yang, Jianfeng; Jin, Jian; Li, Huazhong

2016-04-01

Signal peptides play an important role in directing and efficiently transporting secretory proteins to their proper locations in the endoplasmic reticulum of mammalian cells. The aim of this study was to enhance the expression of recombinant coagulation factor VII (rFVII) in CHO cells by optimizing the signal peptides and type of fed-batch culture medium used. Five sub-clones (O2, I3, H3, G2 and M3) with different signal peptide were selected by western blot (WB) analysis and used for suspension culture. We compared rFVII expression levels of 5 sub-clones and found that the highest rFVII expression level was obtained with the IgK signal peptide instead of Ori, the native signal peptide of rFVII. The high protein expression of rFVII with signal peptide IgK was mirrored by a high transcription level during suspension culture. After analyzing culture and feed media, the combination of M4 and F4 media yielded the highest rFVII expression of 20 mg/L during a 10-day suspension culture. After analyzing cell density and cell cycle, CHO cells feeding by F4 had a similar percentage of cells in G0/G1 and a higher cell density compared to F2 and F3. This may be the reason for high rFVII expression in M4+F4. In summary, rFVII expression was successfully enhanced by optimizing the signal peptide and fed-batch medium used in CHO suspension culture. Our data may be used to improve the production of other therapeutic proteins in fed-batch culture.

Immunoglobulin D (IgD)-deficient mice reveal an auxiliary receptor function for IgD in antigen-mediated recruitment of B cells

PubMed Central

1993-01-01

To assess the role of immunoglobulin D (IgD) in vivo we generated IgD- deficient mice by gene targeting and studied B cell development and function in the absence of IgD expression. In the mutant animals, conventional and CD5-positive (B1) B cells are present in normal numbers, and the expression of the surface markers CD22 and CD23 in the compartment of conventional B cells indicates acquisition of a mature phenotype. As in wild-type animals, most of the peripheral B cells are resting cells. The IgD-deficient mice respond well to T cell- independent and -dependent antigens. However, in heterozygous mutant animals, B cells expressing the wild type IgH locus are overrepresented in the peripheral B cell pool, and T cell-dependent IgG1 responses are further dominated by B cells expressing the wild-type allele. Similarly, in homozygous mutant (IgD-deficient) animals, affinity maturation is delayed in the early primary response compared to control animals, although the mutants are capable of generating high affinity B cell memory. Thus, rather than being involved in major regulatory processes as had been suggested, IgD seems to function as an antigen receptor optimized for efficient recruitment of B cells into antigen- driven responses. The IgD-mediated acceleration of affinity maturation in the early phase of the T cell-dependent primary response may confer to the animal a critical advantage in the defense against pathogens. PMID:8418208

Type I Interferons as Stimulators of DC-Mediated Cross-Priming: Impact on Anti-Tumor Response.

PubMed

Schiavoni, Giovanna; Mattei, Fabrizio; Gabriele, Lucia

2013-12-25

Induction of potent tumor-specific cytotoxic T-cell responses is a fundamental objective in anticancer therapeutic strategies. This event requires that antigen-presenting cells present tumor-associated antigens (Ag) on their MHC class-I molecule, in a process termed cross-presentation. Dendritic cells (DC) are particularly keen on this task and can induce the cross-priming of CD8(+) T cells, when exposed to danger or inflammatory signals that stimulate their activation. Type I interferons (IFN-I), a family of long-known immunostimulatory cytokines, have been proven to produce optimal activation signal for DC-induced cross-priming. Recent in vitro and in vivo evidences have suggested that IFN-I-stimulated cross-priming by DC against tumor-associated Ag is a key mechanism for cancer immunosurveillance and may be usefully exploited to boost anti-tumor CD8(+) T-cell responses. Here, we will review the cross-presentation properties of different DC subsets, with special focus on cell-associated and tumor Ag, and discuss how IFN-I can modify this function, with the aim of identifying more specific and effective strategies for improving anticancer responses.

Development path and current status of the NANIVID: a new device for cancer cell studies

NASA Astrophysics Data System (ADS)

Raja, Waseem Khan; Padgen, Michael R.; Williams, James K.; Wyckoff, Jeffrey; Condeelis, John; Castracane, James

2011-02-01

Cancer cells create a unique microenvironment in vivo which enables migration to distant organs. To better understand the tumor microenvironment, special tools and devices are required to monitor the interactions between different cell types and the effects of particular chemical gradients. This study presents the design and optimization of a new, versatile chemotaxis device called the NANIVID (NANo IntraVital Device). The device is fabricated using BioMEMS techniques and consists of etched and bonded Pyrex substrates, a soluble factor reservoir, fluorescent tracking beads and a microelectrode array for cell quantification. The reservoir contains a customized hydrogel blend loaded with EGF which diffuses out of the hydrogel to create a chemotactic gradient. This reservoir sustains a steady release of growth factor into the surrounding environment for many hours and establishes a concentration gradient that attracts specific cells to the device. In addition to a cell collection tool, the NANIVID can be modified to act as a delivery vehicle for the local generation of alternate soluble factor gradients to initiate controlled changes to the microenvironment such as hypoxia, ECM stiffness and etc. The focus of this study is to design and optimize the new device for wide ranging studies of breast cancer cell dynamics in vitro and ultimately, implantation for in vivo work.

Effects of duration of electric pulse on in vitro development of cloned cat embryos with human artificial chromosome vector.

PubMed

Do, Ltk; Wittayarat, M; Terazono, T; Sato, Y; Taniguchi, M; Tanihara, F; Takemoto, T; Kazuki, Y; Kazuki, K; Oshimura, M; Otoi, T

2016-12-01

The current applications for cat cloning include production of models for the study of human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a human artificial chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0 kV/cm for either 30 or 60 μs. Low rates of fusion and embryo development to the blastocyst stage were observed in the reconstructed HAC-transchromosomic embryos, when the duration of fusion was prolonged to 60 μs. In contrast, the prolongation of electric pulse duration improved the embryo development and quality in the reconstructed control embryos without HAC vector. Our results suggested that the optimal parameters of electric pulses for fusion in cat somatic cell nuclear transfer vary among the types used for donor cells. © 2016 Blackwell Verlag GmbH.

Establishment of optimized MDCK cell lines for reliable efflux transport studies.

PubMed

Gartzke, Dominik; Fricker, Gert

2014-04-01

Madin-Darby canine kidney (MDCK) cells transfected with human MDR1 gene (MDCK-MDR1) encoding for P-glycoprotein (hPgp, ABCB1) are widely used for transport studies to identify drug candidates as substrates of this efflux protein. Therefore, it is necessary to rely on constant and comparable expression levels of Pgp to avoid false negative or positive results. We generated a cell line with homogenously high and stable expression of hPgp through sorting single clones from a MDCK-MDR1 cell pool using fluorescence-activated cell sorting (FACS). To obtain control cell lines for evaluation of cross-interactions with endogenous canine Pgp (cPgp) wild-type cells were sorted with a low expression pattern of cPgp in comparison with the MDCK-MDR1. Expression of other transporters was also characterized in both cell lines by quantitative real-time PCR and Western blot. Pgp function was investigated applying the Calcein-AM assay as well as bidirectional transport assays using (3) H-Digoxin, (3) H-Vinblastine, and (3) H-Quinidine as substrates. Generated MDCK-MDR1 cell lines showed high expression of hPgp. Control MDCK-WT cells were optimized in showing a comparable expression level of cPgp in comparison with MDCK-MDR1 cell lines. Generated cell lines showed higher and more selective Pgp transport compared with parental cells. Therefore, they provide a significant improvement in the performance of efflux studies yielding more reliable results. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Optimizing the dosing schedule of l-asparaginase improves its anti-tumor activity in breast tumor-bearing mice.

PubMed

Shiromizu, Shoya; Kusunose, Naoki; Matsunaga, Naoya; Koyanagi, Satoru; Ohdo, Shigehiro

2018-04-01

Proliferation of acute lymphoblastic leukemic cells is nutritionally dependent on the external supply of asparagine. l-asparaginase, an enzyme hydrolyzing l-asparagine in blood, is used for treatment of acute lymphoblastic leukemic and other related blood cancers. Although previous studies demonstrated that l-asparaginase suppresses the proliferation of cultured solid tumor cells, it remains unclear whether this enzyme prevents the growth of solid tumors in vivo. In this study, we demonstrated the importance of optimizing dosing schedules for the anti-tumor activity of l-asparaginase in 4T1 breast tumor-bearing mice. Cultures of several types of murine solid tumor cells were dependent on the external supply of asparagine. Among them, we selected murine 4T1 breast cancer cells and implanted them into BALB/c female mice kept under standardized light/dark cycle conditions. The growth of 4T1 tumor cells implanted in mice was significantly suppressed by intravenous administration of l-asparaginase during the light phase, whereas its administration during the dark phase failed to show significant anti-tumor activity. Decreases in plasma asparagine levels due to the administration of l-asparaginase were closely related to the dosing time-dependency of its anti-tumor effects. These results suggest that the anti-tumor efficacy of l-asparaginase in breast tumor-bearing mice is improved by optimizing the dosing schedule. Copyright © 2018 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Process development for the mass production of Ehrlichia ruminantium.

PubMed

Marcelino, Isabel; Sousa, Marcos F Q; Veríssimo, Célia; Cunha, António E; Carrondo, Manuel J T; Alves, Paula M

2006-03-06

This work describes the optimization of a cost-effective process for the production of an inactivated bacterial vaccine against heartwater and the first attempt to produce the causative agent of this disease, the rickettsia Ehrlichia ruminantium (ER), using stirred tanks. In vitro, it is possible to produce ER using cultures of ruminant endothelial cells. Herein, mass production of these cells was optimized for stirring conditions. The effect of inoculum size, microcarrier type, concentration of serum at inoculation time and agitation rate upon maximum cell concentration were evaluated. Several strategies for the scale-up of cell inoculum were also tested. Afterwards, using the optimized parameters for cell growth, ER production in stirred tanks was validated for two ER strains (Gardel and Welgevonden). Critical parameters related with the infection strategy such as serum concentration at infection time, multiplicity and time of infection, and medium refeed strategy were analyzed. The results indicate that it is possible to produce ER in stirred tank bioreactors, under serum-free culture conditions, reaching a 6.5-fold increase in ER production yields. The suitability of this process was validated up to a 2-l scale and a preliminary cost estimation has shown that the stirred tanks are the least expensive culture method. Overall, these results are crucial to define a scaleable and fully controlled process for the production of a heartwater vaccine and open "new avenues" for the production of vaccines against other ehrlichial species, with emerging impact in human and animal health.

FLASH X-RAY (FXR) LINEAR INDUCTION ACCELERATOR (LIA) OPTIMIZATION Sensor Delay Correction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ong, M M; Houck, T L; Kreitzer, B R

2006-05-01

The radiographic goal of the FXR Optimization Project is to generate an x-ray pulse with peak energy of 19 MeV, spot-size of 1.5 mm, a dose of 500 rad, and duration of 60 ns. The electrical objectives are to generate a 3 kA electron-beam and refine our 16 MV accelerator so that the voltage does not vary more than 1%-rms. In a multi-cell linear induction accelerator, like FXR, the timing of the acceleration pulses relative to the beam is critical. The pulses must be timed optimally so that a cell is at full voltage before the beam arrives and doesmore » not drop until the beam passes. In order to stay within the energy-variation budget, the synchronization between the cells and beam arrival must be controlled to a couple of nanoseconds. Therefore, temporal measurements must be accurate to a fraction of a nanosecond. FXR Optimization Project developed a one-giga-sample per second (gs/s) data acquisition system to record beam sensor data. Signal processing algorithms were written to determine cell timing with an uncertainty of a fraction of a nanosecond. However, the uncertainty in the sensor delay was still a few nanoseconds. This error had to be reduced if we are to improve the quality of the electron beam. Two types of sensors are used to align the cell voltage pulse against the beam current. The beam current is measured with resistive-wall sensors. The cell voltages are read with capacitive voltage monitors. Sensor delays can be traced to two mechanisms: (1) the sensors are not co-located at the beam and cell interaction points, and (2) the sensors have different length jumper cables and other components that connect them to the standard-length coaxial cables of the data acquisition system. Using the physical locations and dimensions of the sensor components, and the dielectric constant of the materials, delay times were computed. Relative to the cell voltage, the beam current was theoretically reporting late by 7.7 ns. Two experiments were performed to verify and refine the sensor delay correction. In the first experiment, the beam was allowed to drift through a cell that was not pulsed. The beam induces a potential into the cell that is read by the voltage monitor. Analysis of the data indicated that the beam sensor signal was likely 7.1 ns late. In the second experiment, the beam current is calculated from the injector diode voltage that is the sum of the cell voltages. A 7 ns correction produced a very good match between the signals from the two types of sensors. For simplicity, we selected a correction factor that advanced the current signals by 7 ns. This should reduce the uncertainty in the temporal measurements to less than 1 ns.« less

Hydrocarbons Are Essential for Optimal Cell Size, Division, and Growth of Cyanobacteria.

PubMed

Lea-Smith, David J; Ortiz-Suarez, Maite L; Lenn, Tchern; Nürnberg, Dennis J; Baers, Laura L; Davey, Matthew P; Parolini, Lucia; Huber, Roland G; Cotton, Charles A R; Mastroianni, Giulia; Bombelli, Paolo; Ungerer, Petra; Stevens, Tim J; Smith, Alison G; Bond, Peter J; Mullineaux, Conrad W; Howe, Christopher J

2016-11-01

Cyanobacteria are intricately organized, incorporating an array of internal thylakoid membranes, the site of photosynthesis, into cells no larger than other bacteria. They also synthesize C15-C19 alkanes and alkenes, which results in substantial production of hydrocarbons in the environment. All sequenced cyanobacteria encode hydrocarbon biosynthesis pathways, suggesting an important, undefined physiological role for these compounds. Here, we demonstrate that hydrocarbon-deficient mutants of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 exhibit significant phenotypic differences from wild type, including enlarged cell size, reduced growth, and increased division defects. Photosynthetic rates were similar between strains, although a minor reduction in energy transfer between the soluble light harvesting phycobilisome complex and membrane-bound photosystems was observed. Hydrocarbons were shown to accumulate in thylakoid and cytoplasmic membranes. Modeling of membranes suggests these compounds aggregate in the center of the lipid bilayer, potentially promoting membrane flexibility and facilitating curvature. In vivo measurements confirmed that Synechococcus sp. PCC 7002 mutants lacking hydrocarbons exhibit reduced thylakoid membrane curvature compared to wild type. We propose that hydrocarbons may have a role in inducing the flexibility in membranes required for optimal cell division, size, and growth, and efficient association of soluble and membrane bound proteins. The recent identification of C15-C17 alkanes and alkenes in microalgal species suggests hydrocarbons may serve a similar function in a broad range of photosynthetic organisms. © 2016 American Society of Plant Biologists. All Rights Reserved.

Folate-conjugated chitosan-polylactide nanoparticles for enhanced intracellular uptake of anticancer drug

NASA Astrophysics Data System (ADS)

Huang, Shengtang; Wan, Ying; Wang, Zheng; Wu, Jiliang

2013-12-01

Chitosan was conjugated with folic acid (FA) and the resulting chitosan derivatives with a FA-substitution degree of around 6 % was used to synthesize FA-conjugated chitosan-polylactide (FA-CH-PLA) copolymers to build a drug carrier with active targeting characteristics for the anticancer drug of paclitaxel (PTX). Selected FA-CH-PLAs with various polylactide percentages of about 40 wt% or lower were employed to fabricate nanoparticles using sodium tripolyphosphate as a crosslinker, and different types of nanoparticles were endued with similar average particle-sizes located in a range between 100 and 200 nm. Certain types of PTX-loaded FA-CH-PLA nanoparticles having encapsulation efficiency of around 90 % and initial load of about 12 % were able to release PTX in a controlled manner with significant regulation by polylactide content in FA-CH-PLAs. Targeting characteristic of achieved nanoparticles was confirmed using FA-receptor-expressed MCF-7 breast cancer cells. The uptake of PTX revealed that optimized FA-CH-PLA nanoparticles with an equivalent PTX-dose of around 1 μg/mL could have more than sixfold increasing abilities to facilitate intracellular paclitaxel accumulation in MCF-7 cells after 24 h treatment as compared to free PTX. At a relatively safe equivalent PTX-dose for normal MCF-10A mammary epithelial cells, the obtained results from Hoechst 33342 staining indicated that optimized PTX-loaded FA-CH-PLA nanoparticles had more than threefold increasing abilities to induce MCF-7 cell apoptosis in comparison to free PTX.

Performance analysis of nanodisk and core/shell/shell-nanowire type III-Nitride heterojunction solar cell for efficient energy harvesting

NASA Astrophysics Data System (ADS)

Routray, S. R.; Lenka, T. R.

2017-11-01

Now-a-days III-Nitride nanowires with axial (nanodisk) and radial (core/shell/shell-nanowire) junctions are two unique and potential methods for solar energy harvesting adopted by worldwide researchers. In this paper, polarization behavior of GaN/InGaN/GaN junction and its effect on carrier dynamics of nanodisk and CSS-nanowire type solar cells are intensively studied and compared with its planar counterpart by numerical simulations using commercially available Victory TCAD. It is observed that CSS-NW with hexagonal geometrical shapes are robust to detrimental impact of polarization charges and could be good enough to accelerate carrier collection efficiency as compared to nanodisk and planar solar cells. This numerical study provides an innovative aspect of fundamental device physics with respect to polarization charges in CSS-NW and nanodisk type junction towards photovoltaic applications. The internal quantum efficiencies (IQE) are also discussed to evaluate carrier collection mechanisms and recombination losses in each type of junctions of solar cell. Finally, it is interesting to observe a maximum conversion efficiency of 6.46% with 91.6% fill factor from n-GaN/i-In0.1Ga0.9N/p-GaN CSS-nanowire solar cell with an optimized thickness of 180 nm InGaN layer under one Sun AM1.5 illumination.

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes.

PubMed

Traba, Javier; Miozzo, Pietro; Akkaya, Billur; Pierce, Susan K; Akkaya, Munir

2016-11-21

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

Screening attenuation of coaxial cables determined in GTEM-cells

NASA Astrophysics Data System (ADS)

Knobloch, A.; Garbe, H.

2004-05-01

This paper describes the determination of the screening attenuation with a GTEM cell. An analytical part gives the link between the voltage at the cell port and the total radiated power. The next section investigates the optimal cable setup in the cell. With a measurement of the common mode current on the cable and a simulation of the radiation resistance the loop antenna characteristic of the cable setup could be verified. It is shown that the use of ferrit cores decrease the difference between the maximum and the minimum screening attenuation. The determination of great screening attenuation could be improved with the use of N-type measurement cables. A comparison between this GTEM cell method and the standard methods shows a good agreement.

Microgravity

NASA Image and Video Library

2004-04-15

Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. The objective of the research was to define a way to differentiate between effects due to microgravity and those due to possible stress from non-optimal spaceflight conditions. These Jurkat cells, a human acute T-cell leukemia was obtained to evaluate three types of potential experimental stressors: a) Temperature elevation; b) Serum starvation; and c) Centrifugal force. The data from previous spaceflight experiments showed that actin filaments and cell shape are significantly different for the control. These normal cells serve as the baseline for future spaceflight experiments.

Spatial transcriptomics: paving the way for tissue-level systems biology.

PubMed

Moor, Andreas E; Itzkovitz, Shalev

2017-08-01

The tissues in our bodies are complex systems composed of diverse cell types that often interact in highly structured repeating anatomical units. External gradients of morphogens, directional blood flow, as well as the secretion and absorption of materials by cells generate distinct microenvironments at different tissue coordinates. Such spatial heterogeneity enables optimized function through division of labor among cells. Unraveling the design principles that govern this spatial division of labor requires techniques to quantify the entire transcriptomes of cells while accounting for their spatial coordinates. In this review we describe how recent advances in spatial transcriptomics open the way for tissue-level systems biology. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fast globally optimal segmentation of cells in fluorescence microscopy images.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2011-01-01

Accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression in high-throughput screening applications. We propose a new approach for segmenting cell nuclei which is based on active contours and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images of different cell types. We have also performed a quantitative comparison with previous segmentation approaches.

Optimization of the parameters of ITO-CdTe photovoltaic cells

NASA Astrophysics Data System (ADS)

Adib, N.; Simashkevich, A. V.; Sherban, D. A.

The effect of the surface state density at the interface and of the static charge in the intermediate oxide layer on the photoelectric parameters of solar cells based on ITO-nCdTe semiconductor-insulator-semiconductor structures is calculated theoretically. It is shown that,under AMI conditions, the conversion efficiency of such cells can be as high as 12 percent (short-circuit current, 23 mA/sq cm; open-circuit voltage, 0.65 V; fill factor, 0.8), provided that the surface states are acceptors and the oxide is negatively charged. It is concluded that surface states and the dielectric layer charge have a positive effect on the efficiency of solar cells of this type.

Robustness analysis of superpixel algorithms to image blur, additive Gaussian noise, and impulse noise

NASA Astrophysics Data System (ADS)

Brekhna, Brekhna; Mahmood, Arif; Zhou, Yuanfeng; Zhang, Caiming

2017-11-01

Superpixels have gradually become popular in computer vision and image processing applications. However, no comprehensive study has been performed to evaluate the robustness of superpixel algorithms in regard to common forms of noise in natural images. We evaluated the robustness of 11 recently proposed algorithms to different types of noise. The images were corrupted with various degrees of Gaussian blur, additive white Gaussian noise, and impulse noise that either made the object boundaries weak or added extra information to it. We performed a robustness analysis of simple linear iterative clustering (SLIC), Voronoi Cells (VCells), flooding-based superpixel generation (FCCS), bilateral geodesic distance (Bilateral-G), superpixel via geodesic distance (SSS-G), manifold SLIC (M-SLIC), Turbopixels, superpixels extracted via energy-driven sampling (SEEDS), lazy random walk (LRW), real-time superpixel segmentation by DBSCAN clustering, and video supervoxels using partially absorbing random walks (PARW) algorithms. The evaluation process was carried out both qualitatively and quantitatively. For quantitative performance comparison, we used achievable segmentation accuracy (ASA), compactness, under-segmentation error (USE), and boundary recall (BR) on the Berkeley image database. The results demonstrated that all algorithms suffered performance degradation due to noise. For Gaussian blur, Bilateral-G exhibited optimal results for ASA and USE measures, SLIC yielded optimal compactness, whereas FCCS and DBSCAN remained optimal for BR. For the case of additive Gaussian and impulse noises, FCCS exhibited optimal results for ASA, USE, and BR, whereas Bilateral-G remained a close competitor in ASA and USE for Gaussian noise only. Additionally, Turbopixel demonstrated optimal performance for compactness for both types of noise. Thus, no single algorithm was able to yield optimal results for all three types of noise across all performance measures. Conclusively, to solve real-world problems effectively, more robust superpixel algorithms must be developed.

Challenges in cryopreservation of regulatory T cells (Tregs) for clinical therapeutic applications.

PubMed

Golab, Karolina; Leveson-Gower, Dennis; Wang, Xiao-Jun; Grzanka, Jakub; Marek-Trzonkowska, Natalia; Krzystyniak, Adam; Millis, J Michael; Trzonkowski, Piotr; Witkowski, Piotr

2013-07-01

Promising results of initial studies applying ex-vivo expanded regulatory T cell (Treg) as a clinical intervention have increased interest in this type of the cellular therapy and several new clinical trials involving Tregs are currently on the way. Methods of isolation and expansion of Tregs have been studied and optimized to the extent that such therapy is feasible, and allows obtaining sufficient numbers of Tregs in the laboratory following Good Manufacturing Practice (GMP) guidelines. Nevertheless, Treg therapy could even more rapidly evolve if Tregs could be efficiently cryopreserved and stored for future infusion or expansions rather than utilization of only freshly isolated and expanded cells as it is preferred now. Currently, our knowledge regarding the impact of cryopreservation on Treg recovery, viability, and functionality is still limited. Based on experience with cryopreserved peripheral blood mononuclear cells (PBMCs), cryopreservation may have a detrimental effect on Tregs, can decrease Treg viability, cause abnormal cytokine secretion, and compromise expression of surface markers essential for proper Treg function and processing. Therefore, optimal strategies and conditions for Treg cryopreservation in conjunction with cell culture, expansion, and processing for clinical application still need to be investigated and defined. Copyright © 2013 Elsevier B.V. All rights reserved.

Evaluation of telomere length in human cardiac tissues using cardiac quantitative FISH.

PubMed

Sharifi-Sanjani, Maryam; Meeker, Alan K; Mourkioti, Foteini

2017-09-01

Telomere length has been correlated with various diseases, including cardiovascular disease and cancer. The use of currently available telomere-length measurement techniques is often restricted by the requirement of a large amount of cells (Southern-based techniques) or the lack of information on individual cells or telomeres (PCR-based methods). Although several methods have been used to measure telomere length in tissues as a whole, the assessment of cell-type-specific telomere length provides valuable information on individual cell types. The development of fluorescence in situ hybridization (FISH) technologies enables the quantification of telomeres in individual chromosomes, but the use of these methods is dependent on the availability of isolated cells, which prevents their use with fixed archival samples. Here we describe an optimized quantitative FISH (Q-FISH) protocol for measuring telomere length that bypasses the previous limitations by avoiding contributions from undesired cell types. We have used this protocol on small paraffin-embedded cardiac-tissue samples. This protocol describes step-by-step procedures for tissue preparation, permeabilization, cardiac-tissue pretreatment and hybridization with a Cy3-labeled telomeric repeat complementing (CCCTAA) 3 peptide nucleic acid (PNA) probe coupled with cardiac-specific antibody staining. We also describe how to quantify telomere length by means of the fluorescence intensity and area of each telomere within individual nuclei. This protocol provides comparative cell-type-specific telomere-length measurements in relatively small human cardiac samples and offers an attractive technique to test hypotheses implicating telomere length in various cardiac pathologies. The current protocol (from tissue collection to image procurement) takes ∼28 h along with three overnight incubations. We anticipate that the protocol could be easily adapted for use on different tissue types.

Spectroscopic Ellipsometry Studies of Thin Film a-Si:H/nc-Si:H Micromorph Solar Cell Fabrication in the p-i-n Superstrate Configuration

NASA Astrophysics Data System (ADS)

Huang, Zhiquan

Spectroscopic ellipsometry (SE) is a non-invasive optical probe that is capable of accurately and precisely measuring the structure of thin films, such as their thicknesses and void volume fractions, and in addition their optical properties, typically defined by the index of refraction and extinction coefficient spectra. Because multichannel detection systems integrated into SE instrumentation have been available for some time now, the data acquisition time possible for complete SE spectra has been reduced significantly. As a result, real time spectroscopic ellipsometry (RTSE) has become feasible for monitoring thin film nucleation and growth during the deposition of thin films as well as during their removal in processes of thin film etching. Also because of the reduced acquisition time, mapping SE is possible by mounting an SE instrument with a multichannel detector onto a mechanical translation stage. Such an SE system is capable of mapping the thin film structure and its optical properties over the substrate area, and thereby evaluating the spatial uniformity of the component layers. In thin film photovoltaics, such structural and optical property measurements mapped over the substrate area can be applied to guide device optimization by correlating small area device performance with the associated local properties. In this thesis, a detailed ex-situ SE study of hydrogenated amorphous silicon (a-Si:H) thin films and solar cells prepared by plasma enhanced chemical vapor deposition (PECVD) has been presented. An SE analysis procedure with step-by-step error minimization has been applied to obtain accurate measures of the structural and optical properties of the component layers of the solar cells. Growth evolution diagrams were developed as functions of the deposition parameters in PECVD for both p-type and n-type layers to characterize the regimes of accumulated thickness over which a-Si:H, hydrogenated nanocrystalline silicon (nc-Si:H) and mixed phase (a+nc)-Si:H thin films are obtained. The underlying materials for these depositions were newly-deposited intrinsic a-Si:H layers on thermal oxide coated crystalline silicon wafers, designed to simulate specific device configurations. As a result, these growth evolution diagrams can be applied to both p-i-n and n-i-p solar cell optimization. In this thesis, the n-layer growth evolution diagram expressed in terms of hydrogen dilution ratio was applied in correlations with the performance of p-i-n single junction devices in order to optimize these devices. Moreover, ex-situ mapping SE was also employed over the area of multilayer structures in order to achieve better statistics for solar cell optimization by correlating structural parameters locally with small area solar cell performance parameters. In the study of (a-Si:H p-i-n)/(nc-Si:H p-i-n) tandem solar cells, RTSE was successfully applied to monitor the fabrication of the top cell, and efforts to optimize the nanocrystalline p-layer and i-layer of the bottom cell were initiated.

Role of NK cells in immunotherapy and virotherapy of solid tumors.

PubMed

Cantoni, Claudia; Grauwet, Korneel; Pietra, Gabriella; Parodi, Monica; Mingari, Maria Cristina; Maria, Andrea De; Favoreel, Herman; Vitale, Massimo

2015-01-01

Although natural killer (NK) cells are endowed with powerful cytolytic activity against cancer cells, their role in different therapies against solid tumors has not yet been fully elucidated. Their interactions with various elements of the tumor microenvironment as well as their possible effects in contributing to and/or limiting oncolytic virotherapy render this potential immunotherapeutic tool still difficult to exploit at the bedside. Here, we will review the current literature with the aim of providing new hints to manage this powerful cell type in future innovative therapies, such as the use of NK cells in combination with new cytokines, specific mAbs (inducing ADCC), Tyr-Kinase inhibitors, immunomodulatory drugs and/or the design of oncolytic viruses aimed at optimizing the effect of NK cells in virotherapy.

Cell type-dependent variation in paracrine potency determines therapeutic efficacy against neonatal hyperoxic lung injury.

PubMed

Ahn, So Yoon; Chang, Yun Sil; Sung, Dong Kyung; Yoo, Hye Soo; Sung, Se In; Choi, Soo Jin; Park, Won Soon

2015-08-01

The aim of this study was to determine the optimal cell type for transplantation to protect against neonatal hyperoxic lung injury. To this end, the in vitro and in vivo therapeutic efficacies and paracrine potencies of human umbilical cord blood-derived mesenchymal stromal cells (HUMs), human adipose tissue-derived mesenchymal stromal cells (HAMs) and human umbilical cord blood mononuclear cells (HMNs) were compared. Hyperoxic injury was induced in vitro in A549 cells by challenge with H2O2. Alternatively, hyperoxic injury was induced in newborn Sprague-Dawley rats in vivo by exposure to hyperoxia (90% oxygen) for 14 days. HUMs, HAMs or HMNs (5 × 10(5) cells) were given intratracheally at postnatal day 5. Hyperoxia-induced increases in in vitro cell death and in vivo impaired alveolarization were significantly attenuated in both the HUM and HAM groups but not in the HMN group. Hyperoxia impaired angiogenesis, increased the cell death and pulmonary macrophages and elevated inflammatory cytokine levels. These effects were significantly decreased in the HUM group but not in the HAM or HMN groups. The levels of human vascular endothelial growth factor and hepatocyte growth factor produced by donor cells were highest in HUM group, followed by HAM group and then HMN group. HUMs exhibited the best therapeutic efficacy and paracrine potency than HAMs or HMNs in protecting against neonatal hyperoxic lung injury. These cell type-dependent variations in therapeutic efficacy might be associated or mediated with the paracrine potency of the transplanted donor cells. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Two-finger (TF) SPUDT cells.

PubMed

Martin, Guenter; Biryukov, Sergey V; Schmidt, Hagen; Steiner, Bernd; Wall, Bert

2011-03-01

SPUDT cells including two fingers are only known thus far for so-called NSPUDT directions. In that case, usual solid-finger cells are used. The purpose of the present paper is to find SPUDT cell types consisting of two fingers only for pure mode directions. Two-finger (TF) cells for pure mode directions on substrates like 128°YX LiNbO(3) and YZ LiNbO(3) were found by means of an optimization procedure. The forward direction of a TF-cell SPUDT on 128°YX LiNbO(3) was determined experimentally. The properties of the new cells are compared with those of conventional SPUDT cells. The reflectivity of TF cells on 128°YX LiNbO(3) turns out to be two to three times larger than that of distributed acoustic reflection transducer (DART) and Hanma-Hunsinger cells at the same metal layer thickness.

Interplay between transparency and efficiency in dye sensitized solar cells.

PubMed

Tagliaferro, Roberto; Colonna, Daniele; Brown, Thomas M; Reale, Andrea; Di Carlo, Aldo

2013-02-11

In this paper we analyze the interplay between transparency and efficiency in dye sensitized solar cells by varying fabrication parameters such as the thickness of the nano-crystalline TiO(2) layer, the dye loading and the dye type. Both transparency and efficiency show a saturation trend when plotted versus dye loading. By introducing the transparency-efficiency plot, we show that the relation between transparency and efficiency is linear and is almost independent on the TiO(2) thickness for a certain thickness range. On the contrary, the relation between transparency and efficiency depends strongly on the type of the dye. Moreover, we show that co-sensitization techniques can be effectively used to access regions of the transparency-efficiency space that are forbidden for single dye sensitization. The relation found between transparency and efficiency (T&E) can be the general guide for optimization of Dye Solar Cells in building integration applications.

Type-I interferon receptor expression: its circadian rhythm and downregulation after interferon-alpha administration in peripheral blood cells from renal cancer patients.

PubMed

Shiba, Masahiro; Nonomura, Norio; Nakai, Yasutomo; Nakayama, Masashi; Takayama, Hitoshi; Inoue, Hitoshi; Tsujimura, Akira; Nishimura, Kazuo; Okuyama, Akihiko

2009-04-01

To investigate the regulation of interferon-alpha (IFN-alpha) receptor expression in metastatic renal cell carcinoma (RCC) after IFN-alpha administration. Blood sampling was carried out in eight patients with metastatic RCC and six healthy volunteers. Flow-cytometric analysis using a monoclonal antibody against the active subunit of the type-I IFN-alpha receptor (IFNAR2) was carried out to examine the circadian rhythm of IFNAR2 expression in peripheral blood mononuclear cells (PBMC) as well as its downregulation after IFN-alpha administration. According to its circadian rhythm IFNAR2 in PBMC had a peak expression at night. Once IFN-alpha is administered, IFNAR2 levels in PBMC showed downregulation within 48 h and recovered within another 48 h. Our findings might support the establishment of an optimal schedule for IFN-alpha administration.

Designable DNA-binding domains enable construction of logic circuits in mammalian cells.

PubMed

Gaber, Rok; Lebar, Tina; Majerle, Andreja; Šter, Branko; Dobnikar, Andrej; Benčina, Mojca; Jerala, Roman

2014-03-01

Electronic computer circuits consisting of a large number of connected logic gates of the same type, such as NOR, can be easily fabricated and can implement any logic function. In contrast, designed genetic circuits must employ orthogonal information mediators owing to free diffusion within the cell. Combinatorial diversity and orthogonality can be provided by designable DNA- binding domains. Here, we employed the transcription activator-like repressors to optimize the construction of orthogonal functionally complete NOR gates to construct logic circuits. We used transient transfection to implement all 16 two-input logic functions from combinations of the same type of NOR gates within mammalian cells. Additionally, we present a genetic logic circuit where one input is used to select between an AND and OR function to process the data input using the same circuit. This demonstrates the potential of designable modular transcription factors for the construction of complex biological information-processing devices.

A Study of Parameters Affecting Fibroblast Morphology in Response to an Applied Mechanical Force

NASA Technical Reports Server (NTRS)

Grymes, Rosalind A.; Sawyer, Christine

1994-01-01

A precisely controlled stretch/relaxation regimen (20% elongation at 6.6 cycles/min) was applied to normal human fetal, neonatal and aged dermal fibroblasts cultured on flexible membranes. Culture conditions included poly (NH2) or collagen type I coated substrate membranes; control cultures were grown on the same pliable material in the absence of applied stretch. Direct observation and immunofluorescence analyses revealed a progressive change in cell body orientation limited to the stretched dermal fibroblast cultures. Monolayers gradually (over 4 days) acquired a symmetric, radial distribution equivalent to the biaxial array of the applied force. At high seeding density, alignment was inhibited in the fetal cell cultures. This cell strain required collagen type I coating for optimal attachment to the flexible membrane, preferring growth in three-dimensional cell 'balls' on the poly(NH2) coated substrate. Neonatal cells also required the collagen type I coating, but both neonatal and aged dermal fibroblasts aligned efficiently at all seeding densities examined. The randomly oriented neonatal cells on the unstretched control membranes spontaneously detached at confluence, as a single cell sheet. Their aligned counterparts did not detach until the applied stretch stimulus was removed. Low concentrations of cytochalasin D (62.5 ng/ml) disrupted the stretch-related alignment response. Rhodamine phalloidin staining visualized fewer actin stress fibers in stretched, aligned cells than in controls. Both intercellular interactions and cytoskeletal integrity mediate the response to mechanical strain. Normal rabbit corneal stroma fibroblasts (NRC) were also analyzed, and failed to orient under these conditions. This cell type may require a different regimen, or a longer time period, to demonstrate alignment behavior. Supported by NASA Space Biology RTOP 199-40-22 and the NASA-ARC Director's Discretionary Fund.

Enhancement of Lipid Productivity in Oleaginous Colletotrichum Fungus through Genetic Transformation Using the Yeast CtDGAT2b Gene under Model-Optimized Growth Condition

PubMed Central

Dey, Prabuddha; Mall, Nikunj; Chattopadhyay, Atrayee; Chakraborty, Monami; Maiti, Mrinal K.

2014-01-01

Oleaginous fungi are of special interest among microorganisms for the production of lipid feedstocks as they can be cultured on a variety of substrates, particularly waste lingocellulosic materials, and few fungal strains are reported to accumulate inherently higher neutral lipid than bacteria or microalgae. Previously, we have characterized an endophytic filamentous fungus Colletotrichum sp. DM06 that can produce total lipid ranging from 34% to 49% of its dry cell weight (DCW) upon growing with various carbon sources and nutrient-stress conditions. In the present study, we report on the genetic transformation of this fungal strain with the CtDGAT2b gene, which encodes for a catalytically efficient isozyme of type-2 diacylglycerol acyltransferase (DGAT) from oleaginous yeast Candida troplicalis SY005. Besides the increase in size of lipid bodies, total lipid titer by the transformed Colletotrichum (lipid content ∼73% DCW) was found to be ∼1.7-fold more than the wild type (lipid content ∼38% DCW) due to functional activity of the CtDGAT2b transgene when grown under standard condition of growth without imposition of any nutrient-stress. Analysis of lipid fractionation revealed that the neutral lipid titer in transformants increased up to 1.8-, 1.6- and 1.5-fold compared to the wild type when grown under standard, nitrogen stress and phosphorus stress conditions, respectively. Lipid titer of transformed cells was further increased to 1.7-fold following model-based optimization of culture conditions. Taken together, ∼2.9-fold higher lipid titer was achieved in Colletotrichum fungus due to overexpression of a rate-limiting crucial enzyme of lipid biosynthesis coupled with prediction-based bioprocess optimization. PMID:25375973

Model-Based Analysis for Qualitative Data: An Application in Drosophila Germline Stem Cell Regulation

PubMed Central

Pargett, Michael; Rundell, Ann E.; Buzzard, Gregery T.; Umulis, David M.

2014-01-01

Discovery in developmental biology is often driven by intuition that relies on the integration of multiple types of data such as fluorescent images, phenotypes, and the outcomes of biochemical assays. Mathematical modeling helps elucidate the biological mechanisms at play as the networks become increasingly large and complex. However, the available data is frequently under-utilized due to incompatibility with quantitative model tuning techniques. This is the case for stem cell regulation mechanisms explored in the Drosophila germarium through fluorescent immunohistochemistry. To enable better integration of biological data with modeling in this and similar situations, we have developed a general parameter estimation process to quantitatively optimize models with qualitative data. The process employs a modified version of the Optimal Scaling method from social and behavioral sciences, and multi-objective optimization to evaluate the trade-off between fitting different datasets (e.g. wild type vs. mutant). Using only published imaging data in the germarium, we first evaluated support for a published intracellular regulatory network by considering alternative connections of the same regulatory players. Simply screening networks against wild type data identified hundreds of feasible alternatives. Of these, five parsimonious variants were found and compared by multi-objective analysis including mutant data and dynamic constraints. With these data, the current model is supported over the alternatives, but support for a biochemically observed feedback element is weak (i.e. these data do not measure the feedback effect well). When also comparing new hypothetical models, the available data do not discriminate. To begin addressing the limitations in data, we performed a model-based experiment design and provide recommendations for experiments to refine model parameters and discriminate increasingly complex hypotheses. PMID:24626201

Evaluation of endogenous control genes for gene expression studies across multiple tissues and in the specific sets of fat- and muscle-type samples of the pig.

PubMed

Gu, Y R; Li, M Z; Zhang, K; Chen, L; Jiang, A A; Wang, J Y; Li, X W

2011-08-01

To normalize a set of quantitative real-time PCR (q-PCR) data, it is essential to determine an optimal number/set of housekeeping genes, as the abundance of housekeeping genes can vary across tissues or cells during different developmental stages, or even under certain environmental conditions. In this study, of the 20 commonly used endogenous control genes, 13, 18 and 17 genes exhibited credible stability in 56 different tissues, 10 types of adipose tissue and five types of muscle tissue, respectively. Our analysis clearly showed that three optimal housekeeping genes are adequate for an accurate normalization, which correlated well with the theoretical optimal number (r ≥ 0.94). In terms of economical and experimental feasibility, we recommend the use of the three most stable housekeeping genes for calculating the normalization factor. Based on our results, the three most stable housekeeping genes in all analysed samples (TOP2B, HSPCB and YWHAZ) are recommended for accurate normalization of q-PCR data. We also suggest that two different sets of housekeeping genes are appropriate for 10 types of adipose tissue (the HSPCB, ALDOA and GAPDH genes) and five types of muscle tissue (the TOP2B, HSPCB and YWHAZ genes), respectively. Our report will serve as a valuable reference for other studies aimed at measuring tissue-specific mRNA abundance in porcine samples. © 2011 Blackwell Verlag GmbH.

In Vitro Differentiation of First Trimester Human Umbilical Cord Perivascular Cells into Contracting Cardiomyocyte-Like Cells.

PubMed

Szaraz, Peter; Librach, Matthew; Maghen, Leila; Iqbal, Farwah; Barretto, Tanya A; Kenigsberg, Shlomit; Gauthier-Fisher, Andrée; Librach, Clifford L

2016-01-01

Myocardial infarction (MI) causes an extensive loss of heart muscle cells and leads to congestive heart disease (CAD), the leading cause of mortality and morbidity worldwide. Mesenchymal stromal cell- (MSC-) based cell therapy is a promising option to replace invasive interventions. However the optimal cell type providing significant cardiac regeneration after MI is yet to be found. The aim of our study was to investigate the cardiomyogenic differentiation potential of first trimester human umbilical cord perivascular cells (FTM HUCPVCs), a novel, young source of immunoprivileged mesenchymal stromal cells. Based on the expression of cardiomyocyte markers (cTnT, MYH6, SIRPA, and CX43) FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to bone marrow MSCs, while their immunogenicity remained significantly lower as indicated by HLA-A and HLA-G expression and susceptibility to T cell mediated cytotoxicity. When applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells within 1 week of coculture, making them the first MSC type with this ability. Our results indicate that young FTM HUCPVCs have superior cardiomyogenic potential coupled with beneficial immunogenic properties when compared to MSCs of older tissue sources, suggesting that in vitro predifferentiation could be a potential strategy to increase their effectiveness in vivo.

Snowdrift game dynamics and facultative cheating in yeast.

PubMed

Gore, Jeff; Youk, Hyun; van Oudenaarden, Alexander

2009-05-14

The origin of cooperation is a central challenge to our understanding of evolution. The fact that microbial interactions can be manipulated in ways that animal interactions cannot has led to a growing interest in microbial models of cooperation and competition. For the budding yeast Saccharomyces cerevisiae to grow on sucrose, the disaccharide must first be hydrolysed by the enzyme invertase. This hydrolysis reaction is performed outside the cytoplasm in the periplasmic space between the plasma membrane and the cell wall. Here we demonstrate that the vast majority ( approximately 99 per cent) of the monosaccharides created by sucrose hydrolysis diffuse away before they can be imported into the cell, serving to make invertase production and secretion a cooperative behaviour. A mutant cheater strain that does not produce invertase is able to take advantage of and invade a population of wild-type cooperator cells. However, over a wide range of conditions, the wild-type cooperator can also invade a population of cheater cells. Therefore, we observe steady-state coexistence between the two strains in well-mixed culture resulting from the fact that rare strategies outperform common strategies-the defining features of what game theorists call the snowdrift game. A model of the cooperative interaction incorporating nonlinear benefits explains the origin of this coexistence. We are able to alter the outcome of the competition by varying either the cost of cooperation or the glucose concentration in the media. Finally, we note that glucose repression of invertase expression in wild-type cells produces a strategy that is optimal for the snowdrift game-wild-type cells cooperate only when competing against cheater cells.

Brain local and regional neuroglial alterations in Alzheimer's Disease: cell types, responses and implications.

PubMed

Toledano, Adolfo; Álvarez, María-Isabel; Toledano-Díaz, Adolfo; Merino, José-Joaquín; Rodríguez, José Julio

2016-01-01

From birth to death, neurons are dynamically accompanied by neuroglial cells in a very close morphological and functional relationship. Three families have been classically considered within the CNS: astroglia, oligodendroglia and microglia. Many types/subtypes (including NGR2+ cells), with a wide variety of physiological and pathological effects on neurons, have been described using morphological and immunocytochemical criteria. Glio-glial, glio-neuronal and neuro-glial cell signaling and gliotransmission are phenomena that are essential to support brain functions. Morphofunctional changes resulting from the plasticity of all the glial cell types parallel the plastic neuronal changes that optimize the functionality of neuronal circuits. Moreover, neuroglia possesses the ability to adopt a reactive status (gliosis) in which, generally, new functions arise to improve and restore if needed the neural functionality. All these features make neuroglial cells elements of paramount importance when attempting to explain any physiological or pathological processes in the CNS, because they are involved in both, neuroprotection/neurorepair and neurodegeneration. There exist diverse and profound, regional and local, neuroglial changes in all involutive processes (physiological and pathological aging; neurodegenerative disorders, including Alzheimer ´s disease -AD-), but today, the exact meaning of such modifications (the modifications of the different neuroglial types, in time and place), is not well understood. In this review we consider the different neuroglial cells and their responses in order to understand the possible role they fulfill in pathogenesis, diagnosis and treatment (preventive or palliative) of AD. The existence of differentiated and/or concurrent pathogenic and neuro-protective/neuro-restorative astroglial and microglial responses is highlighted.

Optimize radiochemotherapy in pancreatic cancer: PARP inhibitors a new therapeutic opportunity.

PubMed

Porcelli, Letizia; Quatrale, Anna E; Mantuano, Paola; Leo, Maria G; Silvestris, Nicola; Rolland, Jean F; Carioggia, Enza; Lioce, Marco; Paradiso, Angelo; Azzariti, Amalia

2013-06-01

Cancer cells may use PARP enzymes and Homologous Recombination to repair single and double strand breaks caused by genotoxic insults. In this study, the PARP-1 inhibitor Rucaparib was utilized to increase the sensitivity to chemoradiotherapy treatment in BRCA-2-deficient and -proficient pancreatic cancer cells. We used the pancreatic cancer cell lines, Capan-1 with mutated BRCA-2 and Panc-1, AsPC-1 and MiaPaCa-2 with BRCA-1/2 wild type. Cells were treated with Rucaparib and/or radiotherapy (4-10 Gy) plus Gemcitabine then the capability to proliferate was evaluated by colony formation, cell counting and MTT assays. Flow cytometry, immunocytochemistry and western blotting were utilized to assess cell response to Rucaparib plus irradiation. The antitumour effectiveness of combining the PARP-1 inhibitor before, together and after radiotherapy evidenced the first as the optimal schedule in blocking cell growth. Pre-exposure to Rucaparib increased the cytotoxicity of Gemcitabine plus radiotherapy by heavily inducing the accumulation of cells in G2/M phase, impairing mitosis and finally inducing apoptosis and authophagy. The upregulation of p-Akt and downregulation of p53 were evidenced in MiaPaCa-2 which displayed replication stress features. For the first time, the rationale of using a PARP inhibitor as chemoradiosensitizer in pancreatic cancer models has been hypothesized and demonstrated. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A nutrient dependant switch explains mutually exclusive existence of meiosis and mitosis initiation in budding yeast.

PubMed

Wannige, C T; Kulasiri, D; Samarasinghe, S

2014-01-21

Nutrients from living environment are vital for the survival and growth of any organism. Budding yeast diploid cells decide to grow by mitosis type cell division or decide to create unique, stress resistant spores by meiosis type cell division depending on the available nutrient conditions. To gain a molecular systems level understanding of the nutrient dependant switching between meiosis and mitosis initiation in diploid cells of budding yeast, we develop a theoretical model based on ordinary differential equations (ODEs) including the mitosis initiator and its relations to budding yeast meiosis initiation network. Our model accurately and qualitatively predicts the experimentally revealed temporal variations of related proteins under different nutrient conditions as well as the diverse mutant studies related to meiosis and mitosis initiation. Using this model, we show how the meiosis and mitosis initiators form an all-or-none type bistable switch in response to available nutrient level (mainly nitrogen). The transitions to and from meiosis or mitosis initiation states occur via saddle node bifurcation. This bidirectional switch helps the optimal usage of available nutrients and explains the mutually exclusive existence of meiosis and mitosis pathways. © 2013 Elsevier Ltd. All rights reserved.

Effects of cholinergic drugs on receptive field properties of rabbit retinal ganglion cells

PubMed Central

Ariel, M.; Daw, N. W.

1982-01-01

1. Retinal ganglion cells were recorded extracellularly from the rabbit's eye in situ to study the effects of cholinergic drugs on receptive field properties. Physostigmine, an acetylcholinesterase inhibitor, and nicotine increased the spontaneous activity of nearly all retinal ganglion cell types. The effectiveness of physostigmine was roughly correlated with the neurone's inherent level of spontaneous activity. Brisk cells, having high rates of spontaneous firing, showed large increases in their maintained discharge, whereas sluggish cells, with few or no spontaneous spikes, showed small and sometimes transient increases in spontaneous activity during physostigmine. 2. The sensitivity of ganglion cells to spots of optimal size and position did not change substantially during the infusion of physostigmine. However, the responsiveness to light (number of spikes per stimulus above the spontaneous level) increased. This effect occurred with sluggish and more complex cells, rarely with brisk cells. 3. Another effect of physostigmine on sluggish and more complex cells was to make these cells `on—off'. The additional response to the inappropriate change in contrast had a long latency and lacked an initial transient burst. 4. Complex receptive field properties such as orientation sensitivity, radial grating inhibition, speed tuning and size specificity were also examined. These inhibitory properties were still present during infusion of physostigmine and, in most cases, the trigger feature of each cell type remained. 5. These results are consistent with pharmacological results on ACh release from the retina. There appear to be two types of release of ACh, having their most powerful influences on separate classes of cells. One release (transient), occurs at light onset and offset and acts primarily on sluggish and more complex ganglion cells; the other release (tonic) is not light-modulated and acts primarily on brisk cells. A wiring diagram for the ACh cells is suggested. PMID:7097593

A low-background piston-cylinder-type hybrid high pressure cell for muon-spin rotation/relaxation experiments

NASA Astrophysics Data System (ADS)

Shermadini, Z.; Khasanov, R.; Elender, M.; Simutis, G.; Guguchia, Z.; Kamenev, K. V.; Amato, A.

2017-10-01

A low background double-wall piston-cylinder-type pressure cell is developed at the Paul Scherrer Institute. The cell is made from BERYLCO-25 (beryllium copper) and MP35N nonmagnetic alloys with the design and dimensions which are specifically adapted to muon-spin rotation/relaxation (μSR) measurements. The mechanical design and performance of the pressure cell are evaluated using finite-element analysis (FEA). By including the measured stress-strain characteristics of the materials into the finite-element model, the cell dimensions are optimized with the aim to reach the highest possible pressure while maintaining the sample space large (6 mm in diameter and 12 mm high). The presented unconventional design of the double-wall piston-cylinder pressure cell with a harder outer MP35N sleeve and a softer inner CuBe cylinder enables pressures of up to 2.6 GPa to be reached at ambient temperature, corresponding to 2.2 GPa at low temperatures without any irreversible damage to the pressure cell. The nature of the muon stopping distribution, mainly in the sample and in the CuBe cylinder, results in a low-background μSR signal.

Cathepsin B plays a key role in optimal production of the influenza A virus.

PubMed

Coleman, Macon D; Ha, Soon-Duck; Haeryfar, S M Mansour; Barr, Stephen Dominic; Kim, Sung Ouk

2018-01-01

Influenza A virus (IAV) is the etiologic agent of the febrile respiratory illness, commonly referred to as 'flu'. The lysosomal protease cathepsin B (CTSB) has shown to be involved in the lifecycle of various viruses. Here, we examined the role of CTSB in the IAV lifecycle. CTSB-deficient (CTSB -/- ) macrophages and the human lung epithelial cell line A549 cells treated with CA-074Me were infected with the A/Puerto Rico/8/34 strain of IAV (IAV-PR8). Viral entry and propagation were measured through quantitative real-time RT-PCR; production and localization of hemagglutinin (HA) protein in the infected host cells were analysed by Western blots, flow cytometry and confocal microscopy; production of progeny viruses were measured by a hemagglutination assay. CTSB -/- macrophages and CA-074Me-treated A549 cells had no defects in incorporating IAV-PR8 virions and permitting viral RNA synthesis. However, these cells produced significantly lower amounts of HA protein and progeny virions than wild-type or untreated cells. These data indicate that CTSB is involved in the expression of IAV-PR8 HA protein and subsequent optimal production of IAV-PR8 progeny virions. Targeting CTSB can be a novel therapeutic strategy for treating IAV infection.

Cathepsin B plays a key role in optimal production of the influenza A virus

PubMed Central

Coleman, Macon D.; Ha, Soon-Duck; Haeryfar, S.M. Mansour; Barr, Stephen Dominic; Kim, Sung Ouk

2017-01-01

Background Influenza A virus (IAV) is the etiologic agent of the febrile respiratory illness, commonly referred to as ‘flu’. The lysosomal protease cathepsin B (CTSB) has shown to be involved in the lifecycle of various viruses. Here, we examined the role of CTSB in the IAV lifecycle. Methods CTSB-deficient (CTSB−/−) macrophages and the human lung epithelial cell line A549 cells treated with CA-074Me were infected with the A/Puerto Rico/8/34 strain of IAV (IAV-PR8). Viral entry and propagation were measured through quantitative real-time RT-PCR; production and localization of hemagglutinin (HA) protein in the infected host cells were analysed by Western blots, flow cytometry and confocal microscopy; production of progeny viruses were measured by a hemagglutination assay. Results CTSB−/− macrophages and CA-074Me-treated A549 cells had no defects in incorporating IAV-PR8 virions and permitting viral RNA synthesis. However, these cells produced significantly lower amounts of HA protein and progeny virions than wild-type or untreated cells. Conclusion These data indicate that CTSB is involved in the expression of IAV-PR8 HA protein and subsequent optimal production of IAV-PR8 progeny virions. Targeting CTSB can be a novel therapeutic strategy for treating IAV infection. PMID:29349092

A Finite Element Study of Micropipette Aspiration of Single Cells: Effect of Compressibility

PubMed Central

Jafari Bidhendi, Amirhossein; Korhonen, Rami K.

2012-01-01

Micropipette aspiration (MA) technique has been widely used to measure the viscoelastic properties of different cell types. Cells experience nonlinear large deformations during the aspiration procedure. Neo-Hookean viscohyperelastic (NHVH) incompressible and compressible models were used to simulate the creep behavior of cells in MA, particularly accounting for the effect of compressibility, bulk relaxation, and hardening phenomena under large strain. In order to find optimal material parameters, the models were fitted to the experimental data available for mesenchymal stem cells. Finally, through Neo-Hookean porohyperelastic (NHPH) material model for the cell, the influence of fluid flow on the aspiration length of the cell was studied. Based on the results, we suggest that the compressibility and bulk relaxation/fluid flow play a significant role in the deformation behavior of single cells and should be taken into account in the analysis of the mechanics of cells. PMID:22400045

Discovery of a Broad-Spectrum Antiviral Compound That Inhibits Pyrimidine Biosynthesis and Establishes a Type 1 Interferon-Independent Antiviral State

PubMed Central

Adcock, Robert S.; Schroeder, Chad E.; Chu, Yong-Kyu; Sotsky, Julie B.; Cramer, Daniel E.; Chilton, Paula M.; Song, Chisu; Anantpadma, Manu; Davey, Robert A.; Prodhan, Aminul I.; Yin, Xinmin; Zhang, Xiang

2016-01-01

Viral emergence and reemergence underscore the importance of developing efficacious, broad-spectrum antivirals. Here, we report the discovery of tetrahydrobenzothiazole-based compound 1, a novel, broad-spectrum antiviral lead that was optimized from a hit compound derived from a cytopathic effect (CPE)-based antiviral screen using Venezuelan equine encephalitis virus. Compound 1 showed antiviral activity against a broad range of RNA viruses, including alphaviruses, flaviviruses, influenza virus, and ebolavirus. Mechanism-of-action studies with metabolomics and molecular approaches revealed that the compound inhibits host pyrimidine synthesis and establishes an antiviral state by inducing a variety of interferon-stimulated genes (ISGs). Notably, the induction of the ISGs by compound 1 was independent of the production of type 1 interferons. The antiviral activity of compound 1 was cell type dependent with a robust effect observed in human cell lines and no observed antiviral effect in mouse cell lines. Herein, we disclose tetrahydrobenzothiazole compound 1 as a novel lead for the development of a broad-spectrum, antiviral therapeutic and as a molecular probe to study the mechanism of the induction of ISGs that are independent of type 1 interferons. PMID:27185801

A decoy receptor 3 analogue reduces localised defects in phagocyte function in pneumococcal pneumonia.

PubMed

Marriott, Helen M; Daigneault, Marc; Thompson, Alfred A R; Walmsley, Sarah R; Gill, Sharonjit K; Witcher, Derrick R; Wroblewski, Victor J; Hellewell, Paul G; Whyte, Moira K B; Dockrell, David H

2012-11-01

Therapeutic strategies to modulate the host response to bacterial pneumonia are needed to improve outcomes during community-acquired pneumonia. This study used mice with impaired Fas signalling to examine susceptibility to pneumococcal pneumonia and decoy receptor 3 analogue (DcR3-a) to correct factors associated with increased susceptibility. Wild-type mice and those with varying degrees of impairment of Fas (lpr) or Fas ligand signalling (gld) were challenged with Streptococcus pneumoniae and microbiological and immunological outcomes measured in the presence or absence of DcR3-a. During established pneumonia, neutrophils became the predominant cell in the airway and gld mice were less able to clear bacteria from the lungs, demonstrating localised impairment of pulmonary neutrophil function in comparison to lpr or wild-type mice. T-cells from gld mice had enhanced activation and reduced apoptosis in comparison to wild-type and lpr mice during established pneumonia. Treatment with DcR3-a reduced T-cell activation and corrected the defect in pulmonary bacterial clearance in gld mice. The results suggest that imbalance in tumour necrosis factor superfamily signalling and excessive T-cell activation can impair bacterial clearance in the lung but that DcR3-a treatment can reduce T-cell activation, restore optimal pulmonary neutrophil function and enhance bacterial clearance during S pneumoniae infection.

Competition-based phenotyping reveals a fitness cost for maintaining phycobilisomes under fluctuating light in the cyanobacterium Fremyella diplosiphon

DOE PAGES

Agostoni, Marco; Lucker, Ben F.; Smith, Matthew A. Y.; ...

2016-02-21

Phycobilisomes (PBSs) are pigment-rich super-complexes required for efficient harvest and transfer of light energy to photosynthetic reaction centers of cyanobacteria. The model cyanobacterium Fremyella diplosiphon is able to adjust PBS pigmentation and size in response to the prevailing light spectrum through a process called complementary chromatic acclimation to optimize spectral light absorption, concomitantly optimizing photosynthesis and growth. We explored the fitness costs versus advantages of modulating antennae size and composition under sinusoidal continuous and fluctuating light conditions in F. diplosiphon by comparing growth of wild-type (WT) cells with a mutant strain deficient in PBSs in both monoculture and polyculture conditions.more » Comparative analyses of WT and the PBS-deficient FdCh1 strain under continuous vs. fluctuating sinusoidal light suggest a potential fitness advantage for maintaining PBSs in WT cells during continuous light and a fitness cost during transitions to and acclimation under fluctuating light. Here, we explored the physiological changes correlated with the observed differential growth to understand the dynamics and biochemical bases of comparative fitness of distinct strains under defined growth conditions. Wild-type F. diplosiphon appears to accumulate longer PBS rods and exhibits higher oxidative stress under fluctuating light conditions than continuous sinusoidal light, which may impact responses and the fitness of cells that do not adapt to rapid changes in external light.« less

Hyaline Cartilage Tissue Is Formed through the Co-culture of Passaged Human Chondrocytes and Primary Bovine Chondrocytes

PubMed Central

Taylor, Drew W.; Ahmed, Nazish; Hayes, Anthony J.; Ferguson, Peter; Gross, Allan E.; Caterson, Bruce

2012-01-01

To circumvent the problem of a sufficient number of cells for cartilage engineering, the authors previously developed a two-stage culture system to redifferentiate monolayer culture-expanded dedifferentiated human articular chondrocytes by co-culture with primary bovine chondrocytes (bP0). The aim of this study was to analyze the composition of the cartilage tissue formed in stage 1 and compare it with bP0 grown alone to determine the optimal length of the co-culture stage of the system. Biochemical data show that extracellular matrix accumulation was evident after 2 weeks of co-culture, which was 1 week behind the bP0 control culture. By 3 to 4 weeks, the amounts of accumulated proteoglycans and collagens were comparable. Expression of chondrogenic genes, Sox 9, aggrecan, and collagen type II, was also at similar levels by week 3 of culture. Immunohistochemical staining of both co-culture and control tissues showed accumulation of type II collagen, aggrecan, biglycan, decorin, and chondroitin sulfate in appropriate zonal distributions. These data indicate that co-cultured cells form cartilaginous tissue that starts to resemble that formed by bP0 after 3 weeks, suggesting that the optimal time to terminate the co-culture stage, isolate the now redifferentiated cells, and start stage 2 is just after 3 weeks. PMID:22610463

High-efficiency thin-film GaAs solar cells, phase2

NASA Technical Reports Server (NTRS)

Yeh, Y. C. M.

1981-01-01

Thin GaAs epi-layers with good crystallographic quality were grown using a (100) Si-substrate on which a thin Ge epi-interlayer was grown by CVD from germane. Both antireflection-coated metal oxide semiconductor (AMOS) and n(+)/p homojunction structures were studied. The AMOS cells were fabricated on undoped-GaAs epi-layers deposited on bulk poly-Ge substrates using organo-metallic CVD film-growth, with the best achieved AM1 conversion efficiency being 9.1%. Both p-type and n(+)-type GaAs growth were optimized using 50 ppm dimethyl zinc and 1% hydrogen sulfide, respectively. A direct GaAs deposition method in fabricating ultra-thin top layer, epitaxial n(+)/p shallow homojunction solar cells on (100) GaAs substrates (without anodic thinning) was developed to produce large area (1 sq/cm) cells, with 19.4% AM1 conversion efficiency achieved. Additionally, an AM1 conversion efficiency of 18.4% (17.5% with 5% grid coverage) was achieved for a single crystal GaAs n(+)/p cell grown by OM-CVD on a Ge wafer.

Isolation of metallothionein from cells derived from aggressive form of high-grade prostate carcinoma using paramagnetic antibody-modified microbeads off-line coupled with electrochemical and electrophoretic analysis.

PubMed

Masarik, Michal; Gumulec, Jaromir; Sztalmachova, Marketa; Hlavna, Marian; Babula, Petr; Krizkova, Sona; Ryvolova, Marketa; Jurajda, Michal; Sochor, Jiri; Adam, Vojtech; Kizek, Rene

2011-12-01

Prostate cancer with altered zinc(II) cell metabolism is the second most frequently diagnosed cancer in developed countries. The alterations of zinc(II) metabolism can influence metabolism of other metal ions and can also be associated with the expression and translation of metal-binding proteins including metallothioneins. The aim of this article was to optimize immunoseparation protocol based on paramagnetic beads conjugated with protein G for the isolation of metallothionein. Isolated metallothionein was determined by differential pulse voltammetry Brdicka reaction and SDS-PAGE. Optimal conditions: antigen-binding time - 60 min, temperature - 70°C, and buffer composition and pH - acetate buffer, pH 4.3, were determined. Under the optimized conditions, lysates from 22Rv1 prostate cancer cells treated with various concentrations of cadmium(II) and copper(II) ions were analyzed. We observed strong correlation in all experimental groups and all lysate types (r>0.83 at p<0.041) between metallothionein concentration related to viability and concentration of copper(II) ions and cadmium(II) ions in medium. Moreover, the results were compared with standard sample preparation as heat treatment and SDS-PAGE analysis. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Automation and Optimization of Multipulse Laser Zona Drilling of Mouse Embryos During Embryo Biopsy.

PubMed

Wong, Christopher Yee; Mills, James K

2017-03-01

Laser zona drilling (LZD) is a required step in many embryonic surgical procedures, for example, assisted hatching and preimplantation genetic diagnosis. LZD involves the ablation of the zona pellucida (ZP) using a laser while minimizing potentially harmful thermal effects on critical internal cell structures. Develop a method for the automation and optimization of multipulse LZD, applied to cleavage-stage embryos. A two-stage optimization is used. The first stage uses computer vision algorithms to identify embryonic structures and determines the optimal ablation zone farthest away from critical structures such as blastomeres. The second stage combines a genetic algorithm with a previously reported thermal analysis of LZD to optimize the combination of laser pulse locations and pulse durations. The goal is to minimize the peak temperature experienced by the blastomeres while creating the desired opening in the ZP. A proof of concept of the proposed LZD automation and optimization method is demonstrated through experiments on mouse embryos with positive results, as adequately sized openings are created. Automation of LZD is feasible and is a viable step toward the automation of embryo biopsy procedures. LZD is a common but delicate procedure performed by human operators using subjective methods to gauge proper LZD procedure. Automation of LZD removes human error to increase the success rate of LZD. Although the proposed methods are developed for cleavage-stage embryos, the same methods may be applied to most types LZD procedures, embryos at different developmental stages, or nonembryonic cells.

Single cell gene expression profiling in Alzheimer's disease.

PubMed

Ginsberg, Stephen D; Che, Shaoli; Counts, Scott E; Mufson, Elliott J

2006-07-01

Development and implementation of microarray techniques to quantify expression levels of dozens to hundreds to thousands of transcripts simultaneously within select tissue samples from normal control subjects and neurodegenerative diseased brains has enabled scientists to create molecular fingerprints of vulnerable neuronal populations in Alzheimer's disease (AD) and related disorders. A goal is to sample gene expression from homogeneous cell types within a defined region without potential contamination by expression profiles of adjacent neuronal subpopulations and nonneuronal cells. The precise resolution afforded by single cell and population cell RNA analysis in combination with microarrays and real-time quantitative polymerase chain reaction (qPCR)-based analyses allows for relative gene expression level comparisons across cell types under different experimental conditions and disease progression. The ability to analyze single cells is an important distinction from global and regional assessments of mRNA expression and can be applied to optimally prepared tissues from animal models of neurodegeneration as well as postmortem human brain tissues. Gene expression analysis in postmortem AD brain regions including the hippocampal formation and neocortex reveals selectively vulnerable cell types share putative pathogenetic alterations in common classes of transcripts, for example, markers of glutamatergic neurotransmission, synaptic-related markers, protein phosphatases and kinases, and neurotrophins/neurotrophin receptors. Expression profiles of vulnerable regions and neurons may reveal important clues toward the understanding of the molecular pathogenesis of various neurological diseases and aid in identifying rational targets toward pharmacotherapeutic interventions for progressive, late-onset neurodegenerative disorders such as mild cognitive impairment (MCI) and AD.

Optimal Cytoplasmic Transport in Viral Infections

PubMed Central

D'Orsogna, Maria R.; Chou, Tom

2009-01-01

For many viruses, the ability to infect eukaryotic cells depends on their transport through the cytoplasm and across the nuclear membrane of the host cell. During this journey, viral contents are biochemically processed into complexes capable of both nuclear penetration and genomic integration. We develop a stochastic model of viral entry that incorporates all relevant aspects of transport, including convection along microtubules, biochemical conversion, degradation, and nuclear entry. Analysis of the nuclear infection probabilities in terms of the transport velocity, degradation, and biochemical conversion rates shows how certain values of key parameters can maximize the nuclear entry probability of the viral material. The existence of such “optimal” infection scenarios depends on the details of the biochemical conversion process and implies potentially counterintuitive effects in viral infection, suggesting new avenues for antiviral treatment. Such optimal parameter values provide a plausible transport-based explanation of the action of restriction factors and of experimentally observed optimal capsid stability. Finally, we propose a new interpretation of how genetic mutations unrelated to the mechanism of drug action may nonetheless confer novel types of overall drug resistance. PMID:20046829

Selective targeting of alveolar type II respiratory epithelial cells by anti-surfactant protein-C antibody-conjugated lipoplexes

PubMed Central

Wu, Yun; Ma, Junyu; Woods, Parker S.; Chesarino, Nicholas M.; Liu, Chang; Lee, L. James; Nana-Sinkam, Serge P.; Davis, Ian C.

2015-01-01

Alveolar type II (ATII) respiratory epithelial cells are essential to normal lung function. They may be also central to the pathogenesis of diseases such as acute lung injury, pulmonary fibrosis, and pulmonary adenocarcinoma. Hence, ATII cells are important therapeutic targets. However, effective ATII cell-specific drug delivery in vivo requires carriers of an appropriate size, which can cross the hydrophobic alveolar surfactant film and polar aqueous layer overlying ATII cells, and be taken up without inducing ATII cell dysfunction, pulmonary inflammation, lung damage, or excessive systemic spread and side-effects. We have developed lipoplexes as a versatile nanoparticle carrier system for drug/RNA delivery. To optimize their pulmonary localization and ATII cell specificity, lipoplexes were conjugated to an antibody directed against the ATII cell-specific antigen surfactant protein-C (SP-C) then administered to C57BL/6 mice via the nares. Intranasally-administered, anti-SP-C-conjugated lipoplexes targeted mouse ATII cells with >70% specificity in vivo, were retained within ATII cells for at least 48 hours, and did not accumulate at significant levels in other lung cell types or viscera. 48 hours after treatment with anti-SP-C-conjugated lipoplexes containing the test microRNA miR-486, expression of mature miR-486 was approximately 4-fold higher in ATII cells than whole lung by qRT-PCR, and was undetectable in other viscera. Lipoplexes induced no weight loss, hypoxemia, lung dysfunction, pulmonary edema, or pulmonary inflammation over a 6-day period. These findings indicate that ATII cell-targeted lipoplexes exhibit all the desired characteristics of an effective drug delivery system for treatment of pulmonary diseases that result primarily from ATII cell dysfunction. PMID:25687308

Comparisons of human amniotic mesenchymal stem cell viability in FDA-approved collagen-based scaffolds: Implications for engineered diaphragmatic replacement.

PubMed

Shieh, Hester F; Graham, Christopher D; Brazzo, Joseph A; Zurakowski, David; Fauza, Dario O

2017-06-01

We sought to examine amniotic fluid mesenchymal stem cell (afMSC) viability within two FDA-approved collagen-based scaffolds, as a prerequisite to clinical translation of afMSC-based engineered diaphragmatic repair. Human afMSCs were seeded in a human-derived collagen hydrogel and in a bovine-derived collagen sheet at 3 matching densities. Cell viability was analyzed at 1, 3, and 5days using an ATP-based 3D bioluminescence assay. Statistical comparisons were by ANOVA (P<0.05). There was a highly significant 3-way interaction between scaffold type, seeding density, and time in 3D culture as determinants of cell viability, clearly favoring the human hydrogel (P<0.001). In both scaffolds, cell viability was highest at the highest seeding density of 150,000 cells/mL. Time in 3D culture impacted cell viability at the optimal seeding density in the human hydrogel, with the highest levels on days 1 (P<0.001) and 5 (P=0.05) with no significant effect in the bovine sheet (P=0.39-0.96). Among clinically-approved cell delivery vehicles, mesenchymal stem cell viability is significantly enhanced in a collagen hydrogel when compared with a collagen sheet. Cell viability can be further optimized by seeding density and time in 3D culture. These data further support the regulatory viability of clinical trials of engineered diaphragmatic repair. N/A (animal and laboratory study). Copyright © 2017 Elsevier Inc. All rights reserved.

Analysis of Major Histocompatibility Complex-Bound HIV Peptides Identified from Various Cell Types Reveals Common Nested Peptides and Novel T Cell Responses

PubMed Central

Rucevic, Marijana; Kourjian, Georgio; Boucau, Julie; Blatnik, Renata; Garcia Bertran, Wilfredo; Berberich, Matthew J.; Walker, Bruce D.; Riemer, Angelika B.

2016-01-01

ABSTRACT Despite the critical role of epitope presentation for immune recognition, we still lack a comprehensive definition of HIV peptides presented by HIV-infected cells. Here we identified 107 major histocompatibility complex (MHC)-bound HIV peptides directly from the surface of live HIV-transfected 293T cells, HIV-infected B cells, and primary CD4 T cells expressing a variety of HLAs. The majority of peptides were 8 to 12 amino acids (aa) long and mostly derived from Gag and Pol. The analysis of the total MHC-peptidome and of HLA-A02-bound peptides identified new noncanonical HIV peptides of up to 16 aa that could not be predicted by HLA anchor scanning and revealed an heterogeneous surface peptidome. Nested sets of surface HIV peptides included optimal and extended HIV epitopes and peptides partly overlapping or distinct from known epitopes, revealing new immune responses in HIV-infected persons. Surprisingly, in all three cell types, a majority of Gag peptides derived from p15 rather than from the most immunogenic p24. The cytosolic degradation of peptide precursors in corresponding cells confirmed the generation of identified surface-nested peptides. Cytosolic degradation revealed peptides commonly produced in all cell types and displayed by various HLAs, peptides commonly produced in all cell types and selectively displayed by specific HLAs, and peptides produced in only one cell type. Importantly, we identified areas of proteins leading to common presentations of noncanonical peptides by several cell types with distinct HLAs. These peptides may benefit the design of immunogens, focusing T cell responses on relevant markers of HIV infection in the context of HLA diversity. IMPORTANCE The recognition of HIV-infected cells by immune T cells relies on the presentation of HIV-derived peptides by diverse HLA molecules at the surface of cells. The landscape of HIV peptides displayed by HIV-infected cells is not well defined. Considering the diversity of HLA molecules in the human population, it is critical for vaccine design to identify HIV peptides that may be displayed despite the HLA diversity. We identified 107 HIV peptides directly from the surface of three cell types infected with HIV. They corresponded to nested sets of HIV peptides of canonical and novel noncanonical lengths not predictable by the presence of HLA anchors. Importantly, we identified areas of HIV proteins leading to presentation of noncanonical peptides by several cell types with distinct HLAs. Including such peptides in vaccine immunogen may help to focus immune responses on common markers of HIV infection in the context of HLA diversity. PMID:27440904

Engineering CHO cells with an oncogenic KIT improves cells growth, resilience to stress, and productivity.

PubMed

Mahameed, Mohamed; Tirosh, Boaz

2017-11-01

An optimized biomanufacturing process in mammalian cells is contingent on the ability of the producing cells to reach high viable cell densities. In addition, at the peak of growth, cells need to continue producing the biological entity at a consistent quality. Thus, engineering cells with robust growth performance and resilience to variable stress conditions is highly desirable. The tyrosine kinase receptor, KIT, plays a key role in cell differentiation and the survival of several immune cell types. Its oncogenic mutant, D816V, endows cells with high proliferation capacity, and resistance to kinase inhibitors. Importantly, this onco-KIT mutant when introduced into various cell types is arrested in the endoplasmic reticulum in a constitutively active form. Here, we investigated the effect of oncogenic D816V KIT on the performance of CHO-K1 cells under conventional tissue culture growth settings and when adapted, to shaking conditions. The onco-KIT promoted global protein synthesis, elevated the expression of a secretable transgene, enhanced proliferation, and improved the overall titers of a model glycoprotein. Moreover, the expression of the onco-KIT endowed the cells with a remarkable resistance to various stress conditions. Our data suggest that the introduction of onco-KIT can serve as a strategy for improving glycoprotein biomanufacturing. Biotechnol. Bioeng. 2017;114: 2560-2570. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

ESR (electron spin resonance)-determined osmotic behavior of bull spermatozoa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, J.; Kleinhans, F.W.; Spitzer, V.J.

1990-01-01

Our laboratories are pursuing a fundamental approach to the problems of semen cryopreservation. For many cell types (human red cells, yeast, HeLa) it has been demonstrated that there is an optimum cooling rate for cryopreservation. Faster rates allow insufficient time for cell dehydration and result in intracellular ice formation and cell death. It is possible to predict this optimal rate provided that the cell acts as an ideal osmometer and several other cell parameters are known such as the membrane hydraulic conductivity. It is the purpose of this work to examine the osmotic response of bull sperm to sucrose andmore » NaCl utilizing electron spin resonance (ESR) to measure cell volume. For calibration purposes we also measured the ESR response of human red cells (RBC), the osmotic response of which is well documented with other methods. 15 refs., 1 fig.« less

Somatic cell cloning: the ultimate form of nuclear reprogramming?

PubMed

Piedrahita, Jorge A; Mir, Bashir; Dindot, Scott; Walker, Shawn

2004-05-01

With the increasing difficulties associated with meeting the required needs for organs used in transplantation, alternative approaches need to be considered. These include the use of stem cells as potential sources of specialized cells, the ability to transdifferentiate cell types in culture, and the development of complete organs that can be used in humans. All of the above goals will require a complete understanding of the factors affecting cell differentiation and nuclear reprogramming. To make this a reality, however, techniques associated with cloning and genetic modifications in somatic cells need to be continued to be developed and optimized. This includes not only an enhancement of the rate of homologous recombination in somatic cells, but also a thorough understanding of the nuclear reprogramming process taking place during nuclear transfer. The understanding of this process is likely to have an effect beyond the area of nuclear transfer and assist with better methods for transdifferentiation of mammalian cells.

Cell stiffness is a biomarker of the metastatic potential of ovarian cancer cells

NASA Astrophysics Data System (ADS)

Xu, Wenwei; Mezencev, Roman; Kim, Byungkyu; Wang, Lijuan; McDonald, John; Sulchek, Todd; Sulchek Team; McDonald Team

2013-03-01

The metastatic potential of cells is an important parameter in the design of optimal strategies for the personalized treatment of cancer. Using atomic force microscopy (AFM), we show that ovarian cancer cells are generally softer and display lower intrinsic variability in cell stiffness than non-malignant ovarian epithelial cells. A detailed study of highly invasive ovarian cancer cells (HEY A8) and their less invasive parental cells (HEY), demonstrates that deformability can serve as an accurate biomarker of metastatic potential. Comparative gene expression profiling indicate that the reduced stiffness of highly metastatic HEY A8 cells is associated with actin cytoskeleton remodeling, microscopic examination of actin fiber structure in these cell lines is consistent with this prediction. Our results indicate that cell stiffness not only distinguishes ovarian cancer cells from non-malignant cells, but may also be a useful biomarker to evaluate the relative metastatic potential of ovarian and perhaps other types of cancer cells.

Strategies to optimize lithium-ion supercapacitors achieving high-performance: Cathode configurations, lithium loadings on anode, and types of separator

NASA Astrophysics Data System (ADS)

Cao, Wanjun; Li, Yangxing; Fitch, Brian; Shih, Jonathan; Doung, Tien; Zheng, Jim

2014-12-01

The Li-ion capacitor (LIC) is composed of a lithium-doped carbon anode and an activated carbon cathode, which is a half Li-ion battery (LIB) and a half electrochemical double-layer capacitor (EDLC). LICs can achieve much more energy density than EDLC without sacrificing the high power performance advantage of capacitors over batteries. LIC pouch cells were assembled using activated carbon (AC) cathode and hard carbon (HC) + stabilized lithium metal power (SLMP®) anode. Different cathode configurations, various SLMP loadings on HC anode, and two types of separators were investigated to achieve the optimal electrochemical performance of the LIC. Firstly, the cathode binders study suggests that the PTFE binder offers improved energy and power performances for LIC in comparison to PVDF. Secondly, the mass ratio of SLMP to HC is at 1:7 to obtain the optimized electrochemical performance for LIC among all the various studied mass ratios between lithium loading amounts and active anode material. Finally, compared to the separator Celgard PP 3501, cellulose based TF40-30 is proven to be a preferred separator for LIC.

Serum-free differentiation of murine embryonic stem cells into alveolar type II epithelial cells.

PubMed

Winkler, Monica E; Mauritz, Christina; Groos, Stephanie; Kispert, Andreas; Menke, Sandra; Hoffmann, Anika; Gruh, Ina; Schwanke, Kristin; Haverich, Axel; Martin, Ulrich

2008-03-01

Alveolar type II (AT2) epithelial cells have important functions including the production of surfactant and regeneration of lost alveolar type I epithelial cells. The ability of in vitro production of AT2 cells would offer new therapeutic options in treating pulmonary injuries and disorders including genetically based surfactant deficiencies. Aiming at the generation of AT2-like cells, the differentiation of murine embryonic stem cells (mESCs) toward mesendodermal progenitors (MEPs) was optimized using a "Brachyury-eGFP-knock in" mESC line. eGFP expression demonstrated generation of up to 65% MEPs at day 4 after formation of embryoid bodies (EBs) under serum-free conditions. Plated EBs were further differentiated into AT2-like cells for a total of 25 days in serum-free media resulting in the expression of endodermal marker genes (FoxA2, Sox17, TTR, TTF-1) and of markers for distal lung epithelium (surfactant proteins (SP-) A, B, C, and D, CCSP, aquaporin 5). Notably, expression of SP-C as the only known AT2 cell specific marker could be detected after serum-induction as well as under serum-free conditions. Cytoplasmic localization of SP-C was demonstrated by confocal microscopy. The presence of AT2-like cells was confirmed by electron microscopy providing evidence for polarized cells with apical microvilli and lamellar body-like structures. Our results demonstrate the differentiation of AT2-like cells from mESCs after serum-induction and under serum-free conditions. The established serum-free differentiation protocol will facilitate the identification of key differentiation factors leading to a more specific and effective generation of AT2-like cells from ESCs.

An efficient auto TPT stitch guidance generation for optimized standard cell design

NASA Astrophysics Data System (ADS)

Samboju, Nagaraj C.; Choi, Soo-Han; Arikati, Srini; Cilingir, Erdem

2015-03-01

As the technology continues to shrink below 14nm, triple patterning lithography (TPT) is a worthwhile lithography methodology for printing dense layers such as Metal1. However, this increases the complexity of standard cell design, as it is very difficult to develop a TPT compliant layout without compromising on the area. Hence, this emphasizes the importance to have an accurate stitch generation methodology to meet the standard cell area requirement as defined by the technology shrink factor. In this paper, we present an efficient auto TPT stitch guidance generation technique for optimized standard cell design. The basic idea here is to first identify the conflicting polygons based on the Fix Guidance [1] solution developed by Synopsys. Fix Guidance is a reduced sub-graph containing minimum set of edges along with the connecting polygons; by eliminating these edges in a design 3-color conflicts can be resolved. Once the conflicting polygons are identified using this method, they are categorized into four types [2] - (Type 1 to 4). The categorization is based on number of interactions a polygon has with the coloring links and the triangle loops of fix guidance. For each type a certain criteria for keep-out region is defined, based on which the final stitch guidance locations are generated. This technique provides various possible stitch locations to the user and helps the user to select the best stitch location considering both design flexibility (max. pin access/small area) and process-preferences. Based on this technique, a standard cell library for place and route (P and R) can be developed with colorless data and a stitch marker defined by designer using our proposed method. After P and R, the full chip (block) would contain the colorless data and standard cell stitch markers only. These stitch markers are considered as "must be stitch" candidates. Hence during full chip decomposition it is not required to generate and select the stitch markers again for the complete data; therefore, the proposed method reduces the decomposition time significantly.

Improved efficiency of NiOx-based p-i-n perovskite solar cells by using PTEG-1 as electron transport layer

NASA Astrophysics Data System (ADS)

Groeneveld, Bart G. H. M.; Najafi, Mehrdad; Steensma, Bauke; Adjokatse, Sampson; Fang, Hong-Hua; Jahani, Fatemeh; Qiu, Li; ten Brink, Gert H.; Hummelen, Jan C.; Loi, Maria Antonietta

2017-07-01

We present efficient p-i-n type perovskite solar cells using NiOx as the hole transport layer and a fulleropyrrolidine with a triethylene glycol monoethyl ether side chain (PTEG-1) as electron transport layer. This electron transport layer leads to higher power conversion efficiencies compared to perovskite solar cells with PCBM (phenyl-C61-butyric acid methyl ester). The improved performance of PTEG-1 devices is attributed to the reduced trap-assisted recombination and improved charge extraction in these solar cells, as determined by light intensity dependence and photoluminescence measurements. Through optimization of the hole and electron transport layers, the power conversion efficiency of the NiOx/perovskite/PTEG-1 solar cells was increased up to 16.1%.

Accuracy of cytology in sub typing non small cell lung carcinomas.

PubMed

Patel, Trupti S; Shah, Majal G; Gandhi, Jahnavi S; Patel, Pratik

2017-07-01

Sub typing of non small cell lung carcinoma (NSCLC) has an important task in the era of molecular and targeted therapies. Differentiating between squamous cell carcinoma (SQCC) and adenocarcinoma (ADC) is challenging when limited material is available in lung carcinoma. We investigated the accuracy and feasibility of sub typing NSCLCs in cytology and small biopsy material. Concurrent cytology and biopsy material obtained in a single CT- guided procedure in lung carcinoma over a year period retrospectively. Both materials were individually sub typed and analyzed. Immunohistochemistry (IHC) was performed. Accuracy was determined by comparing the results with IHC. Total 107 of 126 cases of NSCLCs were included for analysis, where both cytology and biopsy material were adequate for interpretation. FNAC allowed tumor typing in 83 (77.6%) cases; 36 (33.6%) were ADC, 47 (43.9%) cases were SQCC and 24 (22.4%) cases diagnosed as Non-small cell carcinoma not otherwise specified (NSCLC-NOS). In biopsy, 86 cases (80.4%) were typed, among which 34 (31.8%) were ADC, 52 (48.6%) were SQCC and 21 (19.6%) were of NSCLC-NOS type. The result of Chi-square index was significant. With the aid of IHC, NSCLC-NOS reduced from 14 (13%) cases to 2 (1.9%) cases. Cytology and small biopsy specimens achieved comparable specificity and accuracy in sub-typing NSCLC and optimal results were obtain when findings from both modalities combine. The advantage of paired specimens is to maximize overall diagnostic yield and the remaining material will be available for ancillary technique like IHC or for molecular testing. Diagn. Cytopathol. 2017;45:598-603. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Narrow Band Gap Lead Sulfide Hole Transport Layers for Quantum Dot Photovoltaics.

PubMed

Zhang, Nanlin; Neo, Darren C J; Tazawa, Yujiro; Li, Xiuting; Assender, Hazel E; Compton, Richard G; Watt, Andrew A R

2016-08-24

The band structure of colloidal quantum dot (CQD) bilayer heterojunction solar cells is optimized using a combination of ligand modification and QD band gap control. Solar cells with power conversion efficiencies of up to 9.33 ± 0.50% are demonstrated by aligning the absorber and hole transport layers (HTL). Key to achieving high efficiencies is optimizing the relative position of both the valence band and Fermi energy at the CQD bilayer interface. By comparing different band gap CQDs with different ligands, we find that a smaller band gap CQD HTL in combination with a more p-type-inducing CQD ligand is found to enhance hole extraction and hence device performance. We postulate that the efficiency improvements observed are largely due to the synergistic effects of narrower band gap QDs, causing an upshift of valence band position due to 1,2-ethanedithiol (EDT) ligands and a lowering of the Fermi level due to oxidation.

Establishment of an immortalized mouse dermal papilla cell strain with optimized culture strategy.

PubMed

Guo, Haiying; Xing, Yizhan; Zhang, Yiming; He, Long; Deng, Fang; Ma, Xiaogen; Li, Yuhong

2018-01-01

Dermal papilla (DP) plays important roles in hair follicle regeneration. Long-term culture of mouse DP cells can provide enough cells for research and application of DP cells. We optimized the culture strategy for DP cells from three dimensions: stepwise dissection, collagen I coating, and optimized culture medium. Based on the optimized culture strategy, we immortalized primary DP cells with SV40 large T antigen, and established several immortalized DP cell strains. By comparing molecular expression and morphologic characteristics with primary DP cells, we found one cell strain named iDP6 was similar with primary DP cells. Further identifications illustrate that iDP6 expresses FGF7 and α-SMA, and has activity of alkaline phosphatase. During the process of characterization of immortalized DP cell strains, we also found that cells in DP were heterogeneous. We successfully optimized culture strategy for DP cells, and established an immortalized DP cell strain suitable for research and application of DP cells.

Establishment of an immortalized mouse dermal papilla cell strain with optimized culture strategy

PubMed Central

Zhang, Yiming; He, Long; Deng, Fang; Ma, Xiaogen

2018-01-01

Dermal papilla (DP) plays important roles in hair follicle regeneration. Long-term culture of mouse DP cells can provide enough cells for research and application of DP cells. We optimized the culture strategy for DP cells from three dimensions: stepwise dissection, collagen I coating, and optimized culture medium. Based on the optimized culture strategy, we immortalized primary DP cells with SV40 large T antigen, and established several immortalized DP cell strains. By comparing molecular expression and morphologic characteristics with primary DP cells, we found one cell strain named iDP6 was similar with primary DP cells. Further identifications illustrate that iDP6 expresses FGF7 and α-SMA, and has activity of alkaline phosphatase. During the process of characterization of immortalized DP cell strains, we also found that cells in DP were heterogeneous. We successfully optimized culture strategy for DP cells, and established an immortalized DP cell strain suitable for research and application of DP cells. PMID:29383288

Investigation of impact of water type on borate ore flotation.

PubMed

Ozkan, S G; Acar, A

2004-04-01

In this work, the impact of water type on borate ore flotation was investigated, while various physical parameters during flotation were considered in order to compare the results. Two different colemanite samples from Emet deposits of Turkey, named as Emet-A and Emet-B contained 44% B(2)O(3) and 40% B(2)O(3), respectively. The flotation tests were performed at feed particle size range of -210 +20 microm. Optimal consumption values for the reagents were determined as 2000 gt(-1) for AeroPromoter R825 from Cytec Company, a sulphonate type collector, 1500 gt(-1) for Procol CA927 from Allied Colloids Company, a sulphosuccinamate type collector and 100 gt(-1) for AeroFrother 70 from Cytec Company, an alcohol-type frother. In the tests, the impeller speed of the Denver-type flotation machine was set to 1200 rpm and the samples were fed into a litre cell at 25% solid/liquid ratio and at natural pH value of the slurry at room temperature. The flotation results obtained from the tests with use of tap water, demineralised water and the artificial water prepared with Ca(2+) and Mg(2+) cations deliberately added into demineralised water were compared to each other in optimal flotation conditions.

Quantification of three-dimensional cell-mediated collagen remodeling using graph theory.

PubMed

Bilgin, Cemal Cagatay; Lund, Amanda W; Can, Ali; Plopper, George E; Yener, Bülent

2010-09-30

Cell cooperation is a critical event during tissue development. We present the first precise metrics to quantify the interaction between mesenchymal stem cells (MSCs) and extra cellular matrix (ECM). In particular, we describe cooperative collagen alignment process with respect to the spatio-temporal organization and function of mesenchymal stem cells in three dimensions. We defined two precise metrics: Collagen Alignment Index and Cell Dissatisfaction Level, for quantitatively tracking type I collagen and fibrillogenesis remodeling by mesenchymal stem cells over time. Computation of these metrics was based on graph theory and vector calculus. The cells and their three dimensional type I collagen microenvironment were modeled by three dimensional cell-graphs and collagen fiber organization was calculated from gradient vectors. With the enhancement of mesenchymal stem cell differentiation, acceleration through different phases was quantitatively demonstrated. The phases were clustered in a statistically significant manner based on collagen organization, with late phases of remodeling by untreated cells clustering strongly with early phases of remodeling by differentiating cells. The experiments were repeated three times to conclude that the metrics could successfully identify critical phases of collagen remodeling that were dependent upon cooperativity within the cell population. Definition of early metrics that are able to predict long-term functionality by linking engineered tissue structure to function is an important step toward optimizing biomaterials for the purposes of regenerative medicine.

The distribution of HIV DNA and RNA in cell subsets differs in gut and blood of HIV-positive patients on ART: implications for viral persistence.

PubMed

Yukl, Steven A; Shergill, Amandeep K; Ho, Terence; Killian, Maudi; Girling, Valerie; Epling, Lorrie; Li, Peilin; Wong, Lisa K; Crouch, Pierre; Deeks, Steven G; Havlir, Diane V; McQuaid, Kenneth; Sinclair, Elizabeth; Wong, Joseph K

2013-10-15

Even with optimal antiretroviral therapy, human immunodeficiency virus (HIV) persists in plasma, blood cells, and tissues. To develop new therapies, it is essential to know what cell types harbor residual HIV. We measured levels of HIV DNA, RNA, and RNA/DNA ratios in sorted subsets of CD4+ T cells (CCR7+, transitional memory, and effector memory) and non-CD4+ T leukocytes from blood, ileum, and rectum of 8 ART-suppressed HIV-positive subjects. Levels of HIV DNA/million cells in CCR7+ and effector memory cells were higher in the ileum than blood. When normalized by cell frequencies, most HIV DNA and RNA in the blood were found in CCR7+ cells, whereas in both gut sites, most HIV DNA and RNA were found in effector memory cells. HIV DNA and RNA were observed in non-CD4+ T leukocytes at low levels, particularly in gut tissues. Compared to the blood, the ileum had higher levels of HIV DNA and RNA in both CD4+ T cells and non-CD4+ T leukocytes, whereas the rectum had higher HIV DNA levels in both cell types but lower RNA levels in CD4+ T cells. Future studies should determine whether different mechanisms allow HIV to persist in these distinct reservoirs, and the degree to which different therapies can affect each reservoir.

High-efficiency indium tin oxide/indium phosphide solar cells

NASA Technical Reports Server (NTRS)

Li, X.; Wanlass, M. W.; Gessert, T. A.; Emery, K. A.; Coutts, T. J.

1989-01-01

Improvements in the performance of indium tin oxide (ITO)/indium phosphide solar cells have been realized by the dc magnetron sputter deposition of n-ITO onto an epitaxial p/p(+) structure grown on commercial p(+) bulk substrates. The highest efficiency cells were achieved when the surface of the epilayer was exposed to an Ar/H2 plasma before depositing the bulk of the ITO in a more typical Ar/O2 plasma. With H2 processing, global efficiencies of 18.9 percent were achieved. It is suggested that the excellent performance of these solar cells results from the optimization of the doping, thickness, transport, and surface properties of the p-type base, as well as from better control over the ITO deposition procedure.

Detection of honeycomb cell walls from measurement data based on Harris corner detection algorithm

NASA Astrophysics Data System (ADS)

Qin, Yan; Dong, Zhigang; Kang, Renke; Yang, Jie; Ayinde, Babajide O.

2018-06-01

A honeycomb core is a discontinuous material with a thin-wall structure—a characteristic that makes accurate surface measurement difficult. This paper presents a cell wall detection method based on the Harris corner detection algorithm using laser measurement data. The vertexes of honeycomb cores are recognized with two different methods: one method is the reduction of data density, and the other is the optimization of the threshold of the Harris corner detection algorithm. Each cell wall is then identified in accordance with the neighboring relationships of its vertexes. Experiments were carried out for different types and surface shapes of honeycomb cores, where the proposed method was proved effective in dealing with noise due to burrs and/or deformation of cell walls.

Bone Marrow Mesenchymal Stromal Cells to Treat Complications Following Allogeneic Stem Cell Transplantation

PubMed Central

Battiwalla, Minoo

2014-01-01

Allogeneic hematopoietic stem cell transplantation (HSCT) is a technologically complicated procedure that represents the only cure for many hematologic malignancies. However, HSCT is often complicated by life-threatening toxicities related to the chemo-radiation conditioning regimen, poor engraftment of donor HSCs, the hyperinflammatory syndrome of graft-versus-host disease (GVHD), infection risks from immunosuppression, and end-organ damage. Bone marrow stromal cells (MSCs), also known as “mesenchymal stromal cells,” not only play a nurturing role in the hematopoietic microenvironment but also can differentiate into other cell types of mesenchymal origin. MSCs are poorly immunogenic, and they can modulate immunological responses through interactions with a wide range of innate and adaptive immune cells to reduce inflammation. They are easily expanded ex vivo and after infusion, home to sites of injury and inflammation to promote tissue repair. Despite promising early trial results in HSCT with significant responses that have translated into survival benefits, there have been significant barriers to successful commercialization as an off-the-shelf therapy. Current efforts with MSCs in the HSCT setting are geared toward determining the factors determining potency, understanding the precise mechanisms of action in human HSCT, knowing their kinetics and fate, optimizing dose and schedule, incorporating biomarkers as response surrogates, addressing concerns about safety, optimizing clinical trial design, and negotiating the uncharted regulatory landscape for licensable cellular therapy. PMID:24410434

Achieving 14.4% Alcohol-Based Solution-Processed Cu(In,Ga)(S,Se)2 Thin Film Solar Cell through Interface Engineering.

PubMed

Park, Gi Soon; Chu, Van Ben; Kim, Byoung Woo; Kim, Dong-Wook; Oh, Hyung-Suk; Hwang, Yun Jeong; Min, Byoung Koun

2018-03-28

An optimization of band alignment at the p-n junction interface is realized on alcohol-based solution-processed Cu(In,Ga)(S,Se) 2 (CIGS) thin film solar cells, achieving a power-conversion-efficiency (PCE) of 14.4%. To obtain a CIGS thin film suitable for interface engineering, we designed a novel "3-step chalcogenization process" for Cu 2- x Se-derived grain growth and a double band gap grading structure. Considering S-rich surface of the CIGS thin film, an alternative ternary (Cd,Zn)S buffer layer is adopted to build favorable "spike" type conduction band alignment instead of "cliff" type. Suppression of interface recombination is elucidated by comparing recombination activation energies using a dark J- V- T analysis.

Brain mesenchymal stem cells: physiology and pathological implications.

PubMed

Pombero, Ana; Garcia-Lopez, Raquel; Martinez, Salvador

2016-06-01

Mesenchymal stem cells (MSCs) are defined as progenitor cells that give rise to a number of unique, differentiated mesenchymal cell types. This concept has progressively evolved towards an all-encompassing concept including multipotent perivascular cells of almost any tissue. In central nervous system, pericytes are involved in blood-brain barrier, and angiogenesis and vascular tone regulation. They form the neurovascular unit (NVU) together with endothelial cells, astrocytes and neurons. This functional structure provides an optimal microenvironment for neural proliferation in the adult brain. Neurovascular niche include both diffusible signals and direct contact with endothelial and pericytes, which are a source of diffusible neurotrophic signals that affect neural precursors. Therefore, MSCs/pericyte properties such as differentiation capability, as well as immunoregulatory and paracrine effects make them a potential resource in regenerative medicine. © 2016 Japanese Society of Developmental Biologists.

Type I Interferons as Stimulators of DC-Mediated Cross-Priming: Impact on Anti-Tumor Response

PubMed Central

Schiavoni, Giovanna; Mattei, Fabrizio; Gabriele, Lucia

2013-01-01

Induction of potent tumor-specific cytotoxic T-cell responses is a fundamental objective in anticancer therapeutic strategies. This event requires that antigen-presenting cells present tumor-associated antigens (Ag) on their MHC class-I molecule, in a process termed cross-presentation. Dendritic cells (DC) are particularly keen on this task and can induce the cross-priming of CD8+ T cells, when exposed to danger or inflammatory signals that stimulate their activation. Type I interferons (IFN-I), a family of long-known immunostimulatory cytokines, have been proven to produce optimal activation signal for DC-induced cross-priming. Recent in vitro and in vivo evidences have suggested that IFN-I-stimulated cross-priming by DC against tumor-associated Ag is a key mechanism for cancer immunosurveillance and may be usefully exploited to boost anti-tumor CD8+ T-cell responses. Here, we will review the cross-presentation properties of different DC subsets, with special focus on cell-associated and tumor Ag, and discuss how IFN-I can modify this function, with the aim of identifying more specific and effective strategies for improving anticancer responses. PMID:24400008

Dynamic optimal control of homeostasis: an integrative system approach for modeling of the central nitrogen metabolism in Saccharomyces cerevisiae.

PubMed

van Riel, N A; Giuseppin, M L; Verrips, C T

2000-01-01

The theory of dynamic optimal metabolic control (DOMC), as developed by Giuseppin and Van Riel (Metab. Eng., 2000), is applied to model the central nitrogen metabolism (CNM) in Saccharomyces cerevisiae. The CNM represents a typical system encountered in advanced metabolic engineering. The CNM is the source of the cellular amino acids and proteins, including flavors and potentially valuable biomolecules; therefore, it is also of industrial interest. In the DOMC approach the cell is regarded as an optimally controlled system. Given the metabolic genotype, the cell faces a control problem to maintain an optimal flux distribution in a changing environment. The regulation is based on strategies and balances feedback control of homeostasis and feedforward regulation for adaptation. The DOMC approach is an integrative, holistic approach, not based on mechanistic descriptions and (therefore) not biased by the variation present in biochemical and molecular biological data. It is an effective tool to structure the rapidly increasing amount of data on the function of genes and pathways. The DOMC model is used successfully to predict the responses of pulses of ammonia and glutamine to nitrogen-limited continuous cultures of a wild-type strain and a glutamine synthetase-negative mutant. The simulation results are validated with experimental data.

Three-dimensional Invasion of Human Glioblastoma Cells Remains Unchanged by X-ray and Carbon Ion Irradiation In Vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eke, Iris; Storch, Katja; Kaestner, Ina

Purpose: Cell invasion represents one of the major determinants that treatment has failed for patients suffering from glioblastoma. Contrary findings have been reported for cell migration upon exposure to ionizing radiation. Here, the migration and invasion capability of glioblastoma cells on and in collagen type I were evaluated upon irradiation with X-rays or carbon ions. Methods and Materials: Migration on and invasion in collagen type I were evaluated in four established human glioblastoma cell lines exposed to either X-rays or carbon ions. Furthermore, clonogenic radiation survival, proliferation (5-bromo-2-deoxyuridine positivity), DNA double-strand breaks ({gamma}H2AX/53BP1-positive foci), and expression of invasion-relevant proteins (eg,more » {beta}1 integrin, FAK, MMP2, and MMP9) were explored. Migration and invasion assays for primary glioblastoma cells also were carried out with X-ray irradiation. Results: Neither X-ray nor carbon ion irradiation affected glioblastoma cell migration and invasion, a finding similarly observed in primary glioblastoma cells. Intriguingly, irradiated cells migrated unhampered, despite DNA double-strand breaks and reduced proliferation. Clonogenic radiation survival was increased when cells had contact with extracellular matrix. Specific inhibition of the {beta}1 integrin or proliferation-associated signaling molecules revealed a critical function of JNK, PI3K, and p38 MAPK in glioblastoma cell invasion. Conclusions: These findings indicate that X-rays and carbon ion irradiation effectively reduce proliferation and clonogenic survival without modifying the migration and invasion ability of glioblastoma cells in a collagen type I environment. Addition of targeted agents against members of the MAPK and PI3K signaling axis to conventional chemoradiation therapy seems potentially useful to optimize glioblastoma therapy.« less

Comparison of different cooling rates for fibroblast and keratinocyte cryopreservation.

PubMed

Naaldijk, Yahaira; Friedrich-Stöckigt, Annett; Sethe, Sebastian; Stolzing, Alexandra

2016-10-01

Easy, cost-effective and reliable cryopreservation protocols are crucial for the successful and effective application of tissue engineering. Several different protocols are in use, but no comprehensive comparisons across different machine-based and manual methods have been made. Here, we compare the effects of different cooling rates on the post-thaw survival and proliferative capacity of two basic cell lines for skin tissue engineering fibroblasts and keratinocytes, cultured and frozen in suspension or as a monolayer. We demonstrate that effectiveness of cryopreservation cannot be reliably determined immediately after thawing: the results at this stage were not indicative of cell growth in culture 3 days post-thaw. Cryopreservation of fibroblasts in an adherent state greatly diminishes their subsequent growth potential. This was not observed when freezing in suspension. In keratinocytes, however, adherent freezing is as effective as freezing in suspension, which could lead to significant cost and labour savings in a tissue-engineering environment. The 'optimal' cryopreservation protocol depends on cell type and intended use. Where time, ease and cost are dominant factors, the direct freezing into a nitrogen tank (straight freeze) approach remains a viable method. The most effective solution across the board, as measured by viability 3 days post-thaw, was the commonly used, freezing container method. Where machine-controlled cryopreservation is deemed important for tissue-engineering Good Manufacturing Practice, we present results using a portfolio of different cooling rates, identifying the 'optimal' protocol depending on cell type and culture method. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.

Mesoporous silica nanoparticle-based intelligent drug delivery system for bienzyme-responsive tumour targeting and controlled release.

PubMed

Zhang, Yang; Xu, Juan

2018-01-01

This paper proposes a novel type of multifunctional envelope-type mesoporous silica nanoparticle (MSN) to achieve cancer cell targeting and drug-controlled release. In this system, MSNs were first modified by active targeting moiety hyaluronic acid (HA) for breast cancer cell targeting and hyaluronidases (Hyal)-induced intracellular drug release. Then gelatin, a proteinaceous biopolymer, was grafted onto the MSNs to form a capping layer via glutaraldehyde-mediated cross-linking. To shield against unspecific uptake of cells and prolong circulation time, the nanoparticles were further decorated with poly(ethylene glycol) polymers (PEG) to obtain MSN@HA-gelatin-PEG (MHGP). Doxorubicin (DOX), as a model drug, was loaded into PEMSN to assess the breast cancer cell targeting and drug release behaviours. In vitro study revealed that PEG chains protect the targeting ligand and shield against normal cells. After reaching the breast cancer cells, MMP-2 overpressed by cells hydrolyses gelatin layer to deshield PEG and switch on the function of HA. As a result, DOX-loaded MHGP was selectively trapped by cancer cells through HA receptor-mediated endocytosis and subsequently release DOX due to Hyal-catalysed degradation of HA. This system presents successful bienzyme-responsive targeting drug delivery in an optimal fashion and provides potential applications for targeted cancer therapy.

Perspectives on testicular germ cell neoplasms.

PubMed

Cheng, Liang; Lyu, Bingjian; Roth, Lawrence M

2017-01-01

Our knowledge of testicular germ cell neoplasms has progressed in the last few decades due to the description of germ cell neoplasia in situ (GCNIS) and a variety of specific forms of intratubular germ cell neoplasia, the discovery of isochromosome 12p and its importance in the development of invasiveness in germ cell tumors (GCTs), the identification of specific transcription factors for GCTs, and the recognition that a teratomatous component in mixed GCT represents terminal differentiation. Isochromosome 12p and 12p overrepresentation, collectively referred to as 12p amplification, are fundamental abnormalities that account for many types of malignant GCTs of the testis. Embryonal carcinoma is common in the testis but rare in the ovary, whereas the converse is true for mature cystic teratoma. Spermatocytic tumor occurs only in the testis; it has not been described in the ovary or extragonadal sites. The origin of ovarian mature cystic teratoma is similar to that of prepubertal-type testicular teratoma and dermoid cyst at any age in that it arises from a nontransformed germ cell, whereas postpubertal-type testicular teratoma arises from a malignant germ cell, most commonly through the intermediary of GCNIS. Somatic neoplasms, often referred to as monodermal teratomas, arise not infrequently from mature cystic teratoma of the ovary, whereas such neoplasms are rare in testicular teratoma with the exception of carcinoid. Integration of classical morphologic observations and emerging novel molecular studies will result in better understanding of the pathogenesis of GCTs and will optimize patient therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation

PubMed Central

Lim, Chinten James; Spiegelman, George B.; Weeks, Gerald

2001-01-01

Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC– cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC– cells stimulated by 2′-deoxy-cAMP, but is produced in response to GTPγS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC– cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC– cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC– cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC– cells, suggesting that AleA may activate RasC. PMID:11500376

RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation.

PubMed

Lim, C J; Spiegelman, G B; Weeks, G

2001-08-15

Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC(-) cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC(-) cells stimulated by 2'-deoxy-cAMP, but is produced in response to GTPgammaS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC(-) cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC(-) cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC(-) cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC(-) cells, suggesting that AleA may activate RasC.

The Spleen as an Optimal Site for Islet Transplantation and a Source of Mesenchymal Stem Cells.

PubMed

Sakata, Naoaki; Yoshimatsu, Gumpei; Kodama, Shohta

2018-05-07

This review demonstrates the unique potential of the spleen as an optimal site for islet transplantation and as a source of mesenchymal stem cells. Islet transplantation is a cellular replacement therapy used to treat severe diabetes mellitus; however, its clinical outcome is currently unsatisfactory. Selection of the most appropriate transplantation site is a major factor affecting the clinical success of this therapy. The spleen has long been studied as a candidate site for islet transplantation. Its advantages include physiological insulin drainage and regulation of immunity, and it has recently also been shown to contribute to the regeneration of transplanted islets. However, the efficacy of transplantation in the spleen is lower than that of intraportal transplantation, which is the current representative method of clinical islet transplantation. Safer and more effective methods of islet transplantation need to be established to allow the spleen to be used for clinical transplantation. The spleen is also of interest as a mesenchymal stem cell reservoir. Splenic mesenchymal stem cells contribute to the repair of damaged tissue, and their infusion may thus be a promising therapy for autoimmune diseases, including type 1 diabetes mellitus and Sjogren’s syndrome.

Optimal Resting-Growth Strategies of Microbial Populations in Fluctuating Environments

PubMed Central

Geisel, Nico; Vilar, Jose M. G.; Rubi, J. Miguel

2011-01-01

Bacteria spend most of their lifetime in non-growing states which allow them to survive extended periods of stress and starvation. When environments improve, they must quickly resume growth to maximize their share of limited nutrients. Cells with higher stress resistance often survive longer stress durations at the cost of needing more time to resume growth, a strong disadvantage in competitive environments. Here we analyze the basis of optimal strategies that microorganisms can use to cope with this tradeoff. We explicitly show that the prototypical inverse relation between stress resistance and growth rate can explain much of the different types of behavior observed in stressed microbial populations. Using analytical mathematical methods, we determine the environmental parameters that decide whether cells should remain vegetative upon stress exposure, downregulate their metabolism to an intermediate optimum level, or become dormant. We find that cell-cell variability, or intercellular noise, is consistently beneficial in the presence of extreme environmental fluctuations, and that it provides an efficient population-level mechanism for adaption in a deteriorating environment. Our results reveal key novel aspects of responsive phenotype switching and its role as an adaptive strategy in changing environments. PMID:21525975

Towards optimization of odonto/osteogenic bioengineering: in vitro comparison of simvastatin, sodium fluoride, melanocyte-stimulating hormone.

PubMed

Zijah, Vahid; Salehi, Roya; Aghazadeh, Marziyeh; Samiei, Mohammad; Alizadeh, Effat; Davaran, Soodabeh

2017-06-01

Tissue engineering has emerged as a potential therapeutic option for dental problems in recent years. One of the policies in tissue engineering is to use both scaffolds and additive factors for enhancing cell responses. This study aims to evaluate and compare the effect of three types of biofactors on poly-caprolactone-poly-ethylene glycol-poly caprolactone (PCL-PEG-PCL) nanofibrous scaffold on human dental pulp stem cell (hDPSCs) engineering. The PCL-PEG-PCL copolymer was synthesized with ring opening polymerization method, and its nanofiber scaffold was prepared by electrospinning method. Nanofibrous scaffold-seeded hDPSCs were treated with sodium fluoride (NaF), melanocyte-stimulating hormone (MSH), or simvastatin (SIM). Non-treated nanofiber seeded cells were utilized as control. The viability, biocompatibility, adhesion, proliferation rate, morphology, osteo/odontogenic potential, and the expression of tissue-specific genes were studied. The results showed that significant higher results demonstrated significant higher adhesive behavior, viability, alizarin red activity, and dentin specific gene expression in MSH- and SIM-treated cells (p < 0.05). This study is unique; in that, it compares the effects of different treatments for optimization of dental tissue engineering.

The optimization of Ga (1-x)Al (x)As-GaAs solar cells for air mass zero operation and a study of Ga (1-x)Al (x)As-GaAs solar cells at high temperatures, phase 1

NASA Technical Reports Server (NTRS)

Hovel, H. J.; Woodall, J. M.

1976-01-01

The three types of solar cells investigated were: (1) one consisting of a nGaAs substrate, a Zn doped pGaAs region, and a Zn doped Ga(1-x)Al(x)As layer, (2) one consisting of an nGaAs substrate, a Ge doped pGaAs region, and a pGa(1-x)Al(x)As upper layer, and (3) one consisting of an n+GaAs substrate, an nGa(1-x)Al(X)As region, a pGa(1-x)Bl(X) As region, and a pGa(1-y)Al(y)As upper layer. In all three cases, the upper alloy layer is thin and of high Al composition in order to obtain high spectral response over the widest possible range of photon energies. Spectral response, capacitance-voltage, current-voltage, diffusion length, sunlight (or the equivalent)-efficiency, and efficiency-temperature measurements were made as a function of device parameters in order to analyze and optimize the solar cell behavior.

Selective destruction of mouse islet beta cells by human T lymphocytes in a newly-established humanized type 1 diabetic model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Yong, E-mail: yongzhao@uic.edu; Guo, Chengshan; Hwang, David

2010-09-03

Research highlights: {yields} Establish a human immune-mediated type 1 diabetic model in NOD-scid IL2r{gamma}{sup null} mice. {yields} Using the irradiated diabetic NOD mouse spleen mononuclear cells as trigger. {yields} The islet {beta} cells were selectively destroyed by infiltrated human T cells. {yields} The model can facilitate translational research to find a cure for type 1 diabetes. -- Abstract: Type 1 diabetes (T1D) is caused by a T cell-mediated autoimmune response that leads to the loss of insulin-producing {beta} cells. The optimal preclinical testing of promising therapies would be aided by a humanized immune-mediated T1D model. We develop this model inmore » NOD-scid IL2r{gamma}{sup null} mice. The selective destruction of pancreatic islet {beta} cells was mediated by human T lymphocytes after an initial trigger was supplied by the injection of irradiated spleen mononuclear cells (SMC) from diabetic nonobese diabetic (NOD) mice. This resulted in severe insulitis, a marked loss of total {beta}-cell mass, and other related phenotypes of T1D. The migration of human T cells to pancreatic islets was controlled by the {beta} cell-produced highly conserved chemokine stromal cell-derived factor 1 (SDF-1) and its receptor C-X-C chemokine receptor (CXCR) 4, as demonstrated by in vivo blocking experiments using antibody to CXCR4. The specificity of humanized T cell-mediated immune responses against islet {beta} cells was generated by the local inflammatory microenvironment in pancreatic islets including human CD4{sup +} T cell infiltration and clonal expansion, and the mouse islet {beta}-cell-derived CD1d-mediated human iNKT activation. The selective destruction of mouse islet {beta} cells by a human T cell-mediated immune response in this humanized T1D model can mimic those observed in T1D patients. This model can provide a valuable tool for translational research into T1D.« less

Surface free energy predominates in cell adhesion to hydroxyapatite through wettability.

PubMed

Nakamura, Miho; Hori, Naoko; Ando, Hiroshi; Namba, Saki; Toyama, Takeshi; Nishimiya, Nobuyuki; Yamashita, Kimihiro

2016-05-01

The initial adhesion of cells to biomaterials is critical in the regulation of subsequent cell behaviors. The purpose of this study was to investigate a mechanism through which the surface wettability of biomaterials can be improved and determine the effects of biomaterial surface characteristics on cellular behaviors. We investigated the surface characteristics of various types of hydroxyapatite after sintering in different atmospheres and examined the effects of various surface characteristics on cell adhesion to study cell-biomaterial interactions. Sintering atmosphere affects the polarization capacity of hydroxyapatite by changing hydroxide ion content and grain size. Compared with hydroxyapatite sintered in air, hydroxyapatite sintered in saturated water vapor had a higher polarization capacity that increased surface free energy and improved wettability, which in turn accelerated cell adhesion. We determined the optimal conditions of hydroxyapatite polarization for the improvement of surface wettability and acceleration of cell adhesion. Copyright © 2016 Elsevier B.V. All rights reserved.

A Review of the Cell to Graphene-Based Nanomaterial Interface

NASA Astrophysics Data System (ADS)

Darbandi, Arash; Gottardo, Erik; Huff, Joshua; Stroscio, Michael; Shokuhfar, Tolou

2018-04-01

The area of cellular interactions of nanomaterials is an important research interest. The sensitivity of cells toward their extracellular matrix allows researchers to create microenvironments for guided stem cell differentiation. Among nanomaterials, graphene, often called the "wonder material," and its derivatives are at the forefront of such endeavors. Graphene's carbon backbone, paired with its biocompatibility and ease of functionalization, has been used as an enhanced method of controlled cell proliferation. Graphene's honeycomb nature allows for compatibility with polymers and biological material for the creation of nanocomposite scaffolds that help differentiation into cell types that have otherwise been proven difficult. Such materials and their role in guiding cell growth can aid the construction of tissue grafts where shortages and patient compatibility create a low success rate. This review will bring together novel studies and techniques used to understand and optimizes graphene's role in cell growth mechanisms.

Interface Optoelectronics Engineering for Mechanically Stacked Tandem Solar Cells Based on Perovskite and Silicon.

PubMed

Kanda, Hiroyuki; Uzum, Abdullah; Nishino, Hitoshi; Umeyama, Tomokazu; Imahori, Hiroshi; Ishikawa, Yasuaki; Uraoka, Yukiharu; Ito, Seigo

2016-12-14

Engineering of photonics for antireflection and electronics for extraction of the hole using 2.5 nm of a thin Au layer have been performed for two- and four-terminal tandem solar cells using CH 3 NH 3 PbI 3 perovskite (top cell) and p-type single crystal silicon (c-Si) (bottom cell) by mechanically stacking. Highly transparent connection multilayers of evaporated-Au and sputtered-ITO films were fabricated at the interface to be a point-contact tunneling junction between the rough perovskite and flat silicon solar cells. The mechanically stacked tandem solar cell with an optimized tunneling junction structure was ⟨perovskite for the top cell/Au (2.5 nm)/ITO (154 nm) stacked-on ITO (108 nm)/c-Si for the bottom cell⟩. It was confirmed the best efficiency of 13.7% and 14.4% as two- and four-terminal devices, respectively.

Rear surface effects in high efficiency silicon solar cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wenham, S.R.; Robinson, S.J.; Dai, X.

1994-12-31

Rear surface effects in PERL solar cells can lead not only to degradation in the short circuit current and open circuit voltage, but also fill factor. Three mechanisms capable of changing the effective rear surface recombination velocity with injection level are identified, two associated with oxidized p-type surfaces, and the third with two dimensional effects associated with a rear floating junction. Each of these will degrade the fill factor if the range of junction biases corresponding to the rear surface transition, coincides with the maximum power point. Despite the identified non idealities, PERL cells with rear floating junctions (PERF cells)more » have achieved record open circuit voltages for silicon solar cells, while simultaneously achieving fill factor improvements relative to standard PERL solar cells. Without optimization, a record efficiency of 22% has been demonstrated for a cell with a rear floating junction. The results of both theoretical and experimental studies are provided.« less

Exosome-derived microRNAs in cancer metabolism: possible implications in cancer diagnostics and therapy.

PubMed

Tomasetti, Marco; Lee, Wan; Santarelli, Lory; Neuzil, Jiri

2017-01-20

Malignant progression is greatly affected by dynamic cross-talk between stromal and cancer cells. Exosomes are secreted nanovesicles that have key roles in cell-cell communication by transferring nucleic acids and proteins to target cells and tissues. Recently, MicroRNAs (miRs) and their delivery in exosomes have been implicated in physiological and pathological processes. Tumor-delivered miRs, interacting with stromal cells in the tumor microenvironment, modulate tumor progression, angiogenesis, metastasis and immune escape. Altered cell metabolism is one of the hallmarks of cancer. A number of different types of tumor rely on mitochondrial metabolism by triggering adaptive mechanisms to optimize their oxidative phosphorylation in relation to their substrate supply and energy demands. Exogenous exosomes can induce metabolic reprogramming by restoring the respiration of cancer cells and supress tumor growth. The exosomal miRs involved in the modulation of cancer metabolism may be potentially utilized for better diagnostics and therapy.

Effect of Stapling Architecture on Physiochemical Properties and Cell Permeability of Stapled α-Helical Peptides: A Comparative Study.

PubMed

Tian, Yuan; Jiang, Yanhong; Li, Jingxu; Wang, Dongyuan; Zhao, Hui; Li, Zigang

2017-11-02

Stapled peptides have emerged as a new class of targeting molecules with high binding affinity and specificity for intracellular undruggable targets. Their ability to penetrate cell membranes is exceptionally intriguing but remains elusively and controversially discussed. To understand the effect of stapling architectures on their physiochemical properties and to aid in promoting their cell permeability, we report herein a comparative study on the physiochemical properties and cell permeability of stapled α-helical peptides with different types of crosslinks. We highlight the decisive impact of the intrinsic properties of the crosslinks on cell permeability rather than the helical contents of the peptides in model amphipathic sequences targeting estrogen receptor-coactivator interaction. We envision this finding to shed further light on the chemical optimization of stapled α-helical peptides or macrocyclic cell-penetrating peptides for enhanced cell penetration. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

N-type compensated silicon: resistivity, crystal growth, carrier lifetime, and relevant application for HIT solar cells

NASA Astrophysics Data System (ADS)

Li, Shuai; Gao, Wenxiu; Li, Zhen; Cheng, Haoran; Lin, Jinxia; Cheng, Qijin

2017-05-01

N-type compensated silicon shows unusual distribution of resistivity as crystal grows compared to the n-type uncompensated silicon. In this paper, evolutions of resistivities with varied concentrations of boron and varied starting resistivities of the n-type silicon are intensively calculated. Moreover, reduction of carrier mobility is taken into account by Schindler’s modified model of carrier mobility for the calculation of resistivity of the compensated silicon. As for substrates of solar cells, optimized starting resistivity and corresponding concentration of boron are suggested for better uniformity of resistivity and higher yield (fraction with ρ >0.5 ~ Ω \\centerdot \\text{cm} ) of the n-type compensated Cz crystal rod. A two-step growth method is investigated to obtain better uniformity of resistivity of crystal rod, and this method is very practical especially for the n-type compensated silicon. Regarding the carrier lifetime, the recombination by shallow energy-level dopants is taken into account for the compensated silicon, and evolution of carrier lifetime is simulated by considering all main recombination centers which agrees well with our measured carrier lifetimes as crystal grows. The n-type compensated silicon shows a larger reduction of carrier lifetime compared to the uncompensated silicon at the beginning of crystal growth, and recombination with a oxygen-related deep defect is sufficient to describe the reduction of degraded lifetime. Finally, standard heterojunction with intrinsic thin-layer (HIT) solar cells are made with substrates from the n-type compensated silicon rod, and a high efficiency of 22.1% is obtained with a high concentration (0.8× {{10}16}~\\text{c}{{\\text{m}}-3} ) of boron in the n-type compensated silicon feedstock. However, experimental efficiencies of HIT solar cells based on the n-type compensated silicon show an average reduction of 4% along with the crystal length compared to the uncompensated silicon. The obtained results enrich our knowledge on the n-type compensated silicon and contribute to the development of n-type compensated silicon-based solar cells for commercial application.

Effects of icotinib on advanced non-small cell lung cancer with different EGFR phenotypes.

PubMed

Pan, Huiyun; Liu, Rong; Li, Shengjie; Fang, Hui; Wang, Ziwei; Huang, Sheng; Zhou, Jianying

2014-09-01

Icotinib is the first oral epidermal growth factor receptor (EGFR) tyrosine kinase receptor inhibitor, which has been proven to exert significant inhibitory effects on non-small cell lung cancer in vitro. Clinical evidence has showed that the efficacy of Icotinib on retreating advanced non-small cell lung cancer is comparable to Gefitinib. However, different phenotypes of EGFR can affect the therapeutic outcomes of EGFR tyrosine kinase receptor inhibitor. Therefore, our study focused on efficacy and safety of Icotinib in patients with advanced non-small cell lung cancer of different EGPR phenotypes. Clinical data of patients with advanced non-small cell lung cancer who received Icotinib treatment from August, 2011 to May, 2013 were retrospectively analyzed. Kaplan-Meier analysis was used for survival analysis and comparison. 18 wild-type EGFR and 51 mutant type were found in a total of 69 patients. Objective response rate of patients with mutant type EGFR was 54.9 % and disease control rate was 86.3 %. Objective response rate of wild-type patients was 11.1 % (P = 0.0013 vs mutant type), disease control rate was 50.0 % (P = 0.0017). Median progression-free survival (PFS) of mutant type and wild-type patients were 9.7 and 2.6 months, respectively (P < 0.001). Median PFS of exon 19 mutated mutant patients was 11.3 months, mean PFS of exon 21 L858R mutated mutant patients was 8.7 months (P = 0.3145). Median overall survival (OS) of EGFR mutated patients had not reached. OS time of 13 wild-type patients was 12.9 months (P < 0.001). The common adverse reactions of Icotinib included rash, diarrhea, itching skin with occurrence rates of 24.6 % (17/69), 13.0 % (9/69), and 11.6 % (8/69), respectively. Most adverse reactions were grade I-II. Icotinib has great efficacy in EGFR mutated patients, making it an optimal regimen to treat EGFR mutated patients. Furthermore, most of adverse reactions associated with Icotinib treatment were tolerable.

Improved Low-Temperature Performance of Li-Ion Cells Using New Electrolytes

NASA Technical Reports Server (NTRS)

Smart, Marshall C.; Buga, Ratnakumar V.; Gozdz, Antoni S.; Mani, Suresh

2010-01-01

As part of the continuing efforts to develop advanced electrolytes to improve the performance of lithium-ion cells, especially at low temperatures, a number of electrolyte formulations have been developed that result in improved low-temperature performance (down to 60 C) of 26650 A123Systems commercial lithium-ion cells. The cell type/design, in which the new technology has been demonstrated, has found wide application in the commercial sector (i.e., these cells are currently being used in commercial portable power tools). In addition, the technology is actively being considered for hybrid electric vehicle (HEV) and electric vehicle (EV) applications. In current work, a number of low-temperature electrolytes have been developed based on advances involving lithium hexafluorophosphate-based solutions in carbonate and carbonate + ester solvent blends, which have been further optimized in the context of the technology and targeted applications. The approaches employed, which include the use of ternary mixtures of carbonates, the use of ester co-solvents [e.g., methyl butyrate (MB)], and optimized lithium salt concentrations (e.g., LiPF6), were compared with the commercial baseline electrolyte, as well as an electrolyte being actively considered for DoE HEV applications and previously developed by a commercial enterprise, namely LiPF6 in ethylene carbonate (EC) + ethyl methyl carbonate (EMC)(30:70%).

REAC technology and hyaluron synthase 2, an interesting network to slow down stem cell senescence.

PubMed

Maioli, Margherita; Rinaldi, Salvatore; Pigliaru, Gianfranco; Santaniello, Sara; Basoli, Valentina; Castagna, Alessandro; Fontani, Vania; Ventura, Carlo

2016-06-24

Hyaluronic acid (HA) plays a fundamental role in cell polarity and hydrodynamic processes, affording significant modulation of proliferation, migration, morphogenesis and senescence, with deep implication in the ability of stem cells to execute their differentiating plans. The Radio Electric Asymmetric Conveyer (REAC) technology is aimed to optimize the ions fluxes at the molecular level in order to optimize the molecular mechanisms driving cellular asymmetry and polarization. Here, we show that treatment with 4-methylumbelliferone (4-MU), a potent repressor of type 2 HA synthase and endogenous HA synthesis, dramatically antagonized the ability of REAC to recover the gene and protein expression of Bmi1, Oct4, Sox2, and Nanog in ADhMSCs that had been made senescent by prolonged culture up to the 30(th) passage. In senescent ADhMSCs, 4-MU also counteracted the REAC ability to rescue the gene expression of TERT, and the associated resumption of telomerase activity. Hence, the anti-senescence action of REAC is largely dependent upon the availability of endogenous HA synthesis. Endogenous HA and HA-binding proteins with REAC technology create an interesting network that acts on the modulation of cell polarity and intracellular environment. This suggests that REAC technology is effective on an intracellular niche level of stem cell regulation.

Synergistic responses of superficial chemistry and micro topography of titanium created by wire-type electric discharge machining.

PubMed

Kataoka, Yu; Tamaki, Yukimichi; Miyazaki, Takashi

2011-01-01

Wire-type electric discharge machining has been applied to the manufacture of endosseous titanium implants as this computer associated technique allows extremely accurate complex sample shaping with an optimal micro textured surface during the processing. Since the titanium oxide layer is sensitively altered by each processing, the authors hypothesized that this technique also up-regulates biological responses through the synergistic effects of the superficial chemistry and micro topography. To evaluate the respective in vitro cellular responses on the superficial chemistry and micro topography of titanium surface processed by wire-type electric discharge, we used titanium-coated epoxy resin replica of the surface. An oxide layer on the titanium surface processed by wire-type electric discharge activated the initial responses of osteoblastic cells through an integrin-mediated mechanism. Since the mRNA expression of ALP on those replicas was up-regulated compared to smooth titanium samples, the micro topography of a titanium surface processed by wire-type electric discharge promotes the osteogenic potential of cells. The synergistic response of the superficial chemistry and micro topography of titanium processed by wire-type electric discharge was demonstrated in this study.

Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood–brain barrier model for drug permeability studies

PubMed Central

2013-01-01

Background Reliable human in vitro blood–brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. Methods Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (CCL) in real-time. Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. Results The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells. Conclusions Under the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products. PMID:24262108

Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies.

PubMed

Eigenmann, Daniela E; Xue, Gongda; Kim, Kwang S; Moses, Ashlee V; Hamburger, Matthias; Oufir, Mouhssin

2013-11-22

Reliable human in vitro blood-brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (CCL) in real-time.Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells. Under the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products.

Selection of optimal sensors for predicting performance of polymer electrolyte membrane fuel cell

NASA Astrophysics Data System (ADS)

Mao, Lei; Jackson, Lisa

2016-10-01

In this paper, sensor selection algorithms are investigated based on a sensitivity analysis, and the capability of optimal sensors in predicting PEM fuel cell performance is also studied using test data. The fuel cell model is developed for generating the sensitivity matrix relating sensor measurements and fuel cell health parameters. From the sensitivity matrix, two sensor selection approaches, including the largest gap method, and exhaustive brute force searching technique, are applied to find the optimal sensors providing reliable predictions. Based on the results, a sensor selection approach considering both sensor sensitivity and noise resistance is proposed to find the optimal sensor set with minimum size. Furthermore, the performance of the optimal sensor set is studied to predict fuel cell performance using test data from a PEM fuel cell system. Results demonstrate that with optimal sensors, the performance of PEM fuel cell can be predicted with good quality.

Study on Pyroelectric Harvesters with Various Geometry

PubMed Central

Siao, An-Shen; Chao, Ching-Kong; Hsiao, Chun-Ching

2015-01-01

Pyroelectric harvesters convert time-dependent temperature variations into electric current. The appropriate geometry of the pyroelectric cells, coupled with the optimal period of temperature fluctuations, is key to driving the optimal load resistance, which enhances the performance of pyroelectric harvesters. The induced charge increases when the thickness of the pyroelectric cells decreases. Moreover, the induced charge is extremely reduced for the thinner pyroelectric cell when not used for the optimal period. The maximum harvested power is achieved when a 100 μm-thick PZT (Lead zirconate titanate) cell is used to drive the optimal load resistance of about 40 MΩ. Moreover, the harvested power is greatly reduced when the working resistance diverges even slightly from the optimal load resistance. The stored voltage generated from the 75 μm-thick PZT cell is less than that from the 400 μm-thick PZT cell for a period longer than 64 s. Although the thinner PZT cell is advantageous in that it enhances the efficiency of the pyroelectric harvester, the much thinner 75 μm-thick PZT cell and the divergence from the optimal period further diminish the performance of the pyroelectric cell. Therefore, the designers of pyroelectric harvesters need to consider the coupling effect between the geometry of the pyroelectric cells and the optimal period of temperature fluctuations to drive the optimal load resistance. PMID:26270666

Evaluation of the performance of Human Papillomavirus testing in paired urine and clinician-collected cervical samples among women aged over 30 years in Bhutan.

PubMed

Tshomo, Ugyen; Franceschi, Silvia; Tshokey, Tshokey; Tobgay, Tashi; Baussano, Iacopo; Tenet, Vanessa; Snijders, Peter J F; Gheit, Tarik; Tommasino, Massimo; Vorsters, Alex; Clifford, Gary M

2017-04-08

Urine sampling may offer a less invasive solution than cervical sampling to test for human papillomavirus (HPV) for HPV vaccine impact monitoring. Paired samples of urine and exfoliated cervical cells were obtained for 89 women with history of high-risk (HR) HPV-positive normal cytology in Bhutan. Urine sampling protocol included self-collection of first-void urine immediately into a conservation medium and procedures to optimize DNA yield. Colposcopical abnormalities were biopsied. Two HPV assays were used: a multiplex type-specific PCR (E7-MPG) and a less analytically sensitive GP5+/6+ PCR followed by reverse line blot. HPV positivity for 21 types common to both assays was similar in urine and cells by E7-MPG (62.9% and 57.3%, respectively, p = 0.32) but lower in urine by GP5+/6+ (30.3% and 40.4%, p = 0.05). HPV6/11/16/18 positivity did not significantly differ between urine and cells by either assay. Sensitivity of urine (using cells as gold standard) to detect 21 HPV types was 80% and 58% for E7-MPG and GP5+/6+, respectively, with specificity 61% and 89%. HPV type distribution in urine and cells was similar, regardless of assay. The 5 detected CIN3+ were HR-HPV positive in cells by both assays, compared to 4 and 3 by E7-MPG and GP5+/6+, respectively, in urine samples. For the monitoring of vaccine impact, we demonstrate validity of a urine sampling protocol to obtain HPV prevalence data that are broadly comparable to that from cervical cells. However, detection of HPV in urine varies according to assay sensitivity, presumably because low level infections are frequent.

Enhanced tumor metastasis in response to blockade of the chemokine receptor CXCR6 is overcome by NKT cell activation.

PubMed

Cullen, Robyn; Germanov, Elitza; Shimaoka, Takeshi; Johnston, Brent

2009-11-01

Invariant NKT (iNKT) cells can induce potent antitumor responses in vivo. However, the mechanisms that regulate the effects of iNKT cells are unclear. The chemokine receptor CXCR6, and its ligand CXCL16, have been shown to play critical roles in iNKT cell homeostasis and activation. Thus we investigated the role of CXCR6 in protection against experimental metastasis of B16-F10 melanoma (B16) and Lewis lung carcinoma (LLC) cells to the liver and lungs. Wild-type and CXCR6(-/-) mice exhibited no differences in tumor cell metastasis to the lungs. However, metastasis of LLC and B16 tumor cells to the liver was enhanced in CXCR6(-/-) mice. Liver metastasis was also increased in wild-type mice treated with a CXCL16 neutralizing Ab. As Ab treatments did not alter iNKT cell numbers, this implicates a direct role for CXCR6/CXCL16 in regulating antitumor immunity. Cytokine induction was significantly attenuated in CXCR6(-/-) mice upon systemic iNKT cell activation with the glycolipid Ags alpha-galactosylceramide (alpha-GalCer), alpha-C-GalCer (a Th1 polarizing derivative), or OCH (a Th2 polarizing derivative). Despite differences in the levels of cytokine production, liver and lung metastasis were inhibited significantly in both wild-type and CXCR6(-/-) mice treated with glycolipids. Single doses of alpha-GalCer, alpha-C-GalCer, or OCH were sufficient to prevent liver metastasis and subsequent doses failed to elicit optimal cytokine responses. Our findings implicate a role for CXCR6 in natural immunosurveillance against liver metastasis. However, CXCR6 deficiency could be overcome by systemic iNKT cell activation, demonstrating that even suboptimal iNKT cell activation can protect against metastasis.

The Role of Laboratory Testing in Differentiating Type 1 Diabetes from Type 2 Diabetes in Patients Undergoing Bariatric Surgery.

PubMed

Pilla, Scott J; Maruthur, Nisa M; Schweitzer, Michael A; Magnuson, Thomas H; Potter, James J; Clark, Jeanne M; Lee, Clare J

2018-01-01

It may be difficult to distinguish between adults with type 1 diabetes and type 2 diabetes by clinical assessment. In patients undergoing bariatric surgery, it is critical to correctly classify diabetes subtype to prevent adverse perioperative outcomes including diabetic ketoacidosis. This study aimed to determine whether testing for C-peptide and islet cell antibodies during preoperative evaluation for bariatric surgery could improve the classification of type 1 versus type 2 diabetes compared to clinical assessment alone. This is a retrospective analysis of the Improving Diabetes through Lifestyle and Surgery trial, which randomized patients with clinically diagnosed type 2 diabetes and BMI 30-40 kg/m 2 to medical weight loss or bariatric surgery; one participant was discovered to have type 1 diabetes after experiencing postoperative diabetic ketoacidosis. Using blood samples collected prior to study interventions, we measured islet cell antibodies and fasting/meal-stimulated C-peptide in all participants. The participant with type 1 diabetes was similar to the 11 participants with type 2 diabetes in age at diagnosis, adiposity, and glycemic control but had the lowest C-peptide levels. Among insulin-treated participants, fasting and stimulated C-peptide correlated strongly with the C-peptide area-under-the-curve on mixed meal tolerance testing (R = 0.86 and 0.88, respectively). Three participants, including the one with type 1 diabetes, were islet cell antibody positive. Clinical characteristics did not correctly identify type 1 diabetes in this study. Preoperative C-peptide testing may improve diabetes classification in patients undergoing bariatric surgery; further research is needed to define the optimal C-peptide thresholds.

Practical cell labeling with magnetite cationic liposomes for cell manipulation.

PubMed

Ito, Hiroshi; Nonogaki, Yurika; Kato, Ryuji; Honda, Hiroyuki

2010-07-01

Personalization of the cell culture process for cell therapy is an ideal strategy to obtain maximum treatment effects. In a previous report, we proposed a strategy using a magnetic manipulation device that combined a palm-top size device and a cell-labeling method using magnetite cationic liposomes (MCLs) to enable feasible personalized cell processing. In the present study, we focused on optimizing the MCL-labeling technique with respect to cell manipulation in small devices. From detailed analysis with different cell types, 4 pg/cell of MCL-label was found to be obtained immediately after mixing with MCLs, which was sufficient for magnetic cell manipulation. The amount of label increased within 24 h depending on cell type, although in all cases it decreased along with cell doubling, indicating that the labeling potential of MCLs was limited. The role of free MCLs not involved in labeling was also investigated; MCLs' role was found to be a supportive one that maximized the manipulation performance up to 100%. We also determined optimum conditions to manipulate adherent cells by MCL labeling using the MCL dispersed in trypsin solution. Considering labeling feasibility and practical performance with 10(3)-10(5) cells for personalized cell processing, we determined that 10 microg/ml of label without incubation time (0 h incubation) was the universal MCL-labeling condition. We propose the optimum specifications for a device to be combined with this method. 2010. Published by Elsevier B.V.

Extracellular matrix components direct porcine muscle stem cell behavior

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilschut, Karlijn J.; Haagsman, Henk P.; Roelen, Bernard A.J., E-mail: b.a.j.roelen@uu.nl

2010-02-01

In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatinmore » and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.« less

Sources of Hematopoietic Stem and Progenitor Cells and Methods to Optimize Yields for Clinical Cell Therapy.

PubMed

Panch, Sandhya R; Szymanski, James; Savani, Bipin N; Stroncek, David F

2017-08-01

Bone marrow (BM) aspirates, mobilized peripheral blood, and umbilical cord blood (UCB) have developed as graft sources for hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation and other cellular therapeutics. Individualized techniques are necessary to enhance graft HSPC yields and cell quality from each graft source. BM aspirates yield adequate CD34 + cells but can result in relative delays in engraftment. Granulocyte colony-stimulating factor (G-CSF)-primed BM HSPCs may facilitate faster engraftment while minimizing graft-versus-host disease in certain patient subsets. The levels of circulating HSPCs are enhanced using mobilizing agents, such as G-CSF and/or plerixafor, which act via the stromal cell-derived factor 1/C-X-C chemokine receptor type 4 axis. Alternate niche pathway mediators, including very late antigen-4/vascular cell adhesion molecule-1, heparan sulfate proteoglycans, parathyroid hormone, and coagulation cascade intermediates, may offer promising alternatives for graft enhancement. UCB grafts have been expanded ex vivo with cytokines, notch-ligand, or mesenchymal stromal cells, and most studies demonstrated greater quantities of CD34 + cells ex vivo and improved short-term engraftment. No significant changes were observed in long-term repopulating potential or in patient survival. Early phase clinical trials using nicotinamide and StemReginin1 may offer improved short- and long-term repopulating ability. Breakthroughs in genome editing and stem cell reprogramming technologies may hasten the generation of pooled, third-party HSPC grafts. This review elucidates past, present, and potential future approaches to HSPC graft optimization. Published by Elsevier Inc.

Symmetric vs. Asymmetric Stem Cell Divisions: An Adaptation against Cancer?

PubMed Central

Shahriyari, Leili; Komarova, Natalia L.

2013-01-01

Traditionally, it has been held that a central characteristic of stem cells is their ability to divide asymmetrically. Recent advances in inducible genetic labeling provided ample evidence that symmetric stem cell divisions play an important role in adult mammalian homeostasis. It is well understood that the two types of cell divisions differ in terms of the stem cells' flexibility to expand when needed. On the contrary, the implications of symmetric and asymmetric divisions for mutation accumulation are still poorly understood. In this paper we study a stochastic model of a renewing tissue, and address the optimization problem of tissue architecture in the context of mutant production. Specifically, we study the process of tumor suppressor gene inactivation which usually takes place as a consequence of two “hits”, and which is one of the most common patterns in carcinogenesis. We compare and contrast symmetric and asymmetric (and mixed) stem cell divisions, and focus on the rate at which double-hit mutants are generated. It turns out that symmetrically-dividing cells generate such mutants at a rate which is significantly lower than that of asymmetrically-dividing cells. This result holds whether single-hit (intermediate) mutants are disadvantageous, neutral, or advantageous. It is also independent on whether the carcinogenic double-hit mutants are produced only among the stem cells or also among more specialized cells. We argue that symmetric stem cell divisions in mammals could be an adaptation which helps delay the onset of cancers. We further investigate the question of the optimal fraction of stem cells in the tissue, and quantify the contribution of non-stem cells in mutant production. Our work provides a hypothesis to explain the observation that in mammalian cells, symmetric patterns of stem cell division seem to be very common. PMID:24204602

Immunomodulatory Nature and Site Specific Affinity of Mesenchymal Stem Cells: a Hope in Cell Therapy

PubMed Central

Lotfinegad, Parisa; Shamsasenjan, karim; Movassaghpour, Aliakbar; Majidi, Jafar; Baradaran, Behzad

2014-01-01

Immunosuppressive ability of mesenchymal stem cells (MSCs), their differentiation properties to various specialized tissue types, ease of in vitro and in vivo expansion and specific migration capacity, make them to be tested in different clinical trials for the treatment of various diseases. The immunomodulatory effects of MSCs are less identified which probably has high clinically significance. The clinical trials based on primary research will cause better understanding the ability of MSCs in immunomodulatory applications and site specific migration in the optimization of therapy. So, this review focus on MSCs functional role in modulating immune responses, their ability in homing to tumor, their potency as delivery vehicle and their medical importance. PMID:24409403

Dynamic reciprocity in cell-scaffold interactions.

PubMed

Mauney, Joshua R; Adam, Rosalyn M

2015-03-01

Tissue engineering in urology has shown considerable promise. However, there is still much to understand, particularly regarding the interactions between scaffolds and their host environment, how these interactions regulate regeneration and how they may be enhanced for optimal tissue repair. In this review, we discuss the concept of dynamic reciprocity as applied to tissue engineering, i.e. how bi-directional signaling between implanted scaffolds and host tissues such as the bladder drives the process of constructive remodeling to ensure successful graft integration and tissue repair. The impact of scaffold content and configuration, the contribution of endogenous and exogenous bioactive factors, the influence of the host immune response and the functional interaction with mechanical stimulation are all considered. In addition, the temporal relationships of host tissue ingrowth, bioactive factor mobilization, scaffold degradation and immune cell infiltration, as well as the reciprocal signaling between discrete cell types and scaffolds are discussed. Improved understanding of these aspects of tissue repair will identify opportunities for optimization of repair that could be exploited to enhance regenerative medicine strategies for urology in future studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Examining FKBP5 mRNA expression in human iPSC-derived neural cells

PubMed Central

Lieberman, Richard; Kranzler, Henry R.; Levine, Eric S.; Covault, Jonathan

2016-01-01

In peripheral blood leukocytes, FKBP5 mRNA expression is upregulated following glucocorticoid receptor activation. The single nucleotide polymorphism rs1360780 in FKBP5 is associated with psychiatric illness and has functional molecular effects. However, examination of FKBP5 regulation has largely been limited to peripheral cells, which may not reflect regulation in neural cells. We used 27 human induced pluripotent stem cell lines (iPSCs) derived from 20 subjects to examine FKBP5 mRNA expression following GR activation. Following differentiation into forebrain-lineage neural cultures, cells were exposed to 1μM dexamethasone and mRNA expression of FKBP5 and NR3C1 analyzed. Results from the iPSC-derived neural cells were compared with those from 15 donor matched fibroblast lines. Following dexamethasone treatment, there was a 670% increase in FKBP5 expression in fibroblasts, mimicking findings in peripheral blood-derived cells, but only a 23% increase in iPSC-derived neural cultures. FKBP5 rs1360780 genotype did not affect the induction of FKBP5 mRNA in either fibroblasts or neural cells. These results suggest that iPSC-derived forebrain-lineage neurons may not be an optimal neural cell type in which to examine relationships between GR activation, FKBP5 expression, and genetic variation in human subjects. Further, FKBP5 induction following GR activation may differ between cell types derived from the same individual. PMID:27915167

Algorithms for optimization of the transport system in living and artificial cells.

PubMed

Melkikh, A V; Sutormina, M I

2011-06-01

An optimization of the transport system in a cell has been considered from the viewpoint of the operations research. Algorithms for an optimization of the transport system of a cell in terms of both the efficiency and a weak sensitivity of a cell to environmental changes have been proposed. The switching of various systems of transport is considered as the mechanism of weak sensitivity of a cell to changes in environment. The use of the algorithms for an optimization of a cardiac cell has been considered by way of example. We received theoretically for a cell of a cardiac muscle that at the increase of potassium concentration in the environment switching of transport systems for this ion takes place. This conclusion qualitatively coincides with experiments. The problem of synthesizing an optimal system in an artificial cell has been stated.

Analysis of optimality in natural and perturbed metabolic networks

PubMed Central

Segrè, Daniel; Vitkup, Dennis; Church, George M.

2002-01-01

An important goal of whole-cell computational modeling is to integrate detailed biochemical information with biological intuition to produce testable predictions. Based on the premise that prokaryotes such as Escherichia coli have maximized their growth performance along evolution, flux balance analysis (FBA) predicts metabolic flux distributions at steady state by using linear programming. Corroborating earlier results, we show that recent intracellular flux data for wild-type E. coli JM101 display excellent agreement with FBA predictions. Although the assumption of optimality for a wild-type bacterium is justifiable, the same argument may not be valid for genetically engineered knockouts or other bacterial strains that were not exposed to long-term evolutionary pressure. We address this point by introducing the method of minimization of metabolic adjustment (MOMA), whereby we test the hypothesis that knockout metabolic fluxes undergo a minimal redistribution with respect to the flux configuration of the wild type. MOMA employs quadratic programming to identify a point in flux space, which is closest to the wild-type point, compatibly with the gene deletion constraint. Comparing MOMA and FBA predictions to experimental flux data for E. coli pyruvate kinase mutant PB25, we find that MOMA displays a significantly higher correlation than FBA. Our method is further supported by experimental data for E. coli knockout growth rates. It can therefore be used for predicting the behavior of perturbed metabolic networks, whose growth performance is in general suboptimal. MOMA and its possible future extensions may be useful in understanding the evolutionary optimization of metabolism. PMID:12415116

CMV-promoter driven codon-optimized expression alters the assembly type and morphology of a reconstituted HERV-K(HML-2).

PubMed

Hohn, Oliver; Hanke, Kirsten; Lausch, Veronika; Zimmermann, Anja; Mostafa, Saeed; Bannert, Norbert

2014-11-11

The HERV-K(HML-2) family contains the most recently integrated and best preserved endogenized proviral sequences in the human genome. All known elements have nevertheless been subjected to mutations or deletions that render expressed particles non-infectious. Moreover, these post-insertional mutations hamper the analysis of the general biological properties of this ancient virus family. The expression of consensus sequences and sequences of elements with reverted post-insertional mutations has therefore been very instrumental in overcoming this limitation. We investigated the particle morphology of a recently reconstituted HERV-K113 element termed oriHERV-K113 using thin-section electron microscopy (EM) and could demonstrate that strong overexpression by substitution of the 5'LTR for a CMV promoter and partial codon optimization altered the virus assembly type and morphology. This included a conversion from the regular C-type to an A-type morphology with a mass of cytoplasmic immature cores tethered to the cell membrane and the membranes of vesicles. Overexpression permitted the release and maturation of virions but reduced the envelope content. A weaker boost of virus expression by Staufen-1 was not sufficient to induce these morphological alterations.

CMV-Promoter Driven Codon-Optimized Expression Alters the Assembly Type and Morphology of a Reconstituted HERV-K(HML-2)

PubMed Central

Hohn, Oliver; Hanke, Kirsten; Lausch, Veronika; Zimmermann, Anja; Mostafa, Saeed; Bannert, Norbert

2014-01-01

The HERV-K(HML-2) family contains the most recently integrated and best preserved endogenized proviral sequences in the human genome. All known elements have nevertheless been subjected to mutations or deletions that render expressed particles non-infectious. Moreover, these post-insertional mutations hamper the analysis of the general biological properties of this ancient virus family. The expression of consensus sequences and sequences of elements with reverted post-insertional mutations has therefore been very instrumental in overcoming this limitation. We investigated the particle morphology of a recently reconstituted HERV-K113 element termed oriHERV-K113 using thin-section electron microscopy (EM) and could demonstrate that strong overexpression by substitution of the 5'LTR for a CMV promoter and partial codon optimization altered the virus assembly type and morphology. This included a conversion from the regular C-type to an A-type morphology with a mass of cytoplasmic immature cores tethered to the cell membrane and the membranes of vesicles. Overexpression permitted the release and maturation of virions but reduced the envelope content. A weaker boost of virus expression by Staufen-1 was not sufficient to induce these morphological alterations. PMID:25393897

Shaping electromagnetic waves using software-automatically-designed metasurfaces.

PubMed

Zhang, Qian; Wan, Xiang; Liu, Shuo; Yuan Yin, Jia; Zhang, Lei; Jun Cui, Tie

2017-06-15

We present a fully digital procedure of designing reflective coding metasurfaces to shape reflected electromagnetic waves. The design procedure is completely automatic, controlled by a personal computer. In details, the macro coding units of metasurface are automatically divided into several types (e.g. two types for 1-bit coding, four types for 2-bit coding, etc.), and each type of the macro coding units is formed by discretely random arrangement of micro coding units. By combining an optimization algorithm and commercial electromagnetic software, the digital patterns of the macro coding units are optimized to possess constant phase difference for the reflected waves. The apertures of the designed reflective metasurfaces are formed by arranging the macro coding units with certain coding sequence. To experimentally verify the performance, a coding metasurface is fabricated by automatically designing two digital 1-bit unit cells, which are arranged in array to constitute a periodic coding metasurface to generate the required four-beam radiations with specific directions. Two complicated functional metasurfaces with circularly- and elliptically-shaped radiation beams are realized by automatically designing 4-bit macro coding units, showing excellent performance of the automatic designs by software. The proposed method provides a smart tool to realize various functional devices and systems automatically.

Myogenic Progenitor Cells Control Extracellular Matrix Production by Fibroblasts during Skeletal Muscle Hypertrophy.

PubMed

Fry, Christopher S; Kirby, Tyler J; Kosmac, Kate; McCarthy, John J; Peterson, Charlotte A

2017-01-05

Satellite cells, the predominant stem cell population in adult skeletal muscle, are activated in response to hypertrophic stimuli and give rise to myogenic progenitor cells (MPCs) within the extracellular matrix (ECM) that surrounds myofibers. This ECM is composed largely of collagens secreted by interstitial fibrogenic cells, which influence satellite cell activity and muscle repair during hypertrophy and aging. Here we show that MPCs interact with interstitial fibrogenic cells to ensure proper ECM deposition and optimal muscle remodeling in response to hypertrophic stimuli. MPC-dependent ECM remodeling during the first week of a growth stimulus is sufficient to ensure long-term myofiber hypertrophy. MPCs secrete exosomes containing miR-206, which represses Rrbp1, a master regulator of collagen biosynthesis, in fibrogenic cells to prevent excessive ECM deposition. These findings provide insights into how skeletal stem and progenitor cells interact with other cell types to actively regulate their extracellular environments for tissue maintenance and adaptation. Copyright © 2017 Elsevier Inc. All rights reserved.

Proton channel HVCN1 is required for effector functions of mouse eosinophils

PubMed Central

2013-01-01

Background Proton currents are required for optimal respiratory burst in phagocytes. Recently, HVCN1 was identified as the molecule required for the voltage-gated proton channel activity associated with the respiratory burst in neutrophils. Although there are similarities between eosinophils and neutrophils regarding their mechanism for respiratory burst, the role of proton channels in eosinophil functions has not been fully understood. Results In the present study, we first identified the expression of the proton channel HVCN1 in mouse eosinophils. Furthermore, using HVCN1-deficient eosinophils, we demonstrated important cell-specific effector functions for HVCN1. Similar to HVCN1-deficient neutrophils, HVCN1-deficient eosinophils produced significantly less reactive oxygen species (ROS) upon phorbol myristate acetate (PMA) stimulation compared with WT eosinophils. In contrast to HVCN1-deficient neutrophils, HVCN1-deficient eosinophils did not show impaired calcium mobilization or migration ability compared with wild-type (WT) cells. Uniquely, HVCN1-deficient eosinophils underwent significantly increased cell death induced by PMA stimulation compared with WT eosinophils. The increased cell death was dependent on NADPH oxidase activation, and correlated with the failure of HVCN1-deficient cells to maintain membrane polarization and intracellular pH in the physiological range upon activation. Conclusions Eosinophils require proton channel HVCN1 for optimal ROS generation and prevention of activation-induced cell death. PMID:23705768

Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

PubMed Central

Streng-Ouwehand, Ingeborg; Ho, Nataschja I; Litjens, Manja; Kalay, Hakan; Boks, Martine Annemarie; Cornelissen, Lenneke AM; Kaur Singh, Satwinder; Saeland, Eirikur; Garcia-Vallejo, Juan J; Ossendorp, Ferry A; Unger, Wendy WJ; van Kooyk, Yvette

2016-01-01

Antigen uptake by dendritic cells and intracellular routing of antigens to specific compartments is regulated by C-type lectin receptors that recognize glycan structures. We show that the modification of Ovalbumin (OVA) with the glycan-structure LewisX (LeX) re-directs OVA to the C-type lectin receptor MGL1. LeX-modification of OVA favored Th1 skewing of CD4+ T cells and enhanced cross-priming of CD8+ T cells. While cross-presentation of native OVA requires high antigen dose and TLR stimuli, LeX modification reduces the required amount 100-fold and obviates its dependence on TLR signaling. The OVA-LeX-induced enhancement of T cell cross-priming is MGL1-dependent as shown by reduced CD8+ effector T cell frequencies in MGL1-deficient mice. Moreover, MGL1-mediated cross-presentation of OVA-LeX neither required TAP-transporters nor Cathepsin-S and was still observed after prolonged intracellular storage of antigen in Rab11+LAMP1+ compartments. We conclude that controlled neo-glycosylation of antigens can crucially influence intracellular routing of antigens, the nature and strength of immune responses and should be considered for optimizing current vaccination strategies. DOI: http://dx.doi.org/10.7554/eLife.11765.001 PMID:26999763

Silencing of Endogenous IL-10 in Human Dendritic Cells Leads to the Generation of an Improved CTL Response Against Human Melanoma Associated Antigenic Epitope, MART-127−35

PubMed Central

Chhabra, Arvind; Chakraborty, Nityo G.; Mukherji, Bijay

2008-01-01

Dendritic cells (DC) present antigenic epitopes to and activate T cells. They also polarize the ensuing T cell response to Th1 or Th2 type response, depending on their cytokine production profile. For example, IL-12 producing DC generate Th1 type T cell response whereas IL-10 producing DC is usually tolerogenic. Different strategies -- such as the use of cytokines and anti-cytokine antibodies, dominant negative forms of protein, anti-sense RNA etc. -- have been employed to influence the cytokine synthetic profile of DC as well as to make DC more immunogenic. Utilizing GFP expressing recombinant adenoviruses in association with lipid-mediated transfection of siRNA, we have silenced the endogenous IL-10 gene in DC. We show that IL-10 gene silenced DC produce more IL-12 and also generates a better cytolytic T cell response against the human melanoma associated epitope, MART-127−35, in-vitro. We also show that the GFP expressing adenoviral vector can be used to optimize the parameters for siRNA delivery in primary cells and show that RNA interference methodology can efficiently knock-down virus encoded genes transcribed at very high multiplicity of infection in DC. PMID:18249038

Discovery and Characterization of Novel Nonsubstrate and Substrate NAMPT Inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilsbacher, Julie L.; Cheng, Min; Cheng, Dong

Cancer cells are highly reliant on NAD+-dependent processes, including glucose metabolism, calcium signaling, DNA repair, and regulation of gene expression. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ salvage from nicotinamide, has been investigated as a target for anticancer therapy. Known NAMPT inhibitors with potent cell activity are composed of a nitrogen-containing aromatic group, which is phosphoribosylated by the enzyme. Here, we identified two novel types of NAM-competitive NAMPT inhibitors, only one of which contains a modifiable, aromatic nitrogen that could be a phosphoribosyl acceptor. Both types of compound effectively deplete cellular NAD+, and subsequently ATP, and produce cell deathmore » when NAMPT is inhibited in cultured cells for more than 48 hours. Careful characterization of the kinetics of NAMPT inhibition in vivo allowed us to optimize dosing to produce sufficient NAD+ depletion over time that resulted in efficacy in an HCT116 xenograft model. Our data demonstrate that direct phosphoribosylation of competitive inhibitors by the NAMPT enzyme is not required for potent in vitro cellular activity or in vivo antitumor efficacy. Mol Cancer Ther; 16(7); 1236–45.« less

Analysis of the PEDOT:PSS/Si nanowire hybrid solar cell with a tail state model

NASA Astrophysics Data System (ADS)

Ho, Kuan-Ying; Li, Chi-Kang; Syu, Hong-Jhang; Lai, Yi; Lin, Ching-Fuh; Wu, Yuh-Renn

2016-12-01

In this paper, the electrical properties of the poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT:PSS)/silicon nanowire hybrid solar cell have been analyzed and an optimized structure is proposed. In addition, the planar PEDOT:PSS/c-Si hybrid solar cell is also modeled for comparison. We first developed a simulation software which is capable of modeling organic/inorganic hybrid solar cells by including Gaussian shape density of states into Poisson and drift-diffusion solver to present the tail states and trap states in the organic material. Therefore, the model can handle carrier transport, generation, and recombination in both organic and inorganic materials. Our results show that at the applied voltage near open-circuit voltage (Voc), the recombination rate becomes much higher at the PEDOT:PSS/Si interface region, which limits the fill factor and Voc. Hence, a modified structure with a p-type amorphous silicon (a-Si) layer attached on the interface of Si layer and an n+-type Si layer inserted near the bottom contact are proposed. The highest conversion efficiency of 16.10% can be achieved if both structures are applied.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oh, Seung-Min; Oh, Pilgun; Kim, Sang-Ok

A low-cost sodium-ion full cell with a O3-type layered Na[Cu 0.2(Fe 1/3Mn2/3) 0.8]O 2 cathode and an alloy-type P-TiP2-C anode is presented. The cathode is synthesized by an oxalate coprecipitation method and optimized cathodes shows a high specific capacity of 135 mAh g -1 at 0.1C rate with a high rate capability of 90 mAh g-1 at 1C rate and 70 mAh g -1 at 2C rate with good cyclability. The full cell exhibits better capacity retention than the half cell with the cathode due to the elimination of the degradation caused by sodium-metal anode. The dramatically enhanced electrochemical performancemore » of the Na[Cu 0.2(Fe 1/3Mn 2/3) 0.8]O 2 / P-TiP 2-C full cell compared to that of the sample with no Cu is attributed to the structural stabilization imparted by Cu by suppressing the phase change from the O3 structure to the P3 structure during cycling.« less

A crucial role for plasmacytoid dendritic cells in antiviral protection by CpG ODN–based vaginal microbicide

PubMed Central

Shen, Hong; Iwasaki, Akiko

2006-01-01

Topical microbicides represent a promising new approach to preventing HIV and other sexually transmitted infections. TLR agonists are ideal candidates for microbicides, as they trigger a multitude of antiviral genes effective against a broad range of viruses. Although vaginal application of CpG oligodeoxynucleotides (ODNs) and poly I:C has been shown to protect mice from genital herpes infection, the mechanism by which these agents provide protection remains unclear. Here, we show that plasmacytoid DCs (pDCs) are required for CpG ODN–mediated protection against lethal vaginal challenge with herpes simplex virus type 2 (HSV-2). Moreover, we demonstrate that cells of both the hematopoietic and stromal compartments must respond to CpG ODN via TLR9 and to type I IFNs through IFN-αβ receptor (IFN-αβR) for protection. Thus, crosstalk between pDCs and vaginal stromal cells provides for optimal microbicide efficacy. Our results imply that temporally and spatially controlled targeting of CpG ODN to pDCs and epithelial cells can potentially maximize their effectiveness as microbicides while minimizing the associated inflammatory responses. PMID:16878177

An assessment of alternative fuel cell designs for residential and commercial cogeneration

NASA Technical Reports Server (NTRS)

Wakefield, R. A.

1980-01-01

A comparative assessment of three fuel cell systems for application in different buildings and geographic locations is presented. The study was performed at the NASA Lewis Center and comprised the fuel cell design, performance in different conditions, and the economic parameters. Applications in multifamily housing, stores and hospitals were considered, with a load of 10kW-1 MW. Designs were traced through system sizing, simulation/evaluation, and reliability analysis, and a computer simulation based on a fourth-order representation of a generalized system was performed. The cells were all phosphoric acid type cells, and were found to be incompatible with gas/electric systems and more favorable economically than the gas/electric systems in hospital uses. The methodology used provided an optimized energy-use pattern and minimized back-up system turn-on.

Manufacturing of Proteins and Antibodies: Chapter Downstream Processing Technologies : Harvest Operations.

PubMed

Turner, Richard; Joseph, Adrian; Titchener-Hooker, Nigel; Bender, Jean

2017-08-04

Cell harvesting is the separation or retention of cells and cellular debris from the supernatant containing the target molecule Selection of harvest method strongly depends on the type of cells, mode of bioreactor operation, process scale, and characteristics of the product and cell culture fluid. Most traditional harvesting methods use some form of filtration, centrifugation, or a combination of both for cell separation and/or retention. Filtration methods include normal flow depth filtration and tangential flow microfiltration. The ability to scale down predictably the selected harvest method helps to ensure successful production and is critical for conducting small-scale characterization studies for confirming parameter targets and ranges. In this chapter we describe centrifugation and depth filtration harvesting methods, share strategies for harvest optimization, present recent developments in centrifugation scale-down models, and review alternative harvesting technologies.

Modelling and fabrication of high-efficiency silicon solar cells

NASA Astrophysics Data System (ADS)

Rohatgi, A.; Smith, A. W.; Salami, J.

1991-10-01

This report covers the research conducted on modelling and development of high efficiency silicon solar cells during the period May 1989 to August 1990. First, considerable effort was devoted toward developing a ray tracing program for the photovoltaic community to quantify and optimize surface texturing for solar cells. Second, attempts were made to develop a hydrodynamic model for device simulation. Such a model is somewhat slower than drift-diffusion type models like PC-1D, but it can account for more physical phenomena in the device, such as hot carrier effects, temperature gradients, thermal diffusion, and lattice heat flow. In addition, Fermi-Dirac statistics have been incorporated into the model to deal with heavy doping effects more accurately. The third and final component of the research includes development of silicon cell fabrication capabilities and fabrication of high efficiency silicon cells.

Streptococcus thermophilus Cell Wall-Anchored Proteinase: Release, Purification, and Biochemical and Genetic Characterization

PubMed Central

Fernandez-Espla, María Dolores; Garault, Peggy; Monnet, Véronique; Rul, Françoise

2000-01-01

Streptococcus thermophilus CNRZ 385 expresses a cell envelope proteinase (PrtS), which is characterized in the present work, both at the biochemical and genetic levels. Since PrtS is resistant to most classical methods of extraction from the cell envelopes, we developed a three-step process based on loosening of the cell wall by cultivation of the cells in the presence of glycine (20 mM), mechanical disruption (with alumina powder), and enzymatic treatment (lysozyme). The pure enzyme is a serine proteinase highly activated by Ca2+ ions. Its activity was optimal at 37°C and pH 7.5 with acetyl-Ala-Ala-Pro-Phe-paranitroanilide as substrate. The study of the hydrolysis of the chromogenic and casein substrates indicated that PrtS presented an intermediate specificity between the most divergent types of cell envelope proteinases from lactococci, known as the PI and PIII types. This result was confirmed by the sequence determination of the regions involved in substrate specificity, which were a mix between those of PI and PIII types, and also had unique residues. Sequence analysis of the PrtS encoding gene revealed that PrtS is a member of the subtilase family. It is a multidomain protein which is maturated and tightly anchored to the cell wall via a mechanism involving an LPXTG motif. PrtS bears similarities to cell envelope proteinases from pyogenic streptococci (C5a peptidase and cell surface proteinase) and lactic acid bacteria (PrtP, PrtH, and PrtB). The highest homologies were found with streptococcal proteinases which lack, as PrtS, one domain (the B domain) present in cell envelope proteinases from all other lactic acid bacteria. PMID:11055922

CXCR3 Deficiency Increases Susceptibility to Genital Herpes Simplex Virus Type 2 Infection: Uncoupling of CD8+ T-Cell Effector Function but Not Migration▿

PubMed Central

Thapa, Manoj; Carr, Daniel J. J.

2009-01-01

CXCR3 is a G-protein-coupled receptor preferentially expressed by activated T cells, NK cells, and dendritic cells. Signaling through gamma interferon-regulated chemokines CXCL9, CXCL10, CXCL11, and CXCR3 plays a critical role in the immune response of many viral pathogens. However, the relevance of CXCR3 for optimal T-cell activation and the induction of regulatory transcription factors (i.e., T-bet and eomesodermin) relative to host immune defense against genital herpes simplex virus type 2 (HSV-2) infection have been poorly defined. In this study, we evaluated the requirement of CXCR3 expression during genital HSV-2 infection using mice deficient in CXCR3 (CXCR3−/−) along with wild-type (WT) controls, assessing the resistance of mice to viral infection and focusing on the cytokine/chemokine response, phenotypic analysis of recruited leukocytes, and functional analysis of CD8+ T cells. CXCR3−/− mice showed a heightened sensitivity to infection compared to WT animals in terms of the viral burden in infected tissues as well as elevated mortality. The poor response of CXCR3−/− mice to viral infection was associated with reduced cytotoxic T-lymphocyte activity through the impairment of T-bet, perforin, and granzyme B expression by CD8+ T cells. Corresponding with the defective cytolytic activity, a reduction in recruitment of plasmacytoid dendritic cells and CD80 expression in CD11c+ dendritic cells in the draining lymph nodes of CXCR3−/− mice were detected. Collectively, the results provide a new perspective to CXCR3 signaling for the appropriate activation of CD8+ T cells required for host defense against genital HSV-2 infection. PMID:19587047

Investigation of engineered bacterial adhesins for opportunity to interface cells with abiotic materials

NASA Astrophysics Data System (ADS)

Terrell, Jessica L.; Dong, Hong; Holthoff, Ellen L.; Small, Meagan C.; Sarkes, Deborah A.; Hurley, Margaret M.; Stratis-Cullum, Dimitra N.

2016-05-01

The convenience of cellular genetic engineering has afforded the power to build `smart' synthetic biological tools with novel applications. Here, we have explored opportunities to hybridize engineered cells with inorganic materials toward the development of 'living' device-compatible systems. Cellular structural biology is engineerable based on the ability to rewrite genetic code to generate recombinant, foreign, or even unnatural proteins. With this capability on the biological end, it should be possible to achieve superior abio-compatibility with the inorganic materials that compose current microfabricated technology. This work investigated the hair-like appendages of Escherichia coli known as Type 1 fimbriae that enable natural adhesion to glycosylated substrates. Sequence alterations within the fimbrial gene cluster were found to be well-tolerated, evidenced by tagging the fimbriae with peptide-based probes. As a further development, fimbriae tips could be reconfigured to, in turn, alter cell binding. In particular, the fimbriae were fused with a genetically optimized peptide-for-inorganics to enable metal binding. This work established methodologies to systematically survey cell adhesion properties across a suite of fimbriae-modified cell types as well as to direct patterned cell adhesion. Cell types were further customized for added complexity including turning on secondary gene expression and binding to gold surfaces. The former demonstrates potential for programmable gene switches and the latter for interfacing biology with inorganic materials. In general, the incorporation of 'programmed' cells into devices can be used to provide the feature of dynamic and automated cell response. The outcomes of this study are foundational toward the critical feature of deliberate positioning of cells as configurable biocomponentry. Overall, cellular integration into bioMEMs will yield advanced sensing and actuation.

Optimization methods and silicon solar cell numerical models

NASA Technical Reports Server (NTRS)

Girardini, K.; Jacobsen, S. E.

1986-01-01

An optimization algorithm for use with numerical silicon solar cell models was developed. By coupling an optimization algorithm with a solar cell model, it is possible to simultaneously vary design variables such as impurity concentrations, front junction depth, back junction depth, and cell thickness to maximize the predicted cell efficiency. An optimization algorithm was developed and interfaced with the Solar Cell Analysis Program in 1 Dimension (SCAP1D). SCAP1D uses finite difference methods to solve the differential equations which, along with several relations from the physics of semiconductors, describe mathematically the performance of a solar cell. A major obstacle is that the numerical methods used in SCAP1D require a significant amount of computer time, and during an optimization the model is called iteratively until the design variables converge to the values associated with the maximum efficiency. This problem was alleviated by designing an optimization code specifically for use with numerically intensive simulations, to reduce the number of times the efficiency has to be calculated to achieve convergence to the optimal solution.

An efficient algorithm for function optimization: modified stem cells algorithm

NASA Astrophysics Data System (ADS)

Taherdangkoo, Mohammad; Paziresh, Mahsa; Yazdi, Mehran; Bagheri, Mohammad Hadi

2013-03-01

In this paper, we propose an optimization algorithm based on the intelligent behavior of stem cell swarms in reproduction and self-organization. Optimization algorithms, such as the Genetic Algorithm (GA), Particle Swarm Optimization (PSO) algorithm, Ant Colony Optimization (ACO) algorithm and Artificial Bee Colony (ABC) algorithm, can give solutions to linear and non-linear problems near to the optimum for many applications; however, in some case, they can suffer from becoming trapped in local optima. The Stem Cells Algorithm (SCA) is an optimization algorithm inspired by the natural behavior of stem cells in evolving themselves into new and improved cells. The SCA avoids the local optima problem successfully. In this paper, we have made small changes in the implementation of this algorithm to obtain improved performance over previous versions. Using a series of benchmark functions, we assess the performance of the proposed algorithm and compare it with that of the other aforementioned optimization algorithms. The obtained results prove the superiority of the Modified Stem Cells Algorithm (MSCA).

The optimal density of cellular solids in axial tension.

PubMed

Mihai, L Angela; Alayyash, Khulud; Wyatt, Hayley

2017-05-01

For cellular bodies with uniform cell size, wall thickness, and shape, an important question is whether the same volume of material has the same effect when arranged as many small cells or as fewer large cells. To answer this question, for finite element models of periodic structures of Mooney-type material with different structural geometry and subject to large strain deformations, we identify a nonlinear elastic modulus as the ratio between the mean effective stress and the mean effective strain in the solid cell walls, and show that this modulus increases when the thickness of the walls increases, as well as when the number of cells increases while the volume of solid material remains fixed. Since, under the specified conditions, this nonlinear elastic modulus increases also as the corresponding mean stress increases, either the mean modulus or the mean stress can be employed as indicator when the optimum wall thickness or number of cells is sought.

Gap junction communications influence upon fibroblast synthesis of Type I collagen and fibronectin.

PubMed

Ehrlich, H Paul; Sun, Bonnie; Saggers, Gregory C; Kromath, Fatuma

2006-07-01

In rats polyvinyl alcohol sponge subcutaneous implants treated with gap junctional intercellular communications (GJIC) uncouplers showed reduced deposition of connective tissue. Do uncouplers inhibit the synthesis and deposition of a new connective tissue by fibroblasts? Confluent human dermal fibroblasts in serum-free medium received either endosulfan or oleamide, GJIC uncouplers. Collected media were subjected to Dot Blot analysis for native Type I collagen and fibronectin. Uncoupler-treated fibroblasts released less Type I collagen, while there was no change in fibronectin release. Collagen synthesis was restored to normal, when the uncouplers were removed, showing that these uncouplers were reversible and not toxic to cells. Northern blot analysis revealed procollagen alpha1 (I) mRNA was minimally affected by endosulfan. Oleamide-treated 17-day chick embryo calvaria explants were incubated with Type I collagen antibody, frozen, cryosectioned, and then subjected to rhodamine (Rh) tagged anti-mouse-IgG antibody, to detect newly deposited Type I collagen. Fluorescent antibody-collagen complexes were localized on the periphery of cells in control calvaria, but absent around cells in oleamide-treated calvaria. GJIC optimize collagen synthesis but not fibronectin synthesis. The lack of connective tissue deposited in granulation tissues treated with uncouplers appears related to the inhibition of collagen synthesis. These findings suggest that altering GJIC might control collagen deposition in scarring. 2006 Wiley-Liss, Inc.

Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity

PubMed Central

Calow, Jenny; Bockau, Ulrike; Struwe, Weston B.; Nowaczyk, Marc M.; Loser, Karin; Crispin, Max

2016-01-01

ABSTRACT Antibody glycosylation is a key parameter in the optimization of antibody therapeutics. Here, we describe the production of the anti-cancer monoclonal antibody rituximab in the unicellular ciliate, Tetrahymena thermophila. The resulting antibody demonstrated enhanced antibody-dependent cell-mediated cytotoxicity, which we attribute to unusual N-linked glycosylation. Detailed chromatographic and mass spectrometric analysis revealed afucosylated, oligomannose-type glycans, which, as a whole, displayed isomeric structures that deviate from the typical human counterparts, but whose branches were equivalent to fragments of metabolic intermediates observed in human glycoproteins. From the analysis of deposited crystal structures, we predict that the ciliate glycans adopt protein-carbohydrate interactions with the Fc domain that closely mimic those of native complex-type glycans. In addition, terminal glucose structures were identified that match biosynthetic precursors of human glycosylation. Our results suggest that ciliate-based expression systems offer a route to large-scale production of monoclonal antibodies exhibiting glycosylation that imparts enhanced cell killing activity. PMID:27594301

Expansion and cryopreservation of porcine and human corneal endothelial cells.

PubMed

Marquez-Curtis, Leah A; McGann, Locksley E; Elliott, Janet A W

2017-08-01

Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me 2 SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me 2 SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

In vitro synergistic antitumor efficacy of sequentially combined chemotherapy/icotinib in non‑small cell lung cancer cell lines.

PubMed

Wang, Min-Cong; Liang, Xuan; Liu, Zhi-Yan; Cui, Jie; Liu, Ying; Jing, Li; Jiang, Li-Li; Ma, Jie-Qun; Han, Li-Li; Guo, Qian-Qian; Yang, Cheng-Cheng; Wang, Jing; Wu, Tao; Nan, Ke-Jun; Yao, Yu

2015-01-01

The concurrent administration of chemotherapy and epidermal growth factor receptor‑tyrosine kinase inhibitors (EGFR‑TKIs) has previously produced a negative interaction and failed to confer a survival benefit to non‑small cell lung cancer (NSCLC) patients compared with first‑line cytotoxic chemotherapy. The present study aimed to investigate the optimal schedule of the combined treatment of cisplatin/paclitaxel and icotinib in NSCLC cell lines and clarify the underlying mechanisms. HCC827, H1975, H1299 and A549 human NSCLC cell lines with wild‑type and mutant EGFR genes were used as in vitro models to define the differential effects of various schedules of cisplatin/paclitaxel with icotinib treatments on cell growth, proliferation, cell cycle distribution, apoptosis, and EGFR signaling pathway. Sequence‑dependent antiproliferative effects differed among the four NSCLC cell lines, and were not associated with EGFR mutation, constitutive expression levels of EGFR or downstream signaling molecules. The antiproliferative effect of cisplatin plus paclitaxel followed by icotinib was superior to that of cisplatin or paclitaxel followed by icotinib in the HCC827, H1975, H1299 and A549 cell lines, and induced more cell apoptosis and G0/G1 phase arrest. Cisplatin and paclitaxel significantly increased the expression of EGFR phosphorylation in the HCC827 cell line. However, only paclitaxel increased the expression of EGFR phosphorylation in the H1975 cell line. Cisplatin/paclitaxel followed by icotinib influenced the expression of p‑EGFR and p‑AKT, although the expression of p‑ERK1/2 remained unchanged. The results suggest that the optimal schedule of the combined treatment of cisplatin/paclitaxel and icotinib differed among the NSCLC cell lines. The results also provide molecular evidence to support clinical treatment strategies for NSCLC patients.

Energy-efficient growth of phage Q Beta in Escherichia coli.

PubMed

Kim, Hwijin; Yin, John

2004-10-20

The role of natural selection in the optimal design of organisms is controversial. Optimal forms, functions, or behaviors of organisms have long been claimed without knowledge of how genotype contributes to phenotype, delineation of design constraints, or reference to alternative designs. Moreover, arguments for optimal designs have been often based on models that were difficult, if not impossible, to test. Here, we begin to address these issues by developing and probing a kinetic model for the intracellular growth of bacteriophage Q beta in Escherichia coli. The model accounts for the energetic costs of all template-dependent polymerization reactions, in ATP equivalents, including RNA-dependent RNA elongation by the phage replicase and synthesis of all phage proteins by the translation machinery of the E. coli host cell. We found that translation dominated phage growth, requiring 85% of the total energy expenditure. Only 10% of the total energy was applied to activities other than the direct synthesis of progeny phage components, reflecting primarily the cost of making the negative-strand RNA template that is needed for replication of phage genomic RNA. Further, we defined an energy efficiency of phage growth and showed its direct relationship to the yield of phage progeny. Finally, we performed a sensitivity analysis and found that the growth of wild-type phage was optimized for progeny yield or energy efficiency, suggesting that phage Q beta has evolved to optimally utilize the finite resources of its host cells.

Optimization of dendrimer structure for sentinel lymph node imaging: Effects of generation and terminal group.

PubMed

Niki, Yuichiro; Ogawa, Mikako; Makiura, Rie; Magata, Yasuhiro; Kojima, Chie

2015-11-01

The detection of the sentinel lymph node (SLN), the first lymph node draining tumor cells, is important in cancer diagnosis and therapy. Dendrimers are synthetic macromolecules with highly controllable structures, and are potent multifunctional imaging agents. In this study, 12 types of dendrimer of different generations (G2, G4, G6, and G8) and different terminal groups (amino, carboxyl, and acetyl) were prepared to determine the optimal dendrimer structure for SLN imaging. Radiolabeled dendrimers were intradermally administrated to the right footpads of rats. All G2 dendrimers were predominantly accumulated in the kidney. Amino-terminal, acetyl-terminal, and carboxyl-terminal dendrimers of greater than G4 were mostly located at the injection site, in the blood, and in the SLN, respectively. The carboxyl-terminal dendrimers were largely unrecognized by macrophages and T-cells in the SLN. Finally, SLN detection was successfully performed by single photon emission computed tomography imaging using carboxyl-terminal dendrimers of greater than G4. The early detection of tumor cells in the sentinel draining lymph nodes (SLN) is of utmost importance in terms of determining cancer prognosis and devising treatment. In this article, the authors investigated various formulations of dendrimers to determine the optimal one for tumor detection. The data generated from this study would help clinicians to fight the cancer battle in the near future. Copyright © 2015 Elsevier Inc. All rights reserved.

Self-Assembled ZnO Nanosheet-Based Spherical Structure as Photoanode in Dye-Sensitized Solar Cells

NASA Astrophysics Data System (ADS)

Ameri, Mohsen; Raoufi, Meysam; Zamani-Meymian, M.-R.; Samavat, Feridoun; Fathollahi, M.-R.; Mohajerani, Ezeddin

2018-03-01

High surface area and enhanced light scattering of ZnO nanosheet aggregates have made them a promising active layer candidate material for fabrication of nanostructure dye-sensitized solar cells. Here, we propose a facile preparation method of such ZnO nanosheet structures, and in order to verify their applicability as photoanode material for dye-sensitized solar cells, we employ morphological, optical, structural and electrical measurements. The results reveal the high surface area available for dye molecules for enhancing adsorption, high light scattering and competitive power conversion efficiencies compared to the works in literature. Finally, the device is optimized with respect to the photoanode thickness. The favorable features shown here can extend the application of the structure to other types of sensitization-based perovskite and quantum dot solar cells.

Detection of large deletion in human BRCA1 gene in human breast carcinoma MCF-7 cells by using DNA-Silver Nanoclusters

NASA Astrophysics Data System (ADS)

Borghei, Yasaman-Sadat; Hosseini, Morteza; Ganjali, Mohammad Reza

2018-01-01

Here we describe a label-free detection strategy for large deletion mutation in breast cancer (BC) related gene BRCA1 based on a DNA-silver nanocluster (NC) fluorescence upon recognition-induced hybridization. The specific hybridization of DNA templated silver NCs fluorescent probe to target DNAs can act as effective templates for enhancement of AgNCs fluorescence, which can be used to distinguish the deletion of BRCA1 due to different fluorescence intensities. Under the optimal conditions, the fluorescence intensity of the DNA-AgNCs at emission peaks around 440 nm (upon excitation at 350 nm) increased with the increasing deletion type within a dynamic range from 1.0 × 10-10 to 2.4 × 10-6 M with a detection limit (LOD) of 6.4 × 10-11 M. In this sensing system, the normal type shows no significant fluorescence; on the other hand, the deletion type emits higher fluorescence than normal type. Using this nanobiosensor, we successfully determined mutation using the non-amplified genomic DNAs that were isolated from the BC cell line.

Properties of Neurons in External Globus Pallidus Can Support Optimal Action Selection

PubMed Central

Bogacz, Rafal; Martin Moraud, Eduardo; Abdi, Azzedine; Magill, Peter J.; Baufreton, Jérôme

2016-01-01

The external globus pallidus (GPe) is a key nucleus within basal ganglia circuits that are thought to be involved in action selection. A class of computational models assumes that, during action selection, the basal ganglia compute for all actions available in a given context the probabilities that they should be selected. These models suggest that a network of GPe and subthalamic nucleus (STN) neurons computes the normalization term in Bayes’ equation. In order to perform such computation, the GPe needs to send feedback to the STN equal to a particular function of the activity of STN neurons. However, the complex form of this function makes it unlikely that individual GPe neurons, or even a single GPe cell type, could compute it. Here, we demonstrate how this function could be computed within a network containing two types of GABAergic GPe projection neuron, so-called ‘prototypic’ and ‘arkypallidal’ neurons, that have different response properties in vivo and distinct connections. We compare our model predictions with the experimentally-reported connectivity and input-output functions (f-I curves) of the two populations of GPe neurons. We show that, together, these dichotomous cell types fulfil the requirements necessary to compute the function needed for optimal action selection. We conclude that, by virtue of their distinct response properties and connectivities, a network of arkypallidal and prototypic GPe neurons comprises a neural substrate capable of supporting the computation of the posterior probabilities of actions. PMID:27389780

Sensitivity of human embryonic stem cells to different conditions during cryopreservation.

PubMed

Xu, Yanqing; Zhang, Liang; Xu, Jiandong; Wei, Yuping; Xu, Xia

2015-12-01

Low cell recovery rate of human embryonic stem cells (hESCs) resulting from cryopreservation damages leads to the difficulty in their successful commercialization of clinical applications. Hence in this study, sensitivity of human embryonic stem cells (hESCs) to different cooling rates, ice seeding and cryoprotective agent (CPA) types was compared and cell viability and recovery after cryopreservation under different cooling conditions were assessed. Both extracellular and intracellular ice formation were observed. Reactive oxidative species (ROS) accumulation of hESCs was determined. Cryopreservation of hESCs at 1 °C/min with the ice seeding and at the theoretically predicted optimal cooling rate (TPOCR) led to lower level of intracellular ROS, and prevented irregular and big ice clump formation compared with cryopreservation at 1 °C/min. This strategy further resulted in a significant increase in the hESC recovery when glycerol and 1,2-propanediol were used as the CPAs, but no increase for Me2SO. hESCs after cryopreservation under all the tested conditions still maintained their pluripotency. Our results provide guidance for improving the hESC cryopreservation recovery through the combination of CPA type, cooling rate and ice seeding. Copyright © 2015 Elsevier Inc. All rights reserved.

A High-Performance Sodium-Ion Full Cell with a Layered Oxide Cathode and a Phosphorous-Based Composite Anode

DOE PAGES

Oh, Seung-Min; Oh, Pilgun; Kim, Sang-Ok; ...

2016-12-29

A low-cost sodium-ion full cell with a O3-type layered Na[Cu 0.2(Fe 1/3Mn2/3) 0.8]O 2 cathode and an alloy-type P-TiP2-C anode is presented. The cathode is synthesized by an oxalate coprecipitation method and optimized cathodes shows a high specific capacity of 135 mAh g -1 at 0.1C rate with a high rate capability of 90 mAh g-1 at 1C rate and 70 mAh g -1 at 2C rate with good cyclability. The full cell exhibits better capacity retention than the half cell with the cathode due to the elimination of the degradation caused by sodium-metal anode. The dramatically enhanced electrochemical performancemore » of the Na[Cu 0.2(Fe 1/3Mn 2/3) 0.8]O 2 / P-TiP 2-C full cell compared to that of the sample with no Cu is attributed to the structural stabilization imparted by Cu by suppressing the phase change from the O3 structure to the P3 structure during cycling.« less

Efficient and Stable Photovoltaic Characteristics of Quasi-Solid State DSSC using Polymer Gel Electrolyte Based on Ionic Liquid in Organosiloxane Polymer Gels

NASA Astrophysics Data System (ADS)

Pujiarti, H.; Arsyad, W. S.; Shobih; Muliani, L.; Hidayat, R.

2018-04-01

Dye-Sensitized Solar Cell (DSSC) is still one of the promising solar cell types among the third generation of solar cells because of easiness of fabrication and variety of available materials. In this type of solar cell, the electrolyte is one of the important components for regenerating excited dyes and transporting electric charge carriers to the counter electrode. Indeed, the power conversion efficiency of DSSC can be then significantly affected by the chemical and physical properties of the electrolyte. The simplest electrolyte system of an I-/I3 - redox couple in an organic solvent, however, has some drawbacks due to corrosive properties, volatile and leakage problem. Use of solid phase or gel phase electrolyte may overcome those problems, but it is often considered to suppress the efficiency due to low ion diffusion. Here, we report the photovoltaic characteristics of DSSC using polymer gel electrolyte (PGE), which is composed of ionic liquid and an organosiloxane polymer gel. The better cell performance with power conversion efficiency of about 6% has been obtained by optimizing the mesoporous size of the TiO2 layer and the PGE viscosity.

Intermittent Ca2+ signals mediated by Orai1 regulate basal T cell motility

PubMed Central

Greenberg, Milton L; Jairaman, Amit; Akunwafo, Chijioke; Leverrier, Sabrina; Yu, Ying; Parker, Ian; Dynes, Joseph L

2017-01-01

Ca2+ influx through Orai1 channels is crucial for several T cell functions, but a role in regulating basal cellular motility has not been described. Here, we show that inhibition of Orai1 channel activity increases average cell velocities by reducing the frequency of pauses in human T cells migrating through confined spaces, even in the absence of extrinsic cell contacts or antigen recognition. Utilizing a novel ratiometric genetically encoded cytosolic Ca2+ indicator, Salsa6f, which permits real-time monitoring of cytosolic Ca2+ along with cell motility, we show that spontaneous pauses during T cell motility in vitro and in vivo coincide with episodes of cytosolic Ca2+ signaling. Furthermore, lymph node T cells exhibited two types of spontaneous Ca2+ transients: short-duration ‘sparkles’ and longer duration global signals. Our results demonstrate that spontaneous and self-peptide MHC-dependent activation of Orai1 ensures random walk behavior in T cells to optimize immune surveillance. PMID:29239723

Fabrication of p-type CuO thin films using chemical bath deposition technique and their solar cell applications with Si nanowires

NASA Astrophysics Data System (ADS)

Akgul, Funda Aksoy; Akgul, Guvenc

2017-02-01

Recently, CuO has attracted much interest owing to its suitable material properties, inexpensive fabrication cost and potential applications for optoelectronic devices. In this study, CuO thin films were deposited on glass substrates using chemical bath deposition technique and post-deposition annealing effect on the properties of the prepared samples were investigated. p-n heterojunction solar cells were then constructed by coating of p-type CuO films onto the vertically well-aligned n-type Si nanowires synthesized through MACE method. Photovoltaic performance of the fabricated devices were determined with current-voltage (I-V) measurements under AM 1.5 G illumination. The optimal short-circuit current density, open-circuit voltage, fill factor and power conversion efficiency were found to be 3.2 mA/cm-2, 337 mV, 37.9 and 0.45%, respectively. The observed performance clearly indicates that the investigated device structure could be a promising candidate for high-performance low-cost new-generation photovoltaic diodes.

XanoMatrix surfaces as scaffolds for mesenchymal stem cell culture and growth

PubMed Central

Bhardwaj, Garima; Webster, Thomas J

2016-01-01

Stem cells are being widely investigated for a wide variety of applications in tissue engineering due to their ability to differentiate into a number of cells such as neurons, osteoblasts, and fibroblasts. This ability of stem cells to differentiate into different types of cells is greatly based on mechanical and chemical cues received from their three-dimensional environments. All organs are formed by a number of cells linked together via an extracellular matrix (ECM). The ECM is a complex network of proteins and carbohydrates, which occupies intercellular spaces and regulates cellular activity by controlling cell adhesion, migration, proliferation, and differentiation. The ECM is composed of two main types of macromolecules, namely, polysaccharide glycosaminoglycans, which are covalently attached to proteins in the form of proteoglycans and fibrous proteins belonging to two functional groups, structural (collagen and elastin) and adhesive (fibronectin, laminin, vitronectin, etc). Tissue engineering is a multidisciplinary field that aims to develop biomimetic scaffolds that emulate properties of the ECM to help repair or regenerate diseased or damaged tissue. This study introduces one of these matrices, XanoMatrix, as an optimal scaffold for tissue engineering applications, in particular, for stem cell research, based on its composition, nanofibrous structure, and porosity. Results of this study suggest that XanoMatrix scaffolds are promising for stem cell tissue engineering applications and as improved cell culture inserts for studying stem cell functions (compared to traditional Corning and Falcon cell culture plates) and, thus, should be further studied. PMID:27354795

Dendritic cells for active immunotherapy: optimizing design and manufacture in order to develop commercially and clinically viable products.

PubMed

Nicolette, C A; Healey, D; Tcherepanova, I; Whelton, P; Monesmith, T; Coombs, L; Finke, L H; Whiteside, T; Miesowicz, F

2007-09-27

Dendritic cell (DC) active immunotherapy is potentially efficacious in a broad array of malignant disease settings. However, challenges remain in optimizing DC-based therapy for maximum clinical efficacy within manufacturing processes that permit quality control and scale-up of consistent products. In this review we discuss the critical issues that must be addressed in order to optimize DC-based product design and manufacture, and highlight the DC based platforms currently addressing these issues. Variables in DC-based product design include the type of antigenic payload used, DC maturation steps and activation processes, and functional assays. Issues to consider in development include: (a) minimizing the invasiveness of patient biological material collection; (b) minimizing handling and manipulations of tissue at the clinical site; (c) centralized product manufacturing and standardized processing and capacity for commercial-scale production; (d) rapid product release turnaround time; (e) the ability to manufacture sufficient product from limited starting material; and (f) standardized release criteria for DC phenotype and function. Improvements in the design and manufacture of DC products have resulted in a handful of promising leads currently in clinical development.

Microenvironments and Signaling Pathways Regulating Early Dissemination, Dormancy, and Metastasis

DTIC Science & Technology

2016-09-01

all these cell types in all tissues and we have used intravital imaging to document intravasation in early cancer lesions (see also partnering PI...report we showed how we optimized a mammary gland imaging window to perform intravital imaging and detect P-TMEM function during early stages of...MECs) assemble primary Tumor Microenvironment of Metastasis structures (P-TMEM) during early dissemination. SA1.1. Objective: Use intravital

Precision Sensing by Two Opposing Gradient Sensors: How Does Escherichia coli Find its Preferred pH Level?

PubMed Central

Hu, Bo; Tu, Yuhai

2013-01-01

It is essential for bacteria to find optimal conditions for their growth and survival. The optimal levels of certain environmental factors (such as pH and temperature) often correspond to some intermediate points of the respective gradients. This requires the ability of bacteria to navigate from both directions toward the optimum location and is distinct from the conventional unidirectional chemotactic strategy. Remarkably, Escherichia coli cells can perform such a precision sensing task in pH taxis by using the same chemotaxis machinery, but with opposite pH responses from two different chemoreceptors (Tar and Tsr). To understand bacterial pH sensing, we developed an Ising-type model for a mixed cluster of opposing receptors based on the push-pull mechanism. Our model can quantitatively explain experimental observations in pH taxis for various mutants and wild-type cells. We show how the preferred pH level depends on the relative abundance of the competing sensors and how the sensory activity regulates the behavioral response. Our model allows us to make quantitative predictions on signal integration of pH and chemoattractant stimuli. Our study reveals two general conditions and a robust push-pull scheme for precision sensing, which should be applicable in other adaptive sensory systems with opposing gradient sensors. PMID:23823247

Oxygen requirement for denitrification by the fungus Fusarium oxysporum.

PubMed

Zhou, Z; Takaya, N; Sakairi, M A; Shoun, H

2001-01-01

The effects of dioxygen (O2) on the denitrification activity of the fungus Fusarium oxysporum MT-811 in fed-batch culture in a stirred jar fermentor were examined. The results revealed that fungal denitrifying activity requires a minimal amount of O2 for induction, which is repressed by excess O2. The optimal O2 supply differed between the denitrification substrates : 690 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrate (NO3-) and about 250 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrite (NO2-). The reduction of NO3- required more O2 than that of NO2- . With an optimal O2 supply, 80% and 52% of nitrogen atoms in NO3- and NO2-, respectively, were recovered as the denitrification product N2O. These features of F. oxysporum differ from those of bacterial denitrifiers that work exclusively under anoxic conditions. The denitrification activity of F. oxysporum MT-811 mutants with impaired NO3- assimilation was about double that of the wild-type strain, suggesting competition for the substrate between assimilatory and dissimilatory types of NO3- reduction. These results showed that denitrification by F. oxysporum has unique features, namely, a minimal O2 requirement and competition with assimilatory NO3-.

Carrying photosynthesis genes increases ecological fitness of cyanophage in silico.

PubMed

Hellweger, Ferdi L

2009-06-01

Several viruses infecting marine cyanobacteria carry photosynthesis genes (e.g. psbA, hli) that are expressed, yield proteins (D1, HLIP) and help maintain the cell's photosynthesis apparatus during the latent period. This increases energy and speeds up virus production, allowing for a reduced latent period (a fitness benefit), but it also increases the DNA size, which slows down new virus production and reduces burst size (a fitness cost). How do these genes affect the net ecological fitness of the virus? Here, this question is explored using a combined systems biology and systems ecology ('systems bioecology') approach. A novel agent-based model simulates individual cyanobacteria cells and virus particles, each with their own genes, transcripts, proteins and other properties. The effect of D1 and HLIP proteins is explicitly considered using a mechanistic photosynthesis component. The model is calibrated to the available database for Prochlorococcus ecotype MED4 and podovirus P-SSP7. Laboratory- and field-scale in silico survival, competition and evolution (gene packaging error) experiments with wild type and genetically engineered viruses are performed to develop vertical survival and fitness profiles, and to determine the optimal gene content. The results suggest that photosynthesis genes are nonessential, increase fitness in a manner correlated with irradiance, and that the wild type has an optimal gene content.

Monitoring Cell Proliferation by Dye Dilution: Considerations for Probe Selection

PubMed Central

Tario, Joseph D.; Conway, Alexis N.; Muirhead, Katharine A.; Wallace, Paul K.

2018-01-01

In the third edition of this series, we described protocols for labeling cell populations with tracking dyes, and addressed issues to be considered when combining two different tracking dyes with other phenotypic and viability probes for the assessment of cytotoxic effector activity and regulatory T cell functions. We summarized key characteristics of and differences between general protein and membrane labeling dyes, discussed determination of optimal staining concentrations, and provided detailed labeling protocols for both dye types. Examples of the advantages of two color cell tracking were provided in the form of protocols for: (a) independent enumeration of viable effector and target cells in a direct cytotoxicity assay; and (b) an in vitro suppression assay for simultaneous proliferation monitoring of effector and regulatory T cells. The number of commercially available fluorescent cell tracking dyes has expanded significantly since the last edition, with new suppliers and/or new spectral properties being added at least annually. In this fourth edition, we describe evaluations to be performed by the supplier and/or user when characterizing a new cell tracking dye and by the user when selecting one for use in multicolor proliferation monitoring. These include methods for: Assessment of the dye’s spectral profile on the laboratory’s flow cytometer(s) to optimize compatibility with other employed fluorochromes and minimize compensation problems;Evaluating the effect of labeling on cell growth rate;Testing the fidelity with which dye dilution reports cell division;Determining the maximum number of generations to be included when using dye dilution profiles to estimate fold population expansion or frequency of responder cells; andVerifying that relevant cell functions (e.g., effector activity) remain unaltered by tracking dye labeling. PMID:29071683

Deformation simulation of cells seeded on a collagen-GAG scaffold in a flow perfusion bioreactor using a sequential 3D CFD-elastostatics model.

PubMed

Jungreuthmayer, C; Jaasma, M J; Al-Munajjed, A A; Zanghellini, J; Kelly, D J; O'Brien, F J

2009-05-01

Tissue-engineered bone shows promise in meeting the huge demand for bone grafts caused by up to 4 million bone replacement procedures per year, worldwide. State-of-the-art bone tissue engineering strategies use flow perfusion bioreactors to apply biophysical stimuli to cells seeded on scaffolds and to grow tissue suitable for implantation into the patient's body. The aim of this study was to quantify the deformation of cells seeded on a collagen-GAG scaffold which was perfused by culture medium inside a flow perfusion bioreactor. Using a microCT scan of an unseeded collagen-GAG scaffold, a sequential 3D CFD-deformation model was developed. The wall shear stress and the hydrostatic wall pressure acting on the cells were computed through the use of a CFD simulation and fed into a linear elastostatics model in order to calculate the deformation of the cells. The model used numerically seeded cells of two common morphologies where cells are either attached flatly on the scaffold wall or bridging two struts of the scaffold. Our study showed that the displacement of the cells is primarily determined by the cell morphology. Although cells of both attachment profiles were subjected to the same mechanical load, cells bridging two struts experienced a deformation up to 500 times higher than cells only attached to one strut. As the scaffold's pore size determines both the mechanical load and the type of attachment, the design of an optimal scaffold must take into account the interplay of these two features and requires a design process that optimizes both parameters at the same time.

mTOR Complex Signaling through the SEMA4A-Plexin B2 Axis Is Required for Optimal Activation and Differentiation of CD8+ T Cells.

PubMed

Ito, Daisuke; Nojima, Satoshi; Nishide, Masayuki; Okuno, Tatsusada; Takamatsu, Hyota; Kang, Sujin; Kimura, Tetsuya; Yoshida, Yuji; Morimoto, Keiko; Maeda, Yohei; Hosokawa, Takashi; Toyofuku, Toshihiko; Ohshima, Jun; Kamimura, Daisuke; Yamamoto, Masahiro; Murakami, Masaaki; Morii, Eiichi; Rakugi, Hiromi; Isaka, Yoshitaka; Kumanogoh, Atsushi

2015-08-01

Mammalian target of rapamycin (mTOR) plays crucial roles in activation and differentiation of diverse types of immune cells. Although several lines of evidence have demonstrated the importance of mTOR-mediated signals in CD4(+) T cell responses, the involvement of mTOR in CD8(+) T cell responses is not fully understood. In this study, we show that a class IV semaphorin, SEMA4A, regulates CD8(+) T cell activation and differentiation through activation of mTOR complex (mTORC) 1. SEMA4A(-/-) CD8(+) T cells exhibited impairments in production of IFN-γ and TNF-α and induction of the effector molecules granzyme B, perforin, and FAS-L. Upon infection with OVA-expressing Listeria monocytogenes, pathogen-specific effector CD8(+) T cell responses were significantly impaired in SEMA4A(-/-) mice. Furthermore, SEMA4A(-/-) CD8(+) T cells exhibited reduced mTORC1 activity and elevated mTORC2 activity, suggesting that SEMA4A is required for optimal activation of mTORC1 in CD8(+) T cells. IFN-γ production and mTORC1 activity in SEMA4A(-/-) CD8(+) T cells were restored by administration of recombinant Sema4A protein. In addition, we show that plexin B2 is a functional receptor of SEMA4A in CD8(+) T cells. Collectively, these results not only demonstrate the role of SEMA4A in CD8(+) T cells, but also reveal a novel link between a semaphorin and mTOR signaling. Copyright © 2015 by The American Association of Immunologists, Inc.

Design and optimization of a tissue-engineered bone graft substitute

NASA Astrophysics Data System (ADS)

Shimko, Daniel Andrew

2004-12-01

In 2000, 3.1 million surgical procedures on the musculoskeletal system were reported in the United States. For many of these cases, bone grafting was essential for successful fracture stabilization. Current techniques use intact bone obtained either from the patient (autograft) or a cadaver (allograft) to repair large defects, however, neither source is optimal. Allografts suffer integration problems, and for autografts, the tissue supply is limited. Because of these shortcomings, and the high demand for graft tissues, alternatives are being explored. To successfully engineer a bone graft replacement, one must employ a three pronged research approach, addressing (1) the cells that will inhabit the new tissue, (2) the culture environment that these cells will be exposed to, and (3) the scaffold in which these cells will reside. The work herein examines each of these three aspects in great detail. Both adult and embryonic stem cells (ESCs) were considered for the tissue-engineered bone graft. Both exhibited desirable qualities, however, neither were optimal in all categories examined. In the end, the possibility of teratoma formation and ethical issues surrounding ESCs, made the use of adult marrow-derived stem cells in the remaining experiments obligatory. In subsequent experiments, the adult stem cells' ability to form bone was optimized. Basic fibroblast growth factor, fetal bovine serum, and extracellular calcium supplementation studies were all performed. Ultimately, adult stem cells cultured in alpha-MEM supplemented with 10% fetal bovine serum, 10mM beta-glycerophosphate, 10nM dexamethasone, 50mug/ml ascorbic acid, 1%(v/v) antibiotic/antimycotic, and 10.4mM CaCl2 performed the best, producing nearly four times more mineral than any other medium formulation. Several scaffolds were then investigated including those fabricated from poly(alpha-hydroxy esters), tantalum, and poly-methylmethacrylate. In the final study, the most appealing cell type, medium formulation, and scaffold material from all preceding studies were combined and a tissue-engineered bone graft was fabricated. The graft was exposed to long-term in vitro culture, and then mechanically evaluated to determine its clinical potential. The studies contained herein constitute the first steps in the conception and development of a viable tissue-engineered bone graft substitute and establish a solid scientific foundation for future in vivo experimentation utilizing this design.

Interleukin 6 Influences Germinal Center Development and Antibody Production via a Contribution of C3 Complement Component

PubMed Central

Kopf, Manfred; Herren, Suzanne; Wiles, Michael V.; Pepys, Mark B.; Kosco-Vilbois, Marie H.

1998-01-01

Mice rendered deficient for interleukin (IL) 6 by gene targeting were evaluated for their response to T cell–dependent antigens. Antigen-specific immunoglobulin (Ig)M levels were unaffected whereas all IgG isotypes showed varying degrees of alteration. Germinal center reactions occurred but remained physically smaller in comparison to those in the wild-type mice. This concurred with the observations that molecules involved in initial signaling events leading to germinal center formation were not altered (e.g., B7.2, CD40 and tumor necrosis factor R1). T cell priming was not impaired nor was a gross imbalance of T helper cell (Th) 1 versus Th2 cytokines observed. However, B7.1 molecules, absent from wild-type counterparts, were detected on germinal center B cells isolated from the deficient mice suggesting a modification of costimulatory signaling. A second alteration involved impaired de novo synthesis of C3 both in serum and germinal center cells from IL-6–deficient mice. Indeed, C3 provided an essential stimulatory signal for wild-type germinal center cells as both monoclonal antibodies that interrupted C3-CD21 interactions and sheep anti–mouse C3 antibodies caused a significant decrease in antigen-specific antibody production. In addition, germinal center cells isolated from C3–deficient mice produced a similar defect in isotype production. Low density cells with dendritic morphology were the local source of IL-6 and not the germinal center lymphocytes. Adding IL-6 in vitro to IL-6–deficient germinal center cells stimulated cell cycle progression and increased levels of antibody production. These findings reveal that the germinal center produces and uses molecules of the innate immune system, evolutionarily pirating them in order to optimally generate high affinity antibody responses. PMID:9815267

Comparative cell-specific transcriptomics reveals differentiation of C4 photosynthesis pathways in switchgrass and other C4 lineages

PubMed Central

Rao, Xiaolan; Lu, Nan; Li, Guifen; Nakashima, Jin; Tang, Yuhong; Dixon, Richard A.

2016-01-01

Almost all C4 plants require the co-ordination of the adjacent and fully differentiated cell types, mesophyll (M) and bundle sheath (BS). The C4 photosynthetic pathway operates through two distinct subtypes based on how malate is decarboxylated in BS cells; through NAD-malic enzyme (NAD-ME) or NADP-malic enzyme (NADP-ME). The diverse or unique cell-specific molecular features of M and BS cells from separate C4 subtypes of independent lineages remain to be determined. We here provide an M/BS cell type-specific transcriptome data set from the monocot NAD-ME subtype switchgrass (Panicum virgatum). A comparative transcriptomics approach was then applied to compare the M/BS mRNA profiles of switchgrass, monocot NADP-ME subtype C4 plants maize and Setaria viridis, and dicot NAD-ME subtype Cleome gynandra. We evaluated the convergence in the transcript abundance of core components in C4 photosynthesis and transcription factors to establish Kranz anatomy, as well as gene distribution of biological functions, in these four independent C4 lineages. We also estimated the divergence between NAD-ME and NADP-ME subtypes of C4 photosynthesis in the two cell types within C4 species, including differences in genes encoding decarboxylating enzymes, aminotransferases, and metabolite transporters, and differences in the cell-specific functional enrichment of RNA regulation and protein biogenesis/homeostasis. We suggest that C4 plants of independent lineages in both monocots and dicots underwent convergent evolution to establish C4 photosynthesis, while distinct C4 subtypes also underwent divergent processes for the optimization of M and BS cell co-ordination. The comprehensive data sets in our study provide a basis for further research on evolution of C4 species. PMID:26896851

Three-Dimensional Cell Behavior in Microgels

NASA Astrophysics Data System (ADS)

Bhattacharjee, Tapomoy; Palmer, Glyn; Ghivizzani, Steven; Keselowsky, Benjamin; Sawyer, W. Gregory; Angelini, Thomas

The number of dimensions in which particles can freely move strongly influences the collective behavior that emerges from their individual fluctuations. Thus, in 2D systems of cells in petri-dishes, our growing understanding of collective migration may be insufficient to explain cell behavior in 3D tissues. To study cell behavior in 3D, polymer scaffolds are used. Contemporary designs of 3D cell growth scaffolds enable cell migration and proliferative expansion by incorporating of degradable motifs. Matrix degradation creates space for cells to move and proliferate. However, different cell types and experimental conditions require the design of different scaffolds to optimize degradation with specific cell behaviors. By contrast, liquid like solids made from packed microgels can yield under cell generated stresses, allowing for cell motion without the need for scaffold degradation. Moreover, the use of microgels as 3D culture media allows arranging cells in arbitrary structures, harvesting cells, and delivering drugs and nutrients. Preliminary data describing cell behavior in 3D microgel culture will be presented. This material is based on work supported by the National Science Foundation under Grant No. DMR-1352043.

Geometry of the Gene Expression Space of Individual Cells

PubMed Central

Korem, Yael; Szekely, Pablo; Hart, Yuval; Sheftel, Hila; Hausser, Jean; Mayo, Avi; Rothenberg, Michael E.; Kalisky, Tomer; Alon, Uri

2015-01-01

There is a revolution in the ability to analyze gene expression of single cells in a tissue. To understand this data we must comprehend how cells are distributed in a high-dimensional gene expression space. One open question is whether cell types form discrete clusters or whether gene expression forms a continuum of states. If such a continuum exists, what is its geometry? Recent theory on evolutionary trade-offs suggests that cells that need to perform multiple tasks are arranged in a polygon or polyhedron (line, triangle, tetrahedron and so on, generally called polytopes) in gene expression space, whose vertices are the expression profiles optimal for each task. Here, we analyze single-cell data from human and mouse tissues profiled using a variety of single-cell technologies. We fit the data to shapes with different numbers of vertices, compute their statistical significance, and infer their tasks. We find cases in which single cells fill out a continuum of expression states within a polyhedron. This occurs in intestinal progenitor cells, which fill out a tetrahedron in gene expression space. The four vertices of this tetrahedron are each enriched with genes for a specific task related to stemness and early differentiation. A polyhedral continuum of states is also found in spleen dendritic cells, known to perform multiple immune tasks: cells fill out a tetrahedron whose vertices correspond to key tasks related to maturation, pathogen sensing and communication with lymphocytes. A mixture of continuum-like distributions and discrete clusters is found in other cell types, including bone marrow and differentiated intestinal crypt cells. This approach can be used to understand the geometry and biological tasks of a wide range of single-cell datasets. The present results suggest that the concept of cell type may be expanded. In addition to discreet clusters in gene-expression space, we suggest a new possibility: a continuum of states within a polyhedron, in which the vertices represent specialists at key tasks. PMID:26161936

Hairy Root Transformation Using Agrobacterium rhizogenes as a Tool for Exploring Cell Type-Specific Gene Expression and Function Using Tomato as a Model1[W][OPEN

PubMed Central

Ron, Mily; Kajala, Kaisa; Pauluzzi, Germain; Wang, Dongxue; Reynoso, Mauricio A.; Zumstein, Kristina; Garcha, Jasmine; Winte, Sonja; Masson, Helen; Inagaki, Soichi; Federici, Fernán; Sinha, Neelima; Deal, Roger B.; Bailey-Serres, Julia; Brady, Siobhan M.

2014-01-01

Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type- and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomato-optimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato. PMID:24868032

Electronic structure, defect formation energy, and photovoltaic properties of wurtzite-derived CuGaO2

NASA Astrophysics Data System (ADS)

Okumura, H.; Sato, K.; Kakeshita, T.

2018-04-01

Wurtzite-derived CuGaO2 (β-CuGaO2) is a recently synthesized oxide and expected as a candidate material for photovoltaic solar cells. In this paper, we propose computational material design concerning β-CuGaO2 based on the first-principles calculations. We perform hybrid calculations by using the VASP code. It is predicted that β-CuGaO2 has a direct bandgap (Eg = 1.56 eV), which is nearly optimal for high efficiency solar cells. The calculated formation energy of Cu vacancy (VCu) is very small and can be negative depending on the Fermi level. This result reasonably explains the observed p-type conduction in this material. As for the n-type doping, Cd doping could be suitable; however, VCu formation needs to be repressed in order to realize n-type β-CuGaO2. It is also shown that halogen impurities are not suitable for n-type β-CuGaO2 because of their large formation energy. Band alignment between β-CuGaO2 and ZnO is predicted to be type-II, leading to a suggestion of photovoltaic device based on the heterojunction.

The fluctuation of blood glucose, insulin and glucagon concentrations before and after insulin therapy in type 1 diabetes

NASA Astrophysics Data System (ADS)

Arif, Idam; Nasir, Zulfa

2015-09-01

A dynamical-systems model of plasma glucose, insulin and glucagon concentrations has been developed to investigate the effects of insulin therapy on blood glucose, insulin and glucagon regulations in type 1 diabetic patients. Simulation results show that the normal regulation of blood glucose concentration depends on insulin and glucagon concentrations. On type 1 diabetic case, the role of insulin on regulating blood glucose is not optimal because of the destruction of β cells in pancreas. These β cells destructions cause hyperglycemic episode affecting the whole body metabolism. To get over this, type 1 diabetic patients need insulin therapy to control the blood glucose level. This research has been done by using rapid acting insulin (lispro), long-acting insulin (glargine) and the combination between them to know the effects of insulin therapy on blood glucose, insulin and glucagon concentrations. Simulation results show that these different types of insulin have different effects on blood glucose concentration. Insulin therapy using lispro shows better blood glucose control after consumption of meals. Glargin gives better blood glucose control between meals and during sleep. Combination between lispro and glargine shows better glycemic control for whole day blood glucose level.

Beam-energy-spread minimization using cell-timing optimization

NASA Astrophysics Data System (ADS)

Rose, C. R.; Ekdahl, C.; Schulze, M.

2012-04-01

Beam energy spread, and related beam motion, increase the difficulty in tuning for multipulse radiographic experiments at the dual-axis radiographic hydrodynamic test facility’s axis-II linear induction accelerator (LIA). In this article, we describe an optimization method to reduce the energy spread by adjusting the timing of the cell voltages (both unloaded and loaded), either advancing or retarding, such that the injector voltage and summed cell voltages in the LIA result in a flatter energy profile. We developed a nonlinear optimization routine which accepts as inputs the 74 cell-voltage, injector voltage, and beam current waveforms. It optimizes cell timing per user-selected groups of cells and outputs timing adjustments, one for each of the selected groups. To verify the theory, we acquired and present data for both unloaded and loaded cell-timing optimizations. For the unloaded cells, the preoptimization baseline energy spread was reduced by 34% and 31% for two shots as compared to baseline. For the loaded-cell case, the measured energy spread was reduced by 49% compared to baseline.

High-throughput autofluorescence flow cytometry of breast cancer metabolism (Conference Presentation)

NASA Astrophysics Data System (ADS)

Shah, Amy T.; Cannon, Taylor M.; Higginbotham, Jim N.; Skala, Melissa C.

2016-02-01

Tumor heterogeneity poses challenges for devising optimal treatment regimens for cancer patients. In particular, subpopulations of cells can escape treatment and cause relapse. There is a need for methods to characterize tumor heterogeneity of treatment response. Cell metabolism is altered in cancer (Warburg effect), and cells use the autofluorescent cofactor NADH in numerous metabolic reactions. Previous studies have shown that microscopy measurements of NADH autofluorescence are sensitive to treatment response in breast cancer, and these techniques typically assess hundreds of cells per group. An alternative approach is flow cytometry, which measures fluorescence on a single-cell level and is attractive for characterizing tumor heterogeneity because it achieves high-throughput analysis and cell sorting in millions of cells per group. Current applications for flow cytometry rely on staining with fluorophores. This study characterizes flow cytometry measurements of NADH autofluorescence in breast cancer cells. Preliminary results indicate flow cytometry of NADH is sensitive to cyanide perturbation, which inhibits oxidative phosphorylation, in nonmalignant MCF10A cells. Additionally, flow cytometry is sensitive to higher NADH intensity for HER2-positive SKBr3 cells compared with triple-negative MDA-MB-231 cells. These results agree with previous microscopy studies. Finally, a mixture of SKBr3 and MDA-MB-231 cells were sorted into each cell type using NADH intensity. Sorted cells were cultured, and microscopy validation showed the expected morphology for each cell type. Ultimately, flow cytometry could be applied to characterize tumor heterogeneity based on treatment response and sort cell subpopulations based on metabolic profile. These achievements could enable individualized treatment strategies and improved patient outcomes.

Polyisocyanopeptide hydrogels: A novel thermo-responsive hydrogel supporting pre-vascularization and the development of organotypic structures.

PubMed

Zimoch, Jakub; Padial, Joan Simó; Klar, Agnes S; Vallmajo-Martin, Queralt; Meuli, Martin; Biedermann, Thomas; Wilson, Christopher J; Rowan, Alan; Reichmann, Ernst

2018-04-01

Molecular and mechanical interactions with the 3D extracellular matrix are essential for cell functions such as survival, proliferation, migration, and differentiation. Thermo-responsive biomimetic polyisocyanopeptide (PIC) hydrogels are promising new candidates for 3D cell, tissue, and organ cultures. This is a synthetic, thermo-responsive and stress-stiffening material synthesized via polymerization of the corresponding monomers using a nickel perchlorate as a catalyst. It can be tailored to meet various demands of cells by modulating its stiffness and through the decoration of the polymer with short GRGDS peptides using copper free click chemistry. These peptides make the hydrogels biocompatible by mimicking the binding sites of certain integrins. This study focuses on the optimization of the PIC polymer properties for efficient cell, tissue and organ development. Screening for the optimal stiffness of the hydrogel and the ideal concentration of the GRGDS ligand conjugated with the polymer, enabled cell proliferation, migration and differentiation of various primary cell types of human origin. We demonstrate that fibroblasts, endothelial cells, adipose-derived stem cells and melanoma cells, do survive, thrive and differentiate in optimized PIC hydrogels. Importantly, these hydrogels support the spontaneous formation of complex structures like blood capillaries in vitro. Additionally, we utilized the thermo-responsive properties of the hydrogels for a rapid and gentle recovery of viable cells. Finally, we show that organotypic structures of human origin grown in PIC hydrogels can be successfully transplanted subcutaneously onto immune-compromised rats, on which they survive and integrate into the surrounding tissue. Molecular and mechanical interactions with the surrounding environment are essential for cell functions. Although 2D culture systems greatly contributed to our understanding of complex biological phenomena, they cannot substitute for crucial interaction that take place in 3D. 3D culture systems aim to overcome limitations of the 2D cultures and answer new questions about cell functions. Thermo-responsive biomimetic polyisocyanopeptide (PIC) hydrogels are promising new candidates for 3D cell, tissue, and organ cultures. They are synthetic and can be tailor to meet certain experimental demands. Additionally, they are characterized by strain-stiffening, a feature crucial for cell behaviour, but rare in hydrogels. Their thermos-responsive properties enable quick recovery of the cells by a simple procedure of lowering the temperature. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Transient responses of phosphoric acid fuel cell power plant system. Ph.D. Thesis

NASA Technical Reports Server (NTRS)

Lu, Cheng-Yi

1983-01-01

An analytical and computerized study of the steady state and transient response of a phosphoric acid fuel cell (PAFC) system was completed. Parametric studies and sensitivity analyses of the PAFC system's operation were accomplished. Four non-linear dynamic models of the fuel cell stack, reformer, shift converters, and heat exchangers were developed based on nonhomogeneous non-linear partial differential equations, which include the material, component, energy balance, and electrochemical kinetic features. Due to a lack of experimental data for the dynamic response of the components only the steady state results were compared with data from other sources, indicating reasonably good agreement. A steady state simulation of the entire system was developed using, nonlinear ordinary differential equations. The finite difference method and trial-and-error procedures were used to obtain a solution. Using the model, a PAFC system, that was developed under NASA Grant, NCC3-17, was improved through the optimization of the heat exchanger network. Three types of cooling configurations for cell plates were evaluated to obtain the best current density and temperature distributions. The steady state solutions were used as the initial conditions in the dynamic model. The transient response of a simplified PAFC system, which included all of the major components, subjected to a load change was obtained. Due to the length of the computation time for the transient response calculations, analysis on a real-time computer was not possible. A simulation of the real-time calculations was developed on a batch type computer. The transient response characteristics are needed for the optimization of the design and control of the whole PAFC system. All of the models, procedures and simulations were programmed in Fortran and run on IBM 370 computers at Cleveland State University and the NASA Lewis Research Center.

Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid.

PubMed

Qin, Y M; Marttila, M S; Haapalainen, A M; Siivari, K M; Glumoff, T; Hiltunen, J K

1999-10-01

The yeast peroxisomal (3R)-hydroxyacyl-CoA dehydrogenase/2-enoyl-CoA hydratase 2 (multifunctional enzyme type 2; MFE-2) has two N-terminal domains belonging to the short chain alcohol dehydrogenase/reductase superfamily. To investigate the physiological roles of these domains, here called A and B, Saccharomyces cerevisiae fox-2 cells (devoid of Sc MFE-2) were taken as a model system. Gly(16) and Gly(329) of the S. cerevisiae A and B domains, corresponding to Gly(16), which is mutated in the human MFE-2 deficiency, were mutated to serine and cloned into the yeast expression plasmid pYE352. In oleic acid medium, fox-2 cells transformed with pYE352:: ScMFE-2(aDelta) and pYE352::ScMFE-2(bDelta) grew slower than cells transformed with pYE352::ScMFE-2, whereas cells transformed with pYE352::ScMFE-2(aDeltabDelta) failed to grow. Candida tropicalis MFE-2 with a deleted hydratase 2 domain (Ct MFE- 2(h2Delta)) and mutational variants of the A and B domains (Ct MFE- 2(h2DeltaaDelta), Ct MFE- 2(h2DeltabDelta), and Ct MFE- 2(h2DeltaaDeltabDelta)) were overexpressed and characterized. All proteins were dimers with similar secondary structure elements. Both wild type domains were enzymatically active, with the B domain showing the highest activity with short chain and the A domain with medium and long chain (3R)-hydroxyacyl-CoA substrates. The data show that the dehydrogenase domains of yeast MFE-2 have different substrate specificities required to allow the yeast to propagate optimally on fatty acids as the carbon source.

A standardized staining protocol for L1CAM on formalin-fixed, paraffin-embedded tissues using automated platforms.

PubMed

Fogel, Mina; Harari, Ayelet; Müller-Holzner, Elisabeth; Zeimet, Alain G; Moldenhauer, Gerhard; Altevogt, Peter

2014-06-25

The L1 cell adhesion molecule (L1CAM) is overexpressed in many human cancers and can serve as a biomarker for prognosis in most of these cancers (including type I endometrial carcinomas). Here we provide an optimized immunohistochemical staining procedure for a widely used automated platform (VENTANA™), which has recourse to commercially available primary antibody and detection reagents. In parallel, we optimized the staining on a semi-automated BioGenix (i6000) 
immunostainer. These protocols yield good stainings and should represent the basis for a reliable and standardized immunohistochemical detection of L1CAM in a variety of malignancies in different laboratories.

Identification of mutated driver pathways in cancer using a multi-objective optimization model.

PubMed

Zheng, Chun-Hou; Yang, Wu; Chong, Yan-Wen; Xia, Jun-Feng

2016-05-01

New-generation high-throughput technologies, including next-generation sequencing technology, have been extensively applied to solve biological problems. As a result, large cancer genomics projects such as the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium are producing large amount of rich and diverse data in multiple cancer types. The identification of mutated driver genes and driver pathways from these data is a significant challenge. Genome aberrations in cancer cells can be divided into two types: random 'passenger mutation' and functional 'driver mutation'. In this paper, we introduced a Multi-objective Optimization model based on a Genetic Algorithm (MOGA) to solve the maximum weight submatrix problem, which can be employed to identify driver genes and driver pathways promoting cancer proliferation. The maximum weight submatrix problem defined to find mutated driver pathways is based on two specific properties, i.e., high coverage and high exclusivity. The multi-objective optimization model can adjust the trade-off between high coverage and high exclusivity. We proposed an integrative model by combining gene expression data and mutation data to improve the performance of the MOGA algorithm in a biological context. Copyright © 2016 Elsevier Ltd. All rights reserved.

Design and testing of regulatory cassettes for optimal activity in skeletal and cardiac muscles.

PubMed

Himeda, Charis L; Chen, Xiaolan; Hauschka, Stephen D

2011-01-01

Gene therapy for muscular dystrophies requires efficient gene delivery to the striated musculature and specific, high-level expression of the therapeutic gene in a physiologically diverse array of muscles. This can be achieved by the use of recombinant adeno-associated virus vectors in conjunction with muscle-specific regulatory cassettes. We have constructed several generations of regulatory cassettes based on the enhancer and promoter of the muscle creatine kinase gene, some of which include heterologous enhancers and individual elements from other muscle genes. Since the relative importance of many control elements varies among different anatomical muscles, we are aiming to tailor these cassettes for high-level expression in cardiac muscle, and in fast and slow skeletal muscles. With the achievement of efficient intravascular gene delivery to isolated limbs, selected muscle groups, and heart in large animal models, the design of cassettes optimized for activity in different muscle types is now a practical goal. In this protocol, we outline the key steps involved in the design of regulatory cassettes for optimal activity in skeletal and cardiac muscle, and testing in mature muscle fiber cultures. The basic principles described here can also be applied to engineering tissue-specific regulatory cassettes for other cell types.

Reprogramming cells with synthetic proteins

PubMed Central

Yang, Xiaoxiao; Malik, Vikas; Jauch, Ralf

2015-01-01

Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs) has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to “read” genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivo counterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies. PMID:25652623

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies.

PubMed

Feng, Xuesong; Naz, Faiza; Juan, Aster H; Dell'Orso, Stefania; Sartorelli, Vittorio

2018-04-19

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs

PubMed Central

Chen, Yen-Ming; Chen, Li-Hua; Li, Meng-Pei; Li, Hsing-Fen; Higuchi, Akon; Kumar, S. Suresh; Ling, Qing-Dong; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Chang, Yung; Benelli, Giovanni; Murugan, Kadarkarai; Umezawa, Akihiro

2017-01-01

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity, and prepared them in formations of single chain, single chain with joint segment, dual chain with joint segment, and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain, which has a storage modulus of 25 kPa, supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells. PMID:28332572

Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs.

PubMed

Chen, Yen-Ming; Chen, Li-Hua; Li, Meng-Pei; Li, Hsing-Fen; Higuchi, Akon; Kumar, S Suresh; Ling, Qing-Dong; Alarfaj, Abdullah A; Munusamy, Murugan A; Chang, Yung; Benelli, Giovanni; Murugan, Kadarkarai; Umezawa, Akihiro

2017-03-23

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity, and prepared them in formations of single chain, single chain with joint segment, dual chain with joint segment, and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain, which has a storage modulus of 25 kPa, supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells.

Generation of cell type-specific monoclonal antibodies for the planarian and optimization of sample processing for immunolabeling.

PubMed

Forsthoefel, David J; Waters, Forrest A; Newmark, Phillip A

2014-12-21

Efforts to elucidate the cellular and molecular mechanisms of regeneration have required the application of methods to detect specific cell types and tissues in a growing cohort of experimental animal models. For example, in the planarian Schmidtea mediterranea, substantial improvements to nucleic acid hybridization and electron microscopy protocols have facilitated the visualization of regenerative events at the cellular level. By contrast, immunological resources have been slower to emerge. Specifically, the repertoire of antibodies recognizing planarian antigens remains limited, and a more systematic approach is needed to evaluate the effects of processing steps required during sample preparation for immunolabeling. To address these issues and to facilitate studies of planarian digestive system regeneration, we conducted a monoclonal antibody (mAb) screen using phagocytic intestinal cells purified from the digestive tracts of living planarians as immunogens. This approach yielded ten antibodies that recognized intestinal epitopes, as well as markers for the central nervous system, musculature, secretory cells, and epidermis. In order to improve signal intensity and reduce non-specific background for a subset of mAbs, we evaluated the effects of fixation and other steps during sample processing. We found that fixative choice, treatments to remove mucus and bleach pigment, as well as methods for tissue permeabilization and antigen retrieval profoundly influenced labeling by individual antibodies. These experiments led to the development of a step-by-step workflow for determining optimal specimen preparation for labeling whole planarians as well as unbleached histological sections. We generated a collection of monoclonal antibodies recognizing the planarian intestine and other tissues; these antibodies will facilitate studies of planarian tissue morphogenesis. We also developed a protocol for optimizing specimen processing that will accelerate future efforts to generate planarian-specific antibodies, and to extend functional genetic studies of regeneration to post-transcriptional aspects of gene expression, such as protein localization or modification. Our efforts demonstrate the importance of systematically testing multiple approaches to species-specific idiosyncracies, such as mucus removal and pigment bleaching, and may serve as a template for the development of immunological resources in other emerging model organisms.

Suppression of lethal autoimmunity by regulatory T cells with a single TCR specificity

PubMed Central

Hemmers, Saskia; Schizas, Michail; Faire, Mehlika B.; Konopacki, Catherine; Schmidt-Supprian, Marc; Germain, Ronald N.

2017-01-01

The regulatory T cell (T reg cell) T cell receptor (TCR) repertoire is highly diverse and skewed toward recognition of self-antigens. TCR expression by T reg cells is continuously required for maintenance of immune tolerance and for a major part of their characteristic gene expression signature; however, it remains unknown to what degree diverse TCR-mediated interactions with cognate self-antigens are required for these processes. In this study, by experimentally switching the T reg cell TCR repertoire to a single T reg cell TCR, we demonstrate that T reg cell function and gene expression can be partially uncoupled from TCR diversity. An induced switch of the T reg cell TCR repertoire to a random repertoire also preserved, albeit to a limited degree, the ability to suppress lymphadenopathy and T helper cell type 2 activation. At the same time, these perturbations of the T reg cell TCR repertoire led to marked immune cell activation, tissue inflammation, and an ultimately severe autoimmunity, indicating the importance of diversity and specificity for optimal T reg cell function. PMID:28130403

Full space device optimization for solar cells.

PubMed

Baloch, Ahmer A B; Aly, Shahzada P; Hossain, Mohammad I; El-Mellouhi, Fedwa; Tabet, Nouar; Alharbi, Fahhad H

2017-09-20

Advances in computational materials have paved a way to design efficient solar cells by identifying the optimal properties of the device layers. Conventionally, the device optimization has been governed by single or double descriptors for an individual layer; mostly the absorbing layer. However, the performance of the device depends collectively on all the properties of the material and the geometry of each layer in the cell. To address this issue of multi-property optimization and to avoid the paradigm of reoccurring materials in the solar cell field, a full space material-independent optimization approach is developed and presented in this paper. The method is employed to obtain an optimized material data set for maximum efficiency and for targeted functionality for each layer. To ensure the robustness of the method, two cases are studied; namely perovskite solar cells device optimization and cadmium-free CIGS solar cell. The implementation determines the desirable optoelectronic properties of transport mediums and contacts that can maximize the efficiency for both cases. The resulted data sets of material properties can be matched with those in materials databases or by further microscopic material design. Moreover, the presented multi-property optimization framework can be extended to design any solid-state device.

Protons, osmolytes, and fitness of internal milieu for protein function.

PubMed

Somero, G N

1986-08-01

The composition of the intracellular milieu shows striking similarities among widely different species. Only certain values of intracellular pH, values that generally reflect alphastat regulation, and only narrow ranges of inorganic ion concentrations are found in the cytoplasm of the cells of most animals, plants, and microorganisms. In water-stressed organisms only a few types of low-molecular-weight organic molecules (osmolytes) are accumulated. These highly conserved characteristics of the intracellular fluids reflect the need to maintain critical features of macromolecules within narrow ranges optimal for life. For proteins these features include maintaining adequate rates of catalysis, a high level of regulatory responsiveness, and a precise balance between stability and lability of structure (tertiary conformation, subunit assembly, and multiprotein complexes). The optimal values for these functional and structural features of proteins often lie near the midrange of possible values for these properties, and only under specific conditions of intracellular pH, ionic strength, and osmolyte composition are these optimal midrange values conserved. In dormant cells the departure of solution conditions from values that are optimal for protein function and structure may be instrumental in reducing or shutting down metabolic functions. Seen from a broad evolutionary perspective, the evolution of the intracellular milieu is an important complement to macromolecular evolution. In certain instances appropriate modifications of the internal milieu may reduce the need for adaptive amino acid replacements in proteins.

Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines.

PubMed

Kowarz, Eric; Löscher, Denise; Marschalek, Rolf

2015-04-01

Stable gene expression in mammalian cells is a prerequisite for many in vitro and in vivo experiments. However, either the integration of plasmids into mammalian genomes or the use of retro-/lentiviral systems have intrinsic limitations. The use of transposable elements, e.g. the Sleeping Beauty system (SB), circumvents most of these drawbacks (integration sites, size limitations) and allows the quick generation of stable cell lines. The integration process of SB is catalyzed by a transposase and the handling of this gene transfer system is easy, fast and safe. Here, we report our improvements made to the existing SB vector system and present two new vector types for robust constitutive or inducible expression of any gene of interest. Both types are available in 16 variants with different selection marker (puromycin, hygromycin, blasticidin, neomycin) and fluorescent protein expression (GFP, RFP, BFP) to fit most experimental requirements. With this system it is possible to generate cell lines from stable transfected cells quickly and reliably in a medium-throughput setting (three to five days). Cell lines robustly express any gene-of-interest, either constitutively or tightly regulated by doxycycline. This allows many laboratory experiments to speed up generation of data in a rapid and robust manner. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cell wall-bound silicon optimizes ammonium uptake and metabolism in rice cells.

PubMed

Sheng, Huachun; Ma, Jie; Pu, Junbao; Wang, Lijun

2018-05-16

Turgor-driven plant cell growth depends on cell wall structure and mechanics. Strengthening of cell walls on the basis of an association and interaction with silicon (Si) could lead to improved nutrient uptake and optimized growth and metabolism in rice (Oryza sativa). However, the structural basis and physiological mechanisms of nutrient uptake and metabolism optimization under Si assistance remain obscure. Single-cell level biophysical measurements, including in situ non-invasive micro-testing (NMT) of NH4+ ion fluxes, atomic force microscopy (AFM) of cell walls, and electrolyte leakage and membrane potential, as well as whole-cell proteomics using isobaric tags for relative and absolute quantification (iTRAQ), were performed. The altered cell wall structure increases the uptake rate of the main nutrient NH4+ in Si-accumulating cells, whereas the rate is only half in Si-deprived counterparts. Rigid cell walls enhanced by a wall-bound form of Si as the structural basis stabilize cell membranes. This, in turn, optimizes nutrient uptake of the cells in the same growth phase without any requirement for up-regulation of transmembrane ammonium transporters. Optimization of cellular nutrient acquisition strategies can substantially improve performance in terms of growth, metabolism and stress resistance.

Normalization of Tumor Microenvironment by Neem Leaf Glycoprotein Potentiates Effector T Cell Functions and Therapeutically Intervenes in the Growth of Mouse Sarcoma

PubMed Central

Barik, Subhasis; Banerjee, Saptak; Mallick, Atanu; Goswami, Kuntal Kanti; Roy, Soumyabrata; Bose, Anamika; Baral, Rathindranath

2013-01-01

We have observed restriction of the murine sarcoma growth by therapeutic intervention of neem leaf glycoprotein (NLGP). In order to evaluate the mechanism of tumor growth restriction, here, we have analyzed tumor microenvironment (TME) from sarcoma bearing mice with NLGP therapy (NLGP-TME, in comparison to PBS-TME). Analysis of cytokine milieu within TME revealed IL-10, TGFβ, IL-6 rich type 2 characters was switched to type 1 microenvironment with dominance of IFNγ secretion within NLGP-TME. Proportion of CD8+ T cells was increased within NLGP-TME and these T cells were protected from TME-induced anergy by NLGP, as indicated by higher expression of pNFAT and inhibit related downstream signaling. Moreover, low expression of FasR+ cells within CD8+ T cell population denotes prevention from activation induced cell death. Using CFSE as a probe, better migration of T cells was noted within TME from NLGP treated mice than PBS cohort. CD8+ T cells isolated from NLGP-TME exhibited greater cytotoxicity to sarcoma cells in vitro and these cells show higher expression of cytotoxicity related molecules, perforin and granzyme B. Adoptive transfer of NLGP-TME exposed T cells, but not PBS-TME exposed cells in mice, is able to significantly inhibit the growth of sarcoma in vivo. Such tumor growth inhibition by NLGP-TME exposed T cells was not observed when mice were depleted for CD8+ T cells. Accumulated evidences strongly suggest NLGP mediated normalization of TME allows T cells to perform optimally to inhibit the tumor growth. PMID:23785504

Final Report - Stationary and Emerging Market Fuel Cell System Cost Assessment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Contini, Vince; Heinrichs, Mike; George, Paul

The U.S. Department of Energy (DOE) is focused on providing a portfolio of technology solutions to meet energy security challenges of the future. Fuel cells are a part of this portfolio of technology offerings. To help meet these challenges and supplement the understanding of the current research, Battelle has executed a five-year program that evaluated the total system costs and total ownership costs of two technologies: (1) an ~80 °C polymer electrolyte membrane fuel cell (PEMFC) technology and (2) a solid oxide fuel cell (SOFC) technology, operating with hydrogen or reformate for different applications. Previous research conducted by Battelle, andmore » more recently by other research institutes, suggests that fuel cells can offer customers significant fuel and emission savings along with other benefits compared to incumbent alternatives. For this project, Battelle has applied a proven cost assessment approach to assist the DOE Fuel Cell Technologies Program in making decisions regarding research and development, scale-up, and deployment of fuel cell technology. The cost studies and subsequent reports provide accurate projections of current system costs and the cost impact of state-of-the-art technologies in manufacturing, increases in production volume, and changes to system design on system cost and life cycle cost for several near-term and emerging fuel cell markets. The studies also provide information on types of manufacturing processes that must be developed to commercialize fuel cells and also provide insights into the optimization needed for use of off-the-shelf components in fuel cell systems. Battelle’s analysis is intended to help DOE prioritize investments in research and development of components to reduce the costs of fuel cell systems while considering systems optimization.« less

Coherent organization in gene regulation: a study on six networks

NASA Astrophysics Data System (ADS)

Aral, Neşe; Kabakçıoğlu, Alkan

2016-04-01

Structural and dynamical fingerprints of evolutionary optimization in biological networks are still unclear. Here we analyze the dynamics of genetic regulatory networks responsible for the regulation of cell cycle and cell differentiation in three organisms or cell types each, and show that they follow a version of Hebb's rule which we have termed coherence. More precisely, we find that simultaneously expressed genes with a common target are less likely to act antagonistically at the attractors of the regulatory dynamics. We then investigate the dependence of coherence on structural parameters, such as the mean number of inputs per node and the activatory/repressory interaction ratio, as well as on dynamically determined quantities, such as the basin size and the number of expressed genes.

Anti-freezing-protein type III strongly influences the expression of relevant genes in cryopreserved potato shoot tips.

PubMed

Seo, Ji Hyang; Naing, Aung Htay; Jeon, Su Min; Kim, Chang Kil

2018-06-04

AFP improved cryopreservation efficiency of potato (Solanum tuberosum cv. Superior) by regulating transcript levels of CBF1 and DHN1. However, the optimal AFP concentration required for strong induction of the genes was dependent on the type of vitrification solution to which the AFP was added: This finding suggests that AFP increased cryopreservation efficiency by transcriptional regulation of these genes, which might protect plant cell membranes from cold stress during cryopreservation. Despite the availability of many studies reporting the benefits of anti-freeze protein III (AFP III) as a cryoprotectant, the role of AFP III in this process has not been well demonstrated using molecular analysis. In addition, AFP III has not been exploited in the cryopreservation of potato thus far. Therefore, we studied the effects of AFP III on the cryopreservation of potato (Solanum tuberosum cv. Superior). We found that CBF1 and DHN1 genes are low temperature-inducible in potato leaves (S. tuberosum cv. Superior). Transcript levels of these genes expressed in shoot tips cryopreserved with AFP III were higher than those of the controls. However, the optimal AFP III concentration required for strong induction of the genes was dependent on the type of cryoprotection solution to which the AFP III was added: 500 ng/mL worked best for PVS2, while 1500 ng/mL was optimal for LS. Interestingly, the involvement of AFP III in the cryoprotection solutions improved cryopreservation efficiency as compared to the control, and expression levels of the detected genes were associated with cryopreservation efficiency. This finding suggests that AFP III increased cryopreservation efficiency by transcriptional regulation of these genes, which might protect plant cell membranes from cold stress during cryopreservation. Therefore, we expect that our findings will lead to the successful application of AFP III as a potent cryoprotectant in the cryopreservation of rare and commercially important plant germplasms.

Aliicoccus persicus gen. nov., sp. nov., a halophilic member of the Firmicutes isolated from a hypersaline lake.

PubMed

Amoozegar, Mohammad Ali; Bagheri, Maryam; Makhdoumi-Kakhki, Ali; Didari, Maryam; Schumann, Peter; Nikou, Mahdi Moshtaghi; Sánchez-Porro, Cristina; Ventosa, Antonio

2014-06-01

A novel Gram-staining-positive, moderately halophilic bacterium, designated strain A76(T), was isolated from a brine sample of the hypersaline lake Aran-Bidgol in Iran. Cells were strictly aerobic, coccus-shaped, non-motile, non-sporulating, and catalase- and oxidase-positive. Strain A76(T) grew between pH 7.0 and 10.0 (optimal growth at pH 8.0), between 20 and 45 °C (optimal growth at 35 °C) and at salinities of 0.5 to 12.5% (w/v) NaCl (optimal growth at 7.5%, w/v, NaCl). On the basis of 16S rRNA gene sequence analysis, strain A76(T) was shown to belong to the phylum Firmicutes with sequence similarities of 94.1, 93.1 and 91.1%, to the type species of the genera Jeotgalicoccus, Salinicoccus and Nosocomiicoccus, respectively. The DNA G+C content of this new isolate was 38.8 mol%. The major cellular fatty acids of strain A76(T) were anteiso-C(15 : 0) and iso-C(15 : 0), and its polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol, a glycolipid, an unknown lipid and two unknown phospholipids. The isoprenoid quinones were MK-6 (94%), MK-5 (3%) and MK-7 (3%). The amino acid constituents of the cell wall were Lys, Asp, Gly, Glu and Ala. The physiological, biochemical and phylogenetic differences between strain A76(T) and type strains of taxa with validly published names suggest that this strain represents a novel species in a novel genus within the family Staphylococcaceae, for which the name Aliicoccus persicus gen. nov., sp. nov. is proposed. The type strain of Aliicoccus persicus is strain A76(T) ( = CECT 8508(T) = DSM 28306(T) = IBRC-M 10081(T)). © 2014 IUMS.

Single-Cell Sequencing of the Healthy and Diseased Heart Reveals Ckap4 as a New Modulator of Fibroblasts Activation.

PubMed

Gladka, Monika M; Molenaar, Bas; de Ruiter, Hesther; van der Elst, Stefan; Tsui, Hoyee; Versteeg, Danielle; Lacraz, Grègory P A; Huibers, Manon M H; van Oudenaarden, Alexander; van Rooij, Eva

2018-01-31

Background -Genome-wide transcriptome analysis has greatly advanced our understanding of the regulatory networks underlying basic cardiac biology and mechanisms driving disease. However, so far, the resolution of studying gene expression patterns in the adult heart has been limited to the level of extracts from whole tissues. The use of tissue homogenates inherently causes the loss of any information on cellular origin or cell type-specific changes in gene expression. Recent developments in RNA amplification strategies provide a unique opportunity to use small amounts of input RNA for genome-wide sequencing of single cells. Methods -Here, we present a method to obtain high quality RNA from digested cardiac tissue from adult mice for automated single-cell sequencing of both the healthy and diseased heart. Results -After optimization, we were able to perform single-cell sequencing on adult cardiac tissue under both homeostatic conditions and after ischemic injury. Clustering analysis based on differential gene expression unveiled known and novel markers of all main cardiac cell types. Based on differential gene expression we were also able to identify multiple subpopulations within a certain cell type. Furthermore, applying single-cell sequencing on both the healthy and the injured heart indicated the presence of disease-specific cell subpopulations. As such, we identified cytoskeleton associated protein 4 ( Ckap4 ) as a novel marker for activated fibroblasts that positively correlates with known myofibroblast markers in both mouse and human cardiac tissue. Ckap4 inhibition in activated fibroblasts treated with TGFβ triggered a greater increase in the expression of genes related to activated fibroblasts compared to control, suggesting a role of Ckap4 in modulating fibroblast activation in the injured heart. Conclusions -Single-cell sequencing on both the healthy and diseased adult heart allows us to study transcriptomic differences between cardiac cells, as well as cell type-specific changes in gene expression during cardiac disease. This new approach provides a wealth of novel insights into molecular changes that underlie the cellular processes relevant for cardiac biology and pathophysiology. Applying this technology could lead to the discovery of new therapeutic targets relevant for heart disease.

Tendon biomechanics and mechanobiology - a mini-review of basic concepts and recent advancements

PubMed Central

Wang, James H-C.; Guo, Qianping; Li, Bin

2011-01-01

Due to their unique hierarchical structure and composition, tendons possess characteristic biomechanical properties, including high mechanical strength and viscoelasticity, which enable them to carry and transmit mechanical loads (muscular forces) effectively. Tendons are also mechano-responsive by adaptively changing their structure and function in response to altered mechanical loading conditions. In general, mechanical loading at physiological levels is beneficial to tendons, but excessive loading or disuse of tendons is detrimental. This mechano-adaptability is due to the cells present in tendons. Tendon fibroblasts (tenocytes) are the dominant tendon cells responsible for tendon homeostasis and repair. Tendon stem cells (TSCs), which were recently discovered, also play a vital role in tendon maintenance and repair by virtue of their ability to self-renew and differentiate into tenocytes. TSCs may also be responsible for chronic tendon injury, or tendinopathy, by undergoing aberrant differentiation into non-tenocytes in response to excessive mechanical loading. Thus, it is necessary to devise optimal rehabilitation protocols in order to enhance tendon healing while reducing scar tissue formation and tendon adhesions. Moreover, along with scaffolds that can mimic tendon matrix environments and platelet-rich plasma (PRP), which serves as a source of growth factors, TSCs may be the optimal cell type for enhancing repair of injured tendons. PMID:21925835

Refined protocols of tamoxifen injection for inducible DNA recombination in mouse astroglia.

PubMed

Jahn, Hannah M; Kasakow, Carmen V; Helfer, Andreas; Michely, Julian; Verkhratsky, Alexei; Maurer, Hans H; Scheller, Anja; Kirchhoff, Frank

2018-04-12

Inducible DNA recombination of floxed alleles in vivo by liver metabolites of tamoxifen (TAM) is an important tool to study gene functions. Here, we describe protocols for optimal DNA recombination in astrocytes, based on the GLAST-Cre ERT2 /loxP system. In addition, we demonstrate that quantification of genomic recombination allows to determine the proportion of cell types in various brain regions. We analyzed the presence and clearance of TAM and its metabolites (N-desmethyl-tamoxifen, 4-hydroxytamoxifen and endoxifen) in brain and serum of mice by liquid chromatographic-high resolution-tandem mass spectrometry (LC-HR-MS/MS) and assessed optimal injection protocols by quantitative RT-PCR of several floxed target genes (p2ry1, gria1, gabbr1 and Rosa26-tdTomato locus). Maximal recombination could be achieved in cortex and cerebellum by single daily injections for five and three consecutive days, respectively. Furthermore, quantifying the loss of floxed alleles predicted the percentage of GLAST-positive cells (astroglia) per brain region. We found that astrocytes contributed 20 to 30% of the total cell number in cortex, hippocampus, brainstem and optic nerve, while in the cerebellum Bergmann glia, velate astrocytes and white matter astrocytes accounted only for 8% of all cells.

Tendon biomechanics and mechanobiology--a minireview of basic concepts and recent advancements.

PubMed

Wang, James H-C; Guo, Qianping; Li, Bin

2012-01-01

Due to their unique hierarchical structure and composition, tendons possess characteristic biomechanical properties, including high mechanical strength and viscoelasticity, which enable them to carry and transmit mechanical loads (muscular forces) effectively. Tendons are also mechanoresponsive by adaptively changing their structure and function in response to altered mechanical loading conditions. In general, mechanical loading at physiological levels is beneficial to tendons, but excessive loading or disuse of tendons is detrimental. This mechanoadaptability is due to the cells present in tendons. Tendon fibroblasts (tenocytes) are the dominant tendon cells responsible for tendon homeostasis and repair. Tendon stem cells (TSCs), which were recently discovered, also play a vital role in tendon maintenance and repair by virtue of their ability to self-renew and differentiate into tenocytes. TSCs may also be responsible for chronic tendon injury, or tendinopathy, by undergoing aberrant differentiation into nontenocytes in response to excessive mechanical loading. Thus, it is necessary to devise optimal rehabilitation protocols to enhance tendon healing while reducing scar tissue formation and tendon adhesions. Moreover, along with scaffolds that can mimic tendon matrix environments and platelet-rich plasma, which serves as a source of growth factors, TSCs may be the optimal cell type for enhancing repair of injured tendons. Copyright © 2012 Hanley & Belfus. Published by Elsevier Inc. All rights reserved.

Possibility of Exosome-Based Therapeutics and Challenges in Production of Exosomes Eligible for Therapeutic Application.

PubMed

Yamashita, Takuma; Takahashi, Yuki; Takakura, Yoshinobu

2018-01-01

Exosomes are cell-derived vesicles with a diameter 30-120 nm. Exosomes contain endogenous proteins and nucleic acids; delivery of these molecules to exosome-recipient cells causes biological effects. Exosomes derived from some types of cells such as mesenchymal stem cells and dendritic cells have therapeutic potential and may be biocompatible and efficient agents against various disorders such as organ injury. However, there are many challenges for the development of exosome-based therapeutics. In particular, producing exosomal formulations is the major barrier for therapeutic application because of their heterogeneity and low productivity. Development and optimization of producing methods, including methods for isolation and storage of exosome formulations, are required for realizing exosome-based therapeutics. In addition, improvement of therapeutic potential and delivery efficiency of exosomes are important for their therapeutic application. In this review, we summarize current knowledge about therapeutic application of exosomes and discuss some challenges in their successful use.

InGaP Heterojunction Barrier Solar Cells

NASA Technical Reports Server (NTRS)

Welser, Roger E. (Inventor)

2014-01-01

A new solar cell structure called a heterojunction barrier solar cell is described. As with previously reported quantum-well and quantum-dot solar cell structures, a layer of narrow band-gap material, such as GaAs or indium-rich InGaP, is inserted into the depletion region of a wide band-gap PN junction. Rather than being thin, however, the layer of narrow band-gap material is about 400-430 nm wide and forms a single, ultrawide well in the depletion region. Thin (e.g., 20-50 nm), wide band-gap InGaP barrier layers in the depletion region reduce the diode dark current. Engineering the electric field and barrier profile of the absorber layer, barrier layer, and p-type layer of the PN junction maximizes photogenerated carrier escape. This new twist on nanostructured solar cell design allows the separate optimization of current and voltage to maximize conversion efficiency.

A sealable ultrathin window sample cell for the study of liquids by means of soft X-ray spectroscopy

NASA Astrophysics Data System (ADS)

Grötzsch, D.; Streeck, C.; Nietzold, C.; Malzer, W.; Mantouvalou, I.; Nutsch, A.; Dietrich, P.; Unger, W.; Beckhoff, B.; Kanngießer, B.

2017-12-01

A new sample cell concept for the analysis of liquids or solid-liquid interfaces using soft X-ray spectroscopy is presented, which enables the complete sealing of the cell as well as the transport into vacuum via, for example, a load-lock system. The cell uses pressure monitoring and active as well as passive pressure regulation systems, thereby facilitating the full control over the pressure during filling, sealing, evacuation, and measurement. The cell design and sample preparation as well as the crucial sealing procedure are explained in detail. As a first proof-of-principle experiment, successful nitrogen K-edge fluorescence yield near-edge X-ray absorption fine structure experiments of a biomolecular solution are presented. For this purpose, it is shown that the careful evaluation of all involved parameters, such as window type or photon flux, is desirable for optimizing the experimental result.

Efficient globally optimal segmentation of cells in fluorescence microscopy images using level sets and convex energy functionals.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2012-10-01

In high-throughput applications, accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression and the understanding of cell function. We propose an approach for segmenting cell nuclei which is based on active contours using level sets and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We consider three different well-known energy functionals for active contour-based segmentation and introduce convex formulations of these functionals. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images from different experiments comprising different cell types. We have also performed a quantitative comparison with previous segmentation approaches. Copyright © 2012 Elsevier B.V. All rights reserved.

A systemic study on key parameters affecting nanocomposite coatings on magnesium substrates.

PubMed

Johnson, Ian; Wang, Sebo Michelle; Silken, Christine; Liu, Huinan

2016-05-01

Nanocomposite coatings offer multiple functions simultaneously to improve the interfacial properties of magnesium (Mg) alloys for skeletal implant applications, e.g., controlling the degradation rate of Mg substrates, improving bone cell functions, and providing drug delivery capability. However, the effective service time of nanocomposite coatings may be limited due to their early delamination from the Mg-based substrates. Therefore, the objective of this study was to address the delamination issue of nanocomposite coatings, improve the coating properties for reducing the degradation of Mg-based substrates, and thus improve their cytocompatibility with bone marrow derived mesenchymal stem cells (BMSCs). The surface conditions of the substrates, polymer component type of the nanocomposite coatings, and post-deposition processing are the key parameters that contribute to the efficacy of the nanocomposite coatings in regulating substrate degradation and bone cell responses. Specifically, the effects of metallic surface versus alkaline heat-treated hydroxide surface of the substrates on coating quality were investigated. For the nanocomposite coatings, nanophase hydroxyapatite (nHA) was dispersed in three types of biodegradable polymers, i.e., poly(lactic-co-glycolic acid) (PLGA), poly(l-lactic acid) (PLLA), or poly(caprolactone) (PCL) to determine which polymer component could provide integrated properties for slowest Mg degradation. The nanocomposite coatings with or without post-deposition processing, i.e., melting, annealing, were compared to determine which processing route improved the properties of the nanocomposite coatings most significantly. The results showed that optimizing the coating processes addressed the delamination issue. The melted then annealed nHA/PCL coating on the metallic Mg substrates showed the slowest degradation and the best coating adhesion, among all the combinations of conditions studied; and, it improved the adhesion density of BMSCs. This study elucidated the key parameters for optimizing nanocomposite coatings on Mg-based substrates for skeletal implant applications, and provided rational design guidelines for the nanocomposite coatings on Mg alloys for potential clinical translation of biodegradable Mg-based implants. This manuscript describes the systemic optimization of nanocomposite coatings to control the degradation and bioactivity of magnesium for skeletal implant applications. The key parameters influencing the integrity and functions of the nanocomposite coatings on magnesium were identified, guidelines for the optimization of the coatings were established, and the benefits of coating optimization were demonstrated through reduced magnesium degradation and increased bone marrow derived mesenchymal stem cell (BMSC) adhesion in vitro. The guidelines developed in this manuscript are valuable for the biometal field to improve the design of bioresorbable implants and devices, which will advance the clinical translation of magnesium-based implants. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Stimulation of GABA-Induced Ca2+ Influx Enhances Maturation of Human Induced Pluripotent Stem Cell-Derived Neurons

PubMed Central

Rushton, David J.; Mattis, Virginia B.; Svendsen, Clive N.; Allen, Nicholas D.; Kemp, Paul J.

2013-01-01

Optimal use of patient-derived, induced pluripotent stem cells for modeling neuronal diseases is crucially dependent upon the proper physiological maturation of derived neurons. As a strategy to develop defined differentiation protocols that optimize electrophysiological function, we investigated the role of Ca2+ channel regulation by astrocyte conditioned medium in neuronal maturation, using whole-cell patch clamp and Ca2+ imaging. Standard control medium supported basic differentiation of induced pluripotent stem cell-derived neurons, as assayed by the ability to fire simple, single, induced action potentials. In contrast, treatment with astrocyte conditioned medium elicited complex and spontaneous neuronal activity, often with rhythmic and biphasic characteristics. Such augmented spontaneous activity correlated with astrocyte conditioned medium-evoked hyperpolarization and was dependent upon regulated function of L-, N- and R-type Ca2+ channels. The requirement for astrocyte conditioned medium could be substituted by simply supplementing control differentiation medium with high Ca2+ or γ-amino butyric acid (GABA). Importantly, even in the absence of GABA signalling, opening Ca2+ channels directly using Bay K8644 was able to hyperpolarise neurons and enhance excitability, producing fully functional neurons. These data provide mechanistic insight into how secreted astrocyte factors control differentiation and, importantly, suggest that pharmacological modulation of Ca2+ channel function leads to the development of a defined protocol for improved maturation of induced pluripotent stem cell-derived neurons. PMID:24278369

Validation of a Candida albicans delayed-type hypersensitivity (DTH) model in female juvenile rats for use in immunotoxicity assessments.

PubMed

Collinge, Mark; Thorn, Mitchell; Peachee, Vanessa; White, Kimber

2013-01-01

Establishing an in vivo cell-mediated immunity (CMI) assay, such as the delayed-type hypersensitivity (DTH) assay, has been identified as an important gap and recommended to receive highest priority for new model development in several workshops on developmental immunotoxicity. A Candida albicans DTH model has recently been developed that has the advantage over other DTH models, which use alternative sensitizing antigens, in that antigen-specific antibodies, which may interfere with the assay, are not produced. In addition, the in vivo C. albicans DTH model was demonstrated to be more sensitive in detecting immunosuppression than DTH models using keyhole limpet hemocyanin (KLH) or sheep red blood cells as antigens, as well as some ex vivo CMI assays. While KLH and sheep red blood cells are non-physiological immunogens, C. albicans is an important human pathogen. The present studies were conducted in order to optimize and validate the C. albicans DTH model for use in developmental immunotoxicity studies using juvenile rats. Three known immunosuppressive compounds with different mechanisms of action were tested in this model, cyclosprorin A (CsA), cyclophosphamide (CPS), and dexamethasone (DEX). Animals were sensitized with formalin-fixed C. albicans on postnatal day (PND) 28 and challenged with chitosan on PND 38. Drug was administered beginning on PND 23 and continued until PND 37. Exposure to each of the three immunotoxicants resulted in statistically significant decreases in the DTH response to C. albicans-derived chitosan. Decreases in footpad swelling were observed at ≥10 mg CsA/kg/day, ≥5 mg CPS/kg/day, and ≥0.03 mg DEX/kg/day. These results demonstrate that the C. albicans DTH model, optimized for use in juvenile rats, can be used to identify immunotoxic compounds, and fills the need for a sensitive in vivo CMI model for assessments of developmental immunotoxicity. Abbreviations Ab, antibody APC, antigen presenting cell BSA, bovine serum albumin C. albicans, Candida albicans CI, challenge interval CMI, cell-mediated immunity CO, challenge only CPS, cyclophosphamide CsA, cyclosporin A CTL, cytotoxic T lymphocyte DEX, dexamethasone DIT, developmental immunotoxicity DTH, delayed-type hypersensitivity ip, intraperitoneal KLH, keyhole limpet hemocyanin MLR, mixed lymphocyte reaction OVA, ovalbumin PBS, phosphate-buffered saline PND, postnatal day sc, subcutaneous SEM, standard error of the mean SRBC, sheep red blood cells.

Effects of Hydrogel Stiffness and Extracellular Compositions on Modulating Cartilage Regeneration by Mixed Populations of Stem Cells and Chondrocytes In Vivo.

PubMed

Wang, Tianyi; Lai, Janice H; Yang, Fan

2016-12-01

Cell-based therapies offer great promise for repairing cartilage. Previous strategies often involved using a single cell population such as stem cells or chondrocytes. A mixed cell population may offer an alternative strategy for cartilage regeneration while overcoming donor scarcity. We have recently reported that adipose-derived stem cells (ADSCs) can catalyze neocartilage formation by neonatal chondrocytes (NChons) when mixed co-cultured in 3D hydrogels in vitro. However, it remains unknown how the biochemical and mechanical cues of hydrogels modulate cartilage formation by mixed cell populations in vivo. The present study seeks to answer this question by co-encapsulating ADSCs and NChons in 3D hydrogels with tunable stiffness (∼1-33 kPa) and biochemical cues, and evaluating cartilage formation in vivo using a mouse subcutaneous model. Three extracellular matrix molecules were examined, including chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). Our results showed that the type of biochemical cue played a dominant role in modulating neocartilage deposition. CS and HA enhanced type II collagen deposition, a desirable phenotype for articular cartilage. In contrast, HS promoted fibrocartilage phenotype with the upregulation of type I collagen and failed to retain newly deposited matrix. Hydrogels with stiffnesses of ∼7-33 kPa led to a comparable degree of neocartilage formation, and a minimal initial stiffness was required to retain hydrogel integrity over time. Results from this study highlight the important role of matrix cues in directing neocartilage formation, and they offer valuable insights in guiding optimal scaffold design for cartilage regeneration by using mixed cell populations.

Optical dissection of odor information processing in vivo using GCaMPs expressed in specified cell types of the olfactory bulb

PubMed Central

Wachowiak, Matt; Economo, Michael N.; Díaz-Quesada, Marta; Brunert, Daniela; Wesson, Daniel W.; White, John. A.; Rothermel, Markus

2013-01-01

Understanding central processing requires precise monitoring of neural activity across populations of identified neurons in the intact brain. Here we used recently-optimized variants of the genetically-encoded calcium sensor GCaMP (GCaMP3 and GCaMPG5G) to image activity among genetically- and anatomically-defined neuronal populations in the olfactory bulb (OB), including two types of GABA-ergic interneurons (periglomerular (PG) and short axon (SA) cells) and OB output neurons (mitral/tufted (MT) cells) projecting to piriform cortex. We first established that changes in neuronal spiking can be accurately related to GCaMP fluorescence changes via a simple quantitative relationship over a large dynamic range. We next used in vivo two-photon imaging from individual neurons and epifluorescence signals reflecting population-level activity to investigate the spatiotemporal representation of odorants across these neuron types in anesthetized and awake mice. Under anesthesia, individual PG and SA cells showed temporally simple responses and little spontaneous activity, while MT cells were spontaneously active and showed diverse temporal responses. At the population level, response patterns of PG, SA and MT cells were surprisingly similar to those imaged from sensory inputs, with shared odorant-specific topography across the dorsal OB and inhalation-coupled temporal dynamics. During wakefulness, PG and SA cell responses increased in magnitude but remained temporally simple while those of MT cells changed to complex spatiotemporal patterns reflecting restricted excitation and widespread inhibition. These results point to multiple circuit elements with distinct roles in transforming odor representations in the OB and provide a framework for further dissecting early olfactory processing using optical and genetic tools. PMID:23516293

Hollow-core photonic-crystal-fiber-based optical frequency references

NASA Astrophysics Data System (ADS)

Holá, Miroslava; Hrabina, Jan; Mikel, Břetislav; Lazar, Josef; Číp, Ondřej

2016-12-01

This research deals with preparation of an optical frequency references based on hollow-core photonic crystal fibers (HC-PCF). This fiber-based type of absorption cells represents a effiecient way how to replace classic bulky and fragile glass made tubes references with low-weight and low-volume optical fibers. This approach allows not only to increase possible interaction length between incident light and absorption media but it also carries a possibility of manufacturing of easy-operable reference which is set up just by plugging-in of optical connectors into the optical setup. We present the results of preparation, manufacturing and filling of a set of fiber-based cells intended for lasers frequency stabilization. The work deals with setting and optimalization of HC-PCF splicing processes, minimalization of optical losses between HC-PCF and SMF fiber transitions and finishing of HC-PCF spliced ends with special care for optimal closing of hollow-core structure needed for avoiding of absorption media leakage.

Metabolic Complementation in Bacterial Communities: Necessary Conditions and Optimality

PubMed Central

Mori, Matteo; Ponce-de-León, Miguel; Peretó, Juli; Montero, Francisco

2016-01-01

Bacterial communities may display metabolic complementation, in which different members of the association partially contribute to the same biosynthetic pathway. In this way, the end product of the pathway is synthesized by the community as a whole. However, the emergence and the benefits of such complementation are poorly understood. Herein, we present a simple model to analyze the metabolic interactions among bacteria, including the host in the case of endosymbiotic bacteria. The model considers two cell populations, with both cell types encoding for the same linear biosynthetic pathway. We have found that, for metabolic complementation to emerge as an optimal strategy, both product inhibition and large permeabilities are needed. In the light of these results, we then consider the patterns found in the case of tryptophan biosynthesis in the endosymbiont consortium hosted by the aphid Cinara cedri. Using in-silico computed physicochemical properties of metabolites of this and other biosynthetic pathways, we verified that the splitting point of the pathway corresponds to the most permeable intermediate. PMID:27774085

Biotherapies in stroke.

PubMed

Detante, O; Jaillard, A; Moisan, A; Barbieux, M; Favre, I M; Garambois, K; Hommel, M; Remy, C

2014-12-01

Stroke is the second leading cause of death worldwide and the most common cause of severe disability. Neuroprotection and repair mechanisms supporting endogenous brain plasticity are often insufficient to allow complete recovery. While numerous neuroprotective drugs trials have failed to demonstrate benefits for patients, they have provided interesting translational research lessons related to neurorestorative therapy mechanisms in stroke. Stroke damage is not limited to neurons but involve all brain cell type including the extracellular matrix in a "glio-neurovascular niche". Targeting a range of host brain cells, biotherapies such as growth factors and therapeutic cells, currently hold great promise as a regenerative medical strategy for stroke. These techniques can promote both neuroprotection and delayed neural repair through neuro-synaptogenesis, angiogenesis, oligodendrogliogenesis, axonal sprouting and immunomodulatory effects. Their complex mechanisms of action are interdependent and vary according to the particular growth factor or grafted cell type. For example, while "peripheral" stem or stromal cells can provide paracrine trophic support, neural stem/progenitor cells (NSC) or mature neurons can act as more direct neural replacements. With a wide therapeutic time window after stroke, biotherapies could be used to treat many patients. However, guidelines for selecting the optimal time window, and the best delivery routes and doses are still debated and the answers may depend on the chosen product and its expected mechanism including early neuroprotection, delayed neural repair, trophic systemic transient effects or graft survival and integration. Currently, the great variety of growth factors, cell sources and cell therapy products form a therapeutic arsenal that is available for stroke treatment. Their effective clinical use will require prior careful considerations regarding safety (e.g. tumorgenicity, immunogenicity), potential efficacy, cell characterization, delivery route and in vivo biodistribution. Bone marrow-derived cell populations such as mesenchymal stromal/stem cells (MSC) or mononuclear cells (MNC), umbilical cord stem cells and NSC are most investigated notably in clinical trials. Finally, we discuss perspectives concerning potential novel biotherapies such as combinatorial approaches (growth factor combined with cell therapy, in vitro optimization of cell products, or co-transplantation) and the development of biomaterials, which could be used as injectable hydrogel scaffold matrices that could protect a cell graft or selectively deliver drugs and growth factors into the post-stroke cavity at chronic stages. Considering the remaining questions about the best procedure and the safety cautions, we can hope that future translational research about biotherapies will bring more efficient treatments that will decrease post-stroke disability for many patients. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Continuous 4-1BB co-stimulatory signals for the optimal expansion of tumor-infiltrating lymphocytes for adoptive T-cell therapy.

PubMed

Chacon, Jessica Ann; Pilon-Thomas, Shari; Sarnaik, Amod A; Radvanyi, Laszlo G

2013-09-01

Co-stimulation through members of the tumor necrosis factor receptor (TNFR) family appears to be critical for the generation of T cells with optimal effector-memory properties for adoptive cell therapy. Our work suggests that continuous 4-1BB/CD137 co-stimulation is required for the expansion of T cells with an optimal therapeutic profile and that the administration of 4-1BB agonists upon adoptive cell transfer further improves antitumor T-cell functions.

Macrophage involvement affects matrix stiffness-related influences on cell osteogenesis under three-dimensional culture conditions.

PubMed

He, Xiao-Tao; Wu, Rui-Xin; Xu, Xin-Yue; Wang, Jia; Yin, Yuan; Chen, Fa-Ming

2018-04-15

Accumulating evidence indicates that the physicochemical properties of biomaterials exert profound influences on stem cell fate decisions. However, matrix-based regulation selected through in vitro analyses based on a given cell population do not genuinely reflect the in vivo conditions, in which multiple cell types are involved and interact dynamically. This study constitutes the first investigation of how macrophages (Mφs) in stiffness-tunable transglutaminase cross-linked gelatin (TG-gel) affect the osteogenesis of bone marrow-derived mesenchymal stem cells (BMMSCs). When a single cell type was cultured, low-stiffness TG-gels promoted BMMSC proliferation, whereas high-stiffness TG-gels supported cell osteogenic differentiation. However, Mφs in high-stiffness TG-gels were more likely to polarize toward the pro-inflammatory M1 phenotype. Using either conditioned medium (CM)-based incubation or Transwell-based co-culture, we found that Mφs encapsulated in the low-stiffness matrix exerted a positive effect on the osteogenesis of co-cultured BMMSCs. Conversely, Mφs in high-stiffness TG-gels negatively affected cell osteogenic differentiation. When both cell types were cultured in the same TG-gel type and placed into the Transwell system, the stiffness-related influences of Mφs on BMMSCs were significantly altered; both the low- and high-stiffness matrix induced similar levels of BMMSC osteogenesis. Although the best material parameter for synergistically affecting Mφs and BMMSCs remains unknown, our data suggest that Mφ involvement in the co-culture system alters previously identified material-related influences on BMMSCs, such as matrix stiffness-related effects, which were identified based on a culture system involving a single cell type. Such Mφ-stem cell interactions should be considered when establishing proper matrix parameter-associated cell regulation in the development of biomimetic biomaterials for regenerative applications. The substrate stiffness of a scaffold plays critical roles in modulating both reparative cells, such as mesenchymal stem cells (MSCs), and immune cells, such as macrophages (Mφs). Although the influences of material stiffness on either Mφs or MSCs, have been extensively described, how the two cell types respond to matrix cues to dynamically affect each other in a three-dimensional (3D) biosystem remains largely unknown. Here, we report our findings that, in a platform wherein Mφs and bone marrow-derived MSCs coexist, matrix stiffness can influence stem cell fate through both direct matrix-associated regulation and indirect Mφ-based modulation. Our data support future studies of the MSC-Mφ-matrix interplay in the 3D context to optimize matrix parameters for the development of the next biomaterial. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

STEPS: a grid search methodology for optimized peptide identification filtering of MS/MS database search results.

PubMed

Piehowski, Paul D; Petyuk, Vladislav A; Sandoval, John D; Burnum, Kristin E; Kiebel, Gary R; Monroe, Matthew E; Anderson, Gordon A; Camp, David G; Smith, Richard D

2013-03-01

For bottom-up proteomics, there are wide variety of database-searching algorithms in use for matching peptide sequences to tandem MS spectra. Likewise, there are numerous strategies being employed to produce a confident list of peptide identifications from the different search algorithm outputs. Here we introduce a grid-search approach for determining optimal database filtering criteria in shotgun proteomics data analyses that is easily adaptable to any search. Systematic Trial and Error Parameter Selection--referred to as STEPS--utilizes user-defined parameter ranges to test a wide array of parameter combinations to arrive at an optimal "parameter set" for data filtering, thus maximizing confident identifications. The benefits of this approach in terms of numbers of true-positive identifications are demonstrated using datasets derived from immunoaffinity-depleted blood serum and a bacterial cell lysate, two common proteomics sample types. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Stimulation of hair follicle stem cell proliferation through an IL-1 dependent activation of γδT-cells

PubMed Central

Dutta, Abhik; Pincha, Neha; Rana, Isha; Ghosh, Subhasri; Witherden, Deborah; Kandyba, Eve; MacLeod, Amanda; Kobielak, Krzysztof; Havran, Wendy L

2017-01-01

The cutaneous wound-healing program is a product of a complex interplay among diverse cell types within the skin. One fundamental process that is mediated by these reciprocal interactions is the mobilization of local stem cell pools to promote tissue regeneration and repair. Using the ablation of epidermal caspase-8 as a model of wound healing in Mus musculus, we analyzed the signaling components responsible for epithelial stem cell proliferation. We found that IL-1α and IL-7 secreted from keratinocytes work in tandem to expand the activated population of resident epidermal γδT-cells. A downstream effect of activated γδT-cells is the preferential proliferation of hair follicle stem cells. By contrast, IL-1α-dependent stimulation of dermal fibroblasts optimally stimulates epidermal stem cell proliferation. These findings provide new mechanistic insights into the regulation and function of epidermal cell–immune cell interactions and into how components that are classically associated with inflammation can differentially influence distinct stem cell niches within a tissue. PMID:29199946

Treatment of oral cancer cells with nonthermal atmospheric pressure plasma jet

NASA Astrophysics Data System (ADS)

Yurkovich, James; Han, Xu; Coffey, Benjamin; Klas, Matej; Ptasinska, Sylwia

2012-10-01

Non-thermal atmospheric pressure plasmas are specialized types of plasma that are proposed as a new agent to induce death in cancer cells. The experimental phase of this study will test the application of such plasma to SCC-25 oral cancer cells to determine if it is possible to induce apoptosis or necrosis. Different sources are used on the cells to find a configuration which kills cancer cells but has no effect on normal cells. The sources have been developed based on the dielectric barrier discharge between two external electrodes surrounding a dielectric tube; such a configuration has been shown to induce breaks in DNA strands. Each configuration is characterized using an optical emission spectrophotometer and iCCD camera to determine the optimal conditions for inducing cell death. The cells are incubated after irradiation with plasma, and cell death is determined using microscopy imaging to identify antibody interaction within the cells. These studies are important for better understanding of plasma species interactions with cancer cells and mechanisms of DNA damage and at latter stage they will be useful for the development of advanced cancer therapy.

Independent and high-level dual-gene expression in adult stem-progenitor cells from a single lentiviral vector.

PubMed

Tian, J; Andreadis, S T

2009-07-01

Expression of multiple genes from the same target cell is required in several technological and therapeutic applications such as quantitative measurements of promoter activity or in vivo tracking of stem cells. In spite of such need, reaching independent and high-level dual-gene expression cannot be reliably accomplished by current gene transfer vehicles. To address this issue, we designed a lentiviral vector carrying two transcriptional units separated by polyadenylation, terminator and insulator sequences. With this design, the expression level of both genes was as high as that yielded from lentiviral vectors containing only a single transcriptional unit. Similar results were observed with several promoters and cell types including epidermal keratinocytes, bone marrow mesenchymal stem cells and hair follicle stem cells. Notably, we demonstrated quantitative dynamic monitoring of gene expression in primary cells with no need for selection protocols suggesting that this optimized lentivirus may be useful in high-throughput gene expression profiling studies.

Modelling breast cancer requires identification and correction of a critical cell lineage-dependent transduction bias

DOE PAGES

Hines, William C.; Yaswen, Paul; Bissell, Mina J.

2015-04-21

When trying to explore the biology and etiology of human cancers, clinically relevant human culture models are essential. Current breast tumour models, such as those from oncogenically transformed primary breast cells, produce predominantly basal-like properties, whereas the more common phenotype expressed by the vast majority of breast tumours are luminal. Reasons for this puzzling, yet important phenomenon, are not understood. We show here that luminal epithelial cells are significantly more resistant to viral transduction than their myoepithelial counterparts. Here, we suggest that this is a significant barrier to generating luminal cell lines and experimental tumours in vivo and to accuratemore » interpretation of results. We show that the resistance is due to lower affinity of luminal cells for virus attachment, which can be overcome by pretreating cells—or virus—with neuraminidase. We present an analytical method for quantifying transductional differences between cell types and an optimized protocol for transducing unsorted primary human breast cells in context.« less

Simulation and optimization performance of GaAs/GaAs0.5Sb0.5/GaSb mechanically stacked tandem solar cells

NASA Astrophysics Data System (ADS)

Tayubi, Y. R.; Suhandi, A.; Samsudin, A.; Arifin, P.; Supriyatman

2018-05-01

Different approaches have been made in order to reach higher solar cells efficiencies. Concepts for multilayer solar cells have been developed. This can be realised if multiple individual single junction solar cells with different suitably chosen band gaps are connected in series in multi-junction solar cells. In our work, we have simulated and optimized solar cells based on the system mechanically stacked using computer simulation and predict their maximum performance. The structures of solar cells are based on the single junction GaAs, GaAs0.5Sb0.5 and GaSb cells. We have simulated each cell individually and extracted their optimal parameters (layer thickness, carrier concentration, the recombination velocity, etc), also, we calculated the efficiency of each cells optimized by separation of the solar spectrum in bands where the cell is sensible for the absorption. The optimal values of conversion efficiency have obtained for the three individual solar cells and the GaAs/GaAs0.5Sb0.5/GaSb tandem solar cells, that are: η = 19,76% for GaAs solar cell, η = 8,42% for GaAs0,5Sb0,5 solar cell, η = 4, 84% for GaSb solar cell and η = 33,02% for GaAs/GaAs0.5Sb0.5/GaSb tandem solar cell.

Dynamic PID loop control

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pei, L.; Klebaner, A.; Theilacker, J.

2011-06-01

The Horizontal Test Stand (HTS) SRF Cavity and Cryomodule 1 (CM1) of eight 9-cell, 1.3GHz SRF cavities are operating at Fermilab. For the cryogenic control system, how to hold liquid level constant in the cryostat by regulation of its Joule-Thompson JT-valve is very important after cryostat cool down to 2.0 K. The 72-cell cryostat liquid level response generally takes a long time delay after regulating its JT-valve; therefore, typical PID control loop should result in some cryostat parameter oscillations. This paper presents a type of PID parameter self-optimal and Time-Delay control method used to reduce cryogenic system parameters oscillation.

Solar concentrator with diffuser segments

NASA Astrophysics Data System (ADS)

Esparza, Diego; Moreno, Ivan

2011-08-01

Solar energy systems use concentrating optics with photovoltaic cells for optimizing the performance. Advanced concentrators are designed to maximize both the light collection and the spatial uniformity of radiation. This is important because irradiance uniformity is critical for all types of photovoltaic cells. This is difficult to achieve with traditional concentrators, which are built with polished optical surfaces. In this work we propose a new concept of solar concentrator which uses small diffuser segments in key points to increase the irradiation uniformity. We experimentally demonstrate this new concept by analyzing the effects on both efficiency and irradiance uniformity due to the incorporation of scattering ribbons in a compound parabolic concentrator.

Dementia of frontal lobe type and motor neuron disease. A Golgi study of the frontal cortex.

PubMed Central

Ferrer, I; Roig, C; Espino, A; Peiro, G; Matias Guiu, X

1991-01-01

Neuropathological findings in a 38 year old patient with dementia of frontal lobe type and motor neuron disease included pyramidal tracts, myelin pallor and neuron loss, gliosis and chromatolysis in the hypoglossal nucleus, together with frontal atrophy, neuron loss, gliosis and spongiosis in the upper cortical layers of the frontal (and temporal) lobes. Most remaining pyramidal and non-pyramidal neurons (multipolar, bitufted and bipolar cells) in the upper layers (layers II and III) of the frontal cortex (area B) had reduced dendritic arbors, proximal dendritic varicosities and amputation of dendrites as revealed in optimally stained rapid Golgi sections. Pyramidal cells in these layers also showed depletion of dendritic spines. Neurons in the inner layers were preserved. Loss of receptive surfaces in neurons of the upper cortical layers in the frontal cortex are indicative of neuronal disconnection, and are "hidden" contributory morphological substrates for the development of dementia. Images PMID:1744652

[Aerobic methylobacteria as the basis for a biosensor for dichloromethane detection].

PubMed

Plekhanova, Iu V; Firsova, Iu E; Doronina, N V; Trotsenko, Iu A; Reshetilov, A N

2013-01-01

Cells of dichloromethane (DChM) bacteria-destructors were immobilized by sorption on different types of membranes, which were fixed on the measuring surface of a pH-sensitive field transistor. The presence of DChM in the medium (0.6-8.8 mM) led to a change in the transistor's output signal, which was determined by the appearance of H+ ions in the medium due to DChM utilization by methylobateria. Among four strains of methylobacteria--Methylobacterium dichloromethanicum DM4, Methylobacterium extorquens DM 17, Methylopila helvetica DM6, and Ancylobacter dichloromethanicus DM 16--the highest and most stable activity toward DChM degradation was observed in the strain M. dichloromethanicum DM4. Among 11 types of membranes for cell immobilization, Millipore nitrocellulose membranes and chromatographic fiber paper GF/A, which allow one to obtain stable biosensor signals for 2 weeks without a bioreceptor change, were chosen as optimal carriers.

Cryo-FIB specimen preparation for use in a cartridge-type cryo-TEM.

PubMed

He, Jie; Hsieh, Chyongere; Wu, Yongping; Schmelzer, Thomas; Wang, Pan; Lin, Ying; Marko, Michael; Sui, Haixin

2017-08-01

Cryo-electron tomography (cryo-ET) is a well-established technique for studying 3D structural details of subcellular macromolecular complexes and organelles in their nearly native context in the cell. A primary limitation of the application of cryo-ET is the accessible specimen thickness, which is less than the diameters of almost all eukaryotic cells. It has been shown that focused ion beam (FIB) milling can be used to prepare thin, distortion-free lamellae of frozen biological material for high-resolution cryo-ET. Commercial cryosystems are available for cryo-FIB specimen preparation, however re-engineering and additional fixtures are often essential for reliable results with a particular cryo-FIB and cryo-transmission electron microscope (cryo-TEM). Here, we describe our optimized protocol and modified instrumentation for cryo-FIB milling to produce thin lamellae and subsequent damage-free cryotransfer of the lamellae into our cartridge-type cryo-TEM. Published by Elsevier Inc.