Application of hanging drop technique for stem cell differentiation and cytotoxicity studies.

PubMed

Banerjee, Meenal; Bhonde, Ramesh R

2006-05-01

The aim of our study is to explore the possibility of using an ancient method of culture technique- the hanging drop technique for stem cell differentiation and cytotoxicity testing. We demonstrate here a variety of novel applications of this age old technique not only to harness the differentiation potential of stem cells into specific lineages but also for cytotoxicity studies. Here we have prepared hanging drop cultures by placing 20 microl micro-drops of nutrient media and 10% Fetal Calf Serum (FCS) containing cells of interest on the lids of 60 mm dishes. Bottom plates of the dishes were filled with sterile Phosphate Buffer Saline (PBS) to avoid desiccation of samples. Lids were then placed on the bottom plates to achieve hanging drop cultures. We utilized this technique for cultivation of ciliated epithelia to study cytotoxicity and differentiation of bone marrow stromal cells. Most importantly the modified culture technique presented here is simple, economical and cost effective in terms of the time taken and the reagents required and are amenable to goal specific modification such as cytotoxicity testing. It is advantageous over the existing system in terms of retention of viability and functionality for longer duration and for providing three dimensional growth micro-environment making it useful for organotypic cultures and in vivo simulation.

[Culture of pancreatic progenitor cells in hanging drop and on floating filter].

PubMed

Ma, Feng-xia; Chen, Fang; Chi, Ying; Yang, Shao-guang; Lu, Shi-hong; Han, Zhong-chao

2013-06-01

To construct a method to culture pancreatic progenitor cells in hanging drop and on floating filter,and to examine if pancreatic progenitor cells can differentiate into mature endocrine cells with this method. Murine embryos at day 12.5 were isolated and digested into single cells,which were then cultured in hanging drop for 24h and formed spheres.Spheres were cultured on the filter for 6 days,which floated in the dish containing medium.During culture,the expressions of pancreas duodenum homeobox-1(PDX-1)and neurogenin3(Ngn3)were determined.The expressions of endocrine and exocrine markers,insulin,glucagon,and carboxypeptidase(CPA)were determined on day 7 by immunohistochemistry.Insulin secretion of spheres stimulated by glucose was detected by ELISA.The changes of pancreatic marker expressions during culture were monitored by real-time polymerase chain reaction(PCR). One day after the culture,there were still a large amount of PDX-1 positive cells in pancreatic spheres,and these cells proliferated.On day 3,high expression of Ngn3 was detected,and the Ngn3-positive cells did not proliferate.On day 7,The expressions of endocrine and exocrine markers in the differentiated pancreatic progenitor cells were detected,which were consistent with that in vivo.Insulin was secreted by spheres upon the stimulation of glucose. In hanging drop and on floating filter,pancreatic progenitor cells can differentiate into mature endocrine cells.

The influence of gravity on the distribution of the deposit formed onto a substrate by sessile, hanging, and sandwiched hanging drop evaporation.

PubMed

Sandu, Ion; Fleaca, Claudiu Teodor

2011-06-15

The focus of the present article is the study of the influence of gravity on the particle deposition profiles on a solid substrate during the evaporation of sessile, hanging and sandwiched hanging drops of colloidal particle suspensions. For concentrations of nanoparticles in the colloidal solutions in the range 0.0001-1 wt.%, highly diluted suspensions will preferentially form rings while concentrated suspensions will preferentially form spots in both sessile and hanging drop evaporation. For intermediary concentrations, the particle deposition profiles will depend on the nanoparticle aggregation dynamics in the suspension during the evaporation process, gravity and on the detailed evaporation geometry. The evaporation of a drop of toluene/carbon nanoparticle suspension hanging from a pendant water drop will leave on the substrate a circular spot with no visible external ring. By contrast, a clear external ring is formed on the substrate by the sessile evaporation of a similar drop of suspension sandwiched between a water drop and the substrate. From the application viewpoint, these processes can be used to create preferential electrical conductive carbon networks and contacts for arrays of self-assembled nanostructures fabricated on solid substrates as well as on flexible polymeric substrates. Copyright © 2011 Elsevier Inc. All rights reserved.

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.

Experimental and theoretical analysis of the rate of solvent equilibration in the hanging drop method of protein crystal growth

NASA Technical Reports Server (NTRS)

Fowlis, William W.; Delucas, Lawrence J.; Twigg, Pamela J.; Howard, Sandra B.; Meehan, Edward J.

1988-01-01

The principles of the hanging-drop method of crystal growth are discussed, and the rate of water evaporation in a water droplet (containing protein, buffer, and a precipitating agent) suspended above a well containing a double concentration of precipitating agent is investigated theoretically. It is shown that, on earth, the rate of evaporation may be determined from diffusion theory and the colligative properties of solutions. The parameters affecting the rate of evaporation include the temperature, the vapor pressure of water, the ionization constant of the salt, the volume of the drop, the contact angle between the droplet and the coverslip, the number of moles of salt in the droplet, the number of moles of water and salt in the well, the molar volumes of water and salt, the distance from the droplet to the well, and the coefficient of diffusion of water vapor through air. To test the theoretical equations, hanging-drop experiments were conducted using various reagent concentrations in 25-microliter droplets and measuring the evaporation times at 4 C and 25 C. The results showed good agreement with the theory.

Redifferentiation of in vitro expanded adult articular chondrocytes by combining the hanging-drop cultivation method with hypoxic environment.

PubMed

Martinez, Inigo; Elvenes, Jan; Olsen, Randi; Bertheussen, Kjell; Johansen, Oddmund

2008-01-01

The main purpose of this work has been to establish a new culturing technique to improve the chondrogenic commitment of isolated adult human chondrocytes, with the aim of being used during cell-based therapies or tissue engineering strategies. By using a rather novel technique to generate scaffold-free three-dimensional (3D) structures from in vitro expanded chondrocytes, we have explored the effects of different culture environments on cartilage formation. Three-dimensional chondrospheroids were developed by applying the hanging-drop technique. Cartilage tissue formation was attempted after combining critical factors such as serum-containing or serum-free media and atmospheric (20%) or low (2.5%) oxygen tensions. The quality of the formed microtissues was analyzed by histology, immunohistochemistry, electron microscopy, and real-time PCR, and directly compared with native adult cartilage. Our results revealed highly organized, 3D tissue-like structures developed by the hanging-drop method. All culture conditions allowed formation of 3D spheroids; however, cartilage generated under low oxygen tension had a bigger size, enhanced matrix deposition, and higher quality of cartilage formation. Real-time PCR demonstrated enhanced expression of cartilage-specific genes such us collagen type II and aggrecan in 3D cultures when compared to monolayers. Cartilage-specific matrix proteins and genes expressed in hanging-drop-developed spheroids were comparable to the expression obtained by applying the pellet culture system. In summary, our results indicate that a combination of 3D cultures of chondrocytes in hanging drops and a low oxygen environment represent an easy and convenient way to generate cartilage-like microstructures. We also show that a new specially tailored serum-free medium is suitable for in vitro cartilage tissue formation. This new methodology opens up the possibility of using autogenously produced solid 3D structures with redifferentiated chondrocytes as an attractive alternative to the currently used autologous chondrocyte transplantation for cartilage repair.

Determination of trace arsenic on hanging copper amalgam drop electrode.

PubMed

Piech, Robert; Baś, Bogusław; Niewiara, Ewa; Kubiak, Władysław W

2007-04-30

Hanging copper amalgam drop electrode has been applied for trace determination of arsenic by cathodic stripping analysis. Detection limit for As(III) as low as 0.33nM (0.02mug/L) at deposition time (240s) could be obtained. For seven successive determinations of As(III) at concentration of 5nM relative standard deviation was 2.5% (n=7). Interferences from selected metals and surfactant substances were examined. Absence of copper ions in sample solution causes easier optimization and makes method less vulnerable on contamination. The developed method was validated by analysis of certified reference materials (CRMs) and applied to arsenic determinations in natural water samples.

An optimized protocol for handling and processing fragile acini cultured with the hanging drop technique.

PubMed

Snyman, Celia; Elliott, Edith

2011-12-15

The hanging drop three-dimensional culture technique allows cultivation of functional three-dimensional mammary constructs without exogenous extracellular matrix. The fragile acini are, however, difficult to preserve during processing steps for advanced microscopic investigation. We describe adaptations to the protocol for handling of hanging drop cultures to include investigation using confocal, scanning, and electron microscopy, with minimal loss of cell culture components. Copyright © 2011 Elsevier Inc. All rights reserved.

Adding the 'heart' to hanging drop networks for microphysiological multi-tissue experiments.

PubMed

Rismani Yazdi, Saeed; Shadmani, Amir; Bürgel, Sebastian C; Misun, Patrick M; Hierlemann, Andreas; Frey, Olivier

2015-11-07

Microfluidic hanging-drop networks enable culturing and analysis of 3D microtissue spheroids derived from different cell types under controlled perfusion and investigating inter-tissue communication in multi-tissue formats. In this paper we introduce a compact on-chip pumping approach for flow control in hanging-drop networks. The pump includes one pneumatic chamber located directly above one of the hanging drops and uses the surface tension at the liquid-air-interface for flow actuation. Control of the pneumatic protocol provides a wide range of unidirectional pulsatile and continuous flow profiles. With the proposed concept several independent hanging-drop networks can be operated in parallel with only one single pneumatic actuation line at high fidelity. Closed-loop medium circulation between different organ models for multi-tissue formats and multiple simultaneous assays in parallel are possible. Finally, we implemented a real-time feedback control-loop of the pump actuation based on the beating of a human iPS-derived cardiac microtissue cultured in the same system. This configuration allows for simulating physiological effects on the heart and their impact on flow circulation between the organ models on chip.

Seamless Combination of Fluorescence-Activated Cell Sorting and Hanging-Drop Networks for Individual Handling and Culturing of Stem Cells and Microtissue Spheroids.

PubMed

Birchler, Axel; Berger, Mischa; Jäggin, Verena; Lopes, Telma; Etzrodt, Martin; Misun, Patrick Mark; Pena-Francesch, Maria; Schroeder, Timm; Hierlemann, Andreas; Frey, Olivier

2016-01-19

Open microfluidic cell culturing devices offer new possibilities to simplify loading, culturing, and harvesting of individual cells or microtissues due to the fact that liquids and cells/microtissues are directly accessible. We present a complete workflow for microfluidic handling and culturing of individual cells and microtissue spheroids, which is based on the hanging-drop network concept: The open microfluidic devices are seamlessly combined with fluorescence-activated cell sorting (FACS), so that individual cells, including stem cells, can be directly sorted into specified culturing compartments in a fully automated way and at high accuracy. Moreover, already assembled microtissue spheroids can be loaded into the microfluidic structures by using a conventional pipet. Cell and microtissue culturing is then performed in hanging drops under controlled perfusion. On-chip drop size control measures were applied to stabilize the system. Cells and microtissue spheroids can be retrieved from the chip by using a parallelized transfer method. The presented methodology holds great promise for combinatorial screening of stem-cell and multicellular-spheroid cultures.

Convection effects in protein crystal growth

NASA Technical Reports Server (NTRS)

Roberts, Glyn O.

1988-01-01

Protein crystals for X-ray diffraction study are usually grown resting on the bottom of a hanging drop of a saturated protein solution, with slow evaporation to the air in a small enclosed cell. The evaporation rate is controlled by hanging the drop above a reservoir of water, with its saturation vapor pressure decreased by a low concentration of a passive solute. The drop has a lower solute concentration, and its volume shrinks by evaporation until the molecular concentrations match. Protein crystals can also be grown from a seed crystal suspended or supported in the interior of a supersaturated solution. The main analysis of this report concerns this case because it is less complicated than hanging-drop growth. Convection effects have been suggested as the reason for the apparent cessation of growth at a certain rather small crystal size. It seeems that as the crystal grows, the number of dislocations increases to a point where further growth is hindered. Growth in the microgravity environment of an orbiting space vehicle has been proposed as a method for obtaining larger crystals. Experimental observations of convection effects during the growth of protein crystals have been reported.

The preparation of Drosophila embryos for live-imaging using the hanging drop protocol.

PubMed

Reed, Bruce H; McMillan, Stephanie C; Chaudhary, Roopali

2009-03-13

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

Application of Hanging Drop Technique for Kidney Tissue Culture.

PubMed

Wang, Shaohui; Wang, Ximing; Boone, Jasmine; Wie, Jin; Yip, Kay-Pong; Zhang, Jie; Wang, Lei; Liu, Ruisheng

2017-01-01

The hanging drop technique is a well-established method used in culture of animal tissues. However, this method has not been used in adult kidney tissue culture yet. This study was to explore the feasibility of using this technique for culturing adult kidney cortex to study the time course of RNA viability in the tubules and vasculature, as well as the tissue structural integrity. In each Petri dish with the plate covered with sterile buffer, a section of mouse renal cortex was cultured within a drop of DMEM culture medium on the inner surface of the lip facing downward. The tissue were then harvested at each specific time points for Real-time PCR analysis and histological studies. The results showed that the mRNA level of most Na+ related transporters and cotransporters were stably maintained within 6 hours in culture, and that the mRNA level of most receptors found in the vasculature and glomeruli were stably maintained for up to 9 days in culture. Paraffin sections of the cultured renal cortex indicated that the tubules began to lose tubular integrity after 6 hours, but the glomeruli and vasculatures were still recognizable up to 9 days in culture. We concluded that adult kidney tissue culture by hanging drop method can be used to study gene expressions in vasculature and glomeruli. © 2017 The Author(s). Published by S. Karger AG, Basel.

Expression, purification and crystallization of a human protein SH3BGRL at atomic resolution

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

Yin, Lei; Zhu, De-Yu; Yang, Na

2005-04-01

The protein SH3BGRL, containing both SH3-binding and Homer EVH1-binding motifs, has been crystallized using the hanging-drop vapour-diffusion method. The protein SH3BGRL, containing both SH3-binding and Homer EVH1-binding motifs, has been crystallized using the hanging-drop vapour-diffusion method. The crystals diffract to 0.88 Å resolution and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 28.8886, b = 34.9676, c = 98.0016 Å. Preliminary analysis indicates that the asymmetric unit contains one molecule and has a solvent content of about 34%.

Characterization and reproducibility of HepG2 hanging drop spheroids toxicology in vitro.

PubMed

Hurrell, Tracey; Ellero, Andrea Antonio; Masso, Zelie Flavienne; Cromarty, Allan Duncan

2018-02-21

Hepatotoxicity remains a major challenge in drug development despite preclinical toxicity screening using hepatocytes of human origin. To overcome some limitations of reproducing the hepatic phenotype, more structurally and functionally authentic cultures in vitro can be introduced by growing cells in 3D spheroid cultures. Characterisation and reproducibility of HepG2 spheroid cultures using a high-throughput hanging drop technique was performed and features contributing to potential phenotypic variation highlighted. Cultured HepG2 cells were seeded into Perfecta 3D® 96-well hanging drop plates and assessed over time for morphology, viability, cell cycle distribution, protein content and protein-mass profiles. Divergent aspects which were assessed included cell stocks, seeding density, volume of culture medium and use of extracellular matrix additives. Hanging drops are advantageous due to no complex culture matrix being present, enabling background free extractions for downstream experimentation. Varying characteristics were observed across cell stocks and batches, seeding density, culture medium volume and extracellular matrix when using immortalized HepG2 cells. These factors contribute to wide-ranging cellular responses and highlights concerns with respect to generating a reproducible phenotype in HepG2 hanging drop spheroids. Copyright © 2018 Elsevier Ltd. All rights reserved.

Water collection behavior and hanging ability of bioinspired fiber.

PubMed

Hou, Yongping; Chen, Yuan; Xue, Yan; Zheng, Yongmei; Jiang, Lei

2012-03-13

Since the water-collecting ability of the wetted cribellate spider capture silk is the result of a unique fiber structure, bioinspired fibers have been researched significantly so as to expose a new water-acquiring route in fogging-collection projects. However, the design of the geometry of bioinspired fiber is related to the ability of hanging drops, which has not been investigated in depth so far. Here, we fabricate bioinspired fibers to investigate the water collection behavior and the influence of geometry (i.e., periodicity of spindle knot) on the hanging-drop ability. We especially discuss water collection related to the periodicity of geometry on the bioinspired fiber. We reveal the length of the three phase contact line (TCL) at threshold conditions in conjunction with the maximal volume of a hanging drop at different modes. The study demonstrates that the geometrical structure of bioinspired fiber induces much stronger water hanging ability than that of uniform fiber, attributed to such special geometry that offers effectively an increasing TCL length or limits the contact length to be shorted. In addition, the geometry also improves the fog-collection efficiency by controlling tiny water drops to be collected in the large water drops at a given location.

Adding the ‘heart’ to hanging drop networks for microphysiological multi-tissue experiments†

PubMed Central

Yazdi, Saeed Rismani; Shadmani, Amir; Bürgel, Sebastian C.; Misun, Patrick M.; Hierlemann, Andreas; Frey, Olivier

2017-01-01

Microfluidic hanging-drop networks enable culturing and analysis of 3D microtissue spheroids derived from different cell types under controlled perfusion and investigating inter-tissue communication in multi-tissue formats. In this paper we introduce a compact on-chip pumping approach for flow control in hanging-drop networks. The pump includes one pneumatic chamber located directly above one of the hanging drops and uses the surface tension at the liquid–air-interface for flow actuation. Control of the pneumatic protocol provides a wide range of unidirectional pulsatile and continuous flow profiles. With the proposed concept several independent hanging-drop networks can be operated in parallel with only one single pneumatic actuation line at high fidelity. Closed-loop medium circulation between different organ models for multi-tissue formats and multiple simultaneous assays in parallel are possible. Finally, we implemented a real-time feedback control-loop of the pump actuation based on the beating of a human iPS-derived cardiac microtissue cultured in the same system. This configuration allows for simulating physiological effects on the heart and their impact on flow circulation between the organ models on chip. PMID:26401602

Generation of multicellular tumor spheroids by the hanging-drop method.

PubMed

Timmins, Nicholas E; Nielsen, Lars K

2007-01-01

Owing to their in vivo-like characteristics, three-dimensional (3D) multicellular tumor spheroid (MCTS) cultures are gaining increasing popularity as an in vitro model of tumors. A straightforward and simple approach to the cultivation of these MCTS is the hanging-drop method. Cells are suspended in droplets of medium, where they develop into coherent 3D aggregates and are readily accessed for analysis. In addition to being simple, the method eliminates surface interactions with an underlying substratum (e.g., polystyrene plastic or agarose), requires only a low number of starting cells, and is highly reproducible. This method has also been applied to the co-cultivation of mixed cell populations, including the co-cultivation of endothelial cells and tumor cells as a model of early tumor angiogenesis.

Hanging drop culture enhances differentiation of human adipose-derived stem cells into anterior neuroectodermal cells using small molecules.

PubMed

Amirpour, Noushin; Razavi, Shahnaz; Esfandiari, Ebrahim; Hashemibeni, Batoul; Kazemi, Mohammad; Salehi, Hossein

2017-06-01

Inspired by in vivo developmental process, several studies were conducted to design a protocol for differentiating of mesenchymal stem cells into neural cells in vitro. Human adipose-derived stem cells (hADSCs) as mesenchymal stem cells are a promising source for this purpose. At current study, we applied a defined neural induction medium by using small molecules for direct differentiation of hADSCs into anterior neuroectodermal cells. Anterior neuroectodermal differentiation of hADSCs was performed by hanging drop and monolayer protocols. At these methods, three small molecules were used to suppress the BMP, Nodal, and Wnt signaling pathways in order to obtain anterior neuroectodermal (eye field) cells from hADSCs. After two and three weeks of induction, the differentiated cells with neural morphology expressed anterior neuroectodermal markers such as OTX2, SIX3, β-TUB III and PAX6. The protein expression of such markers was confirmed by real time, RT-PCR and immunocytochemistry methods According to our data, it seems that the hanging drop method is a proper approach for neuroectodermal induction of hADSCs. Considering wide availability and immunosuppressive properties of hADSCs, these cells may open a way for autologous cell therapy of neurodegenerative disorders. Copyright © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.

Mouse ES cells have a potential to differentiate into odontoblast-like cells using hanging drop method.

PubMed

Kawai, R; Ozeki, N; Yamaguchi, H; Tanaka, T; Nakata, K; Mogi, M; Nakamura, H

2014-05-01

We examined whether mouse embryonic stem (ES) cells can differentiate into odontoblast-like cells without epithelial-mesenchymal interaction. Cells were cultured by the 'hanging drop' method using a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). Expression of odontoblast-related mRNA and protein, and cell proliferation were performed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and WST-1 assay, respectively. Cells potently expressed odontoblast-related cell marker mRNAs following induction of odontoblastic differentiation. Dentin sialophosphoprotein, a marker of mature odontoblasts, was strongly expressed in differentiated ES cells. The cells also acquired an odontoblast-like functional phenotype, as evidenced by the appearance of alkaline phosphatase activity and calcification. The cell-surface expression of α2, α6, αV and αVβ3 integrin proteins was rapidly upregulated in differentiated cells. Finally, anti-α2 integrin antibody suppressed the expression of odontoblastic markers in cells grown using this culture system, suggesting that α2 integrin expression in ES cells triggers their differentiation into odontoblast-like cells. Mouse ES cells cultured by the 'hanging drop' method are able to differentiate into cells with odontoblast-specific physiological functions and cell-surface integrin protein expression. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The effect of body position on the 'hanging drop' method for identifying the extradural space in anaesthetized dogs.

PubMed

Naganobu, Kiyokazu; Hagio, Mitsuyoshi

2007-01-01

To assess the accuracy of the 'hanging drop method' for identifying the extradural space in anaesthetized dogs positioned in sternal or lateral recumbency. Prospective randomized-experimental study. Seventeen clinically healthy adult dogs, 10 females and seven males weighing 8.4-26.2 kg. Dogs were positioned in either sternal (n = 8) or lateral (n = 9) recumbency under general anaesthesia. A 20 SWG spinal needle pre-filled with 0.9% saline was advanced through the skin into the lumbosacral extradural space and the response of the saline drop recorded, i.e. whether it: 1) was aspirated from the hub into the needle; 2) remained within the hub, or 3) moved synchronously with i) spontaneous respiration, ii) heart beat or iii) manual lung inflation. The position of the needle tip was ultimately determined by positive contrast radiography. One dog positioned in lateral recumbency was excluded from the study because bleeding occurred from the needle hub. Saline was aspirated into the needle in seven of eight dogs held in sternal recumbency but in none of the dogs positioned in lateral recumbency. Accurate needle tip placement in the extradural space was confirmed by positive contrast radiography in all dogs. The 'hanging drop' method, when performed with a spinal needle, appears to be a useful technique for identifying the location of the extradural space in anaesthetized medium-sized dogs positioned in sternal, but not in lateral recumbency. The technique may yield 'false negative' results when performed in dogs positioned in sternal recumbency.

Micro-Ring Structures Stabilize Microdroplets to Enable Long Term Spheroid Culture in 384 Hanging Drop Array Plates

PubMed Central

Hsiao, Amy Y.; Tung, Yi-Chung; Kuo, Chuan-Hsien; Mosadegh, Bobak; Bedenis, Rachel; Pienta, Kenneth J.; Takayama, Shuichi

2012-01-01

Using stereolithography, 20 different structural variations comprised of millimeter diameter holes surrounded by trenches, plateaus, or micro-ring structures were prepared and tested for their ability to stably hold arrays of microliter sized droplets within the structures over an extended period of time. The micro-ring structures were the most effective in stabilizing droplets against mechanical and chemical perturbations. After confirming the importance of micro-ring structures using rapid prototyping, we developed an injection molding tool for mass production of polystyrene 3D cell culture plates with an array of 384 such micro-ring surrounded through-hole structures. These newly designed and injection molded polystyrene 384 hanging drop array plates with micro-rings were stable and robust against mechanical perturbations as well as surface fouling-facilitated droplet spreading making them capable of long term cell spheroid culture of up to 22 days within the droplet array. This is a significant improvement over previously reported 384 hanging drop array plates which are susceptible to small mechanical shocks and could not reliably maintain hanging drops for longer than a few days. With enhanced droplet stability, the hanging drop array plates with micro-ring structures provide better platforms and open up new opportunities for high-throughput preparation of microscale 3D cell constructs for drug screening and cell analysis. PMID:22057945

Micro-ring structures stabilize microdroplets to enable long term spheroid culture in 384 hanging drop array plates.

PubMed

Hsiao, Amy Y; Tung, Yi-Chung; Kuo, Chuan-Hsien; Mosadegh, Bobak; Bedenis, Rachel; Pienta, Kenneth J; Takayama, Shuichi

2012-04-01

Using stereolithography, 20 different structural variations comprised of millimeter diameter holes surrounded by trenches, plateaus, or micro-ring structures were prepared and tested for their ability to stably hold arrays of microliter sized droplets within the structures over an extended period of time. The micro-ring structures were the most effective in stabilizing droplets against mechanical and chemical perturbations. After confirming the importance of micro-ring structures using rapid prototyping, we developed an injection molding tool for mass production of polystyrene 3D cell culture plates with an array of 384 such micro-ring surrounded through-hole structures. These newly designed and injection molded polystyrene 384 hanging drop array plates with micro-rings were stable and robust against mechanical perturbations as well as surface fouling-facilitated droplet spreading making them capable of long term cell spheroid culture of up to 22 days within the droplet array. This is a significant improvement over previously reported 384 hanging drop array plates which are susceptible to small mechanical shocks and could not reliably maintain hanging drops for longer than a few days. With enhanced droplet stability, the hanging drop array plates with micro-ring structures provide better platforms and open up new opportunities for high-throughput preparation of microscale 3D cell constructs for drug screening and cell analysis.

Crystallization and initial X-ray analysis of polyhydroxyalkanoate granule-associated protein from Aeromonas hydrophila

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

Zhao, Minglian; Li, Zhenguo; Zheng, Wei

The phasin PhaP{sub Ah} from A. hydrophila strain 4AK4 was crystallized using the hanging-drop vapour-diffusion method. Polyhydroxyalkanoate (PHA) granule-associated proteins (phasins) were discovered in PHA-accumulating bacteria. They play a crucial role as a structural protein during initial PHA-granule formation and granule growth and also serve as interfaces for granule stabilization in vivo. The phasin PhaP{sub Ah} from Aeromonas hydrophila strain 4AK4 was crystallized using the hanging-drop vapour-diffusion method. Single crystals were cryocooled for X-ray diffraction analysis. The phasin crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 80.8, b = 108.9, c = 134.4 Å.

Expression, crystallization and phasing of vacuolar H(+)-ATPase subunit C (Vma5p) of Saccharomyces cerevisiae.

PubMed

Drory, Omri; Mor, Adi; Frolow, Felix; Nelson, Nathan

2004-10-01

The expression, crystallization and phasing of subunit C (Vma5p) of the yeast (Saccharomyces cerevisiae) vacuolar proton-translocating ATPase (V-ATPase) is described. The expressed protein consists of 412 residues: 392 from the reading frame of Vma5p and 20 N-terminal residues originating from the plasmid. Diffraction-quality crystals were obtained using the hanging-drop and sitting-drop vapour-diffusion methods assisted by streak-seeding, with PEG 3350 as precipitant. The crystals formed in hanging drops diffracted to 1.80 A and belong to space group P4(3)2(1)2(1), with unit-cell parameters a = b = 62.54, c = 327.37 A, alpha = beta = gamma = 90 degrees. The structure was solved using SIRAS with a Lu(O2C2H3)2 heavy-atom derivative.

A hollow sphere soft lithography approach for long-term hanging drop methods.

PubMed

Lee, Won Gu; Ortmann, Daniel; Hancock, Matthew J; Bae, Hojae; Khademhosseini, Ali

2010-04-01

In conventional hanging drop (HD) methods, embryonic stem cell aggregates or embryoid bodies (EBs) are often maintained in small inverted droplets. Gravity limits the volumes of these droplets to less than 50 microL, and hence such cell cultures can only be sustained for a few days without frequent media changes. Here we present a new approach to performing long-term HD methods (10-15 days) that can provide larger media reservoirs in a HD format to maintain more consistent culture media conditions. To implement this approach, we fabricated hollow sphere (HS) structures by injecting liquid drops into noncured poly(dimethylsiloxane) mixtures. These structures served as cell culture chambers with large media volumes (500 microL in each sphere) where EBs could grow without media depletion. The results showed that the sizes of the EBs cultured in the HS structures in a long-term HD format were approximately twice those of conventional HD methods after 10 days in culture. Further, HS cultures showed multilineage differentiation, similar to EBs cultured in the HD method. Due to its ease of fabrication and enhanced features, this approach may be of potential benefit as a stem cell culture method for regenerative medicine.

A Hollow Sphere Soft Lithography Approach for Long-Term Hanging Drop Methods

PubMed Central

Lee, Won Gu; Ortmann, Daniel; Hancock, Matthew J.; Bae, Hojae

2010-01-01

In conventional hanging drop (HD) methods, embryonic stem cell aggregates or embryoid bodies (EBs) are often maintained in small inverted droplets. Gravity limits the volumes of these droplets to less than 50 μL, and hence such cell cultures can only be sustained for a few days without frequent media changes. Here we present a new approach to performing long-term HD methods (10–15 days) that can provide larger media reservoirs in a HD format to maintain more consistent culture media conditions. To implement this approach, we fabricated hollow sphere (HS) structures by injecting liquid drops into noncured poly(dimethylsiloxane) mixtures. These structures served as cell culture chambers with large media volumes (500 μL in each sphere) where EBs could grow without media depletion. The results showed that the sizes of the EBs cultured in the HS structures in a long-term HD format were approximately twice those of conventional HD methods after 10 days in culture. Further, HS cultures showed multilineage differentiation, similar to EBs cultured in the HD method. Due to its ease of fabrication and enhanced features, this approach may be of potential benefit as a stem cell culture method for regenerative medicine. PMID:19505251

Preparation, crystallization and preliminary crystallographic analysis of old yellow enzyme from Trypanosoma cruzi

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

Sugiyama, Shigeru; Tokuoka, Keiji; Uchiyama, Nahoko

2007-10-01

Old yellow enzyme from Trypanosoma cruzi, has been crystallized using the hanging-drop vapour-diffusion method. Old yellow enzyme (OYE) is an NADPH oxidoreductase that contains a flavin mononucleotide as a prosthetic group. The OYE from Trypanosoma cruzi, which produces prostaglandin F{sub 2α}, a potent mediator of various physiological and pathological processes, from prostaglandin H2. The protein was recombinantly expressed and purified from Escherichia coli and was crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 56.3, b = 78.8, c = 78.8 Å, β = 93.4° and two moleculesmore » per asymmetric unit. The crystals were suitable for X-ray crystallographic studies and diffracted to 1.70 Å resolution. A Patterson search method is in progress using the structure of OYE from Pseudomonas putida as a starting model.« less

Controlling Vapor Pressure In Hanging-Drop Crystallization

NASA Technical Reports Server (NTRS)

Carter, Daniel C.; Smith, Robbie

1988-01-01

Rate of evaporation adjusted to produce larger crystals. Device helps to control vapor pressure of water and other solvents in vicinity of hanging drop of solution containing dissolved enzyme protein. Well of porous frit (sintered glass) holds solution in proximity to drop of solution containing protein or enzyme. Vapor from solution in frit controls evaporation of solvent from drop to control precipitation of protein or enzyme. With device, rate of nucleation limited to decrease number and increase size (and perhaps quality) of crystals - large crystals of higher quality needed for x-ray diffraction studies of macromolecules.

Purification, identification and preliminary crystallographic studies of an allergenic protein from Lathyrus sativus

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

Qureshi, Insaf A.; Sethi, Dhruv K.; Salunke, Dinakar M., E-mail: dinakar@nii.res.in

2006-09-01

A 24 kDa protein was purified from the seeds of L. sativus by ammonium sulfate fractionation and ion-exchange chromatography. Crystals were obtained by the hanging-drop vapour-diffusion method. A 24 kDa protein was purified from the seeds of Lathyrus sativus by ammonium sulfate fractionation and ion-exchange chromatography. The N-terminal amino-acid sequence showed significant homology with the 2S albumin class of seed storage proteins. The protein showed 85% sequence homology with the seed albumin of Pisum sativum within the 40 N-terminal residues. Crystals were obtained by the hanging-drop vapour-diffusion method. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cellmore » parameters a = 43.5, b = 82.7, c = 153.4 Å.« less

Optimization of the formation of embedded multicellular spheroids of MCF-7 cells: How to reliably produce a biomimetic 3D model.

PubMed

Zhang, Wenli; Li, Caibin; Baguley, Bruce C; Zhou, Fang; Zhou, Weisai; Shaw, John P; Wang, Zhen; Wu, Zimei; Liu, Jianping

2016-12-15

To obtain a multicellular MCF-7 spheroid model to mimic the three-dimensional (3D) of tumors, the microwell liquid overlay (A) and hanging-drop/agar (B) methods were first compared for their technical parameters. Then a method for embedding spheroids within collagen was optimized. For method A, centrifugation assisted cells form irregular aggregates but not spheroids. For method B, an extended sedimentation period of over 24 h for cell suspensions and increased viscosity of the culture medium using methylcellulose were necessary to harvest a dense and regular cell spheroid. When the number was less than 5000 cells/drop, embedded spheroids showed no tight cores and higher viability than the unembedded. However, above 5000 cells/drop, cellular viability of embedded spheroids was not significantly different from unembedded spheroids and cells invading through the collagen were in a sun-burst pattern with tight cores. Propidium Iodide staining indicated that spheroids had necrotic cores. The doxorubicin cytotoxicity demonstrated that spheroids were less susceptible to DOX than their monolayer cells. A reliable and reproducible method for embedding spheroids using the hanging-drop/agarose method within collagen is described herein. The cell culture model can be used to guide experimental manipulation of 3D cell cultures and to evaluate anticancer drug efficacy. Copyright © 2016 Elsevier Inc. All rights reserved.

Hanging Drop, A Best Three-Dimensional (3D) Culture Method for Primary Buffalo and Sheep Hepatocytes.

PubMed

Shri, Meena; Agrawal, Himanshu; Rani, Payal; Singh, Dheer; Onteru, Suneel Kumar

2017-04-26

Livestock, having close resemblance to humans, could be a better source of primary hepatocytes than rodents. Herein, we successfully developed three-dimensional (3D) culturing system for primary sheep and buffalo hepatocytes. The 3D-structures of sheep hepatocytes were formed on the fifth-day and maintained until the tenth-day on polyHEMA-coated plates and in hanging drops with William's E media (HDW). Between the cultured and fresh cells, we observed a similar expression of GAPDH, HNF4α, ALB, CYP1A1, CK8 and CK18. Interestingly, a statistically significant increase was noted in the TAT, CPS, AFP, AAT, GSP and PCNA expression. In buffalo hepatocytes culture, 3D-like structures were formed on the third-day and maintained until the sixth-day on polyHEMA and HDW. The expression of HNF4α, GSP, CPS, AFP, AAT, PCNA and CK18 was similar between cultured and fresh cells. Further, a statistically significant increase in the TAT and CK8 expression, and a decrease in the GAPDH, CYP1A1 and ALB expression were noted. Among the culture systems, HDW maintained the liver transcript markers more or less similar to the fresh hepatocytes of the sheep and buffalo for ten and six days, respectively. Taken together, hanging drop is an efficient method for 3D culturing of primary sheep and buffalo hepatocytes.

Reconfigurable microfluidic hanging drop network for multi-tissue interaction and analysis.

PubMed

Frey, Olivier; Misun, Patrick M; Fluri, David A; Hengstler, Jan G; Hierlemann, Andreas

2014-06-30

Integration of multiple three-dimensional microtissues into microfluidic networks enables new insights in how different organs or tissues of an organism interact. Here, we present a platform that extends the hanging-drop technology, used for multi-cellular spheroid formation, to multifunctional complex microfluidic networks. Engineered as completely open, 'hanging' microfluidic system at the bottom of a substrate, the platform features high flexibility in microtissue arrangements and interconnections, while fabrication is simple and operation robust. Multiple spheroids of different cell types are formed in parallel on the same platform; the different tissues are then connected in physiological order for multi-tissue experiments through reconfiguration of the fluidic network. Liquid flow is precisely controlled through the hanging drops, which enable nutrient supply, substance dosage and inter-organ metabolic communication. The possibility to perform parallelized microtissue formation on the same chip that is subsequently used for complex multi-tissue experiments renders the developed platform a promising technology for 'body-on-a-chip'-related research.

Application of hanging drop technique to optimize human IgG formulations.

PubMed

Li, Guohua; Kasha, Purna C; Late, Sameer; Banga, Ajay K

2010-01-01

The purpose of this work is to assess the hanging drop technique in screening excipients to develop optimal formulations for human immunoglobulin G (IgG). A microdrop of human IgG and test solution hanging from a cover slide and undergoing vapour diffusion was monitored by a stereomicroscope. Aqueous solutions of IgG in the presence of different pH, salt concentrations and excipients were prepared and characterized. Low concentration of either sodium/potassium phosphate or McIlvaine buffer favoured the solubility of IgG. Addition of sucrose favoured the stability of this antibody while addition of NaCl caused more aggregation. Antimicrobial preservatives were also screened and a complex effect at different buffer conditions was observed. Dynamic light scattering, differential scanning calorimetry and size exclusion chromatography studies were performed to further validate the results. In conclusion, hanging drop is a very easy and effective approach to screen protein formulations in the early stage of formulation development.

Transfer, Imaging, and Analysis Plate for Facile Handling of 384 Hanging Drop 3D Tissue Spheroids

PubMed Central

Cavnar, Stephen P.; Salomonsson, Emma; Luker, Kathryn E.; Luker, Gary D.; Takayama, Shuichi

2014-01-01

Three-dimensional culture systems bridge the experimental gap between in vivo and in vitro physiology. However, nonstandardized formation and limited downstream adaptability of 3D cultures have hindered mainstream adoption of these systems for biological applications, especially for low- and moderate-throughput assays commonly used in biomedical research. Here we build on our recent development of a 384-well hanging drop plate for spheroid culture to design a complementary spheroid transfer and imaging (TRIM) plate. The low-aspect ratio wells of the TRIM plate facilitated highfidelity, user-independent, contact-based collection of hanging drop spheroids. Using the TRIM plate, we demonstrated several downstream analyses, including bulk tissue collection for flow cytometry, high-resolution low working-distance immersion imaging, and timely reagent delivery for enzymatic studies. Low working-distance multiphoton imaging revealed a cell type–dependent, macroscopic spheroid structure. Unlike ovarian cancer spheroids, which formed loose, disk-shaped spheroids, human mammary fibroblasts formed tight, spherical, and nutrient-limited spheroids. Beyond the applications we describe here, we expect the hanging drop spheroid plate and complementary TRIM plate to facilitate analyses of spheroids across the spectrum of throughput, particularly for bulk collection of spheroids and high-content imaging. PMID:24051516

Transfer, imaging, and analysis plate for facile handling of 384 hanging drop 3D tissue spheroids.

PubMed

Cavnar, Stephen P; Salomonsson, Emma; Luker, Kathryn E; Luker, Gary D; Takayama, Shuichi

2014-04-01

Three-dimensional culture systems bridge the experimental gap between in vivo and in vitro physiology. However, nonstandardized formation and limited downstream adaptability of 3D cultures have hindered mainstream adoption of these systems for biological applications, especially for low- and moderate-throughput assays commonly used in biomedical research. Here we build on our recent development of a 384-well hanging drop plate for spheroid culture to design a complementary spheroid transfer and imaging (TRIM) plate. The low-aspect ratio wells of the TRIM plate facilitated high-fidelity, user-independent, contact-based collection of hanging drop spheroids. Using the TRIM plate, we demonstrated several downstream analyses, including bulk tissue collection for flow cytometry, high-resolution low working-distance immersion imaging, and timely reagent delivery for enzymatic studies. Low working-distance multiphoton imaging revealed a cell type-dependent, macroscopic spheroid structure. Unlike ovarian cancer spheroids, which formed loose, disk-shaped spheroids, human mammary fibroblasts formed tight, spherical, and nutrient-limited spheroids. Beyond the applications we describe here, we expect the hanging drop spheroid plate and complementary TRIM plate to facilitate analyses of spheroids across the spectrum of throughput, particularly for bulk collection of spheroids and high-content imaging.

Crystallization and preliminary crystallographic analysis of recombinant immunoglobulin G-binding protein from Streptococcus suis

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

Khan, Abdul Hamid; Chu, Fuliang; Feng, Youjun

2008-08-01

Crystallization of recombinant IgG-binding protein expressed in Escherichia coli using the hanging-drop vapour-diffusion method is described. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.98, b = 43.94, c = 78.17 Å. Streptococcus suis, an important zoonotic pathogen, expresses immunoglobulin G-binding protein, which is thought to be helpful to the organism in eluding the host defence system. Recombinant IgG-binding protein expressed in Escherichia coli has been crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.98, b = 43.94, c =more » 78.17 Å and one molecule in the asymmetric unit. Diffraction data were collected to 2.60 Å resolution.« less

Determination of nitrobenzene in wastewater using a hanging mercury drop electrode.

PubMed

Liang, Shu-Xuan; Zhang, Huan-Kun; Lu, Da

2007-06-01

The determination of trace amount nitrobenzene in wastewater on a hanging mercury drop electrode was studied. The determination conditions of pH, supporting electrolyte, accumulation potential, accumulation time, and voltammetric response were optimized. The sharp peak of the nitrobenzene was appeared at 0.05 V. The peak electric current was proportional to the concentration of nitrobenzene in the range of 1.47 x 10(-5) approximately 1.0 x 10(-3) mol/l with relative standard deviations of 3.99 approximately 8.94%. The detection limit of the nitrobenzene in water was 5 x 10(-6) mol/l. The proposed method offered low limit of determination, easy operation, the use of simple instrumentation, high sensitivity and good reproducibility. It was applied to the determination of nitrobenzene in wastewater with an average recovery of 94.0% approximately 105%. The proposed method provided fast, sensitive and sometimes real time detection of nitrobenzene.

Hanging drop cultures of human testis and testis cancer samples: a model used to investigate activin treatment effects in a preserved niche

PubMed Central

Jørgensen, A; Young, J; Nielsen, J E; Joensen, U N; Toft, B G; Rajpert-De Meyts, E; Loveland, K L

2014-01-01

Background: Testicular germ cell tumours of young adults, seminoma or non-seminomas, are preceded by a pre-invasive precursor, carcinoma in situ (CIS), understood to arise through differentiation arrest of embryonic germ cells. Knowledge about the malignant transformation of germ cells is currently limited by the lack of experimental models. The aim of this study was to establish an experimental tissue culture model to maintain normal and malignant germ cells within their niche and allow investigation of treatment effects. Methods: Human testis and testis cancer specimens from orchidectomies were cultured in ‘hanging drops' and effects of activin A and follistatin treatment were investigated in seminoma cultures. Results: Testis fragments with normal spermatogenesis or CIS cells were cultured for 14 days with sustained proliferation of germ cells and CIS cells and without increased apoptosis. Seminoma cultures survived 7 days, with proliferating cells detectable during the first 5 days. Activin A treatment significantly reduced KIT transcript and protein levels in seminoma cultures, thereby demonstrating a specific treatment response. Conclusions: Hanging drop cultures of human testis and testis cancer samples can be employed to delineate mechanisms governing growth of normal, CIS and tumorigenic germ cells retained within their niche. PMID:24781282

Energy stability of droplets and dry spots in a thin film model of hanging drops

NASA Astrophysics Data System (ADS)

Cheung, Ka-Luen; Chou, Kai-Seng

2017-10-01

The 2-D thin film equation describing the evolution of hang drops is studied. All radially symmetric steady states are classified, and their energy stability is determined. It is shown that the droplet with zero contact angle is the only global energy minimizer and the dry spot with zero contact angle is a strict local energy minimizer.

Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment.

PubMed

Yoon, Byung Sun; Yoo, Seung Jun; Lee, Jeoung Eun; You, Seungkwon; Lee, Hoon Taek; Yoon, Hyun Soo

2006-04-01

Cell replacement therapy is a promising approach for the treatment of cardiac diseases. It is, however, challenged by a limited supply of appropriate cells. Therefore, we have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem cells (hESCs). In this study, we developed an efficient protocol for the generation of functional cardiomyocytes from hESCs by combining hanging drop culture and 5-azacytidine, a well-known demethylating agent, and then evaluated the expression of cardiac-specific markers. hESCs were cultured both in the medium without or with 0.1, 1, or 10 microM of 5-azacytidine under a hanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR, RT-PCR, immunofluorescence, and confocal microscopy. To verify the structural and functional properties of hESC-derived cardiomyocytes, we performed electron microscopy and electrophysiological recording. The efficiency of beating cell generation was significantly improved in the hanging drop culture compared with that in suspension culture. Treatment of hESCs with 0.1 microM of 5-azacytidine for 1-3 days significantly increased the number of beating cells and simultaneously enhanced the expression of cardiac-specific markers. Transmission electron microscopy and electrophysiological recording showed that hESC-derived cardiomyocytes acquired structural and functional properties of cardiomyocytes. In conclusion, these results suggest that differentiation of hESCs into cardiomyocytes can be enhanced by the combination of hanging drop culture and 5-azacytidine treatment. Also the methylation status of genes related to cardiomyocyte development may play an important role in the differentiation of hESCs into cardiomyocytes.

A Fiber Optic Probe for Monitoring Protein Aggregation, Nucleation, and Crystallization

NASA Technical Reports Server (NTRS)

Ansari, Rafat R.; Suh, Kwang I.; Arabshahi, Alireza; Wilson, William W.; Bray, Terry L.; DeLucas, Lawrence J.

1996-01-01

Protein crystals are experimentally grown in hanging drops in microgravity experiments on-board the Space Shuttle orbiter. The technique of dynamic light scattering (DLS) can be used to monitor crystal growth process in hanging droplets (approx. 30 (L)) in microgravity experiments, but elaborate instrumentation and optical alignment problems have made in-situ applications difficult. In this paper we demonstrate that such experiments are now feasible. We apply a newly developed fiber optic probe to various earth and space (micro- gravity) bound protein crystallization system configurations to test its capability. These include conventional batch (cuvette or capillary) systems, hanging drop method in a six-pack hanging drop vapor diffusion apparatus (HDVDA), a modified HDVDA for temperature- induced nucleation and aggregation studies, and a newly envisioned dynamically controlled vapor diffusion system (DCVDS) configuration. Our compact system exploits the principles of DLS and offers a fast (within a few seconds) means of quantitatively and non-invasively monitoring the various growth stages of protein crystallization. In addition to DLS capability, the probe can also be used for performing single-angle static light scattering measurements. It utilizes extremely low levels of laser power (approx. few (W)) without a need of having any optical alignment and vibration isolation. The compact probe is also equipped with a miniaturized microscope for visualization of macroscopic protein crystals. This new optical diagnostic system opens up enormous opportunity for exploring new ways to grow good quality crystals suitable for x-ray crystallographic analysis and may help develop a concrete scientific basis for understanding the process of crystallization.

A three-dimensional in vitro HepG2 cells liver spheroid model for genotoxicity studies.

PubMed

Shah, Ume-Kulsoom; Mallia, Jefferson de Oliveira; Singh, Neenu; Chapman, Katherine E; Doak, Shareen H; Jenkins, Gareth J S

2018-01-01

The liver's role in metabolism of chemicals makes it an appropriate tissue for toxicity testing. Current testing protocols, such as animal testing and two-dimensional liver cell systems, offer limited resemblance to in vivo liver cell behaviour, in terms of gene expression profiles and metabolic competence; thus, they do not always accurately predict human toxicology. In vitro three-dimensional liver cell models offer an attractive alternative. This study reports on the development of a 3D liver model, using HepG2 cells, by a hanging-drop technique, with a focus on evaluating spheroid growth characteristics and suitability for genotoxicity testing. The cytokinesis-blocked micronucleus assay protocol was adapted to enable micronucleus (MN) detection in the 3D spheroid models. This involved evaluating the difference between hanging vs non-hanging drop positions for dosing of the test agents and comparison of automated Metafer scoring with manual scoring for MN detection in HepG2 spheroids. The initial seeding density, used for all experiments, was 5000 cells/20 μl drop hanging spheroids, harvested on day 4, with >75% cell viability. Albumin secretion (7.8 g/l) and both CYP1A1 and CYP1A2 gene expression were highest in the 3D environment at day 4. Exposure to metabolically activated genotoxicants for 24 h resulted in a 6-fold increase in CYP1A1 enzyme activity (3 μM B[a]P) and a 30-fold increase in CYP1A2 enzyme activity (5 μM PhIP) in 3D hanging spheroids. MN inductions in response to B[a]P or PhIP were 2-fold and 3-fold, respectively, and were greater in 3D hanging spheroids than in 2D format, showing that hanging spheroids are more sensitive to genotoxic agents. HepG2 hanging-drop spheroids are an exciting new alternative system for genotoxicity studies, due to their improved structural and physiological properties, relative to 2D cultures. Copyright © 2017 Elsevier B.V. All rights reserved.

The stopped-drop method: a novel setup for containment-free and time-resolved measurements

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

Schiener, Andreas; Seifert, Soenke; Magerl, Andreas

2016-03-01

A novel setup for containment-free time-resolved experiments at a free-hanging drop is reported. Within a dead-time of 100 ms a drop of mixed reactant solutions is formed and the time evolution of a reaction can be followed from thereon by various techniques. As an example, a small-angle X-ray scattering study on the formation mechanism of EDTA-stabilized CdS both at a synchrotron and a laboratory X-ray source is presented here. While the evolution can be followed with one drop only at a synchrotron source, a stroboscopic mode with many drops is preferable for the laboratory source.

Human HepaRG Cells can be Cultured in Hanging-drop Plates for Cytochrome P450 Induction and Function Assays.

PubMed

Murayama, Norie; Usui, Takashi; Slawny, Nicky; Chesné, Christophe; Yamazaki, Hiroshi

2015-01-01

Recent guidance/guidelines for industry recommend that cytochrome P450 induction can be assessed using human hepatocyte enzyme activity and/or mRNA levels to evaluate potential drug- drug interactions. To evaluate time-dependent cytochrome P450 induction precisely, induction of CYP1A2, CYP2B6, and CYP3A4 mRNA was confirmed (>2-fold) by the treatment with omeprazole, phenobarbital, and rifampicin, respectively, for 24 or 48 h on day 3 from the start of culture. After 24 h, the fold induction of CYP1A2 with 3.6 and 1.8x10(4) HepaRG cells per well was lower than that for 7.2x10(4) cells. CYP1A2 induction levels at 24 h were higher than those after 48 h. In contrast, higher CYP2B6 inductions were confirmed after 48 h exposure than after 24 h, independent of the number of cells per well. To help reduce the use of human cryopreserved hepatocytes, typical P450-dependent enzyme activities were investigated in human HepaRG cells cultured in commercial hanging-drop plates. Newly designed 96-well hanging-drop plates were capable of maintaining human CYP3A-dependent midazolam hydroxylation activities for up to 4 days using only 10% of the recommended initial 7.2x10(4) cells per well. Favorable HepaRG function using hanging-drop plates was confirmed by detecting 1'- hydroxymidazolam O-glucuronide on day 3, suggesting an improvement over traditional control plates in which this metabolite can be detected for 24-well plates. These results suggest that the catalytic function and/or induction of CYP1A2, CYP2B6, and CYP3A4 can be readily assessed with reduced numbers of starting HepaRG cells cultured in three-dimensional cultures in drops prepared with hanging-drop plates.

Increased sensitivity of anodic stripping voltammetry at the hanging mercury drop electrode by ultracathodic deposition.

PubMed

Rodrigues, José A; Rodrigues, Carlos M; Almeida, Paulo J; Valente, Inês M; Gonçalves, Luís M; Compton, Richard G; Barros, Aquiles A

2011-09-09

An improved approach to the anodic stripping voltammetric (ASV) determination of heavy metals, using the hanging mercury drop electrode (HMDE), is reported. It was discovered that using very cathodic accumulation potentials, at which the solvent reduction occurs (overpotential deposition), the voltammetric signals of zinc(II), cadmium(II), lead(II) and copper(II) increase. When compared with the classical methodology a 5 to 10-fold signal increase is obtained. This effect is likely due to both mercury drop oscillation at such cathodic potentials and added local convection at the mercury drop surface caused by the evolution of hydrogen bubbles. Copyright © 2011 Elsevier B.V. All rights reserved.

Effects of hanging drop culture conditions on embryoid body formation and neuronal cell differentiation using mouse embryonic stem cells: optimization of culture conditions for the formation of well-controlled embryoid bodies.

PubMed

Ohnuki, Yoshitsugu; Kurosawa, Hiroshi

2013-05-01

Hanging drop (HD) cultures were carried out with a drop volume of either 20 or 30 μl. An incubation period of 3 days was determined to be appropriate for the formation of well-controlled embryoid bodies (EBs), and the initial cell number was identified as the most critical factor in the growth and neuronal cell differentiation of EBs. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray analysis of Streptococcus mutans dextran glucosidase

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

Saburi, Wataru; Hondoh, Hironori, E-mail: hondoh@abs.agr.hokudai.ac.jp; Unno, Hideaki

2007-09-01

Dextran glucosidase from S. mutans was crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.2 Å resolution. Dextran glucosidase from Streptococcus mutans is an exo-hydrolase that acts on the nonreducing terminal α-1,6-glucosidic linkage of oligosaccharides and dextran with a high degree of transglucosylation. Based on amino-acid sequence similarity, this enzyme is classified into glycoside hydrolase family 13. Recombinant dextran glucosidase was purified and crystallized by the hanging-drop vapour-diffusion technique using polyethylene glycol 6000 as a precipitant. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 72.72, b = 86.47, cmore » = 104.30 Å. A native data set was collected to 2.2 Å resolution from a single crystal.« less

Comparison of the hanging-drop technique and running-drip method for identifying the epidural space in dogs.

PubMed

Martinez-Taboada, Fernando; Redondo, José I

2017-03-01

To compare the running-drip and hanging-drop techniques for locating the epidural space in dogs. Prospective, randomized, clinical trial. Forty-five healthy dogs requiring epidural anaesthesia. Dogs were randomized into four groups and administered epidural anaesthesia in sternal (S) or lateral (L) recumbency. All blocks were performed by the same person using Tuohy needles with either a fluid-prefilled hub (HDo) or connected to a drip set attached to a fluid bag elevated 60 cm (RDi). The number of attempts, 'pop' sensation, clear drop aspiration or fluid dripping, time to locate the epidural space (TTLES) and presence of cerebrospinal fluid (CSF) were recorded. A morphine-bupivacaine combination was injected after positive identification. The success of the block was assessed by experienced observers based on perioperative usage of rescue analgesia. Data were checked for normality. Binomial variables were analysed with the chi-squared or Fisher's exact test as appropriate. Non-parametric data were analysed using Kruskal-Wallis and Mann-Whitney tests. Normal data were studied with an anova followed by a Tukey's means comparison for groups of the same size. A p-value of < 0.05 was considered to indicate statistical significance. Lateral recumbency HDo required more attempts (six of 11 dogs required more than one attempt) than SRDi (none of 11 dogs) (p = 0.0062). Drop aspiration was observed more often in SHDo (nine of 11 dogs) than in LHDo (two of 11 dogs) (p = 0.045). Mean (range) TTLES was longer in LHDo [47 (18-82) seconds] than in SHDo [20 (14-79) seconds] (p = 0.006) and SRDi [(34 (17-53) seconds] (p = 0.038). There were no differences in 'pop' sensation, presence of CSF, rescue analgesia or pain scores between the groups. The running-drip method is a useful and fast alternative technique for identifying the epidural space in dogs. The hanging-drop technique in lateral recumbency was more difficult to perform than the other methods, requiring more time and attempts. Copyright © 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved.

Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture

PubMed Central

Schmal, Olga; Seifert, Jan; Schäffer, Tilman E.; Walter, Christina B.; Aicher, Wilhelm K.; Klein, Gerd

2016-01-01

Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard. PMID:26839560

Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture.

PubMed

Schmal, Olga; Seifert, Jan; Schäffer, Tilman E; Walter, Christina B; Aicher, Wilhelm K; Klein, Gerd

2016-01-01

Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34(+) hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard.

Self-Renewal and CSCs In Vitro Enrichment: Growth as Floating Spheres

PubMed Central

Mehta, Pooja; Novak, Caymen; Raghavan, Shreya; Ward, Maria; Mehta, Geeta

2018-01-01

Cancer stem cells (CSC) are a vital component to the progression and reoccurrence of cancers, making them a primary target of study for both fundamental understanding of cancer biology and the development of effective and targeted treatments. CSCs reside in a complex 3D microenvironment, and the 3D spheroids are an indispensable tool in tumor biology due to their 3D structure and replication of the tumor microenvironment. Within this chapter the methodology for CSC isolation, suspension culture in hanging drop model, and characterization assays for CSC are described. First, the methodology for identifying and isolating CSCs from patient tumors, ascites, or cancer cell lines is described through the use of FACS analysis. Next, a detailed description of 3D hanging drop model for generating CSC spheroids is provided, followed by maintenance and monitoring techniques for extended 3D culture. Analysis methods are described for the quantification of CSC spheroid proliferation and viability tracking, throughout culture by on-plate alamarBlue fluorescence. Additional viability assays are described utilizing confocal microscopy with Live/Dead Viability/Cytotoxicity Kit. The characterization of CSCs populations within spheroids is described through FACS analysis. Further, an immunohistochemistry procedure is described for cell-cell and cell-matrix interaction assessment. Finally, several notes and tips for successful experiments with 3D CSC spheroids on the hanging drop model are provided. These methods are not only applicable to CSCs within a variety of tumor cell types, for not only understanding the fundamental tumor biology, but also for drug screening and development of preclinical chemotherapeutic strategies. PMID:28986887

Instrumentation and Methodology for Generation of an Electrochemical Data Base for Pattern Recognition.

DTIC Science & Technology

1982-05-01

and mercury drop hang time all produced changes in cyclic differential capacity curves and -..-- DD 0A" 1473 EDITION OF 1 NOV 6S IS OBSOLETE S/N 0102...scan rate, and mercury drop hang time all produced changes in cyclic differential capacity curves and cyclic staircase voltammograms which were unique...Faradaic measurements with staircase voltammetry have been enumerated elewhere (24, 25). -4- EXPERIMENTAL Experimental Design The seven variables which

Cervical spine injuries in suicidal hanging without a long-drop--patterns and possible underlying mechanisms of injury: an autopsy study.

PubMed

Nikolić, Slobodan; Zivković, Vladimir

2014-06-01

The incidence of cervical spine injuries in suicidal hangings with a short-drop has been reported to be extremely low or non-existent. The aim of this study was to determine the frequency and pattern of cervical spine injuries in suicidal hanging. A retrospective autopsy study was performed and short-drop suicidal hanging cases with documented cervical spine injuries were identified. This group was further analyzed with regard to the gender and age of the deceased, the position of the ligature knot, the presence of hyoid-laryngeal fractures, and the level of cervical spine injury. Cervical spine injuries were present in 25 of the 766 cases, with an average age of 71.9 ± 10.7 years (range 39-88 years). In 16 of these 25 cases, the ligature knot was in the anterior position. The most common pattern of cervical spine injury included partial or complete disruption of the anterior longitudinal ligament and widening of the lower cervical spine disk spaces, associated with absence of hyoid-laryngeal fractures. Cervical spine injuries are not commonly found in short-drop suicidal hanging, occurring in only 3.3 % of all observed cases. Cervical spine injury may be occurring in 80 % of subjects aged 66.5 years and above. The most common pattern of cervical spine injury included anterior longitudinal ligament disruption of the lower cervical spine, disk space widening, and no vertebral body displacement. These injuries were mainly associated with an anterior knot position, and may be a consequence of loop pressure to the posterior neck and cervical spine hyperextension.

Hanging colloidal drop: A new photonic crystal synthesis route

NASA Astrophysics Data System (ADS)

Sandu, Ion; Dumitru, Marius; Fleaca, Claudiu Teodor; Dumitrache, Florian

2018-05-01

High-quality photonic crystals (hundreds of micrometres in thickness) were grown by the free evaporation of a colloidal drop consisting of silica and polystyrene nanospheres with dimensions of 300 nm, 500 nm, and 1000 nm. The essence of experimental findings is that the drop has to hang on a pillar. This leads to the inhibition of the droplet spreading, the minimisation of the convective force, and the zeroing of the static frictional force between nanospheres and the liquid/air interface, where the first layer is formed. The theoretical essence is the continuous adjustment of nanospheres positions during the growth of photonic crystal, a key condition of the self-assembling phenomenon.

Formation of gut-like structures in vitro from mouse embryonic stem cells.

PubMed

Torihashi, Shigeko

2006-01-01

Embryonic stem (ES) cells have the potential to differentiate into all cell types originating from the three germ layers; however, there are still few reports about the formation of functional organs from embryonic stem cells. Recently, we reported that by hanging drops of mouse ES cells, embryoid bodies (EBs) formed gut-like structures in vitro composed of three layers corresponding to the epithelium, lamina propria, and musculature. The morphological features and the process of formation are similar to gut and its organogenesis in vivo. Thus, this is a good model for development of the gut and a useful tool for analysis of the factors required for gut organogenesis. The protocol basically involves a method of hanging drops to make EBs, which are then plated on coated dishes for outgrowth. EBs develop to form gut-like structures when induced to spontaneously enter a program of differentiation in vitro without addition of any extrinsic factors.

Imprinted polymer-modified hanging mercury drop electrode for differential pulse cathodic stripping voltammetric analysis of creatine.

PubMed

Lakshmi, Dhana; Sharma, Piyush S; Prasad, Bhim B

2007-06-15

The molecularly imprinted polymer [poly(p-aminobenzoicacid-co-1,2-dichloroethane)] film casting was made on the surface of a hanging mercury drop electrode by drop-coating method for the selective and sensitive evaluation of creatine in water, blood serum and pharmaceutical samples. The molecular recognition of creatine by the imprinted polymer was found to be specific via non-covalent (electrostatic) imprinting. The creatine binding could easily be detected by differential pulse, cathodic stripping voltammetric signal at optimised operational conditions: accumulation potential -0.01 V (versus Ag/AgCl), polymer deposition time 15s, template accumulation time 60s, pH 7.1 (supporting electrolyte< or =5 x 10(-4)M NaOH), scan rate 10 mV s(-1), pulse amplitude 25 mV. The modified sensor in the present study was found to be highly reproducible and selective with detection limit 0.11 ng mL(-1) of creatine. Cross-reactivity studies revealed no response to the addition of urea, creatinine and phenylalanine; however, some insignificant magnitude of current was observed for tryptophan and histidine in the test samples.

The formation of intestinal organoids in a hanging drop culture.

PubMed

Panek, Malgorzata; Grabacka, Maja; Pierzchalska, Malgorzata

2018-01-25

Recently organoids have become widely used in vitro models of many tissue and organs. These type of structures, originated from embryonic or adult mammalian intestines, are called "mini guts". They organize spontaneously when intestinal crypts or stem cells are embedded in the extracellular matrix proteins preparation scaffold (Matrigel). This approach has some disadvantages, as Matrigel is undefined (the concentrations of growth factors and other biologically active components in it may vary from batch to batch), difficult to handle and expensive. Here we show that the organoids derived from chicken embryo intestine are formed in a hanging drop without embedding, providing an attractive alternative for currently used protocols. Using this technique we obtained compact structures composed of contiguous organoids, which were generally similar to chicken organoids cultured in Matrigel in terms of morphology and expression of intestinal epithelial markers. Due to the simplicity, high reproducibility and throughput capacity of hanging drop technique our model may be applied in various studies concerning the gut biology.

Hanging drop crystal growth apparatus

NASA Technical Reports Server (NTRS)

Naumann, Robert J. (Inventor); Witherow, William K. (Inventor); Carter, Daniel C. (Inventor); Bugg, Charles E. (Inventor); Suddath, Fred L. (Inventor)

1990-01-01

This invention relates generally to control systems for controlling crystal growth, and more particularly to such a system which uses a beam of light refracted by the fluid in which crystals are growing to detect concentration of solutes in the liquid. In a hanging drop apparatus, a laser beam is directed onto drop which refracts the laser light into primary and secondary bows, respectively, which in turn fall upon linear diode detector arrays. As concentration of solutes in drop increases due to solvent removal, these bows move farther apart on the arrays, with the relative separation being detected by arrays and used by a computer to adjust solvent vapor transport from the drop. A forward scattering detector is used to detect crystal nucleation in drop, and a humidity detector is used, in one embodiment, to detect relative humidity in the enclosure wherein drop is suspended. The novelty of this invention lies in utilizing angular variance of light refracted from drop to infer, by a computer algorithm, concentration of solutes therein. Additional novelty is believed to lie in using a forward scattering detector to detect nucleating crystallites in drop.

3D Hanging Drop Culture to Establish Prostate Cancer Organoids.

PubMed

Eder, Theresa; Eder, Iris E

2017-01-01

Three-dimensional (3D) cell culture enables the growth of cells in a multidimensional and multicellular manner compared to conventional cell culture techniques. Especially in prostate cancer research there is a big need for more tissue-recapitulating models to get a better understanding of the mechanisms driving prostate cancer as well as to screen for more efficient drugs that can be used for treatment. In this chapter we describe a 3D hanging drop system that can be used to culture prostate cancer organoids as tumor epithelial monocultures and as epithelial-stromal cocultures.

Tetrazolium chloride as an indicator of pine pollen germinability

Treesearch

Stanton A. Cook; Robert G. Stanley

1960-01-01

Controlled pollination in forest tree breeding requires pollen of known germination capacity. Methods of determining pollen viability include germination in a hanging drop, in a moist atmosphere, on agar gel, or in a sugar solution (DUFFIELD, 1954; DILLON et al., 1957). Errors commonly arise in the application of these techniques because maximum...

Crystallization and preliminary crystallographic analysis of the catechol 2,3-dioxygenase PheB from Bacillus stearothermophilus BR219

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

Sugimoto, Keisuke; Matsufuzi, Kazuki; Ohnuma, Hiroaki

2006-02-01

PheB, an extradiol-cleaving catecholic dioxygenase, was crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystal belongs to the orthorhombic system, space group P2{sub 1}2{sub 1}2{sub 1}, and diffracts to 2.3 Å resolution. Class II extradiol-cleaving catecholic dioxygenase, a key enzyme of aromatic compound degradation in bacteria, cleaves the aromatic ring of catechol by adding two O atoms. PheB is one of the class II extradiol-cleaving catecholic dioxygenases and shows a high substrate specificity for catechol derivatives, which have one aromatic ring. In order to reveal the mechanism of the substrate specificity of PheB, PheB hasmore » been crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The space group of the obtained crystal was P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 65.5, b = 119.2, c = 158.7 Å. The crystal diffracted to 2.3 Å resolution.« less

Transplantation of co-aggregates of Sertoli cells and islet cells into liver without immunosuppression.

PubMed

Takemoto, Naohiro; Liu, Xibao; Takii, Kento; Teramura, Yuji; Iwata, Hiroo

2014-02-15

Transplantation of islets of Langerhans (islets) was used to treat insulin-dependent diabetes mellitus. However, islet grafts must be maintained by administration of immunosuppressive drugs, which can lead to complications in the long term. An approach that avoids immunosuppressive drug use is desirable. Co-aggregates of Sertoli cells and islet cells from BALB/c mice that were prepared by the hanging drop method were transplanted into C57BL/6 mouse liver through the portal vein as in human clinical islet transplantation. The core part of the aggregates contained mainly Sertoli cells, and these cells were surrounded by islet cells. The co-aggregates retained the functions of both Sertoli and islet cells. When 800 co-aggregates were transplanted into seven C57BL/6 mice via the portal vein, six of seven recipient mice demonstrated quasi-normoglycemia for more than 100 days. The hanging drop method is suitable for preparing aggregates of Sertoli and islet cells for transplantation. Notably, transplantation of these allogeneic co-aggregates into mice with chemically induced diabetes via the portal vein resulted in long-term graft survival without systemic immunosuppression.

Crystallization and preliminary X-ray diffraction studies of choline-binding protein F from Streptococcus pneumoniae

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

Molina, Rafael; González, Ana; Moscoso, Miriam

2007-09-01

The modular choline-binding protein F (CbpF) from S. pneumoniae has been crystallized by the hanging-drop vapour-diffusion method. A SAD data set from a gadolinium-complex derivative has been collected to 2.1 Å resolution. Choline-binding protein F (CbpF) is a modular protein that is bound to the pneumococcal cell wall through noncovalent interactions with choline moieties of the bacterial teichoic and lipoteichoic acids. Despite being one of the more abundant proteins on the surface, along with the murein hydrolases LytA, LytB, LytC and Pce, its function is still unknown. CbpF has been crystallized using the hanging-drop vapour-diffusion method at 291 K. Diffraction-qualitymore » orthorhombic crystals belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 49.13, b = 114.94, c = 75.69 Å. A SAD data set from a Gd-HPDO3A-derivatized CbpF crystal was collected to 2.1 Å resolution at the gadolinium L{sub III} absorption edge using synchrotron radiation.« less

Linear scan voltammetric indirect determination of Al(III) by the catalytic cathodic response of norepinephrine at the hanging mercury drop electrode.

PubMed

Zhang, Fuping; Ji, Ming; Xu, Quan; Yang, Li; Bi, Shuping

2005-09-01

The biological effects of aluminum (Al) have received much attention in recent years. Al is of basic relevance as concern with its reactivity and bioavailability. In this paper, the electrochemical behaviors of norepinephrine (NE) in the absence and presence of Al(III) at the hanging mercury drop electrode have been studied and applied to the practical analysis. Highly selective catalytic cathodic peak of NE is yielded by linear scan voltammetry (LSV) at -1.32 V (vs. SCE). A linear relationship holds between the cathodic peak current and the Al(III) concentration. It has been successfully applied to the determination of Al(III) in real waters and synthetic biological samples with satisfying results, which are in accordance with those obtained by ICP-AES method. The electrochemical properties and the mechanisms of the peaks in the presence and absence of Al(III) have been explored. The results show that they are irreversible adsorptive hydrogen catalytic waves. These studies not only enrich the methods of determining Al, but also lay foundations of further understanding of the mechanisms of neurodementia.

Imaging transport phenomena during lysozyme protein crystal growth by the hanging drop technique

NASA Astrophysics Data System (ADS)

Sethia Gupta, Anamika; Gupta, Rajive; Panigrahi, P. K.; Muralidhar, K.

2013-06-01

The present study reports the transport process that occurs during the growth of lysozyme protein crystals by the hanging drop technique. A rainbow schlieren technique has been employed for imaging changes in salt concentration. A one dimensional color filter is used to record the deflection of the light beam. An optical microscope and an X-ray crystallography unit are used to characterize the size, tetragonal shape and Bravais lattice constants of the grown crystals. A parametric study on the effect of drop composition, drop size, reservoir height and number of drops on the crystal size and quality is reported. Changes in refractive index are not large enough to create a meaningful schlieren image in the air gap between the drop and the reservoir. However, condensation of fresh water over the reservoir solution creates large changes in the concentration of NaCl, giving rise to clear color patterns in the schlieren images. These have been analyzed to obtain salt concentration profiles near the free surface of the reservoir solution as a function of time. The diffusion of fresh water into the reservoir solution at the early stages of crystal growth followed by the mass flux of salt from the bulk solution towards the free surface has been recorded. The overall crystal growth process can be classified into two regimes, as demarcated by the changes in slope of salt concentration within the reservoir. The salt concentration in the reservoir equilibrates at long times when the crystallization process is complete. Thus, transport processes in the reservoir emerge as the route to monitor protein crystal growth in the hanging drop configuration. Results show that crystal growth rate is faster for a higher lysozyme concentration, smaller drops, and larger reservoir heights.

Life-History Traits of the Model Organism Pristionchus pacificus Recorded Using the Hanging Drop Method: Comparison with Caenorhabditis elegans.

PubMed

Gilarte, Patricia; Kreuzinger-Janik, Bianca; Majdi, Nabil; Traunspurger, Walter

2015-01-01

The nematode Pristionchus pacificus is of growing interest as a model organism in evolutionary biology. However, despite multiple studies of its genetics, developmental cues, and ecology, the basic life-history traits (LHTs) of P. pacificus remain unknown. In this study, we used the hanging drop method to follow P. pacificus at the individual level and thereby quantify its LHTs. This approach allowed direct comparisons with the LHTs of Caenorhabditis elegans recently determined using this method. When provided with 5×10(9) Escherichia coli cells ml(-1) at 20°C, the intrinsic rate of natural increase of P. pacificus was 1.125 (individually, per day); mean net production was 115 juveniles produced during the life-time of each individual, and each nematode laid an average of 270 eggs (both fertile and unfertile). The mean age of P. pacificus individuals at first reproduction was 65 h, and the average life span was 22 days. The life cycle of P. pacificus is therefore slightly longer than that of C. elegans, with a longer average life span and hatching time and the production of fewer progeny.

Media additives to promote spheroid circularity and compactness in hanging drop platform.

PubMed

Leung, Brendan M; Lesher-Perez, Sasha Cai; Matsuoka, Toshiki; Moraes, Christopher; Takayama, Shuichi

2015-02-01

Three-dimensional spheroid cultures have become increasingly popular as drug screening platforms, especially with the advent of different high throughput spheroid forming technologies. However, comparing drug efficacy across different cell types in spheroid culture can be difficult due to variations in spheroid morphologies and transport characteristics. Improving the reproducibility of compact, circular spheroids contributes to standardizing and increasing the fidelity of the desired gradient profiles in these drug screening three-dimensional tissue cultures. In this study we discuss the role that circularity and compaction has on spheroids, and demonstrate the impact methylcellulose (MethoCel) and collagen additives in the culture media can contribute to more compact and circular spheroid morphology. We demonstrate that improved spheroid formation is not a simple function of increased viscosity of the different macromolecule additives, suggesting that other macromolecular characteristics contribute to improved spheroid formation. Of the various macromolecular additives tested for hanging drop culture, MethoCel provided the most desirable spheroid formation. Additionally, the higher viscosity of MethoCel-containing media improved the ease of imaging of cellular spheroids within hanging drop cultures by reducing motion-induced image blur.

High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array.

PubMed

Tung, Yi-Chung; Hsiao, Amy Y; Allen, Steven G; Torisawa, Yu-suke; Ho, Mitchell; Takayama, Shuichi

2011-02-07

Culture of cells as three-dimensional (3D) aggregates can enhance in vitro tests for basic biological research as well as for therapeutics development. Such 3D culture models, however, are often more complicated, cumbersome, and expensive than two-dimensional (2D) cultures. This paper describes a 384-well format hanging drop culture plate that makes spheroid formation, culture, and subsequent drug testing on the obtained 3D cellular constructs as straightforward to perform and adapt to existing high-throughput screening (HTS) instruments as conventional 2D cultures. Using this platform, we show that drugs with different modes of action produce distinct responses in the physiological 3D cell spheroids compared to conventional 2D cell monolayers. Specifically, the anticancer drug 5-fluorouracil (5-FU) has higher anti-proliferative effects on 2D cultures whereas the hypoxia activated drug commonly referred to as tirapazamine (TPZ) are more effective against 3D cultures. The multiplexed 3D hanging drop culture and testing plate provides an efficient way to obtain biological insights that are often lost in 2D platforms.

Crystallization, optimization and preliminary X-ray characterization of a metal-dependent PI-PLC from Streptomyces antibioticus

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

Jackson, Michael R.; Selby, Thomas L.

2012-10-30

A recombinant metal-dependent phosphatidylinositol-specific phospholipase C (PI-PLC) fromStreptomyces antibioticushas been crystallized by the hanging-drop method with and without heavy metals. The native crystals belonged to the orthorhombic space groupP222, with unit-cell parametersa= 41.26,b= 51.86,c = 154.78 Å. The X-ray diffraction results showed significant differences in the crystal quality of samples soaked with heavy atoms. Additionally, drop pinning, which increases the surface area of the drops, was also used to improve crystal growth and quality. The combination of heavy-metal soaks and drop pinning was found to be critical for producing high-quality crystals that diffracted to 1.23 Å resolution.

Comparative study of P19 EC stem cell differentiation in between conventional hanging drop and the zebrafish chorion as a bio-derived material.

PubMed

Dae Seok Na; Lee, Hwang; Sun Uk Kim; Chang Nam Hwang; Sang Ho Lee; Ji Yoon Kang; Jai Kyeong Kim; James Jungho Pak

2008-07-01

Various materials including glass and polymers have been widely used for stem cell culture due to their biocompatibility. However, the roles of these materials are fundamentally limited because they cannot realize or imitate the complex biological functions of living tissues, except in very simple cases. Here, the development of a bio-derived material suitable for stem cell culture and improvement of differentiation efficiency to specific cell lineages with no stimulating agents by using a chorion obtained from a fertilized zebrafish egg through the removal of the yolk and embryonic cell mass from the egg is reported. Mouse P19 EC stem cells introduced into the empty chorion form a uniform embryoid body (EB) without addition of any inducing agent. It is demonstrated that the zebrafish chorion with nanopores improves efficiencies greatly in the EB formation, cell proliferation, and lineage-specific differentiations compared to those of the conventional hanging drop culture method.

Electrochemical behavior of phytochelatins and related peptides at the hanging mercury drop electrode in the presence of cobalt(II) ions.

PubMed

Dorcák, Vlastimil; Sestáková, Ivana

2006-01-01

Direct current voltammetry and differential pulse voltammetry have been used to investigate the electrochemical behaviour of two phytochelatins: heptapeptide (gamma-Glu-Cys)3-Gly and pentapeptide (gamma-Glu-Cys)2-Gly, tripeptide glutathione gamma-Glu-Cys-Gly and its fragments: dipeptides Cys-Gly and gamma-Glu-Cys at the hanging mercury drop electrode in the presence of cobalt(II) ions. Most interesting results were obtained with direct current voltammetry in the potential region of -0.80 V up to -1.80 V. Differential pulse voltammetry of the same solutions of Co(II) with peptides gives more complicated voltammograms with overlapping peaks, probably in connection with the influence of adsorption at slow scan rates necessarily used in this method. However, in using Brdicka catalytic currents for analytical purposes, differential pulse voltammograms seem to be more helpful. Presented investigations have shown that particularly the prewave of cobalt(II) allows distinguishing among phytochelatins, glutathione, and its fragments.

A morphological screening of protein crystals for interferon delivery by metal ion-chelate technology.

PubMed

Jiang, Yanbo; Shi, Kai; Wang, Shuo; Li, Xuefeng; Cui, Fude

2010-12-01

This study presents a preliminary exploration on extending the half-life of therapeutic proteins by crystallization strategy without new molecular entities generation. Recombinant human interferon (rhIFN) α-2b, a model protein drug in this case, was crystallized using a hanging-drop vapor diffusion method. A novel chelating technique with metal ions was employed to promote crystals formation. The effects of key factors such as seeding protein concentration, pH of the hanging drop, ionic strength of the equilibration solution, and precipitants were investigated. Size-exclusion liquid chromatography, antiviral activity determination, and enzyme-linked immunosorbent assay indicated that both the molecular integrity and biological potency of rhIFN were not significantly affected by crystallization process. In addition, the in vitro release behavior of rhIFN from crystal lattice was characterized by an initial fast release, followed by a sustained release up to 48 hour. The work described here suggested an exciting possibility of therapeutic protein crystals as a long-acting formulation.

Comparison of the lateral retention forces on sessile and pendant water drops on a solid surface

NASA Astrophysics Data System (ADS)

de la Madrid, Rafael; Whitehead, Taylor; Irwin, George M.

2015-06-01

We present a simple experiment that demonstrates how a water drop hanging from a Plexiglas surface (pendant drop) experiences a lateral retention force that is comparable to, and in some cases larger than, the lateral retention force on a drop resting on top of the surface (sessile drop). The experiment also affords a simple demonstration of the Coriolis effect in two dimensions.

Comparison of spring-loaded, loss of resistance and hanging drop techniques in lumbar epidural blocks.

PubMed

Gülen, Güven; Akkaya, Taylan; Ozkan, Derya; Kaydul, Mehmet; Gözaydin, Orhan; Gümüş, Haluk

2012-01-01

The spring-loaded syringe is a loss of resistance syringe that provide a more objective sign that the epidural space has been entered compared with the traditional techniques. The aim of this study was to compare the time required to locate the epidural space and the backache incidence with the spring-loaded (SL), loss of resistance (LOR) and the hanging drop (HD) techniques for epidural blocks in patients undergoing transurethral resection procedure. Sixty patients undergoing transurethral resections were enrolled in the study. The patients were randomly assigned to one of three groups. Epidural block was performed in the first group with a spring-loaded syringe (n=20), in the second group with loss-of-resistance syringe (n=20), and in the third group with the hanging drop technique (n=20). The required time to locate the epidural space, the number of attempts, the incidence of dural puncture and the backache incidence were assessed during the procedure and for four weeks after the procedure in all patients. The required time to locate the epidural space was 29.1 ± 9.16 seconds in Group 1; 45.25 ± 19.58 seconds in Group 2, and 47.35 ± 11.42 seconds in Group 3 (p<0.001). In Group 1 this was significantly shorter than the other two groups. There was no significant difference in the number of attempts, the incidence of dural puncture and backache incidence between the three groups (p>0.05). The use of SL syringe was found to have a shorter time period to locate the epidural space when compared with the LOR syringe and hanging drop technique.

Retrospective Analysis of Hanging Deaths in Ontario.

PubMed

Tugaleva, Elena; Gorassini, Donald R; Shkrum, Michael J

2016-11-01

Hanging deaths from investigation standpoint are rarely problematic. Unusual circumstances can on occasion raise suspicion of foul play. Associated neck injuries are reported in the literature with variable frequency (from 0% to 76.8%). This study retrospectively analyzed 755 hanging deaths in Ontario (Canada) to evaluate the demographic features and circumstances of hanging fatalities, and the frequency of hanging-related neck injuries. A number of cases showed unusual/special circumstances (e.g., complex, double suicides, restraints). Among 632 cases with complete autopsies, hyoid and larynx fractures were present in 46 cases (7.3%) with the most common being isolated hyoid fractures. The incidence of cricoid fractures was 0.5% and cervical spine injuries, 1.1%. A higher incidence of neck injuries occurred among males, long drop hangings, and in cases with complete suspension. There was a tendency for the number of fractures to increase with increasing age and weight of the deceased. © 2016 American Academy of Forensic Sciences.

Suicidal Decapitation by Hanging-A Population-based Study.

PubMed

Byard, Roger W; Gilbert, John D

2018-05-01

A prospective study was undertaken at Forensic Science SA over a 15-year period from July 2002 to June 2017 for all cases of adult (>18 years) suicidal hangings with decapitation. A total of 1446 cases of suicidal hangings were identified from a general population of approximately 1.5 million (1206 males-age range 18-97 years, average 42.6; and 240 females-age range 18-96 years, average 40.1). Only three cases of decapitation were found, all from long-drop hangings; these consisted of three males (ages 32-55 years; average 45 years). Spinal transections had occurred between the first and second, second and third, and third and fourth cervical vertebrae, respectively. In this study, the number of suicidal hangings with decapitation represented only 0.2% of the total number of hangings. These events are therefore extremely rare, most likely due to most suicidal hangings occurring from relatively low levels in a domestic environment. © 2017 American Academy of Forensic Sciences.

Inhibition of hexokinase-2 with targeted liposomal 3-bromopyruvate in an ovarian tumor spheroid model of aerobic glycolysis

PubMed Central

Gandham, Srujan Kumar; Talekar, Meghna; Singh, Amit; Amiji, Mansoor M

2015-01-01

Background The objective of this study was to evaluate the expression levels of glycolytic markers, especially hexokinase-2 (HK2), using a three-dimensional multicellular spheroid model of human ovarian adenocarcinoma (SKOV-3) cells and to develop an epidermal growth factor receptor-targeted liposomal formulation for improving inhibition of HK2 and the cytotoxicity of 3-bromopyruvate (3-BPA). Methods Multicellular SKOV-3 tumor spheroids were developed using the hanging drop method and expression levels of glycolytic markers were examined. Non-targeted and epidermal growth factor receptor-targeted liposomal formulations of 3-BPA were formulated and characterized. Permeability and cellular uptake of the liposomal formulations in three-dimensional SKOV-3 spheroids was evaluated using confocal microscopy. The cytotoxicity and HK2 inhibition potential of solution form of 3-BPA was compared to the corresponding liposomal formulation by using cell proliferation and HK2 enzymatic assays. Results SKOV-3 spheroids were reproducibly developed using the 96-well hanging drop method, with an average size of 900 µm by day 5. HK2 enzyme activity levels under hypoxic conditions were found to be higher than under normoxic conditions (P<0.0001, Student’s t-test, unpaired and two-tailed). Liposomal formulations (both non-targeted and targeted) of 3-BPA showed a more potent inhibitory effect (P<0.001, Student’s t-test, unpaired and two-tailed) at a dose of 50 µM than the aqueous solution form at 3, 6, and 24 hours post administration. Similarly, the cytotoxic activity 3-BPA at various concentrations (10 µM–100 µM) showed that the liposomal formulations had an enhanced cytotoxic effect of 2–5-fold (P<0.0001, Student’s t-test, unpaired and two-tailed) when compared to the aqueous solution form for both 10 µM and 25 µM concentrations. Conclusion SKOV-3 spheroids developed by the hanging drop method can be used as a tumor aerobic glycolysis model for evaluation of therapies targeting the glycolytic pathway in cancer cells. Encapsulation of 3-BPA in a liposomal formulation improved permeability, HK2 inhibition, and cytotoxicity in the multicellular spheroid model. PMID:26185443

Purification and crystallization of Kokobera virus helicase

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

De Colibus, Luigi; Speroni, Silvia; Coutard, Bruno

2007-03-01

Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method and exhibit a diffraction limit of 2.3 Å. Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. The flavivirus genus is characterized by a positive-sense single-stranded RNA genome. The unique open reading frame of the viral RNA is transcribed and translated as a single polyprotein which is post-translationally cleaved to yield three structural and seven nonstructural proteins, one of which ismore » the NS3 gene that encodes a C-terminal helicase domain consisting of 431 amino acids. Helicase inhibitors are potential antiviral drugs as the helicase is essential to viral replication. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P3{sub 1}21 (or P3{sub 2}21), with unit-cell parameters a = 88.6, c = 138.6 Å, and exhibit a diffraction limit of 2.3 Å.« less

Study of the voltammetric behaviour of the ethalfluralin and methalpropalin and its determination in environmental matrices at hanging mercury drop electrode.

PubMed

Thriveni, T; Kumar, J Rajesh; Lee, Jin Young; Sreedhar, N Y

2009-04-01

An electroanalytical method has been developed for the determination of the herbicides ethalfluralin[N-ethyl-N-(2-methyl-2-propenyl)-2,6-dinitro-4-(trifluoromethyl) bezenamine] and methalpropalin [N-(2-methyl-2-propenyl)-2, 6-dinitro-N-propyl-4 (trifluoromethyl) benzenamine] by differential pulse adsorptive stripping voltammetry (DP-AdSV) on a hanging mercury drop electrode (HMDE) with universal buffer as supporting electrolyte. The optimum adsorption conditions were found to be pH 6.0, an accumulation potential of -0.6 V (HMDE vs SCE), an accumulation time of 80 s. and scan rate 45 mVs(-1). Calibration curve is linear in the range 1.30 x 10(-9) to 1.32 x 10(-5) M of ethalfluralin and 1.13 x 10(-5) to 2.0 x 10(-8) M of methalpropalin with detection limits of 1.08 x 10(-9) and 1.87 x 10(-8) M, respectively. The relative SD and correlation coefficients were found to be 1.24%, 0.998 and 1.34%, 0.995, respectively for ten replicates. The method is applied to the determination of the ethalfluralin and methalpropalin in formulations and environmental matrices.

Expression, purification, crystallization and preliminary X-ray analysis of the ligand-binding domain of metabotropic glutamate receptor 7

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

Muto, Takanori; Tsuchiya, Daisuke; Morikawa, Kosuke, E-mail: morikako@protein.osaka-u.ac.jp

2007-07-01

The ligand-binding domain of metabotropic glutamate receptor 7 has been overexpressed, purified, and crystallized by the hanging-drop vapour-diffusion method. A complete data set has been collected to 3.30 Å. Glutamate is the major excitatory neurotransmitter and its metabotropic glutamate receptor (mGluR) plays an important role in the central nervous system. The ligand-binding domain (LBD) of mGluR subtype 7 (mGluR7) was produced using the baculovirus expression system and purified from the culture medium. The purified protein was characterized by gel-filtration chromatography, SDS–PAGE and a ligand-binding assay. Crystals of mGluR7 LBD were grown at 293 K by the hanging-drop vapour-diffusion method. Themore » crystals diffracted X-rays to 3.30 Å resolution using synchrotron radiation and belong to the trigonal space group P3{sub 1}21, with unit-cell parameters a = b = 92.4, c = 114.3 Å. Assuming the presence of one protomer per crystallographic asymmetric unit, the Matthews coefficient V{sub M} was calculated to be 2.5 Å{sup 3} Da{sup −1} and the solvent content was 51%.« less

Crystallization and X-ray analysis of the salmon-egg lectin SEL24K

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

Murata, Kenji; Fisher, Andrew J.; Hedrick, Jerry L., E-mail: jlhedrick@ucdavis.edu

2007-05-01

The 24 kDa egg lectin of Chinook salmon (Oncorhynchus tshawytscha) was purified by affinity chromatography from salmon eggs and crystallized by the hanging-drop vapor-diffusion method using 15/4 EO/OH (pentaerythritol ethoxylate) as a precipitant. The 24 kDa egg lectin of Chinook salmon (Oncorhynchus tshawytscha) is released from the egg during the cortical reaction. The lectin functions in blocking polyspermy during the fertilization process. The egg lectin was purified by affinity chromatography from salmon eggs and crystallized by the hanging-drop vapor-diffusion method using 15/4 EO/OH (pentaerythritol ethoxylate) as a precipitant. The crystal diffracted synchrotron-radiation X-rays to 1.63 Å resolution. The crystal belongsmore » to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 93.0, b = 73.6, c = 113.6 Å, α = 90, β = 92.82, γ = 90°. The crystal is likely to contain eight molecules in the asymmetric unit (V{sub M} = 2.3 Å{sup 3} Da{sup −1}), corresponding to a solvent content of 45.5%. A self-rotation function suggests an arrangement with 222 point symmetry within the asymmetric unit.« less

Crystallization and preliminary crystallographic analysis of orotidine 5′-monophosphate decarboxylase from the human malaria parasite Plasmodium falciparum

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

Krungkrai, Sudaratana R.; Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871; Tokuoka, Keiji

Orotidine 5′-monophosphate decarboxylase of human malaria parasite P. falciparum was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation. Orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC; EC 4.1.1.23) catalyzes the final step in the de novo synthesis of uridine 5′-monophosphate (UMP) and defects in the enzyme are lethal in the malaria parasite Plasmodium falciparum. Active recombinant P. falciparum OMPDC (PfOMPDC) was crystallized by the seeding method in a hanging drop using PEG 3000 asmore » a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation at the Swiss Light Source. The crystal exhibits trigonal symmetry (space group R3), with hexagonal unit-cell parameters a = b = 201.81, c = 44.03 Å. With a dimer in the asymmetric unit, the solvent content is 46% (V{sub M} = 2.3 Å{sup 3} Da{sup −1})« less

Laser scattering in a hanging drop vapor diffusion apparatus for protein crystal growth in a microgravity environment

NASA Technical Reports Server (NTRS)

Casay, G. A.; Wilson, W. W.

1992-01-01

One type of hardware used to grow protein crystals in the microgravity environment aboard the U.S. Space Shuttle is a hanging drop vapor diffusion apparatus (HDVDA). In order to optimize crystal growth conditions, dynamic control of the HDVDA is desirable. A critical component in the dynamically controlled system is a detector for protein nucleation. We have constructed a laser scattering detector for the HDVDA capable of detecting the nucleation stage. The detector was successfully tested for several scatterers differing in size using dynamic light scattering techniques. In addition, the ability to detect protein nucleation using the HDVDA was demonstrated for lysozyme.

Hanging drop cultures of human testis and testis cancer samples: a model used to investigate activin treatment effects in a preserved niche.

PubMed

Jørgensen, A; Young, J; Nielsen, J E; Joensen, U N; Toft, B G; Rajpert-De Meyts, E; Loveland, K L

2014-05-13

Testicular germ cell tumours of young adults, seminoma or non-seminomas, are preceded by a pre-invasive precursor, carcinoma in situ (CIS), understood to arise through differentiation arrest of embryonic germ cells. Knowledge about the malignant transformation of germ cells is currently limited by the lack of experimental models. The aim of this study was to establish an experimental tissue culture model to maintain normal and malignant germ cells within their niche and allow investigation of treatment effects. Human testis and testis cancer specimens from orchidectomies were cultured in 'hanging drops' and effects of activin A and follistatin treatment were investigated in seminoma cultures. Testis fragments with normal spermatogenesis or CIS cells were cultured for 14 days with sustained proliferation of germ cells and CIS cells and without increased apoptosis. Seminoma cultures survived 7 days, with proliferating cells detectable during the first 5 days. Activin A treatment significantly reduced KIT transcript and protein levels in seminoma cultures, thereby demonstrating a specific treatment response. Hanging drop cultures of human testis and testis cancer samples can be employed to delineate mechanisms governing growth of normal, CIS and tumorigenic germ cells retained within their niche.

Formation of stable small cell number three-dimensional ovarian cancer spheroids using hanging drop arrays for preclinical drug sensitivity assays

PubMed Central

Raghavan, Shreya; Ward, Maria R.; Rowley, Katelyn R.; Wold, Rachel M.; Takayama, Shuichi; Buckanovich, Ronald J.; Mehta, Geeta

2015-01-01

Background Ovarian cancer grows and metastasizes from multicellular spheroidal aggregates within the ascites fluid. Multicellular tumor spheroids are therefore physiologically significant3Din vitro models for ovarian cancer research. Conventional hanging drop cultures require high starting cell numbers, and are tedious for long-term maintenance. In this study, we generate stable, uniform multicellular spheroids using very small number of ovarian cancer cells in a novel 384 well hanging drop array platform. Methods We used novel tumor spheroid platform and two ovarian cancer cell lines (A2780 and OVCAR3) to demonstrate the stable incorporation of as few as 10 cells into a single spheroid. Results Spheroids had uniform geometry, with projected areas (42.60 × 103 μm–475.22 × 103 μm2 for A2780 spheroids and 37.24 × 103 μm2–281.01 × 103 μm2 for OVCAR3 spheroids) that varied as a function of the initial cell seeding density. Phalloidin and nuclear stains indicated cells formed tightly packed spheroids with demarcated boundaries and cell–cell interaction within spheroids. Cells within spheroids demonstrated over 85% viability. 3D tumor spheroids demonstrated greater resistance (70–80% viability) to cisplatin chemotherapy compared to 2D cultures (30–50% viability). Conclusions Ovarian cancer spheroids can be generated from limited cell numbers in high throughput 384 well plates with high viability. Spheroids demonstrate therapeutic resistance relative to cells in traditional 2D culture. Stable incorporation of low cell numbers is advantageous when translating this research to rare patient-derived cells. This system can be used to understand ovarian cancer spheroid biology, as well as carry out preclinical drug sensitivity assays. PMID:25913133

Crystallization and preliminary X-ray crystallographic analysis of BxlE, a xylobiose transporter from Streptomyces thermoviolaceus OPC-520

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

Seike, Kiho; Sato, Junji; Tomoo, Koji, E-mail: tomoo@gly.oups.ac.jp

2007-07-01

To clarify the structural basis of sugar binding by BxlE at the atomic level, recombinant BxlE was crystallized using the hanging-drop vapour-diffusion method at 290 K. Together with the integral membrane proteins BxlF and BxlG, BxlE isolated from Streptomyces thermoviolaceus OPC-520 forms an ATP-binding cassette (ABC) transport system that mediates the uptake of xylan. To clarify the structural basis of sugar binding by BxlE at the atomic level, recombinant BxlE was crystallized using the hanging-drop vapour-diffusion method at 290 K. The crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 44.63, b = 63.27, cmore » = 66.40 Å, β = 103.05°, and contained one 48 kDa molecule per asymmetric unit (V{sub M} = 1.96 Å{sup 3} Da{sup −1}). Diffraction data collected to a resolution of 1.65 Å using a rotating-anode X-ray source gave a data set with an overall R{sub merge} of 2.6% and a completeness of 91.3%. A data set from a platinum derivative is being used for phasing by the SAD method.« less

Transport phenomena in the crystallization of lysozyme by osmotic dewatering and liquid-liquid diffusion in low gravity

NASA Technical Reports Server (NTRS)

Todd, Paul; Sportiello, Michael G.; Gregory, Derek; Cassanto, John M.; Alvarado, Ulises A.; Ostroff, Robert; Korszun, Z. R.

1993-01-01

Two methods of protein crystallization, osmotic dewatering and liquid-liquid diffusion, like the vapor diffusion (hanging-drop and sessile-drop) methods allow a gradual approach to supersaturation conditions. The crystallization of hen egg-white lysozyme, an extensively characterized protein crystal, in the presence of sodium chloride was used as an experimental model with which to compare these two methods in low gravity and in the laboratory. Comparisons of crystal growth rates by the two methods under the two conditions have, to date, indicated that the rate of crystal growth by osmotic dewatering is nearly the same in low gravity and on the ground, while much faster crystal growth rates can be achieved by the liquid-liquid diffusion method in low gravity.

Inhibition of hexokinase-2 with targeted liposomal 3-bromopyruvate in an ovarian tumor spheroid model of aerobic glycolysis.

PubMed

Gandham, Srujan Kumar; Talekar, Meghna; Singh, Amit; Amiji, Mansoor M

2015-01-01

The objective of this study was to evaluate the expression levels of glycolytic markers, especially hexokinase-2 (HK2), using a three-dimensional multicellular spheroid model of human ovarian adenocarcinoma (SKOV-3) cells and to develop an epidermal growth factor receptor-targeted liposomal formulation for improving inhibition of HK2 and the cytotoxicity of 3-bromopyruvate (3-BPA). Multicellular SKOV-3 tumor spheroids were developed using the hanging drop method and expression levels of glycolytic markers were examined. Non-targeted and epidermal growth factor receptor-targeted liposomal formulations of 3-BPA were formulated and characterized. Permeability and cellular uptake of the liposomal formulations in three-dimensional SKOV-3 spheroids was evaluated using confocal microscopy. The cytotoxicity and HK2 inhibition potential of solution form of 3-BPA was compared to the corresponding liposomal formulation by using cell proliferation and HK2 enzymatic assays. SKOV-3 spheroids were reproducibly developed using the 96-well hanging drop method, with an average size of 900 µm by day 5. HK2 enzyme activity levels under hypoxic conditions were found to be higher than under normoxic conditions (P<0.0001, Student's t-test, unpaired and two-tailed). Liposomal formulations (both non-targeted and targeted) of 3-BPA showed a more potent inhibitory effect (P<0.001, Student's t-test, unpaired and two-tailed) at a dose of 50 µM than the aqueous solution form at 3, 6, and 24 hours post administration. Similarly, the cytotoxic activity 3-BPA at various concentrations (10 µM-100 µM) showed that the liposomal formulations had an enhanced cytotoxic effect of 2-5-fold (P<0.0001, Student's t-test, unpaired and two-tailed) when compared to the aqueous solution form for both 10 µM and 25 µM concentrations. SKOV-3 spheroids developed by the hanging drop method can be used as a tumor aerobic glycolysis model for evaluation of therapies targeting the glycolytic pathway in cancer cells. Encapsulation of 3-BPA in a liposomal formulation improved permeability, HK2 inhibition, and cytotoxicity in the multicellular spheroid model.

First steps towards the successful surface-based cultivation of human embryonic stem cells in hanging drop systems.

PubMed

Schulz, Julia C; Stumpf, Patrick S; Katsen-Globa, Alisa; Sachinidis, Agapios; Hescheler, Jürgen; Zimmermann, Heiko

2012-11-01

Miniaturization and parallelization of cell culture procedures are in focus of research in order to develop test platforms with low material consumption and increased standardization for toxicity and drug screenings. The cultivation in hanging drops (HDs) is a convenient and versatile tool for biological applications and represents an interesting model system for the screening applications due to its uniform shape, the advantageous gas supply, and the small volume. However, its application has so far been limited to non-adherent and aggregate forming cells. Here, we describe for the first time the proof-of-principle regarding the adherent cultivation of human embryonic stem cells in HD. For this microcarriers were added to the droplet as dynamic cultivation surfaces resulting in a maintained pluripotency and proliferation capacity for 10 days. This enables the HD technique to be extended to the cultivation of adherence-dependent stem cells. Also, the possible automation of this method by implementation of liquid handling systems opens new possibilities for miniaturized screenings, the improvement of cultivation and differentiation conditions, and toxicity and drug development.

Preparation of anti-inflammatory mesenchymal stem/precursor cells (MSCs) through sphere formation using hanging-drop culture technique.

PubMed

Bartosh, Thomas J; Ylostalo, Joni H

2014-02-06

Herein, we describe a protocol for preparation of pre-activated anti-inflammatory human mesenchymal stem/precursor cells (MSCs) in 3-D culture without addition of exogenous chemicals or gene-transfer approaches. MSCs are an easily procurable source of multipotent adult stem cells with therapeutic potential largely attributed to their paracrine regulation of inflammation and immunity. However, the culture conditions to prepare the ideal MSCs for cell therapy remain elusive. Furthermore, the reported lag time for activation in experimental models has prompted investigations on pre-activating the cells prior to their administration. In this protocol, standard 2-D culture-expanded MSCs are activated by aggregation into 3-D spheres using hanging-drop cultures. MSC activation is evaluated by real-time PCR and/or ELISA for anti-inflammatory factors (TSG-6, STC-1, PGE2), and by a functional assay using lipopolysaccharide-stimulated macrophage cultures. Further, we elucidate methods to prepare MSC-sphere conditioned medium, intact spheres, and suspension of single cells from spheres for experimental and clinical applications. Copyright © 2014 John Wiley & Sons, Inc.

First steps towards the successful surface-based cultivation of human embryonic stem cells in hanging drop systems

PubMed Central

Schulz, Julia C; Stumpf, Patrick S; Katsen-Globa, Alisa; Sachinidis, Agapios; Hescheler, Jürgen; Zimmermann, Heiko

2012-01-01

Miniaturization and parallelization of cell culture procedures are in focus of research in order to develop test platforms with low material consumption and increased standardization for toxicity and drug screenings. The cultivation in hanging drops (HDs) is a convenient and versatile tool for biological applications and represents an interesting model system for the screening applications due to its uniform shape, the advantageous gas supply, and the small volume. However, its application has so far been limited to non‐adherent and aggregate forming cells. Here, we describe for the first time the proof-of-principle regarding the adherent cultivation of human embryonic stem cells in HD. For this microcarriers were added to the droplet as dynamic cultivation surfaces resulting in a maintained pluripotency and proliferation capacity for 10 days. This enables the HD technique to be extended to the cultivation of adherence-dependent stem cells. Also, the possible automation of this method by implementation of liquid handling systems opens new possibilities for miniaturized screenings, the improvement of cultivation and differentiation conditions, and toxicity and drug development. PMID:23486530

Preparation of anti-inflammatory mesenchymal stem/precursor cells (MSCs) through sphere formation using hanging drop culture technique

PubMed Central

Bartosh, Thomas J.

2014-01-01

Herein, we describe a protocol for preparation of pre-activated anti-inflammatory human mesenchymal stem/precursor cells (MSCs) in 3D culture without addition of exogenous chemicals or gene transfer approaches. MSCs are an easily procurable source of multipotent adult stem cells with therapeutic potential largely attributed to their paracrine regulation of inflammation and immunity. However, the culture conditions to prepare the ideal MSCs for cell therapy remain elusive. Furthermore, reported lag time for activation in experimental models have prompted investigations to pre-activate the cells prior to their administration. In this protocol, standard 2D culture expanded MSCs are activated by aggregation into 3D spheres using hanging drop cultures. MSC activation is evaluated by real-time PCR and/or ELISA for anti-inflammatory factors (TSG-6, STC-1, PGE2), and by a functional assay using lipopolysaccharide-stimulated macrophage cultures. Furthermore, we elucidate methods to prepare MSC sphere conditioned medium, intact spheres, and suspension of single cells from spheres for experimental and clinical applications. PMID:24510769

Isolation, expansion, and differentiation of goat adipose-derived stem cells.

PubMed

Ren, Yu; Wu, Haiqing; Zhou, Xueyuan; Wen, Jianxun; Jin, Muzi; Cang, Ming; Guo, Xudong; Wang, Qinglian; Liu, Dongjun; Ma, Yuzhen

2012-08-01

A goat adipose-derived stem cell (ADSC) line was established and compared to a rat line. Goat ADSC cells had normal diploidy after subculture. Proliferation of goat ADSCs was faster than rat cells in the same conditions. Both rat and goat ADSCs stained positively for vimentin, CD49d, CD44 and CD13, but stained negatively for CD34 and CD106. Bone nodules were apparent, and alizarin staining was positive after osteogenic induction. Cells expressing osteocalcin were positive by alkaline phosphatase (ALP) staining. After osteogenic induction, ossification nodules of goat ADSCs were larger than in rats, with dense ALP staining. Adipogenic induction resulting in lipid droplets and peroxisome proliferator-activated receptor (PPARγ2) expression were observed. Cartilage lacunae were formed and COL2A1 was expressed. More cartilage lacunae with better morphology were seen following differentiation of goat ADSC's using the hang-drop method. For goat ADSCs, results with both adherent-induced and hanging-drop induced cultures were better than for three-dimensional cultures. Copyright © 2012. Published by Elsevier India Pvt Ltd.

X-RAY INDUCED INCORPORATION OF TRITIATED THYMIDINE INTO GRASSHOPPER NEUROBLAST CHROMOSOMES

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

McGrath, R.A.

1962-01-01

Neuroblasts of the grasshopper Chortophage viridifasciata (De Geer) were used to study incorporation of tritiated thymidine (H/sub 3/Thd), a deoxyribonucleic acid (DNA) precursor, into chromosomes. Embryos were made into hanging drop cultures which contained H/sub 3/Thd and exposed to 32 or 250 r of x rays. Individual cells were identified and observed by bright field microscopy for periods of time which ranged from 15 min to 6 hrs after irradiation. At the end of the observation period embryos were either sectioned or made into squash preparations. In the former case preselected neuroblasts were reidentified: in the latter they were not.more » Results of observations of living neuroblasts in hanging drop cultures are presented. Autoradiograms were prepared for the assay of H/sub 3/Thd incorporation by grain counting methods. Information obtained from the autoradiograms is discussed. Results are interpreted as a reflection of repair of x-ray-damaged DNA. A correlation is suggested between detectable chromosome breakage, the normal DNA synthesis period, and labeling of delayed, stopped or reverted prophase neuroblasts. (M.P.G.)« less

A comparison between protein crystals grown with vapor diffusion methods in microgravity and protein crystals using a gel liquid-liquid diffusion ground-based method

NASA Technical Reports Server (NTRS)

Miller, Teresa Y.; He, Xiao-Min; Carter, Daniel C.

1992-01-01

Crystals of human serum albumin have been successfully grown in a variety of gels using crystallization conditions otherwise equivalent to those utilized in the popular hanging-drop vapor-equilibrium method. Preliminary comparisons of gel grown crystals with crystals grown by the vapor diffusion method via both ground-based and microgravity methods indicate that crystals superior in size and quality may be grown by limiting solutal convection. Preliminary X-ray diffraction statistics are presented.

Studying electron transfer through alkanethiol self-assembled monolayers on a hanging mercury drop electrode using potentiometric measurements.

PubMed

Cohen-Atiya, Meirav; Mandler, Daniel

2006-10-14

A new approach based on measuring the change of the open-circuit potential (OCP) of a hanging mercury drop electrode (HMDE), modified with alkanethiols of different chain length conducted in a solution containing a mixture of Ru(NH3)6(2+) and Ru(NH3)6(3+) is used for studying electron transfer across the monolayer. Following the time dependence of the OCP allowed the extraction of the kinetic parameters, such as the charge transfer resistance (R(ct)) and the electron transfer rate constant (k(et)), for different alkanethiol monolayers. An electron tunneling coefficient, beta, of 0.9 A(-1) was calculated for the monolayers on Hg.

Thermostatic tissue platform for intravital microscopy: 'the hanging drop' model.

PubMed

Pavlovic, Dragan; Frieling, Helge; Lauer, Kai-Stephan; Bac, Vo Hoai; Richter, Joern; Wendt, Michael; Lehmann, Christian; Usichenko, Taras; Meissner, Konrad; Gruendling, Matthias

2006-11-01

Intravital microscopy imposes the particular problem of the combined control of the body temperature of the animal and the local temperature of the observed organ or tissues. We constructed and tested, in the rat ileum microcirculation preparation, a new organ-support platform. The platform consisted of an organ bath filled with physiological solution, and contained a suction tube, a superfusion tube, an intestine-support hand that was attached to a micromanipulator and a thermometer probe. To cover the intestine we used a cover glass plate with a plastic ring glued on its upper surface. After a routine procedure (anaesthesia, monitoring and surgery), the intestine segment (2-3 cm long) was gently exteriorized and placed on the 'hand' of the organ support. A small part of the intestine formed a small 'island' in the bath that was filled with physiological salt solution. The cover glass was secured in place. The physiological salt solution from the superfusion tube, which was pointed to the lower surface of the cover glass, formed a 'hanging drop'. The objective of the microscope was then immersed into distilled water that was formed by the cover glass plastic ring. The 'hanging drop' technique prevented any tissue quenching, ensured undisturbed microcirculation, provided for stable temperature and humidity, and permitted a clear visual field.

Digital microfluidics for automated hanging drop cell spheroid culture.

PubMed

Aijian, Andrew P; Garrell, Robin L

2015-06-01

Cell spheroids are multicellular aggregates, grown in vitro, that mimic the three-dimensional morphology of physiological tissues. Although there are numerous benefits to using spheroids in cell-based assays, the adoption of spheroids in routine biomedical research has been limited, in part, by the tedious workflow associated with spheroid formation and analysis. Here we describe a digital microfluidic platform that has been developed to automate liquid-handling protocols for the formation, maintenance, and analysis of multicellular spheroids in hanging drop culture. We show that droplets of liquid can be added to and extracted from through-holes, or "wells," and fabricated in the bottom plate of a digital microfluidic device, enabling the formation and assaying of hanging drops. Using this digital microfluidic platform, spheroids of mouse mesenchymal stem cells were formed and maintained in situ for 72 h, exhibiting good viability (>90%) and size uniformity (% coefficient of variation <10% intraexperiment, <20% interexperiment). A proof-of-principle drug screen was performed on human colorectal adenocarcinoma spheroids to demonstrate the ability to recapitulate physiologically relevant phenomena such as insulin-induced drug resistance. With automatable and flexible liquid handling, and a wide range of in situ sample preparation and analysis capabilities, the digital microfluidic platform provides a viable tool for automating cell spheroid culture and analysis. © 2014 Society for Laboratory Automation and Screening.

Sizing of colloidal particle and protein molecules in a hanging fluid drop

NASA Technical Reports Server (NTRS)

Ansari, Rafat R.; Suh, Kwang I.

1995-01-01

We report non-invasive particle size measurements of polystyrene latex colloidal particles and bovine serum albumin (BSA) protein molecules suspended in tiny hanging fluid drops of 30 micro-Liter volume using a newly designed fiber optic probe. The probe is based upon the principles of the technique of dynamic light scattering (DLS). The motivation for this work comes from growing protein crystals in outer space. Protein crystals have been grown previously in hanging drops in microgravity experiments on-board the space shuttle orbiter. However, obtaining quantitative information on nucleation and growth of the protein crystals in real time has always been a desired goal, but hitherto not achieved. Several protein researchers have shown interest in using DLS to monitor crystal growth process in a droplet, but elaborate instrumentation and optical alignment problems have made in-situ applications difficult. We demonstrate that such an experiment is now possible. Our system offers fast (5 seconds) determination of particle size, utilize safe levels of very low laser power (less than or equal to 0.2 mW), a small scattering volume (approximately 2 x 10(exp -5) cu mm) and high spatial coherence (Beta) values. This is a major step forward when compared to currently available DLS systems.

Electrodeless electro-hydrodynamic gentle printing of personalized medicines

NASA Astrophysics Data System (ADS)

Khusid, Boris; Elele, Ezinwa; Shen, Yueyang

2010-11-01

Drop-on-demand (DOD) principle appears to be a particular promising approach for manufacturing personalized treatments carefully tailored to a patient's genetic background. The authors have recently developed a DOD method for gentle printing of personalized medicines. A fluid is infused into an electrically insulating nozzle to form a pendant drop. A sufficiently strong voltage pulse is applied to external electrodes to stretch the pendant drop until it touches an electrically insulating film and forms a liquid bridge. As the liquid bridge is intentionally formed in an unstable configuration, it breaks up, creating two drops, one on the film and the other hanging from the nozzle. To prove the validity and versatility of the method, experiments are conducted on fluids whose viscosity, conductivity, dielectric constant, and surface tension vary over a broad range, respectively: 1-1045 cP, 0.02-290 μS/cm, 9-78, and 41-72 dyn/cm. We present a scaling analysis that captures the essential physics of drop evolution and provides the critical design guidelines. The work was supported by NSF Engineering Research Center on Structured Organic Particulate Systems.

A new approach to stability and oscillations of constrained drops and capillary bridges

NASA Astrophysics Data System (ADS)

Fabre, David; Chireux, Veronique; Risso, Frederic; Tordjeman, Philippe

2014-11-01

Static equilibria of liquid inclusions under the effect of gravity and capillarity is a large class of situations which encompasses drops hanging from a ceiling or from a capillary, sessile drops, liquid bridges, etc... In such equilibria the surface shape is governed by the Yong-Laplace equation, which is usually solved in a local way using a ``shooting'' method. We introduce a new method which solves the Laplace-Young in a global way, using an iterative deformation of the shape towards the equilibrium shape. The method is easy to implement and versatile, and allows to prescribe constraints such as the volume of liquid, the angle of attachment, etc... We subsequently consider the issue of stability and oscillations of such configurations. Using finite elements and considering small-amplitude displacements of the surface with respect to the static configuration previously computed, we introduce a global stability approach which allows to predict the stability limits, the oscillation frequencies and the eigenmode shapes for quite general geometries. The approach will be illustrated and compared with experiments in two situations, namely a drop attached to a capilary and a liquid bridge resulting from the coalescence of two facing millimetric drops.

Factors affecting the morphology of isocitrate lyase crystals

NASA Technical Reports Server (NTRS)

Demattei, Robert C.; Feigelson, Robert S.; Weber, Patricia C.

1992-01-01

Isocitrate lyase crystals have been grown by the hanging drop vapor equilibration method in both 1-g and microgravity and by vapor equilibrium in small capillaries. The crystal morphologies obtained have ranged from dendritic to 'octagonal' prisms. Theoretical evaporation models have been applied to these growth regimes. The results of these analyses along with other experimental results, indicate the factors which must be controlled to produce good growth morphologies.

Electrochemical methods for monitoring of environmental carcinogens.

PubMed

Barek, J; Cvacka, J; Muck, A; Quaiserová, V; Zima, J

2001-04-01

The use of modern electroanalytical techniques, namely differential pulse polarography, differential pulse voltammetry on hanging mercury drop electrode or carbon paste electrode, adsorptive stripping voltammetry and high performance liquid chromatography with electrochemical detection for the determination of trace amounts of carcinogenic N-nitroso compounds, azo compounds, heterocyclic compounds, nitrated polycyclic aromatic hydrocarbons and aromatic and heterocyclic amines is discussed. Scope and limitations of these methods are described and some practical applications based on their combination with liquid-liquid or solid phase extraction are given.

Expression, purification, crystallization and initial crystallographic characterization of the p-hydroxybenzoate hydroxylase from Corynebacterium glutamicum

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

Kwon, Soo-Young; Kang, Beom Sik; Kim, Ghyung-Hwa

2007-11-01

PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH{sub 2}PO{sub 4} and K{sub 2}HPO{sub 4} as precipitants. X-ray diffraction data were collected to a maximum resolution of 2.5 Å on a synchrotron beamline. p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction and plays an important role in the biodegradation of aromatic compounds. PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH{sub 2}PO{sub 4} and K{sub 2}HPO{sub 4} as precipitants. X-ray diffraction data were collectedmore » to a maximum resolution of 2.5 Å on a synchrotron beamline. The crystal belongs to the hexagonal space group P6{sub 3}22, with unit-cell parameters a = b = 94.72, c = 359.68 Å, γ = 120°. The asymmetric unit contains two molecules, corresponding to a packing density of 2.65 Å{sup 3} Da{sup −1}. The structure was solved by molecular replacement. Structure refinement is in progress.« less

Crystallization and preliminary X-ray diffraction studies of the glutaminyl cyclase from Carica papaya latex

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

Azarkan, Mohamed; Clantin, Bernard; Bompard, Coralie

2005-01-01

The glutaminyl cyclase isolated from C. papaya latex has been crystallized using the hanging-drop method. Diffraction data have been collected at ESRF beamline BM14 and processed to 1.7 Å resolution. In living systems, the intramolecular cyclization of N-terminal glutamine residues is accomplished by glutaminyl cyclase enzymes (EC 2.3.2.5). While in mammals these enzymes are involved in the synthesis of hormonal and neurotransmitter peptides, the physiological role played by the corresponding plant enzymes still remains to be unravelled. Papaya glutaminyl cyclase (PQC), a 33 kDa enzyme found in the latex of the tropical tree Carica papaya, displays an exceptional resistance tomore » chemical and thermal denaturation as well as to proteolysis. In order to elucidate its enzymatic mechanism and to gain insights into the structural determinants underlying its remarkable stability, PQC was isolated from papaya latex, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 62.82, b = 81.23, c = 108.17 Å and two molecules per asymmetric unit. Diffraction data have been collected at ESRF beamline BM14 and processed to a resolution of 1.7 Å.« less

Optimal formation of genetically modified and functional pancreatic islet spheroids by using hanging-drop strategy.

PubMed

Kim, H J; Alam, Z; Hwang, J W; Hwang, Y H; Kim, M J; Yoon, S; Byun, Y; Lee, D Y

2013-03-01

Rejection and hypoxia are important factors causing islet loss at an early stage after pancreatic islet transplantation. Recently, islets have been dissociated into single cells for reaggregation into so-called islet spheroids. Herein, we used a hanging-drop strategy to form islet spheroids to achieve functional equivalence to intact islets. To obtain single islet cells, we dissociated islets with trypsin-EDTA digestion for 10 minutes. To obtain spheroids, we dropped various numbers of single cells (125, 250, or 500 cells/30 μL drop) onto a Petri dish, that was inverted for incubation in humidified air containing 5% CO(2) at 37 °C for 7 days. The aggregated spheroids in the droplets were harvested for further culture. The size of the aggregated islet spheroids depended on the number of single cells (125-500 cells/30 μL droplet). Their morphology was similar to that of intact islets without any cellular damage. When treated with various concentrations of glucose to evaluate responsiveness, their glucose-mediated stimulation index value was similar to that of intact islets, an observation that was attributed to strong cell-to-cell interactions in islet spheroids. However, islet spheroids aggregated in general culture dishes showed abnormal glucose responsiveness owing to weak cell-to-cell interactions. Cell-to-cell interactions in islet spheroids were confirmed with an anti-connexin-36 monoclonal antibody. Finally, nonviral poly(ethylene imine)-mediated interleukin-10 cytokine gene delivered beforehand into dissociated single cells before formation of islet spheroids increased the gene transfection efficacy and interleukin-10 secretion from islet spheroids >4-fold compared with intact islets. These results demonstrated the potential application of genetically modified, functional islet spheroids with of controlled size and morphology using an hanging-drop technique. Copyright © 2013 Elsevier Inc. All rights reserved.

Capillary Thinning of Particle-laden Drops

NASA Astrophysics Data System (ADS)

Wagoner, Brayden; Thete, Sumeet; Jahns, Matt; Doshi, Pankaj; Basaran, Osman

2015-11-01

Drop formation is central in many applications such as ink-jet printing, microfluidic devices, and atomization. During drop formation, a thinning filament is created between the about-to-form drop and the fluid hanging from the nozzle. Therefore, the physics of capillary thinning of filaments is key to understanding drop formation and has been thoroughly studied for pure Newtonian fluids. The thinning dynamics is, however, altered completely when the fluid contains particles, the physics of which is not well understood. In this work, we explore the impact of solid particles on filament thinning and drop formation by using a combination of experiments and numerical simulations.

Growth factors mediated differentiation of mesenchymal stem cells to cardiac polymicrotissue using hanging drop and bioreactor.

PubMed

Konstantinou, Dimitrios; Lei, Ming; Xia, Zhidao; Kanamarlapudi, Venkateswarlu

2015-04-01

Heart disease is the major leading cause of death worldwide and the use of stem cells promises new ways for its treatment. The relatively easy and quick acquisition of human umbilical cord matrix mesenchymal stem cells (HUMSCs) and their properties make them useful for the treatment of cardiac diseases. Therefore, the main aim of this investigation was to create cardiac polymicrotissue from HUMSCs using a combination of growth factors [sphingosine-1-phosphate (S1P) and suramin] and techniques (hanging drop and bioreactor). Using designated culture conditions of the growth factors (100 nM S1P and 500 µM suramin), cardiomyocyte differentiation medium (CDM), hanging drop, bioreactor and differentiation for 7 days, a potential specific cardiac polymicrotissue was derived from HUMSCs. The effectiveness of growth factors alone or in combination in differentiation of HUMSCs to cardiac polymicrotissue was analysed by assessing the presence of cardiac markers by immunocytochemistry. This analysis demonstrated the importance of those growth factors for the differentiation. This study for the first time demonstrated the formation of a cardiac polymicrotissue under specific culture conditions. The polymicrotissue thus obtained may be used in future as a 'patch' to cover the injured cardiac region and would thereby be useful for the treatment of heart diseases. © 2014 International Federation for Cell Biology.

Surface tension isotherms of the dioxane-acetone-water and glycerol-ethanol-water ternary systems

NASA Astrophysics Data System (ADS)

Dzhambulatov, R. S.; Dadashev, R. Kh.; Elimkhanov, D. Z.; Dadashev, I. N.

2016-10-01

The results of the experimental and theoretical studies of the concentration dependence of surface tension of aqueous solutions of the 1,4-dioxane-acetone-water and glycerol-ethanol-water ternary systems were given. The studies were performed by the hanging-drop method on a DSA100 tensiometer. The maximum error of surface tension was 1%. The theoretical models for calculating the surface tension of the ternary systems of organic solutions were analyzed.

Crystallization and preliminary X-ray diffraction analysis of central structure domains from mumps virus F protein

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

Liu, Yueyong; Xu, Yanhui; Zhu, Jieqing

2005-09-01

Single crystals of the central structure domains from mumps virus F protein have been obtained by the hanging-drop vapour-diffusion method. A diffraction data set has been collected to 2.2 Å resolution. Fusion of members of the Paramyxoviridae family involves two glycoproteins: the attachment protein and the fusion protein. Changes in the fusion-protein conformation were caused by binding of the attachment protein to the cellular receptor. In the membrane-fusion process, two highly conserved heptad-repeat (HR) regions, HR1 and HR2, are believed to form a stable six-helix coiled-coil bundle. However, no crystal structure has yet been determined for this state in themore » mumps virus (MuV, a member of the Paramyxoviridae family). In this study, a single-chain protein consisting of two HR regions connected by a flexible amino-acid linker (named 2-Helix) was expressed, purified and crystallized by the hanging-drop vapour-diffusion method. A complete X-ray data set was obtained in-house to 2.2 Å resolution from a single crystal. The crystal belongs to space group C2, with unit-cell parameters a = 161.2, b = 60.8, c = 40.1 Å, β = 98.4°. The crystal structure will help in understanding the molecular mechanism of Paramyxoviridae family membrane fusion.« less

Crystallization and preliminary X-ray studies of dUTPase from Mason–Pfizer monkey retrovirus

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

Barabás, Orsolya; Németh, Veronika; Vértessy, Beáta G., E-mail: vertessy@enzim.hu

2006-04-01

Deoxyuridine 5′-triphosphate nucleotidohydrolase from Mason–Pfizer monkey retrovirus (M-PMV dUTPase) is a betaretroviral member of the dUTPase enzyme family. The nucleocapsid-free dUTPase (48426 Da) was co-crystallized with a dUTP substrate analogue using the hanging-drop vapour-diffusion method. Deoxyuridine 5′-triphosphate nucleotidohydrolase from Mason–Pfizer monkey retrovirus (M-PMV dUTPase) is a betaretroviral member of the dUTPase enzyme family. In the mature M-PMV virion, this enzyme is present as the C-terminal domain of the fusion protein nucleocapsid-dUTPase. The homotrimeric organization characteristic of dUTPases is retained in this bifunctional fusion protein. The fusion protein supposedly plays a role in adequate localization of dUTPase activity in the vicinitymore » of nucleic acids during reverse transcription and integration. Here, the nucleocapsid-free dUTPase (48 426 Da) was cocrystallized with a dUTP substrate analogue using the hanging-drop vapour-diffusion method. The obtained crystals belong to the primitive hexagonal space group P6{sub 3}, with unit-cell parameters a = 60.6, b = 60.6, c = 63.6 Å, α = 90, β = 90, γ = 120°. Native and PtCl{sub 4}-derivative data sets were collected using synchrotron radiation to 1.75 and 2.3 Å, respectively. Phasing was successfully performed by isomorphous replacement combined with anomalous scattering.« less

Low-level determination of silicon in steels by anodic stripping voltammetry on a hanging mercury drop electrode.

PubMed

Rahier, A H; Lunardi, S; Nicolle, F; George, S M

2010-10-15

The sensitive differential pulse anodic stripping voltammetry (DPASV) proposed originally by Ishiyama et al. (2001) has been revised and improved to allow the accurate measurement of silicon on a hanging mercury drop electrode (HMDE) instead of a glassy carbon electrode. We assessed the rate of formation of the partially reduced β-silicododecamolybdate and found that metallic mercury promotes the reaction in the presence of a large concentration of Fe(3+). The scope of the method has been broadened by carrying out the measurements in the presence of a constant amount of Fe(3+). The limit of detection (LOD) of the method described in the present paper is 100 μg Sig(-1) of steel, with a relative precision ranging from 5% to 12%. It can be further enhanced to 700 ng Sig(-1) of steel provided the weight of the sample, the dilution factors, the duration of the electrolysis and the ballast of iron are adequately revised. The tolerance to several interfering species has been examined, especially regarding Al(3+), Cr(3+) and Cr VI species. The method was validated using four low-alloy ferritic steels certified by the National Institute of Standards and Technology (NIST). Its application to nickel base alloys as well as to less complicated matrixes is straightforward. It has also been successfully applied to the determination of free silicon into silicon carbide nano-powder. Copyright © 2010 Elsevier B.V. All rights reserved.

High-throughput method for optimum solubility screening for homogeneity and crystallization of proteins

DOEpatents

Kim, Sung-Hou [Moraga, CA; Kim, Rosalind [Moraga, CA; Jancarik, Jamila [Walnut Creek, CA

2012-01-31

An optimum solubility screen in which a panel of buffers and many additives are provided in order to obtain the most homogeneous and monodisperse protein condition for protein crystallization. The present methods are useful for proteins that aggregate and cannot be concentrated prior to setting up crystallization screens. A high-throughput method using the hanging-drop method and vapor diffusion equilibrium and a panel of twenty-four buffers is further provided. Using the present methods, 14 poorly behaving proteins have been screened, resulting in 11 of the proteins having highly improved dynamic light scattering results allowing concentration of the proteins, and 9 were crystallized.

Crystallization and preliminary X-ray diffraction analysis of the lectin from Dioclea rostrata Benth seeds

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

Delatorre, Plínio; Departamento de Ciências Biológicas, Universidade Regional do Cariri, Crato, CE 63195-000; Nascimento, Kyria Santiago

2006-02-01

D. rostrata lectin was crystallized by hanging-drop vapor diffusion. The crystal belongs to the orthorhombic space group I222 and diffracted to 1.87 Å resolution. Lectins from the Diocleinae subtribe (Leguminosae) are highly similar proteins that promote various biological activities with distinctly differing potencies. The structural basis for this experimental data is not yet fully understood. Dioclea rostrata lectin was purified and crystallized by hanging-drop vapour diffusion at 293 K. The crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 61.51, b = 88.22, c = 87.76 Å. Assuming the presence of one monomer per asymmetric unit,more » the solvent content was estimated to be about 47.9%. A complete data set was collected at 1.87 Å resolution.« less

Hanging drop: an in vitro air toxic exposure model using human lung cells in 2D and 3D structures.

PubMed

Liu, Faye F; Peng, Cheng; Escher, Beate I; Fantino, Emmanuelle; Giles, Cindy; Were, Stephen; Duffy, Lesley; Ng, Jack C

2013-10-15

Using benzene as a candidate air toxicant and A549 cells as an in vitro cell model, we have developed and validated a hanging drop (HD) air exposure system that mimics an air liquid interface exposure to the lung for periods of 1h to over 20 days. Dose response curves were highly reproducible for 2D cultures but more variable for 3D cultures. By comparing the HD exposure method with other classically used air exposure systems, we found that the HD exposure method is more sensitive, more reliable and cheaper to run than medium diffusion methods and the CULTEX(®) system. The concentration causing 50% of reduction of cell viability (EC50) for benzene, toluene, p-xylene, m-xylene and o-xylene to A549 cells for 1h exposure in the HD system were similar to previous in vitro static air exposure. Not only cell viability could be assessed but also sub lethal biological endpoints such as DNA damage and interleukin expressions. An advantage of the HD exposure system is that bioavailability and cell concentrations can be derived from published physicochemical properties using a four compartment mass balance model. The modelled cellular effect concentrations EC50cell for 1h exposure were very similar for benzene, toluene and three xylenes and ranged from 5 to 15 mmol/kgdry weight, which corresponds to the intracellular concentration of narcotic chemicals in many aquatic species, confirming the high sensitivity of this exposure method. Copyright © 2013 Elsevier B.V. All rights reserved.

A simple apparatus for controlling nucleation and size in protein crystal growth

NASA Technical Reports Server (NTRS)

Gernert, Kim M.; Smith, Robert; Carter, Daniel C.

1988-01-01

A simple device is described for controlling vapor equilibrium in macromolecular crystallization as applied to the protein crystal growth technique commonly referred to as the 'hanging drop' method. Crystal growth experiments with hen egg white lysozyme have demonstrated control of the nucleation rate. Nucleation rate and final crystal size have been found to be highly dependent upon the rate at which critical supersaturation is approached. Slower approaches show a marked decrease in the nucleation rate and an increase in crystal size.

Compact Apparatus Grows Protein Crystals

NASA Technical Reports Server (NTRS)

Bugg, Charles E.; Delucas, Lawrence J.; Suddath, Fred L.; Snyder, Robert S.; Herren, Blair J.; Carter, Daniel C.; Yost, Vaughn H.

1989-01-01

Laboratory apparatus provides delicately balanced combination of materials and chemical conditions for growth of protein crystals. Apparatus and technique for growth based on hanging-drop method for crystallization of macromolecules. Includes pair of syringes with ganged plungers. One syringe contains protein solution; other contains precipitating-agent solution. Syringes intrude into cavity lined with porous reservoir material saturated with 1 mL or more of similar precipitating-agent solution. Prior to activation, ends of syringes plugged to prevent transport of water vapor among three solutions.

Before the Drop: Engineers Ready Supersonic Decelerator

NASA Image and Video Library

2014-05-21

A saucer-shaped vehicle part of NASA Low-Density Supersonic Decelerator LDSD project designed to test interplanetary landing devices hangs on a tower in preparation for launch at the Pacific Missile Range Facility in Kauai, Hawaii.

HTR8/SVneo cells display trophoblast progenitor cell-like characteristics indicative of self-renewal, repopulation activity, and expression of "stemness-" associated transcription factors.

PubMed

Weber, Maja; Knoefler, Ilka; Schleussner, Ekkehard; Markert, Udo R; Fitzgerald, Justine S

2013-01-01

JEG3 is a choriocarcinoma--and HTR8/SVneo a transformed extravillous trophoblast--cell line often used to model the physiologically invasive extravillous trophoblast. Past studies suggest that these cell lines possess some stem or progenitor cell characteristics. Aim was to study whether these cells fulfill minimum criteria used to identify stem-like (progenitor) cells. In summary, we found that the expression profile of HTR8/SVneo (CDX2+, NOTCH1+, SOX2+, NANOG+, and OCT-) is distinct from JEG3 (CDX2+ and NOTCH1+) as seen only in human-serum blocked immunocytochemistry. This correlates with HTR8/SVneo's self-renewal capacities, as made visible via spheroid formation and multi-passagability in hanging drops protocols paralleling those used to maintain embryoid bodies. JEG3 displayed only low propensity to form and reform spheroids. HTR8/SVneo spheroids migrated to cover and seemingly repopulate human chorionic villi during confrontation cultures with placental explants in hanging drops. We conclude that HTR8/SVneo spheroid cells possess progenitor cell traits that are probably attained through corruption of "stemness-" associated transcription factor networks. Furthermore, trophoblastic cells are highly prone to unspecific binding, which is resistant to conventional blocking methods, but which can be alleviated through blockage with human serum.

Etiology and use of the "hanging drop" technique: a review.

PubMed

Todorov, Ludmil; VadeBoncouer, Timothy

2014-01-01

Background. The hanging drop (HD) technique presumably relies on the presence of subatmospheric epidural pressure. It is not clear whether this negative pressure is intrinsic or an artifact and how it is affected by body position. There are few data to indicate how often HD is currently being used. Methods. We identified studies that measured subatmospheric pressures and looked at the effect of the sitting position. We also looked at the technique used for cervical and thoracic epidural anesthesia in the last 10 years. Results. Intrinsic subatmospheric pressures were measured in the thoracic and cervical spine. Three trials studied the effect of body position, indicating a higher incidence of subatmospheric pressures when sitting. The results show lower epidural pressure (-10.7 mmHg) with the sitting position. 28.8% of trials of cervical and thoracic epidural anesthesia that documented the technique used, utilized the HD technique. When adjusting for possible bias, the rate of HD use can be as low as 11.7%. Conclusions. Intrinsic negative pressure might be present in the cervical and thoracic epidural space. This effect is more pronounced when sitting. This position might be preferable when using HD. Future studies are needed to compare it with the loss of resistance technique.

Direct voltammetric specific recognition of dopamine using AlIII-DA complexes at the hanging mercury drop electrode.

PubMed

Zhang, Fuping; Zhang, Min; Cheng, Jiongjia; Yang, Li; Ji, Ming; Bi, Shuping

2007-11-01

In this paper, we firstly report the direct voltammetric recognition and determination of dopamine (DA) by using Al(III)-DA complexes at the hanging mercury drop electrode (HMDE). A new sensitive cathodic peak of Al(III)-DA can be detected at -900 mV (vs. SCE) in 0.1 M NH(4)Cl-NH(3).H(2)O-0.1 M KCl buffer solution at pH 8.5. This unique -900 mV cathodic peak arises from the specific interaction between Al(III) and DA on the HMDE, whereas other substances with similar structures, such as L-dopa, epinephrine (EP), norepinephrine (NE), catechols, caffeic acid (CA), trihydric phenols and tiron, do not yield any new peak on the voltammograms in the potential range from -100 to -1200 mV when Al(III) is added. The distinct voltammetric characteristic of the recognition of DA can effectively inhibit the interferences of both ascorbic acid and uric acid in the DA determination by the direct electrochemistry, which is a major difficulty when a solid electrode is used. The proposed method can be anticipated as an effective means for the recognition of DA in the elucidation of the mechanisms of Parkinson's disease (PD) and Alzheimer's disease (AD) in the presence of Al(III).

Sequential Coating of Insulin Secreting Beta Cells within Multilayers of Polysaccharide Nanogels.

PubMed

Bal, Tugba; Oran, Dilem Ceren; Sasaki, Yoshihiro; Akiyoshi, Kazunari; Kizilel, Seda

2018-05-01

Pancreatic islet transplantation has emerged as a promising treatment for type-1 diabetes (T1D); however, its clinical application is still limited by the life-long use of immunosuppressive drugs, insufficient number of islets to achieve normoglycemia, and large transplantation volume. This paper reports a unique approach for nanothin coating of insulin secreting beta cell aggregates. The coating is based on hydrophobic and covalent interactions between natural acrylate modified cholesterol bearing pullulan (CHPOA) nanogels and MIN6 beta cell aggregates. Beta cell aggregates are prepared as spheroids through hanging drop method, which is optimized with respect to hanging drop volume and initial number of beta cells. These aggregates, defined as pseudoislets, are coated with sequential layers of nanogels and are evaluated as viable and functional for insulin secretion. Coating experiments are carried out using physiologically compatible medium, where pseudoislets are not brought in contact with toxic prepolymer solutions used in existing approaches. This study offers new opportunities through coating of islets with advanced functional materials under completely physiological conditions for clinical translation of cell transplantation technology. The technique developed here will establish a new paradigm for creating tolerable grafts for other chronic diseases such as anemia, cancer, central nervous system (CNS) diseases. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Formation and hematopoietic differentiation of human embryoid bodies by suspension and hanging drop cultures.

PubMed

Cerdan, Chantal; Hong, Seok Ho; Bhatia, Mickie

2007-10-01

The in vitro aggregation of human embryonic stem cells (hESCs) into clusters termed embryoid bodies (EBs) allows for the spontaneous differentiation of cells representing endoderm, mesoderm, and ectoderm lineages. This stochastic process results however, in the generation of low numbers of differentiated cells, and can be enhanced to some extent by the addition of exogenous growth factors or overexpression of regulatory genes. In the authors' laboratory, the use of hematopoietic cytokines in combination with the mesoderm inducer bone morphogenetic protein-4 (BMP-4) was able to generate up to 90% of CD45(+) hematopoietic cells with colony-forming unit (CFU) activity. This unit describes two protocols that have been successfully applied in the authors' laboratory for the generation of EBs in (1) suspension and (2) hanging drop (HD) cultures from enzymatically digested clumps of undifferentiated hESC colonies.

A novel hanging spherical drop system for the generation of cellular spheroids and high throughput combinatorial drug screening.

PubMed

Neto, A I; Correia, C R; Oliveira, M B; Rial-Hermida, M I; Alvarez-Lorenzo, C; Reis, R L; Mano, J F

2015-04-01

We propose a novel hanging spherical drop system for anchoring arrays of droplets of cell suspension based on the use of biomimetic superhydrophobic flat substrates, with controlled positional adhesion and minimum contact with a solid substrate. By facing down the platform, it was possible to generate independent spheroid bodies in a high throughput manner, in order to mimic in vivo tumour models on the lab-on-chip scale. To validate this system for drug screening purposes, the toxicity of the anti-cancer drug doxorubicin in cell spheroids was tested and compared to cells in 2D culture. The advantages presented by this platform, such as feasibility of the system and the ability to control the size uniformity of the spheroid, emphasize its potential to be used as a new low cost toolbox for high-throughput drug screening and in cell or tissue engineering.

Voltammetric analysis of N-containing drugs using the hanging galinstan drop electrode (HGDE).

PubMed

Channaa, H; Surmann, P

2009-03-01

The electrochemical behaviour of several N-containing voltammetric active drugs such as 1,4-benzodiazepines (chlordiazepoxide, nitrazepam and diazepam) as well as one nitro-compound (nitrofurantoin) and one azo-compound (phenazopyridine) is described using a new kind of liquid electrode, the hanging galinstan drop electrode. Concentrations of 10(-5) - 10(-8) mol L(-1) are generally measurable. Differential pulse and adsorptive stripping voltammograms are recorded in different supporting electrolytes, like 0.1 M KNO3, acetate buffer solution pH = 4.6 and phosphate buffer solution pH = 7.0. The effects of varying the starting potentials, U(start) for DPV and accumulation times, t(acc) for AdSV are considered. Briefly, it is shown that the novel galinstan electrode is suitable for reducing several functional groups in organic substances, here presented for N-oxide-, azomethine-, nitro- and azo-groups.

Factors Associated with Correct and Consistent Insecticide Treated Curtain Use in Iquitos, Peru

PubMed Central

Scott, Thomas W.; Elder, John P.; Alexander, Neal; Halsey, Eric S.; McCall, Philip J.

2016-01-01

Dengue is an arthropod-borne virus of great public health importance, and control of its mosquito vectors is currently the only available method for prevention. Previous research has suggested that insecticide treated curtains (ITCs) can lower dengue vector infestations in houses. This observational study investigated individual and household-level socio-demographic factors associated with correct and consistent use of ITCs in Iquitos, Peru. A baseline knowledge, attitudes, and practices (KAP) survey was administered to 1,333 study participants, and ITCs were then distributed to 593 households as part of a cluster-randomized trial. Follow up KAP surveys and ITC-monitoring checklists were conducted at 9, 18, and 27 months post-ITC distribution. At 9 months post-distribution, almost 70% of ITCs were hanging properly (e.g. hanging fully extended or tied up), particularly those hung on walls compared to other locations. Proper ITC hanging dropped at 18 months to 45.7%. The odds of hanging ITCs correctly and consistently were significantly greater among those participants who were housewives, knew three or more correct symptoms of dengue and at least one correct treatment for dengue, knew a relative or close friend who had had dengue, had children sleeping under a mosquito net, or perceived a change in the amount of mosquitoes in the home. Additionally, the odds of recommending ITCs in the future were significantly greater among those who perceived a change in the amount of mosquitoes in the home (e.g. perceived the ITCs to be effective). Despite various challenges associated with the sustained effectiveness of the selected ITCs, almost half of the ITCs were still hanging at 18 months, suggesting a feasible vector control strategy for sustained community use. PMID:26967157

Hanging drop crystal growth apparatus and method

NASA Technical Reports Server (NTRS)

Carter, Daniel C. (Inventor); Smith, Robbie E. (Inventor)

1989-01-01

An apparatus (10) is constructed having a cylindrical enclosure (16) within which a disc-shaped wicking element (18) is positioned. A well or recess (22) is cut into an upper side (24) of this wicking element, and a glass cover plate or slip (28) having a protein drop disposed thereon is sealably positioned on the wicking element (18), with drop (12) being positioned over well or recess (22). A flow of control fluid is generated by a programmable gradient former (16), with this control fluid having a vapor pressure that is selectively variable. This flow of control fluid is coupled to the wicking element (18) where control fluid vapor diffusing from walls (26) of the recess (22) is exposed to the drop (12), forming a vapor pressure gradient between the drop (12) and the control fluid vapor. Initially, this gradient is adjusted to draw solvent from the drop (12) at a relatively high rate, and as the critical supersaturation point is approached (the point at which crystal nucleation occurs), the gradient is reduced to more slowly draw solvent from the drop (12). This allows discrete protein molecules more time to orient themselves into an ordered crystalline lattice, producing protein crystals which, when processed by X-ray crystallography, possess a high degree of resolution.

Development of a sequential injection-square wave voltammetry method for determination of paraquat in water samples employing the hanging mercury drop electrode.

PubMed

dos Santos, Luciana B O; Infante, Carlos M C; Masini, Jorge C

2010-03-01

This work describes the development and optimization of a sequential injection method to automate the determination of paraquat by square-wave voltammetry employing a hanging mercury drop electrode. Automation by sequential injection enhanced the sampling throughput, improving the sensitivity and precision of the measurements as a consequence of the highly reproducible and efficient conditions of mass transport of the analyte toward the electrode surface. For instance, 212 analyses can be made per hour if the sample/standard solution is prepared off-line and the sequential injection system is used just to inject the solution towards the flow cell. In-line sample conditioning reduces the sampling frequency to 44 h(-1). Experiments were performed in 0.10 M NaCl, which was the carrier solution, using a frequency of 200 Hz, a pulse height of 25 mV, a potential step of 2 mV, and a flow rate of 100 µL s(-1). For a concentration range between 0.010 and 0.25 mg L(-1), the current (i(p), µA) read at the potential corresponding to the peak maximum fitted the following linear equation with the paraquat concentration (mg L(-1)): i(p) = (-20.5 ± 0.3)C (paraquat) - (0.02 ± 0.03). The limits of detection and quantification were 2.0 and 7.0 µg L(-1), respectively. The accuracy of the method was evaluated by recovery studies using spiked water samples that were also analyzed by molecular absorption spectrophotometry after reduction of paraquat with sodium dithionite in an alkaline medium. No evidence of statistically significant differences between the two methods was observed at the 95% confidence level.

Propelling a water drop with the vapor-mediated Marangoni effect

NASA Astrophysics Data System (ADS)

Kim, Seungho; Kim, Ho-Young

2013-11-01

We show that a water drop on solid surfaces can be propelled just by placing a volatile alcohol drop nearby. It is found to be because the water-air interface near the alcohol drop mixes with alcohol vapor, thereby locally lowering the surface tension. The surface-tension-gradient induces the motion of the water drop, enabling the trajectory control of water drops through the motion of remote alcohol drops. This vapor-mediated Marangoni effect also gives rise to other interesting interfacial flow phenomena, such as nucleation of holes on a water film and ballooning of a water drop hanging from a syringe needle with the approach of an alcohol drop. We visualize such interfacial dynamics with a high-speed camera and rationalize their salient features by scaling analysis. This work was supported by the National Research Foundation of Korea (grant no. 2012-008023).

Purification, characterization and preliminary crystallographic studies of a PR-10 protein from Pachyrrhizus erosus seeds.

PubMed

Wu, Fang; Li, Yikun; Chang, Shaojie; Zhou, Zhaocai; Wang, Fang; Song, Xiaomin; Lin, Yujuan; Gong, Weimin

2002-12-01

A 16 kDa protein SPE16 was purified from the seeds of Pachyrrhizus erosus. Its N-terminal amino-acid sequence showed significant sequence homology to pathogenesis-related proteins from the PR-10 family. An activity assay indicated that SPE16 possesses ribonuclease activity as do some other PR-10 proteins. SPE16 crystals were obtained by the hanging-drop vapour-diffusion method. The space group is P2(1)2(1)2(1), with unit-cell parameters a = 53.36, b = 63.70, c = 72.96 A.

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

Imamura, Kayo; Matsuura, Takanori; Ye, Zhengmao

Disproportionating enzyme from potato was crystallized and preliminarily analyzed using X-ray diffraction. Disproportionating enzyme (D-enzyme; EC 2.4.1.25) is a 59 kDa protein that belongs to the α-amylase family. D-enzyme catalyses intramolecular and intermolecular transglycosylation reactions of α-1,4 glucan. A crystal of the D-enzyme from potato was obtained by the hanging-drop vapour-diffusion method. Preliminary X-ray data showed that the crystal diffracts to 2.0 Å resolution and belongs to space group C222{sub 1}, with unit-cell parameters a = 69.7, b = 120.3, c = 174.2 Å.

Protein crystal growth in space

NASA Technical Reports Server (NTRS)

Bugg, C. E.; Clifford, D. W.

1987-01-01

The advantages of protein crystallization in space, and the applications of protein crystallography to drug design, protein engineering, and the design of synthetic vaccines are examined. The steps involved in using protein crystallography to determine the three-dimensional structure of a protein are discussed. The growth chamber design and the hand-held apparatus developed for protein crystal growth by vapor diffusion techniques (hanging-drop method) are described; the experimental data from the four Shuttle missions are utilized to develop hardware for protein crystal growth in space and to evaluate the effects of gravity on protein crystal growth.

Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening.

PubMed

Amaral, Robson L F; Miranda, Mariza; Marcato, Priscyla D; Swiech, Kamilla

2017-01-01

Introduction: Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better. Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates), and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 × 10 4 cells/mL), and then, subjected to drug resistance evaluation. Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300-500 μm) was obtained using cultures initiated with 0.5 and 1.25 × 10 4 cells/mL for the ULA method and 2.5 and 3.75 × 10 4 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC 50 of 1.00 and 0.83 μg/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC 50 ranging from 0.39 to 0.43). Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application.

Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening

PubMed Central

Amaral, Robson L. F.; Miranda, Mariza; Marcato, Priscyla D.; Swiech, Kamilla

2017-01-01

Introduction: Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better. Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates), and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 × 104 cells/mL), and then, subjected to drug resistance evaluation. Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300–500 μm) was obtained using cultures initiated with 0.5 and 1.25 × 104 cells/mL for the ULA method and 2.5 and 3.75 × 104 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC50 of 1.00 and 0.83 μg/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC50 ranging from 0.39 to 0.43). Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application. PMID:28878686

Design of 3-D adipospheres for quantitative metabolic study

PubMed Central

Akama, Takeshi; Leung, Brendan M.; Labuz, Joseph M.; Takayama, Shuichi; Chun, Tae-Hwa

2017-01-01

Quantitative assessment of adipose mitochondrial activity is critical for better understanding of adipose tissue function in obesity and diabetes. While the two-dimensional (2-D) tissue culture method has been sufficient to discover key molecules that regulate adipocyte differentiation and function, the method is insufficient to determine the role of extracellular matrix (ECM) molecules and their modifiers, such as matrix metalloproteinases (MMPs), in regulating adipocyte function in three-dimensional (3-D) in vivo-like microenvironments. By using a 3-D hanging drop tissue culture system, we are able to produce scalable 3-D adipospheres that are suitable for quantitative mitochondrial study in 3-D microenvironment. PMID:28244051

A variational approach to the study of capillary phenomena

NASA Technical Reports Server (NTRS)

Emmer, M.; Gonzalez, E.; Tamanini, I.

1982-01-01

The problem of determining the free surface of a liquid in a capillary tube, and of a liquid drop, sitting first on a horizontal plane and then on more general surfaces is considered. With some modifications, the method applies to the study of pendent drops and of rotating drops as well. The standard capillary problem, i.e. the determination of the free surface of a liquid in a thin tube of general cross section, which resuls from the simultaneous action of surface tension, boundary adhesion and gravity is discussed. It turns out that in this case the existence of the solution surface depends heavily on the validity of a simple geometric condition about the mean curvature of the boundary curve of the cross section of the capillary tube. Some particular examples of physical interest are also be discussed. Liquid drops sitting on or hanging from a fixed horizontal plane are discussed. The symmetry of the solutions (which can actually be proved, as consequence of a general symmetrization argument) now plays the chief role in deriving both the existence and the regularity of energy-minimizing configurations. When symmetry fails (this is the case, for example, when the contact angle between the drop and the plate is not constant, or when the supporting surface is not itself symmetric), then more sophisticated methods must be used. Extensions in this direction are outlined.

In vitro culture of early secondary preantral follicles in hanging drop of ovarian cell-conditioned medium to obtain MII oocytes from outbred deer mice.

PubMed

Choi, Jung Kyu; Agarwal, Pranay; He, Xiaoming

2013-12-01

The ovarian follicle (each contains a single oocyte) is the fundamental functional tissue unit of mammalian ovaries. In humans, it has been long held true that females are born with a maximum number of follicles (or oocytes) that are not only nonrenewable, but also undergoing degeneration with time with a sharply decreased oocyte quality after the age of ∼35. Therefore, it is of importance to isolate and bank ovarian follicles for in vitro culture to obtain fertilizable oocytes later, to preserve the fertility of professional women who may want to delay childbearing, young and unmarried women who may lose gonadal function because of exposure to environmental/occupational hazards or aggressive medical treatments, such as radiation and chemotherapy, and even endangered species and breeds. Although they contributed significantly to the understanding of follicle science and biology, most studies reported to date on this topic were done using the man-made, unnatural inbred animal species. It was found in this study that the conventional two-dimensional microliter drop and three-dimensional hanging drop (HD) methods, reported to be effective for in vitro culture of preantral follicles from inbred mice, are not directly transferrable to outbred deer mice. Therefore, a modified HD method was developed in this study to achieve a much higher (>5 times compared to the best conventional methods) percentage of developing early secondary preantral follicles from the outbred mice to the antral stage, for which, the use of an ovarian cell-conditioned medium and multiple follicles per HD were identified to be crucial. It was further found that the method for in vitro maturation of oocytes in antral follicles obtained by in vitro culture of preantral follicles could be very different from that for oocytes in antral follicles obtained by hormone stimulation in vivo. Therefore, this study should provide important guidance for establishing effective protocols of in vitro follicle culture to preserve the fertility of wildlife and humans outbred by nature.

In Vitro Culture of Early Secondary Preantral Follicles in Hanging Drop of Ovarian Cell-Conditioned Medium to Obtain MII Oocytes from Outbred Deer Mice

PubMed Central

Choi, Jung Kyu; Agarwal, Pranay

2013-01-01

The ovarian follicle (each contains a single oocyte) is the fundamental functional tissue unit of mammalian ovaries. In humans, it has been long held true that females are born with a maximum number of follicles (or oocytes) that are not only nonrenewable, but also undergoing degeneration with time with a sharply decreased oocyte quality after the age of ∼35. Therefore, it is of importance to isolate and bank ovarian follicles for in vitro culture to obtain fertilizable oocytes later, to preserve the fertility of professional women who may want to delay childbearing, young and unmarried women who may lose gonadal function because of exposure to environmental/occupational hazards or aggressive medical treatments, such as radiation and chemotherapy, and even endangered species and breeds. Although they contributed significantly to the understanding of follicle science and biology, most studies reported to date on this topic were done using the man-made, unnatural inbred animal species. It was found in this study that the conventional two-dimensional microliter drop and three-dimensional hanging drop (HD) methods, reported to be effective for in vitro culture of preantral follicles from inbred mice, are not directly transferrable to outbred deer mice. Therefore, a modified HD method was developed in this study to achieve a much higher (>5 times compared to the best conventional methods) percentage of developing early secondary preantral follicles from the outbred mice to the antral stage, for which, the use of an ovarian cell-conditioned medium and multiple follicles per HD were identified to be crucial. It was further found that the method for in vitro maturation of oocytes in antral follicles obtained by in vitro culture of preantral follicles could be very different from that for oocytes in antral follicles obtained by hormone stimulation in vivo. Therefore, this study should provide important guidance for establishing effective protocols of in vitro follicle culture to preserve the fertility of wildlife and humans outbred by nature. PMID:23789595

Formation of stable small cell number three-dimensional ovarian cancer spheroids using hanging drop arrays for preclinical drug sensitivity assays.

PubMed

Raghavan, Shreya; Ward, Maria R; Rowley, Katelyn R; Wold, Rachel M; Takayama, Shuichi; Buckanovich, Ronald J; Mehta, Geeta

2015-07-01

Ovarian cancer grows and metastasizes from multicellular spheroidal aggregates within the ascites fluid. Multicellular tumor spheroids are therefore physiologically significant 3D in vitro models for ovarian cancer research. Conventional hanging drop cultures require high starting cell numbers, and are tedious for long-term maintenance. In this study, we generate stable, uniform multicellular spheroids using very small number of ovarian cancer cells in a novel 384 well hanging drop array platform. We used novel tumor spheroid platform and two ovarian cancer cell lines (A2780 and OVCAR3) to demonstrate the stable incorporation of as few as 10 cells into a single spheroid. Spheroids had uniform geometry, with projected areas (42.60×10(3)μm-475.22×10(3)μm(2) for A2780 spheroids and 37.24×10(3)μm(2)-281.01×10(3)μm(2) for OVCAR3 spheroids) that varied as a function of the initial cell seeding density. Phalloidin and nuclear stains indicated cells formed tightly packed spheroids with demarcated boundaries and cell-cell interaction within spheroids. Cells within spheroids demonstrated over 85% viability. 3D tumor spheroids demonstrated greater resistance (70-80% viability) to cisplatin chemotherapy compared to 2D cultures (30-50% viability). Ovarian cancer spheroids can be generated from limited cell numbers in high throughput 384 well plates with high viability. Spheroids demonstrate therapeutic resistance relative to cells in traditional 2D culture. Stable incorporation of low cell numbers is advantageous when translating this research to rare patient-derived cells. This system can be used to understand ovarian cancer spheroid biology, as well as carry out preclinical drug sensitivity assays. Copyright © 2015 Elsevier Inc. All rights reserved.

Rat pancreatic islet size standardization by the "hanging drop" technique.

PubMed

Cavallari, G; Zuellig, R A; Lehmann, R; Weber, M; Moritz, W

2007-01-01

Rejection and hypoxia are the main factors that limit islet engraftment in the recipient liver in the immediate posttransplant period. Recently authors have reported a negative relationship of graft function and islet size, concluding that small islets are superior to large islets. Islets can be dissociated into single cells and reaggregated into so called "pseudoislets," which are functionally equivalent to intact islets but exhibit reduced immunogenicity. The aim of our study was develop a technique that enabled one to obtain pseudoislets of defined, preferably small, dimensions. Islets were harvested from Lewis rats by the collagenase digestion procedure. After purification, the isolated islets were dissociated into single cells by trypsin digestion. Fractions with different cell numbers were seeded into single drops onto cell culture dishes, which were inverted and incubated for 5 to 8 days under cell culture conditions. Newly formed pseudoislets were analyzed for dimension, morphology, and cellular composition. The volume of reaggregated pseudoislets strongly correlated with the cell number (r(2) = .995). The average diameter of a 250-cell aggregate was 95 +/- 8 microm (mean +/- SD) compared with 122 +/- 46 microm of freshly isolated islets. Islet cell loss may be minimized by performing reaggregation in the presence of medium glucose (11 mmol/L) and the GLP-1 analogue Exendin-4. Morphology, cellular composition, and architecture of reaggregated islets were comparable to intact islets. The "hanging drop" culture method allowed us to obtain pseudoislets of standardized size and regular shape, which did not differ from intact islets in terms of cellular composition or architecture. Further investigations are required to minimize cell loss and test in vivo function of transplanted pseudoislets.

Three-dimensional spheroid culture targeting versatile tissue bioassays using a PDMS-based hanging drop array.

PubMed

Kuo, Ching-Te; Wang, Jong-Yueh; Lin, Yu-Fen; Wo, Andrew M; Chen, Benjamin P C; Lee, Hsinyu

2017-06-29

Biomaterial-based tissue culture platforms have emerged as useful tools to mimic in vivo physiological microenvironments in experimental cell biology and clinical studies. We describe herein a three-dimensional (3D) tissue culture platform using a polydimethylsiloxane (PDMS)-based hanging drop array (PDMS-HDA) methodology. Multicellular spheroids can be achieved within 24 h and further boosted by incorporating collagen fibrils in PDMS-HDA. In addition, the spheroids generated from different human tumor cells exhibited distinct sensitivities toward drug chemotherapeutic agents and radiation as compared with two-dimensional (2D) cultures that often lack in vivo-like biological insights. We also demonstrated that multicellular spheroids may enable key hallmarks of tissue-based bioassays, including drug screening, tumor dissemination, cell co-culture, and tumor invasion. Taken together, these results offer new opportunities not only to achieve the active control of 3D multicellular spheroids on demand, but also to establish a rapid and cost-effective platform to study anti-cancer therapeutics and tumor microenvironments.

A simple hanging drop cell culture protocol for generation of 3D spheroids.

PubMed

Foty, Ramsey

2011-05-06

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels or in biomaterial scaffolds. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model. Copyright © 2011 Journal of Visualized Experiments

Analysis of models for two solution crystal growth problems

NASA Technical Reports Server (NTRS)

Fehribach, Joseph D.; Rosenberger, Franz

1989-01-01

Two diffusive solution crystal growth models are considered which are characterized by two phases separated by an interface, a lack of convective mixing in either phase, and the presence of diffusion components differing widely in diffusivity. The first model describes precipitant-driven solution crystal growth and the second model describes a hanging drop evaporation problem. It is shown that for certain proteins sharp concentration gradients may develop in the drop during evaporation, while under the same conditions the concentrations of other proteins remain uniform.

Crystallization and preliminary X-ray diffraction analysis of red clover necrotic mosaic virus

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

Martin, Stanton L.; Guenther, Richard H.; Sit, Tim L.

2010-11-12

Red clover necrotic mosaic virus (RCNMV) is a species that belongs to the Tombusviridae family of plant viruses with a T = 3 icosahedral capsid. RCNMV virions were purified and were crystallized for X-ray analysis using the hanging-drop vapor-diffusion method. Self-rotation functions and systematic absences identified the space group as I23, with two virions in the unit cell. The crystals diffracted to better than 4 {angstrom} resolution but were very radiation-sensitive, causing rapid decay of the high-resolution reflections. The data were processed to 6 {angstrom} in the analysis presented here.

Crystallization and preliminary X-ray studies of a non-haemorrhagic fibrin(ogen)olytic metalloproteinase from the venom of Agkistrodon acutus

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

Hou, Jing; Li, Ming; Chen, Jiashu

Crystals of a non-haemorrhagic fibrin(ogen)olytic metalloproteinase from the venom of A. acutus have been obtained and characterized by X-ray diffraction. A non-haemorrhagic fibrin(ogen)olytic metalloproteinase from the venom of Agkistrodon acutus has been crystallized by the hanging-drop method. The crystals belong to space group P3{sub 1}21, with unit-cell parameters a = b = 80.57, c = 66.77 Å and one molecule in the asymmetric unit. X-ray diffraction data were collected to 1.86 Å resolution.

Preliminary investigations of protein crystal growth using the Space Shuttle

NASA Technical Reports Server (NTRS)

Delucas, L. J.; Suddath, F. L.; Snyder, R.; Naumann, R.; Broom, M. B.; Pusey, M.; Yost, V.; Herren, B .; Carter, D.

1986-01-01

Four preliminary Shuttle experiments are described which have been used to develop prototype hardware for a more advanced system that will evaluate effects of gravity on protein crystal growth. The first phase of these experiments has centered on the development of micromethods for protein crystal growth by vapor-diffusion techniques (using a space version of the hanging-drop method) and on dialysis using microdialysis cells. Results suggest that the elimination of density-driven sedimentation can effect crystal morphology. In the dialysis experiment, space-grown crystals of concanavalin B were three times longer and 1/3 the thickness of earth-grown crystals.

Purification, crystallization and preliminary crystallographic analysis of biotin protein ligase from Staphylococcus aureus.

PubMed

Pendini, Nicole R; Polyak, Steve W; Booker, Grant W; Wallace, John C; Wilce, Matthew C J

2008-06-01

Biotin protein ligase from Staphylococcus aureus catalyses the biotinylation of acetyl-CoA carboxylase and pyruvate carboxylase. Recombinant biotin protein ligase from S. aureus has been cloned, expressed and purified. Crystals were grown using the hanging-drop vapour-diffusion method using PEG 8000 as the precipitant at 295 K. X-ray diffraction data were collected to 2.3 A resolution from crystals using synchrotron X-ray radiation at 100 K. The diffraction was consistent with the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 93.665, c = 131.95.

Crystallization and X-ray diffraction analysis of a catalytic domain of hyperthermophilic chitinase from Pyrococcus furiosus

PubMed Central

Mine, Shouhei; Nakamura, Tsutomu; Hirata, Kunio; Ishikawa, Kazuhiko; Hagihara, Yoshihisa; Uegaki, Koichi

2006-01-01

The crystallization and preliminary X-ray diffraction analysis of a catalytic domain of chitinase (PF1233 gene) from the hyperthermophilic archaeon Pyrococcus furiosus is reported. The recombinant protein, prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected at the undulator beamline BL44XU at SPring-8 to a resolution of 1.50 Å. The crystals belong to space group P212121, with unit-cell parameters a = 90.0, b = 92.8, c = 107.2 Å. PMID:16880559

Crystallization and diffraction analysis of [beta]-N-acetylhexosaminidase from Aspergillus oryzae

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

Vanek, Ondrej; Brynd, Jirí; Hofbauerová, Katerina

2012-05-08

Fungal {beta}-N-acetylhexosaminidases are enzymes that are used in the chemoenzymatic synthesis of biologically interesting oligosaccharides. The enzyme from Aspergillus oryzae was produced and purified from its natural source and crystallized using the hanging-drop vapor-diffusion method. Diffraction data from two crystal forms (primitive monoclinic and primitive tetragonal) were collected to resolutions of 3.2 and 2.4 {angstrom}, respectively. Electrophoretic and quantitative N-terminal protein-sequencing analyses confirmed that the crystals are formed by a complete biologically active enzyme consisting of a glycosylated catalytic unit and a noncovalently attached propeptide.

Estimation of Tegaserod Maleate by Differential Pulse Polarography

PubMed Central

Rajput, S. J.; Raj, H. A.

2009-01-01

A highly sensitive differential pulse polarographic method has been developed for the estimation of tegaserod maleate after treating it with hydrogen peroxide solution. The oxidation of tegaserod maleate is a reversible process as the oxidized product could be reduced at hanging mercury drop electrode in a quantitative manner using differential pulse polarography mode. The limit of quantification was 0.1ng/ml. The voltametric peak was obtained at -1.05 volts in presence of 0.1M potassium chloride as supporting electrolyte. The technique could be used successfully to analyze tegaserod maleate in its tablet formulation. PMID:20177456

Common Cause Case Study: An Estimated Probability of Four Solid Rocket Booster Hold-down Post Stud Hang-ups

NASA Technical Reports Server (NTRS)

Cross, Robert

2005-01-01

Until Solid Rocket Motor ignition, the Space Shuttle is mated to the Mobil Launch Platform in part via eight (8) Solid Rocket Booster (SRB) hold-down bolts. The bolts are fractured using redundant pyrotechnics, and are designed to drop through a hold-down post on the Mobile Launch Platform before the Space Shuttle begins movement. The Space Shuttle program has experienced numerous failures where a bolt has "hung-up." That is, it did not clear the hold-down post before liftoff and was caught by the SRBs. This places an additional structural load on the vehicle that was not included in the original certification requirements. The Space Shuttle is currently being certified to withstand the loads induced by up to three (3) of eight (8) SRB hold-down post studs experiencing a "hang-up." The results af loads analyses performed for four (4) stud-hang ups indicate that the internal vehicle loads exceed current structural certification limits at several locations. To determine the risk to the vehicle from four (4) stud hang-ups, the likelihood of the scenario occurring must first be evaluated. Prior to the analysis discussed in this paper, the likelihood of occurrence had been estimated assuming that the stud hang-ups were completely independent events. That is, it was assumed that no common causes or factors existed between the individual stud hang-up events. A review of the data associated with the hang-up events, showed that a common factor (timing skew) was present. This paper summarizes a revised likelihood evaluation performed for the four (4) stud hang-ups case considering that there are common factors associated with the stud hang-ups. The results show that explicitly (i.e. not using standard common cause methodologies such as beta factor or Multiple Greek Letter modeling) taking into account the common factor of timing skew results in an increase in the estimated likelihood of four (4) stud hang-ups of an order of magnitude over the independent failure case.

Common Cause Case Study: An Estimated Probability of Four Solid Rocket Booster Hold-Down Post Stud Hang-ups

NASA Technical Reports Server (NTRS)

Cross, Robert

2005-01-01

Until Solid Rocket Motor ignition, the Space Shuttle is mated to the Mobil Launch Platform in part via eight (8) Solid Rocket Booster (SRB) hold-down bolts. The bolts are fractured using redundant pyrotechnics, and are designed to drop through a hold-down post on the Mobile Launch Platform before the Space Shuttle begins movement. The Space Shuttle program has experienced numerous failures where a bolt has hung up. That is, it did not clear the hold-down post before liftoff and was caught by the SRBs. This places an additional structural load on the vehicle that was not included in the original certification requirements. The Space Shuttle is currently being certified to withstand the loads induced by up to three (3) of eight (8) SRB hold-down experiencing a "hang-up". The results of loads analyses performed for (4) stud hang-ups indicate that the internal vehicle loads exceed current structural certification limits at several locations. To determine the risk to the vehicle from four (4) stud hang-ups, the likelihood of the scenario occurring must first be evaluated. Prior to the analysis discussed in this paper, the likelihood of occurrence had been estimated assuming that the stud hang-ups were completely independent events. That is, it was assumed that no common causes or factors existed between the individual stud hang-up events. A review of the data associated with the hang-up events, showed that a common factor (timing skew) was present. This paper summarizes a revised likelihood evaluation performed for the four (4) stud hang-ups case considering that there are common factors associated with the stud hang-ups. The results show that explicitly (i.e. not using standard common cause methodologies such as beta factor or Multiple Greek Letter modeling) taking into account the common factor of timing skew results in an increase in the estimated likelihood of four (4) stud hang-ups of an order of magnitude over the independent failure case.

Hanging drop monoculture for selection of optimal antioxidants during in vitro maturation of porcine oocytes.

PubMed

Ishikawa, S; Machida, R; Hiraga, K; Hiradate, Y; Suda, Y; Tanemura, K

2014-04-01

We analysed the effect of three antioxidants that have different functional mechanisms on the in vitro maturation (IVM) of porcine oocytes. Single oocyte monoculture using the hanging drop (HD) system has some advantages such as improving analysis efficiency brought by the smaller number of samples than the number of oocytes cultured in one drop. Direct effects of ligands on single oocytes could also be detected without considering the effects of paracrine factors from other oocytes. After 22 h of pre-culture, denuded oocytes were cultured for 22 h with 0.01 and 0.1 μg/ml of L-carnitine (LC), lactoferrin (LF) or sulforaphane (SF) in the presence/non-presence of oxidant stress induced by H2O2 supplementation to evaluate the reducing effects against oxidative stress on nuclear maturation. As a result, compared with LC and SF, LF showed effective reduction in oxidative stress at a lower concentration (0.01 μg/ml), suggesting that LF is a more effective antioxidant in porcine oocyte IVM. Additionally, LF also increased maturation rate even in culture without H2O2. Our results clearly suggest that the HD monoculture system is useful for screening the substances that affect porcine oocyte culture. © 2014 Blackwell Verlag GmbH.

In Vitro Culture of Ovarian Follicles from Peromyscus

PubMed Central

He, Xiaoming; Toth, Thomas L.

2016-01-01

The ovarian follicle is the fundamental functional tissue unit of mammalian ovary. Each ovarian follicle contains one single oocyte. Isolation and in vitro culture of ovarian follicles to obtain fertilizable oocytes have been regarded as a promising strategy for women to combat infertility. The follicles from Peromyscus are considered as a better model than that from inbred mice for studying follicle culture. This is because Peromyscus mice are outbred (as with humans) with an increased life span. In this article, we reviewed studies on this subject conducted using Peromyscus follicles. These studies show that the conventional 2D micro-drop and 3D hanging-drop approaches established for in vitro culture of early preantral follicles from inbred mice are not directly applicable for cultivating the follicles from Peromyscus However, the efficiency could be significantly improved by culturing multiple early preantral follicles in one hanging drop of Peromyscus ovarian cell-conditioned medium. It is further revealed that the mechanical heterogeneity in the extracellular matrix of ovary is crucial for developing early preantral follicles to the antral stage and for the subsequent ovulation to release cumulus-oocyte complex. These findings may provide valuable guidance for furthering the technology of in vitro follicle culture to restore fertility in the clinic. PMID:27397871

Differentiation of mouse embryonic stem cells into cardiomyocytes via the hanging-drop and mass culture methods.

PubMed

Fuegemann, Christopher J; Samraj, Ajoy K; Walsh, Stuart; Fleischmann, Bernd K; Jovinge, Stefan; Breitbach, Martin

2010-12-01

Herein, we describe two protocols for the in vitro differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes. mESCs are pluripotent and can be differentiated into cells of all three germ layers, including cardiomyocytes. The methods described here facilitate the differentiation of mESCs into the different cardiac subtypes (atrial-, ventricular-, nodal-like cells). The duration of cell culture determines whether preferentially early- or late-developmental stage cardiomyocytes can be obtained preferentially. This approach allows the investigation of cardiomyocyte development and differentiation in vitro, and also allows for the enrichment and isolation of physiologically intact cardiomyocytes for transplantation purposes. © 2010 by John Wiley & Sons, Inc.

Lysozyme

NASA Technical Reports Server (NTRS)

2004-01-01

Protein isolated from hen egg-white and functions as a bacteriostatic enzyme by degrading bacterial cell walls. First enzyme ever characterized by protein crystallography. It is used as an excellent model system for better understanding parameters involved in microgravity experiments with data from laboratory experiments to study the equilibrium rate of hanging drop experiments in microgravity.

Microgravity

NASA Image and Video Library

2004-04-15

Protein isolated from hen egg-white and functions as a bacteriostatic enzyme by degrading bacterial cell walls. First enzyme ever characterized by protein crystallography. It is used as an excellent model system for better understanding parameters involved in microgravity experiments with data from laboratory experiments to study the equilibrium rate of hanging drop experiments in microgravity.

Implementation of an axisymmetric drop shape apparatus using a Raspberry-Pi single-board computer and a web camera

NASA Astrophysics Data System (ADS)

Carlà, Marcello; Orlando, Antonio

2018-07-01

This paper describes the implementation of an axisymmetric drop shape apparatus for the measurement of surface or interfacial tension of a hanging liquid drop, using only cheap resources like a common web camera and a single-board microcomputer. The mechanics of the apparatus is composed of stubs of commonly available aluminium bar, with all other mechanical parts manufactured with an amateur 3D printer. All of the required software, either for handling the camera and taking the images, or for processing the drop images to get the drop profile and fit it with the Bashforth and Adams equation, is freely available under an open source license. Despite the very limited cost of the whole setup, an extensive test has demonstrated an overall accuracy of ±0.2% or better.

The quaternary structure of the amidase from Geobacillus pallidus RAPc8 is revealed by its crystal packing

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

Agarkar, Vinod B.; Kimani, Serah W.; Cowan, Donald A.

2006-12-01

The amidase from G. pallidus RAPc8, a moderate thermophile, converts amides to the corresponding acids and ammonia and has application as an industrial catalyst. RAPc8 amidase has been cloned, expressed and purified, and then crystallized using the hanging-drop vapour-diffusion method. The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase enzyme superfamily. It converts amides to the corresponding acids and ammonia and has application as an industrial catalyst. RAPc8 amidase has been cloned and functionally expressed in Escherichia coli and has been purified by heat treatment and a number of chromatographic steps. The enzyme wasmore » crystallized using the hanging-drop vapour-diffusion method. Crystals produced in the presence of 1.2 M sodium citrate, 400 mM NaCl, 100 mM sodium acetate pH 5.6 were selected for X-ray diffraction studies. A data set having acceptable statistics to 1.96 Å resolution was collected under cryoconditions using an in-house X-ray source. The space group was determined to be primitive cubic P4{sub 2}32, with unit-cell parameter a = 130.49 (±0.05) Å. The structure was solved by molecular replacement using the backbone of the hypothetical protein PH0642 from Pyrococcus horikoshii (PDB code 1j31) with all non-identical side chains substituted with alanine as a probe. There is one subunit per asymmetric unit. The subunits are packed as trimers of dimers with D3 point-group symmetry around the threefold axis in such a way that the dimer interface seen in the homologues is preserved.« less

Transformation of atmospheric components near a spark discharge at the anode polarization of a metallic electrode hanging over a solution

NASA Astrophysics Data System (ADS)

Orlov, A. M.; Yavtushenko, I. O.; Bodnarskii, D. S.

2013-03-01

The variation of the pressure of a gas phase activated by spark discharges between an aqueous electrolyte solution (liquid cathode) and a metallic electrode (anode) hanging over the solution is studied. A mathematical model of the proceeding reaction kinetics is constructed, and the variation of the partial pressures of all initial and produced components in the gas phase is calculated. Both the Faraday and non-Faraday mechanisms of gas component production from water are confirmed. It is found that a large overhanging drop responsible for additional supply of simultaneously produced H2 and O2 molecules forms rapidly at the end face of the anodically polarized electrode.

Device For Controlling Crystallization Of Protein

NASA Technical Reports Server (NTRS)

Noever, David A.

1993-01-01

Variable sandwich spacer enables optimization of evaporative driving force that governs crystallization of protein from solution. Mechanically more rigid than hanging-drop and sitting-drop devices. Large oscillations and dislodgment of drop of solution in response to vibrations suppressed by glass plates. Other advantages include: suitable for automated delivery, stable handling, and programmable evaporation of protein solution; controlled configuration enables simple and accurate determination of volume of solution without disrupting crystallization; pH and concentration of precipitant controlled dynamically because pH and concentration coupled to rate of evaporation, controllable via adjustment of gap between plates; and enables variation of ratio between surface area and volume of protein solution. Alternative version, plates oriented vertically instead of horizontally.

Hanging an Airplane: A Case Study in Static Equilibrium

ERIC Educational Resources Information Center

Katz, Debora M.

2009-01-01

Our classrooms are filled with engineering majors who take a semester-long course in static equilibrium. Many students find this class too challenging and drop their engineering major. In our introductory physics class, we often breeze through static equilibrium; to physicists equilibrium is just a special case of Newton's second law. While it is…

Purification, Crystallization, and Preliminary Crystallographic Analysis of Deoxyuridine Triphosphate Nucleotidohydrolase from Arabidopsis Thaliana

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

Bajaj,M.; Moriyama, H.

2007-01-01

The deoxyuridine triphosphate nucleotidohydrolase gene from Arabidopsis thaliana was expressed and the gene product was purified. Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.2 Angstroms resolution using Cu K{alpha} radiation. The crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 69.90, b = 70.86 Angstroms, c = 75.55 Angstroms . Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a VM of 1.8 Angstroms 3 Da-1.

Purification, crystallization and preliminary crystallographic analysis of biotin protein ligase from Staphylococcus aureus

PubMed Central

Pendini, Nicole R.; Polyak, Steve W.; Booker, Grant W.; Wallace, John C.; Wilce, Matthew C. J.

2008-01-01

Biotin protein ligase from Staphylococcus aureus catalyses the biotinylation of acetyl-CoA carboxylase and pyruvate carboxylase. Recombinant biotin protein ligase from S. aureus has been cloned, expressed and purified. Crystals were grown using the hanging-drop vapour-diffusion method using PEG 8000 as the precipitant at 295 K. X-ray diffraction data were collected to 2.3 Å resolution from crystals using synchrotron X-ray radiation at 100 K. The diffraction was consistent with the tetragonal space group P42212, with unit-cell parameters a = b = 93.665, c = 131.95. PMID:18540065

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

Guo, Feng; Jin, Tengchuan; Howard, Andrew

The crystallization of the brazil nut allergen Ber e 2 is reported. Peanut and tree-nut allergies have attracted considerable attention because of their frequency and their lifelong persistence. Brazil-nut (Bertholletia excelsa) allergies have been well documented and the 11S legumin-like seed storage protein Ber e 2 (excelsin) is one of the two known brazil-nut allergens. In this study, Ber e 2 was extracted from brazil-nut kernels and purified to high purity by crystalline precipitation and gel-filtration chromatography. Well diffracting single crystals were obtained using the hanging-drop vapour-diffusion method. A molecular-replacement structural solution has been obtained. Refinement of the structure ismore » currently under way.« less

Spore-to-spore agar culture of the myxomycete Physarum globuliferum.

PubMed

Liu, Pu; Wang, Qi; Li, Yu

2010-02-01

The ontogeny of the myxomycete Physarum globuliferum was observed on corn meal agar and hanging drop cultures without adding sterile oat flakes, bacteria or other microorganisms. Its complete life cycle including spore germination, myxamoebae, swarm cells, plasmodial development, and maturity of fructifications was demonstrated. Details of spore-to-spore development are described and illustrated.

Determination of picloram in natural waters employing sequential injection square wave voltammetry using the hanging mercury drop electrode.

PubMed

Dos Santos, Luciana B O; Masini, Jorge C

2007-05-15

This paper describes the development of a sequential injection analysis method to automate the determination of picloram by square wave voltammetry exploiting the concept of monosegmented flow analysis to perform in-line sample conditioning and standard addition. To perform these tasks, an 800muL monosegment is formed, composed by 400muL of sample and 400muL of conditioning/standard solution, in medium of 0.10molL(-1) H(2)SO(4). Homogenization of the monosegment is achieved by three flow reversals. After homogenization the mixture zone is injected toward the flow cell, which is adapted to the capillary of a hanging drop mercury electrode, at a flow rate of 50muLs(-1). After a suitable delay time, the potential is scanned from -0.5 to -1.0V versus Ag/AgCl at frequency of 300Hz and pulse height of 25mV. The linear dynamic range is observed for picloram concentrations between 0.10 and 2.50mgL(-1) fitting to the linear equation I(p)=(-2.19+/-0.03)C(picloram)+(0.096+/-0.039), with R(2)=0.9996, for which the slope is given in muALmg(-1). The detection and quantification limits are 0.036 and 0.12mgL(-1), respectively. The sampling frequency is 37h(-1) when the standard addition protocol is followed, but can be increased to 41h(-1) if the protocol to obtain in-line external calibration curve is used for quantification. The method was applied for determination of picloram in spiked water samples and the accuracy was evaluated by comparison with high performance liquid chromatography using molecular absorption at 220nm for detection. No evidences of statistically significant differences between the two methods were observed.

Derivation and characterization of gut-like structures from embryonic stem cells.

PubMed

Yamada, Takatsugu; Nakajima, Yoshiyuki

2006-01-01

Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages of all three embryonic germ layers in vitro. The hanging drop culture of ES cell suspension in the absence of leukemia inhibitory factor induces aggregation and differentiation of the cells into simple or cystic embryoid bodies (EBs). After 6 d of hanging drop culture, the resulting EBs are plated onto plastic dishes for the outgrowth culture. At d 21 after outgrowth culture, cell populations of EBs can give rise to three-dimensional gut-like structures that exhibit spontaneous contraction and highly coordinated peristalsis. The gut-like structures have large lumens surrounded by three layers: epithelium, lamina propria, and muscularis. Ganglia are scattered along the periphery, and interstitial cells of Cajal are distributed among the smooth muscle cells. The fundamental process of formation of the in vitro organized gut-like structures is similar to embryonic gastrointestinal development in vivo. The EBs at the 6-d egg-cylinder stage may have the potential to regulate developmental programs associated with cell lineage commitment and provide an appropriate microenvironment to differentiate ES cells into enteric derivatives of all three embryonic germ layers and reproduce the gut organization process in vitro.

Numerical analysis of moving contact line with contact angle hysteresis using feedback deceleration technique

NASA Astrophysics Data System (ADS)

Park, Jun Kwon; Kang, Kwan Hyoung

2012-04-01

Contact angle (CA) hysteresis is important in many natural and engineering wetting processes, but predicting it numerically is difficult. We developed an algorithm that considers CA hysteresis when analyzing the motion of the contact line (CL). This algorithm employs feedback control of CA which decelerates CL speed to make the CL stationary in the hysteretic range of CA, and one control coefficient should be heuristically determined depending on characteristic time of the simulated system. The algorithm requires embedding only a simple additional routine with little modification of a code which considers the dynamic CA. The method is non-iterative and explicit, and also has less computational load than other algorithms. For a drop hanging on a wire, the proposed algorithm accurately predicts the theoretical equilibrium CA. For the drop impacting on a dry surface, the results of the proposed algorithm agree well with experimental results including the intermittent occurrence of the pinning of CL. The proposed algorithm is as accurate as other algorithms, but faster.

Surface Tension Guided Hanging-Drop: Producing Controllable 3D Spheroid of High-Passaged Human Dermal Papilla Cells and Forming Inductive Microtissues for Hair-Follicle Regeneration.

PubMed

Lin, Bojie; Miao, Yong; Wang, Jin; Fan, Zhexiang; Du, Lijuan; Su, Yongsheng; Liu, Bingcheng; Hu, Zhiqi; Xing, Malcolm

2016-03-09

Human dermal papilla (DP) cells have been studied extensively when grown in the conventional monolayer. However, because of great deviation from the real in vivo three-dimensional (3D) environment, these two-dimensional (2D) grown cells tend to lose the hair-inducible capability during passaging. Hence, these 2D caused concerns have motivated the development of novel 3D culture techniques to produce cellular microtissues with suitable mimics. The hanging-drop approach is based on surface tension-based technique and the interaction between surface tension and gravity field that makes a convergence of liquid drops. This study used this technique in a converged drop to form cellular spheroids of dermal papilla cells. It leads to a controllable 3Dspheroid model for scalable fabrication of inductive DP microtissues. The optimal conditions for culturing high-passaged (P8) DP spheroids were determined first. Then, the morphological, histological and functional studies were performed. In addition, expressions of hair-inductive markers including alkaline phosphatase, α-smooth muscle actin and neural cell adhesion molecule were also analyzed by quantitative RT-PCR, immunostaining and immunoblotting. Finally, P8-DP microtissues were coimplanted with newborn mouse epidermal cells (EPCs) into nude mice. Our results indicated that the formation of 3D microtissues not only endowed P8-DP microtissues many similarities to primary DP, but also confer these microtissues an enhanced ability to induce hair-follicle (HF) neogenesis in vivo. This model provides a potential to elucidate the native biology of human DP, and also shows the promising for the controllable and scalable production of inductive DP cells applied in future follicle regeneration.

Process modelling for space station experiments

NASA Technical Reports Server (NTRS)

Rosenberger, Franz; Alexander, J. Iwan D.

1988-01-01

The work performed during the first year 1 Oct. 1987 to 30 Sept. 1988 involved analyses of crystal growth from the melt and from solution. The particular melt growth technique under investigation is directional solidification by the Bridgman-Stockbarger method. Two types of solution growth systems are also being studied. One involves growth from solution in a closed container, the other concerns growth of protein crystals by the hanging drop method. Following discussions with Dr. R. J. Naumann of the Low Gravity Science Division at MSFC it was decided to tackle the analysis of crystal growth from the melt earlier than originally proposed. Rapid progress was made in this area. Work is on schedule and full calculations were underway for some time. Progress was also made in the formulation of the two solution growth models.

How far can Tarzan jump?

NASA Astrophysics Data System (ADS)

Shima, Hiroyuki

2012-11-01

The tree-based rope swing is a popular recreational facility, often installed in outdoor areas. Hanging from a rope, users drop from a high platform and then swing at great speed like ‘Tarzan’, finally jumping ahead to land on the ground. The question naturally arises, how far can Tarzan jump using the swing? In this paper, I present an introductory analysis of the mechanics of the Tarzan swing, a large pendulum-like swing with Tarzan himself attached as weight. This enables determination of how much further forward Tarzan can jump using a given swing apparatus. The discussion is based on elementary mechanics and is, therefore, expected to provide rich opportunities for investigations using analytic and numerical methods.

Expression, purification and preliminary crystallographic analysis of sucrose phosphate synthase (SPS) from Halothermothrix orenii

PubMed Central

Huynh, Frederick; Tan, Tien-Chye; Swaminathan, Kunchithapadam; Patel, Bharat K. C.

2005-01-01

This is the first report of the crystallization of a sucrose phosphate synthase (SPS; EC 2.4.1.14). It also constitutes the first study of a sucrose phosphate synthase from a non-photosynthetic thermohalophilic anaerobic bacterium, Halothermothrix orenii. The purified recombinant spsA protein has been crystallized in the monoclinic space group C2, with unit-cell parameters a = 154.2, b = 47.9, c = 72.3 Å, β = 103.16°, using the hanging-drop vapour-diffusion method. The crystal diffracts X-rays to a resolution limit of 3.01 Å. Heavy-metal and halide-soaking trials are currently in progress to solve the structure. PMID:16508108

Creatinine sensor based on a molecularly imprinted polymer-modified hanging mercury drop electrode.

PubMed

Lakshmi, Dhana; Prasad, Bhim Bali; Sharma, Piyush Sindhu

2006-09-15

Molecularly imprinted polymers (MIP) have been elucidated to work as artificial receptors. In our present study, a MIP was applied as a molecular recognition element to a chemical sensor. We have constructed a creatinine sensor based on a MIP layer selective for creatinine and its differential pulse, cathodic stripping voltammetric detection (DPCSV) on a hanging mercury drop electrode (HMDE). The creatinine sensor was fabricated by the drop coating of dimethylformamide (DMF) solution of a creatinine-imprinted polymer onto the surface of HMDE. The modified-HMDE, preanodised in neutral medium at +0.4V versus Ag/AgCl for 120s, exhibited a marked enhancement in DPCSV current in comparison to the less anodised (

The possible equilibrium shapes of static pendant drops

NASA Astrophysics Data System (ADS)

Sumesh, P. T.; Govindarajan, Rama

2010-10-01

Analytical and numerical studies are carried out on the shapes of two-dimensional and axisymmetric pendant drops hanging under gravity from a solid surface. Drop shapes with both pinned and equilibrium contact angles are obtained naturally from a single boundary condition in the analytical energy optimization procedure. The numerical procedure also yields optimum energy shapes, satisfying Young's equation without the explicit imposition of a boundary condition at the plate. It is shown analytically that a static pendant two-dimensional drop can never be longer than 3.42 times the capillary length. A related finding is that a range of existing solutions for long two-dimensional drops correspond to unphysical drop shapes. Therefore, two-dimensional drops of small volume display only one static solution. In contrast, it is known that axisymmetric drops can display multiple solutions for a given volume. We demonstrate numerically that there is no limit to the height of multiple-lobed Kelvin drops, but the total volume is finite, with the volume of successive lobes forming a convergent series. The stability of such drops is in question, though. Drops of small volume can attain large heights. A bifurcation is found within the one-parameter space of Laplacian shapes, with a range of longer drops displaying a minimum in energy in the investigated space. Axisymmetric Kelvin drops exhibit an infinite number of bifurcations.

Scaling during capillary thinning of particle-laden drops

NASA Astrophysics Data System (ADS)

Thete, Sumeet; Wagoner, Brayden; Basaran, Osman

2017-11-01

A fundamental understanding of drop formation is crucial in many applications such as ink-jet printing, microfluidic devices, and atomization. During drop formation, the about-to-form drop is connected to the fluid hanging from the nozzle via a thinning filament. Therefore, the physics of capillary thinning of filaments is key to understanding drop formation and has been thoroughly studied for pure Newtonian fluids using theory, simulations, and experiments. In some of the applications however, the forming drop and hence the thinning filament may contain solid particles. The thinning dynamics of such particle-laden filaments differs radically from that of particle-free filaments. Moreover, our understanding of filament thinning in the former case is poor compared to that in the latter case despite the growing interest in pinch-off of particle-laden filaments. In this work, we go beyond similar studies and experimentally explore the impact of solid particles on filament thinning by measuring both the radial and axial scalings in the neck region. The results are summarized in terms of a phase diagram of capillary thinning of particle-laden filaments.

Investigation for the differentiation process of mouse ES cells by Raman spectroscopy

NASA Astrophysics Data System (ADS)

Yamaguchi, Yoshinori; El-Hagrasy, Maha A.; Shimizu, Eiichi; Saito, Masato; Tamiya, Eiichi

2012-03-01

The arrangement of differentiated pluripotent embryonic stem cells into three-dimensional aggregates, which are known as embryonic bodies, is a main step for progressing the embryonic stem cells differentiation. In this work, embryonic stem cells that were directly produced from the hanging drop step as a three-dimensional structure with no further twodimensional differentiation were diagnosed with Raman spectroscopy as a non-invasive and label-free technique. Raman spectroscopy was employed to discriminate between mouse embryonic bodies of different degrees of maturation. EBs were prepared applying the hanging drop method. The Raman scattering measurements were obtained in vitro with a Nanophoton RAMAN-11 micro-spectrometer (Japan: URL: www.nanophoton.jp equipped with an Olympus XLUM Plan FLN 20X/NA= 1.0 objective lens. Spectral data were smoothed, baseline corrected and normalized to the a welldefined intense 1003 cm-1 band (phenylalanine) which is insensitive to changes in conformation or environment. The differentiation process of embryonic stem cells is initiated by the removal of LIF from culture medium. 1, 7 and 17-dayold embryonic stem cells were collected and investigated by Raman spectroscopy. The main differences involve bands which decreased with maturation such as: 784 cm-1 (U, T, C ring br DNA/RNA, O-P-O str); 1177 cm-1 (cytosine, guanine) and 1578 cm-1 (G, A). It was found that with the progress of differentiation the protein content was amplified. The increase of protein to nucleic acid ratio was also previously observed with the progress of the differentiation process. Raman spectroscopy has the potential to distinguish between the Raman signatures of live embryonic stem cells with different degrees of maturation.

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

Kish, Kevin; McDonnell, Patricia A.; Goldfarb, Valentina

Protein tyrosine phosphatase {gamma} is a membrane-bound receptor and is designated RPTP{gamma}. RPTP{gamma} and two mutants, RPTP{gamma}(V948I, S970T) and RPTP{gamma}(C858S, S970T), were recombinantly expressed and purified for X-ray crystallographic studies. The purified enzymes were crystallized using the hanging-drop vapor-diffusion method. Crystallographic data were obtained from several different crystal forms in the absence and the presence of inhibitor. In this paper, a description is given of how three different crystal forms were obtained that were used with various ligands. An orthorhombic crystal form and a trigonal crystal form were obtained both with and without ligand, and a monoclinic crystal form wasmore » only obtained in the presence of a particularly elaborated inhibitor.« less

Crystallization and preliminary crystallographic analysis of a surface antigen glycoprotein, SAG19, from Eimeria tenella

PubMed Central

Ramly, Nur Zazarina; Rouzheinikov, Sergey N.; Sedelnikova, Svetlana E.; Baker, Patrick J.; Chow, Yock-Ping; Wan, Kiew-Lian; Nathan, Sheila; Rice, David W.

2013-01-01

Coccidiosis in chickens is caused by the apicomplexan parasite Eimeria tenella and is thought to involve a role for a superfamily of more than 20 cysteine-rich surface antigen glycoproteins (SAGs) in host–parasite interactions. A representative member of the family, SAG19, has been overexpressed in Escherichia coli, purified and crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. Crystals of SAG19 diffracted to beyond 1.50 Å resolution and belonged to space group I4, with unit-cell parameters a = b = 108.2, c = 37.5 Å. Calculation of possible values of V M suggests that there is a single molecule in the asymmetric unit. PMID:24316835

Crystallization and preliminary X-ray diffraction analysis of a cold-adapted catalase from Vibrio salmonicida

PubMed Central

Riise, Ellen Kristin; Lorentzen, Marit Sjo; Helland, Ronny; Willassen, Nils Peder

2006-01-01

Catalase (EC 1.11.1.6) catalyses the breakdown of hydrogen peroxide to water and molecular oxygen. Recombinant Vibrio salmonicida catalase (VSC) possesses typical cold-adapted features, with higher catalytic efficiency, lower thermal stability and a lower temperature optimum than its mesophilic counterpart from Proteus mirabilis. Crystals of VSC were produced by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 98.15, b = 217.76, c = 99.28 Å, β = 110.48°. Data were collected to 1.96 Å and a molecular-replacement solution was found with eight molecules in the asymmetric unit. PMID:16511268

Sensitive determination of mercury by a miniaturized spectrophotometer after in situ single-drop microextraction.

PubMed

Yang, Fangwen; Liu, Rui; Tan, Zhiqiang; Wen, Xiaodong; Zheng, Chengbin; Lv, Yi

2010-11-15

An in situ single-drop microextraction (SDME) method was developed for trace mercury determination by a miniaturized spectrophotometer, in which a simple and cheap light-emitting diode (LED) was employed as the light source, and a handheld charge coupled device (CCD) was served as the detector. A droplet of 0.006% dithizone-CCl(4) (m/v) was used as extraction phase and hanged on a rolled PTFE tube. LED light was adjusted carefully to pass through the centre of the droplet and the entrance slit of the CCD detector. The radiation intensities of 475 nm before and after SDME (I(0) and I(i)) were recorded for quantification. Under the optimum conditions, the system provided a linear range of 2-50 μg L(-1), with a correlation coefficient of 0.9983 and a limit of detection (3σ) of 0.2 μg L(-1). The enrichment factor was about 69. The present method showed the merits of high sensitivity, simplicity, rapidity, low reagent consumption and field analysis potential. Finally, this method was successfully applied for the determination of the total mercury in spiked tap water sample, spiked river water sample and certified reference material (GBW (E) 080393, simulated water). Copyright © 2010 Elsevier B.V. All rights reserved.

Newtonian Analysis of a Folded Chain Drop

ERIC Educational Resources Information Center

Mungan, Carl E.

2018-01-01

Consider a chain of length L that hangs in a U shape with end A fixed to a rigid support and free end E released from rest starting from the same initial height (call it y = 0) as A. Figure 1 sketches the chain after end E has fallen a distance y. Points O and A are assumed to be close enough to each other and the chain flexible enough that the…

Reassembly of Anterior Pituitary Organization by Hanging Drop Three-Dimensional Cell Culture

PubMed Central

Tsukada, Takehiro; Kouki, Tom; Fujiwara, Ken; Ramadhani, Dini; Horiguchi, Kotaro; Kikuchi, Motoshi; Yashiro, Takashi

2013-01-01

The anterior pituitary gland comprises 5 types of hormone-producing cells and non-endocrine cells, such as folliculostellate (FS) cells. The cells form a lobular structure surrounded by extracellular matrix (ECM) but are not randomly distributed in each lobule; hormone-producing cells have affinities for specific cell types (topographic affinity), and FS cells form a homotypic meshwork. To determine whether this cell and ECM organization can be reproduced in vitro, we developed a 3-dimensional (3D) model that utilizes hanging drop cell culture. We found that the topographic affinities of hormone-producing cells were indeed maintained (ie, GH to ACTH cells, GH to TSH cells, PRL to LH/FSH cells). Fine structures in hormone-producing cells retained their normal appearance. In addition, FS cells displayed well-developed cytoplasmic protrusions, which interconnected with adjacent FS cells to form a 3D meshwork. In addition, reassembly of gap junctions and pseudofollicles among FS cells was observed in cell aggregates. Major ECM components—collagens and laminin—were deposited and distributed around the cells. In sum, the dissociated anterior pituitary cells largely maintained their in vivo anterior pituitary architectures. This culture system appears to be a powerful experimental tool for detailed analysis of anterior pituitary cell organization. PMID:24023396

Mixture effects of benzene, toluene, ethylbenzene, and xylenes (BTEX) on lung carcinoma cells via a hanging drop air exposure system.

PubMed

Liu, Faye F; Escher, Beate I; Were, Stephen; Duffy, Lesley; Ng, Jack C

2014-06-16

A recently developed hanging drop air exposure system for toxicity studies of volatile chemicals was applied to evaluate the cell viability of lung carcinoma A549 cells after 1 and 24 h of exposure to benzene, toluene, ethylbenzene, and xylenes (BTEX) as individual compounds and as mixtures of four or six components. The cellular chemical concentrations causing 50% reduction of cell viability (EC50) were calculated using a mass balance model and came to 17, 12, 11, 9, 4, and 4 mmol/kg cell dry weight for benzene, toluene, ethylbenzene, m-xylene, o-xylene, and p-xylene, respectively, after 1 h of exposure. The EC50 decreased by a factor of 4 after 24 h of exposure. All mixture effects were best described by the mixture toxicity model of concentration addition, which is valid for chemicals with the same mode of action. Good agreement with the model predictions was found for benzene, toluene, ethylbenzene, and m-xylene at four different representative fixed concentration ratios after 1 h of exposure, but lower agreement with mixture prediction was obtained after 24 h of exposure. A recreated car exhaust mixture, which involved the contribution of the more toxic p-xylene and o-xylene, yielded an acceptable, but lower quality, prediction as well.

Reassembly of anterior pituitary organization by hanging drop three-dimensional cell culture.

PubMed

Tsukada, Takehiro; Kouki, Tom; Fujiwara, Ken; Ramadhani, Dini; Horiguchi, Kotaro; Kikuchi, Motoshi; Yashiro, Takashi

2013-08-29

The anterior pituitary gland comprises 5 types of hormone-producing cells and non-endocrine cells, such as folliculostellate (FS) cells. The cells form a lobular structure surrounded by extracellular matrix (ECM) but are not randomly distributed in each lobule; hormone-producing cells have affinities for specific cell types (topographic affinity), and FS cells form a homotypic meshwork. To determine whether this cell and ECM organization can be reproduced in vitro, we developed a 3-dimensional (3D) model that utilizes hanging drop cell culture. We found that the topographic affinities of hormone-producing cells were indeed maintained (ie, GH to ACTH cells, GH to TSH cells, PRL to LH/FSH cells). Fine structures in hormone-producing cells retained their normal appearance. In addition, FS cells displayed well-developed cytoplasmic protrusions, which interconnected with adjacent FS cells to form a 3D meshwork. In addition, reassembly of gap junctions and pseudofollicles among FS cells was observed in cell aggregates. Major ECM components-collagens and laminin-were deposited and distributed around the cells. In sum, the dissociated anterior pituitary cells largely maintained their in vivo anterior pituitary architectures. This culture system appears to be a powerful experimental tool for detailed analysis of anterior pituitary cell organization.

Linkage between canopy water storage and drop size distributions of leaf drips

NASA Astrophysics Data System (ADS)

Nanko, Kazuki; Watanabe, Ai; Hotta, Norifumi; Suzuki, Masakazu

2013-04-01

Differences in drop size distribution (DSD) of leaf drips among tree species have been estimated and physically interpreted to clarify the leaf drip generation process. Leaf drip generation experiments for nine species were conducted in an indoor location without foliage vibration using an automatic mist spray. Broad-leaved species produced a similar DSD among species whose leaves had a matte surface and a second similar DSD among species whose leaves had a coated surface. The matte broad leaves produced a larger and wider range of DSDs than the coated broad leaves. Coated coniferous needles had a wider range of DSDs than the coated broad leaves and different DSDs were observed for different species. The species with shorter dense needles generated a larger DSD. The leaf drip diameter was calculated through the estimation of a state of equilibrium of a hanging drop on the leaves based on physical theory. The calculations indicated that the maximum diameter of leaf drips was determined by the contact angle, and the range of DSDs was determined by the variation in contact length and the contact diameter at the hanging points. The results revealed that leaf drip DSD changed due to variations in leaf hydrophobicity, leaf roughness, leaf geometry and leaf inclination among the different tree species. This study allows the modelization of throughfall DSD. Furthermore, it indicates the possibility of interpreting canopy water processes from canopy water storage to drainage through the contact angle and leaf drip DSD. The part of this study is published in Nanko et al. (2013, Agric. Forest. Meteorol. 169, 74-84).

Course of Near-hanging Victims Succumbed to Death: A Seven Year Study

PubMed Central

Mugadlimath, Anand B.; Zine, K.U.; Farooqui, Jamebaseer M.; Phalke, Balaji J.

2015-01-01

Introduction: Near hanging refers to victims who survive a hanging injury following attempted hanging, long enough to reach hospital. Delayed deaths in near hanging patients are mostly due to complication of hanging. The purpose of this study was to evaluate the demographics, mortality patterns and cause of delayed deaths in near hanging victims. Materials and Methods: In this study autopsy files over a seven year period from 2007 to 2013 were reviewed, and data of near hanging deaths (attempted hanging cases who succumbed to death and subjected for medicolegal autopsy) was extracted. Records of 14,000 autopsies was reviewed, and 10 deceased having died delayed deaths after near hanging episode were identified. In each case, the patients’ details, including gender, age, type of suspension, type of ligature material used for hanging and subsequent hanging mark produced were reviewed using autopsy reports and photographs taken during autopsy. Results: Demographic and pathological aspects of the each case discussed to throw light on autopsy findings in victims who died following near hanging. Complete suspension was present in 3 cases, while partial suspension was present in 7 cases. Survivals in delayed death after near hanging episode have ranged from 9 h to 72 d. Hypoxic encephalopathy was the most common cause of death, followed by pneumonia. Conclusion: Most of the near hanging patients did succumb to hypoxic encephalopathy; however, consolidation of lungs (pneumonia) was the next common cause of death reflecting need for aggressive oxygen therapy and selective resuscitation should be performed in all such cases. PMID:25954634

Dynamics of contracting surfactant-covered filaments

NASA Astrophysics Data System (ADS)

Kamat, Pritish; Thete, Sumeet; Xu, Qi; Basaran, Osman

2013-11-01

When drops are produced from a nozzle, a thin liquid thread connects the primary drop that is about to form to the rest of the liquid in the nozzle. Often, the thread becomes disconnected from both the primary drop and the remnant liquid mass hanging from the nozzle and thereby gives rise to a free filament. Due to surface tension, the free filament then contracts or recoils. During recoil, the filament can either contract into a single satellite droplet or break up into several small satellites. Such satellite droplets are undesirable in applications where they can, for example, cause misting in a manufacturing environment and mar product quality in ink-jet printing. In many applications, the filaments are coated with a monolayer of surfactant. In this work, we study the dynamics of contraction of slender filaments of a Newtonian fluid that are covered with a monolayer of surfactant when the surrounding fluid is a passive gas. Taking advantage of the fact that the filaments are long and slender, we use a 1D-slender-jet approximation of the governing system of equations consisting of the Navier-Stokes system and the convection-diffusion equation for surfactant transport. We solve the 1D system of equations by a finite element based numerical method.

Etiology and Use of the “Hanging Drop” Technique: A Review

PubMed Central

Todorov, Ludmil; VadeBoncouer, Timothy

2014-01-01

Background. The hanging drop (HD) technique presumably relies on the presence of subatmospheric epidural pressure. It is not clear whether this negative pressure is intrinsic or an artifact and how it is affected by body position. There are few data to indicate how often HD is currently being used. Methods. We identified studies that measured subatmospheric pressures and looked at the effect of the sitting position. We also looked at the technique used for cervical and thoracic epidural anesthesia in the last 10 years. Results. Intrinsic subatmospheric pressures were measured in the thoracic and cervical spine. Three trials studied the effect of body position, indicating a higher incidence of subatmospheric pressures when sitting. The results show lower epidural pressure (−10.7 mmHg) with the sitting position. 28.8% of trials of cervical and thoracic epidural anesthesia that documented the technique used, utilized the HD technique. When adjusting for possible bias, the rate of HD use can be as low as 11.7%. Conclusions. Intrinsic negative pressure might be present in the cervical and thoracic epidural space. This effect is more pronounced when sitting. This position might be preferable when using HD. Future studies are needed to compare it with the loss of resistance technique. PMID:24839558

Systematic Improvement of Protein Crystals by Determining the Supersolubility Curves of Phase Diagrams

PubMed Central

Saridakis, Emmanuel; Chayen, Naomi E.

2003-01-01

A systematic approach for improving protein crystals by growing them in the metastable zone using the vapor diffusion technique is described. This is a simple technique for optimization of crystallization conditions. Screening around known conditions is performed to establish a working phase diagram for the crystallization of the protein. Dilutions of the crystallization drops across the supersolubility curve into the metastable zone are then carried out as follows: the coverslips holding the hanging drops are transferred, after being incubated for some time at conditions normally giving many small crystals, over reservoirs at concentrations which normally yield clear drops. Fewer, much larger crystals are obtained when the incubation times are optimized, compared with conventional crystallization at similar conditions. This systematic approach has led to the structure determination of the light-harvesting protein C-phycocyanin to the highest-ever resolution of 1.45 Å. PMID:12547801

High-Throughput Platform for Patient-Derived, Small Cell Number, Three-Dimensional Ovarian Cancer Spheroids

DTIC Science & Technology

2014-09-01

these small cell number spheroids show 3-D morphology (Figure 3). We also observed differences in the expression of mesenchymal markers when...Scale bar =100 µm. Figure 3: Small cell number spheroids demonstrate 3-D morphology . 3-D reconstructions of confocal z-stacks are shown for...formation was observed with the addition of MSCs, and subsequent co-culture in hanging drop plates preserved spheroid morphology indicated in the phase

Human serum albumin crystals and method of preparation

NASA Technical Reports Server (NTRS)

Carter, Daniel C. (Inventor)

1989-01-01

Human serum albumin (HSA) crystals are provided in the form of tetragonal plates having the space groups P42(sub 1)2, the crystals being grown to sizes in excess of 0.5 mm in two dimensions and a thickness of 0.1 mm. Growth of the crystals is carried out by a hanging drop method wherein a precipitant solution containing polyethylene glycol (PEG) and a phosphate buffer is mixed with an HSA solution, and a droplet of mixed solution is suspended over a well of precipitant solution. Crystals grow to the desired size in 3 to 7 days. Concentration of reagents, pH and other parameters are controlled within prescribed limits. The resulting crystals exhibit a size and quality such as to allow performance of x ray diffraction studies and enable the conduct of drug binding studies as well as genetic engineering studies.

Protein crystal growth in a microgravity environment

NASA Technical Reports Server (NTRS)

Bugg, Charles E.

1988-01-01

Protein crystal growth is a major experimental problem and is the bottleneck in widespread applications of protein crystallography. Research efforts now being pursued and sponsored by NASA are making fundamental contributions to the understanding of the science of protein crystal growth. Microgravity environments offer the possibility of performing new types of experiments that may produce a better understanding of protein crystal growth processes and may permit growth environments that are more favorable for obtaining high quality protein crystals. A series of protein crystal growth experiments using the space shuttle was initiated. The first phase of these experiments was focused on the development of micro-methods for protein crystal growth by vapor diffusion techniques, using a space version of the hanging drop method. The preliminary space experiments were used to evolve prototype hardware that will form the basis for a more advanced system that can be used to evaluate effects of gravity on protein crystal growth.

Crystallization and preliminary X-ray analysis of 2,3-diketo-5-methylthiopentyl-1-phosphate enolase from Bacillus subtilis

PubMed Central

Tamura, Haruka; Ashida, Hiroki; Koga, Shogo; Saito, Yohtaro; Yadani, Tomonori; Kai, Yasushi; Inoue, Tsuyoshi; Yokota, Akiho; Matsumura, Hiroyoshi

2009-01-01

2,3-Diketo-5-methylthiopentyl-1-phosphate enolase (DK-MTP-1P enolase) from Bacillus subtilis was crystallized using the hanging-drop vapour-diffusion method. Crystals grew using PEG 3350 as the precipitant at 293 K. The crystals diffracted to 2.3 Å resolution at 100 K using synchrotron radiation and were found to belong to the monoclinic space group P21, with unit-cell parameters a = 79.3, b = 91.5, c = 107.0 Å, β = 90.8°. The asymmetric unit contained four molecules of DK-MTP-1P enolase, with a V M value of 2.2 Å3 Da−1 and a solvent content of 43%. PMID:19194007

Purification, crystallization and preliminary crystallographic study of a recombinant plant aminoaldehyde dehydrogenase from Pisum sativum

PubMed Central

Tylichová, Martina; Briozzo, Pierre; Kopečný, David; Ferrero, Julien; Moréra, Solange; Joly, Nathalie; Snégaroff, Jacques; Šebela, Marek

2008-01-01

Aminoaldehydes are products of polyamine degradation and are known to be reactive metabolites that are toxic to living cells at high concentrations. These compounds are catabolized by aminoaldehyde dehydrogenases, which are enzymes that contain a nicotinamide adenine dinucleotide coenzyme. Amino­aldehyde dehydrogenase from Pisum sativum was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop method. A complete data set was collected to 2.8 Å resolution at 100 K. Crystals belong to the monoclinic space group P21, with unit-cell parameters a = 86.4, b = 216.6, c = 205.4 Å, β = 98.1°. Molecular replacement was performed and led to the identification of six dimers per asymmetric unit. PMID:18259056

Expression, Purification And Preliminary X-Ray Analysis of the C-Terminal Domain of An Arginine Repressor Protein From Mycobacterium Tuberculosis

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

Lu, G.J.; Garen, C.R.; Cherney, M.M.

2009-06-03

The gene product of an open reading frame Rv1657 from Mycobacterium tuberculosis is a putative arginine repressor protein (ArgR), a transcriptional factor that regulates the expression of arginine-biosynthetic enzymes. Rv1657 was expressed and purified and a C-terminal domain was crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 2.15 {angstrom}. The crystals belong to space group P1 and the Matthews coefficient suggests that the crystals contain six C-terminal domain molecules per unit cell. Previous structural and biochemical studies on the arginine repressor proteins from other organisms have likewise shown the presence of sixmore » molecules per unit cell.« less

Purification, identification and preliminary crystallographic studies of Pru du amandin, an allergenic protein from Prunus dulcis.

PubMed

Gaur, Vineet; Sethi, Dhruv K; Salunke, Dinakar M

2008-01-01

Food allergies appear to be one of the foremost causes of hypersensitivity reactions. Nut allergies account for most food allergies and are often permanent. The 360 kDa hexameric protein Pru du amandin, a known allergen, was purified from almonds (Prunus dulcis) by ammonium sulfate fractionation and ion-exchange chromatography. The protein was identified by a BLAST homology search against the nonredundant sequence database. Pru du amandin belongs to the 11S legumin family of seed storage proteins characterized by the presence of a cupin motif. Crystals were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P4(1) (or P4(3)), with unit-cell parameters a = b = 150.7, c = 164.9 A.

Purification, identification and preliminary crystallographic studies of Pru du amandin, an allergenic protein from Prunus dulcis

PubMed Central

Gaur, Vineet; Sethi, Dhruv K.; Salunke, Dinakar M.

2008-01-01

Food allergies appear to be one of the foremost causes of hypersensitivity reactions. Nut allergies account for most food allergies and are often permanent. The 360 kDa hexameric protein Pru du amandin, a known allergen, was purified from almonds (Prunus dulcis) by ammonium sulfate fractionation and ion-exchange chromatography. The protein was identified by a BLAST homology search against the nonredundant sequence database. Pru du amandin belongs to the 11S legumin family of seed storage proteins characterized by the presence of a cupin motif. Crystals were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P41 (or P43), with unit-cell parameters a = b = 150.7, c = 164.9 Å. PMID:18097098

Purification, crystallization and preliminary crystallographic analysis of a 6-pyruvoyltetrahydropterin synthase homologue from Esherichia coli.

PubMed

Seo, Kyung Hye; Supangat; Kim, Hye Lim; Park, Young Shik; Jeon, Che Ok; Lee, Kon Ho

2008-02-01

6-Pyruvoyltetrahydropterin synthase from E. coli (ePTPS) has been crystallized using the hanging-drop vapour-diffusion method. Hexagonal- and rectangular-shaped crystals were obtained. Diffraction data were collected from the hexagonal and rectangular crystals to 3.0 and 2.3 A resolution, respectively. The hexagonal plate-shaped crystals belonged to space group P321, with unit-cell parameters a = b = 112.59, c = 68.82 A , and contained two molecules in the asymmetric unit. The rectangular crystals belonged to space group I222, with unit-cell parameters a = 112.76, b = 117.66, c = 153.57 A , and contained six molecules in the asymmetric unit. The structure of ePTPS in both crystal forms has been determined by molecular replacement.

Purification, crystallization and preliminary crystallographic analysis of a 6-pyruvoyltetrahydropterin synthase homologue from Esherichia coli

PubMed Central

Seo, Kyung Hye; Supangat; Kim, Hye Lim; Park, Young Shik; Jeon, Che Ok; Lee, Kon Ho

2008-01-01

6-Pyruvoyltetrahydropterin synthase from E. coli (ePTPS) has been crystallized using the hanging-drop vapour-diffusion method. Hexagonal- and rectangular-shaped crystals were obtained. Diffraction data were collected from the hexagonal and rectangular crystals to 3.0 and 2.3 Å resolution, respectively. The hexagonal plate-shaped crystals belonged to space group P321, with unit-cell parameters a = b = 112.59, c = 68.82 Å, and contained two molecules in the asymmetric unit. The rectangular crystals belonged to space group I222, with unit-cell parameters a = 112.76, b = 117.66, c = 153.57 Å, and contained six molecules in the asymmetric unit. The structure of ePTPS in both crystal forms has been determined by molecular replacement. PMID:18271114

Crystallization and preliminary X-ray diffraction analysis of a chitin-binding domain of hyperthermophilic chitinase from Pyrococcus furiosus

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

Nakamura, Tsutomu; Ishikawa, Kazuhiko; Hagihara, Yoshihisa

The expression, purification and preliminary X-ray diffraction studies of a chitin-binding domain of the chitinase from P. furiosus are reported. The crystallization and preliminary X-ray diffraction analysis of the chitin-binding domain of chitinase from a hyperthermophilic archaeon, Pyrococcus furiosus, are reported. The recombinant protein was prepared using an Escherichia coli overexpression system and was crystallized by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected to 1.70 Å resolution. The crystal belonged to space group P4{sub 3}2{sub 1}2 or P4{sub 1}2{sub 1}2. The unit-cell parameters were determined to be a = b = 48.8, c = 85.0 Å.

The effects analysis of two neonicotinoid insecticides on in vitro maturation of porcine oocytes using hanging drop monoculture method.

PubMed

Ishikawa, Sadamasa; Hiraga, Kou; Hiradate, Yuuki; Tanemura, Kentaro

2015-06-01

Acetamiprid (ACE) and imidacroprid (IMI) are known neonicotinoid insecticides with strong affinities for the insect-selective nicotinic acetylcholine receptor. These provide insect control by hyperstimulating insect nerves and are used for agricultural pest management. However, it has also been reported that ACE and IMI affect mammalian reproductive function. We determined the effects of ACE and IMI on the in vitro maturation of porcine oocytes. Significant decreases in nuclear maturation rates were observed in the ACE or IMI-exposed groups. Also, in matured oocytes from the ACE or IMI-exposed groups, irregular chromosomes were observed. Our results suggest that ACE and IMI exposure was detrimental to porcine oocytes and the extent of the effects depends on the concentration of exposure.

Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of SET/TAF-Iß δN from Homo sapiens.

PubMed

Xu, Zhen; Yang, Weili; Shi, Nuo; Gao, Yongxiang; Teng, Maikun; Niu, Liwen

2010-08-01

The histone chaperone SET encoded by the SET gene, which is also known as template-activating factor Iß (TAF-Iß), is a multifunctional molecule that is involved in many biological phenomena such as histone binding, nucleosome assembly, chromatin remodelling, replication, transcription and apoptosis. A truncated SET/TAF-Iß ΔN protein that lacked the first 22 residues of the N-terminus but contained the C-terminal acidic domain and an additional His6 tag at the C-terminus was overexpressed in Escherichia coli and crystallized by the hanging-drop vapour-diffusion method using sodium acetate as precipitant at 283 K. The crystals diffracted to 2.7 A resolution and belonged to space group P4(3)2(1)2.

Crystallization and preliminary X-ray diffraction studies of a novel ferredoxin involved in the dioxygenation of carbazole by Novosphingobium sp. KA1

PubMed Central

Umeda, Takashi; Katsuki, Junichi; Usami, Yusuke; Inoue, Kengo; Noguchi, Haruko; Fujimoto, Zui; Ashikawa, Yuji; Yamane, Hisakazu; Nojiri, Hideaki

2008-01-01

Novosphingobium sp. KA1 uses carbazole 1,9a-dioxygenase (CARDO) as the first dioxygenase in its carbazole-degradation pathway. The CARDO of KA1 contains a terminal oxygenase component and two electron-transfer components: ferredoxin and ferredoxin reductase. In contrast to the CARDO systems of other species, the ferredoxin component of KA1 is a putidaredoxin-type protein. This novel ferredoxin was crystallized at 293 K by the hanging-drop vapour-diffusion method using PEG MME 550 as the precipitant under anaerobic conditions. The crystals belong to space group C2221 and diffraction data were collected to a resolution of 1.9 Å (the diffraction limit was 1.6 Å). PMID:18607094

D.R.O.P: The Durable Reconnaissance and Observation Platform

NASA Technical Reports Server (NTRS)

McKenzie, Clifford; Parness, Aaron

2011-01-01

Robots can provide a remote presence in areas that are either inaccessible or too dangerous for humans. However, robots are often limited by their ability to adapt to the terrain or resist environmental factors. The Durable Reconnaissance and Observation Platform (DROP) is a lightweight robot that addresses these challenges with the capability to survive falls from significant heights, carry a useable payload, and traverse a variety of surfaces, including climbing vertical surfaces like wood, stone, and concrete. DROP is manufactured using a combination of rapid prototyping and shape deposition manufacturing. It uses microspine technology to create a new wheel-like design for vertical climbing. To date, DROP has successfully engaged several vertical surfaces, hanging statically without assistance, and traversed horizontal surfaces at approximately 30 cm/s. Unassisted vertical climbing is capable on surfaces up to 85deg at a rate of approximately 25cm*s(sup -1). DROP can also survive falls from up to 3 meters and has the ability to be thrown off of and onto rooftops. Future efforts will focus on improving the microspine wheels, selecting more resilient materials, customizing the controls, and performing more rigorous and quantifiable testing.

3-D simulation of hanging wall effect at dam site

NASA Astrophysics Data System (ADS)

Zhang, L.; Xu, Y.

2017-12-01

Hanging wall effect is one of the near fault effects. This paper focuses on the difference of the ground motions on the hanging wall side between the footwall side of the fault at dam site considering the key factors, such as actual topography, the rupture process. For this purpose, 3-D ground motions are numerically simulated by the spectrum element method (SEM), which takes into account the physical mechanism of generation and propagation of seismic waves. With the SEM model of 548 million DOFs, excitation and propagation of seismic waves are simulated to compare the difference between the ground motion on the hanging wall side and that on the footwall side. Take Dagangshan region located in China as an example, several seismogenic finite faults with different dip angle are simulated to investigate the hanging wall effect. Furthermore, by comparing the ground motions of the receiving points, the influence of several factors on hanging wall effect is investigated, such as the dip of the fault and the fault type (strike slip fault or dip-slip fault). The peak acceleration on the hanging wall side is obviously larger than those on the footwall side, which numerically evidences the hanging wall effect. Besides, the simulation shows that only when the dip is less than 70° does the hanging wall effect deserve attention.

STUDIES UPON THE EFFECT OF LIGHT ON BLOOD AND TISSUE CELLS

PubMed Central

Earle, W. R.

1928-01-01

1. An extreme and rapid degeneration which occurred in tissue cultures of leucocytes from the blood of cats, guinea pigs, and rabbits, is described in detail. 2. This degeneration was found to appear in the culture when the cells were planted in any of the culture media tried, some of which were autogenous heparin plasma, autogenous plasma, autogenous serum, Tyrode solution, and mixtures of these with embryo juice. 3. The specific cellular changes which occurred are described for the different leucocytes. In general, there was first a latent period during which no change could be observed in the cell. Following this there was a period of stimulation during which the motion of the cell was greatly accelerated. This second stage has been observed in all cells except the lymphocyte, in which it may possibly occur to a slight degree. Finally there was the terminal stage, the stage of degeneration, in which the cell rounded up, lost its motility, and either became badly swollen or else underwent a more or less complete coagulation. 4. The factor causing this degeneration was found to be exposure of the culture to light, as, for example, during microscopic examination. 5. By a reduction of the infrared part of the spectrum, it was indicated that the effect was not due to a heat coagulation of the cells. 6. This degeneration was also found to occur in the complete absence of ultra-violet wave-lengths. 7. Further, it was shown that this degeneration was caused by light which lay within each of the three wave-length zones (1) 430µµ to 550µµ; infra-red; (2) 475µµ to 630µµ; 690µµ to infra-red; (3) 600µµ to infra-red. 8. No indication was given as to whether all regions of these zones were active in causing the degeneration, or whether the active rays are limited to certain wave-length bands lying within these zones. 9. This degeneration of the leucocytes under the action of light was also found to occur upon irradiation of hanging drops of whole blood. This is interpreted as showing conclusively that the degeneration was not dependent upon the additional factors of centrifugation, continued lowering of temperature, or the presence of abnormal saline solution. 10. It was noted, however, that the leucocytes in hanging drop cultures required a markedly longer time for their degeneration under the action of light than did the leucocytes in cultures prepared from the buffy coat and inoculated in serum. This is considered as possibly due, either to injury to the cell during centrifugation and subsequent handling, or to some action of the red blood cells present in large amounts in the hanging drops of whole blood. 11. In these hanging drop cultures of whole blood degeneration of the leucocytes was also found to occur when the light reaching the culture was first freed from the larger part of its infra-red and from all of its ultra-violet. 12. It was also shown that the same degeneration was produced by wave-lengths of light lying within each of the three wave-length zones defined in Section 6 of this summary. PMID:19869498

Expression, purification, crystallization and preliminary X-ray analysis of the Met244Ala variant of catalase–peroxidase (KatG) from the haloarchaeon Haloarcula marismortui

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

Ten-i, Tomomi; Kumasaka, Takashi; Higuchi, Wataru

2007-11-01

The Met244Ala variant of the H. marismortui KatG enzyme was expressed in haloarchaeal host cells and purified to homogeneity. The variant was crystallized using the hanging-drop vapour-diffusion method with ammonium sulfate and NaCl as precipitants. The reddish-brown rod-shaped crystals obtained belong to the monoclinic space group C2, with unit-cell parameters a = 315.24, b = 81.04, c = 74.77 Å, β = 99.81°. The covalent modification of the side chains of Trp95, Tyr218 and Met244 within the active site of Haloarcula marismortui catalase–peroxidase (KatG) appears to be common to all KatGs and has been demonstrated to be particularly significant formore » its bifunctionality [Smulevich et al. (2006 ▶), J. Inorg. Biochem.100, 568–585; Jakopitsch, Kolarich et al. (2003 ▶), FEBS Lett.552, 135–140; Jakopitsch, Auer et al. (2003 ▶), J. Biol. Chem.278, 20185–20191; Jakopitsch et al. (2004 ▶), J. Biol. Chem.279, 46082–46095; Regelsberger et al. (2001 ▶), Biochem. Soc. Trans.29, 99–105; Ghiladi, Knudsen et al. (2005 ▶), J. Biol. Chem.280, 22651–22663; Ghiladi, Medzihradzky et al. (2005 ▶), Biochemistry, 44, 15093–15105]. The Met244Ala variant of the H. marismortui KatG enzyme was expressed in haloarchaeal host cells and purified to homogeneity. The variant showed a complete loss of catalase activity, whereas the peroxidase activity of this mutant was highly enhanced owing to an increase in its affinity for the peroxidatic substrate. The variant was crystallized using the hanging-drop vapour-diffusion method with ammonium sulfate and NaCl as precipitants. The reddish-brown rod-shaped crystals obtained belong to the monoclinic space group C2, with unit-cell parameters a = 315.24, b = 81.04, c = 74.77 Å, β = 99.81°. A crystal frozen using lithium sulfate as the cryoprotectant diffracted to beyond 2.0 Å resolution. Preliminary X-ray analysis suggests the presence of a dimer in the asymmetric unit.« less

Gender differences in suicide methods.

PubMed

Callanan, Valerie J; Davis, Mark S

2012-06-01

Gender differences in suicide completion rates have been attributed to the differences in lethality of suicide methods chosen by men and women, but few empirical studies have investigated factors other than demographic characteristics that might explain this differential. Data from the 621 suicides in Summit County, Ohio during 1997-2006 were disaggregated by gender to compare known correlates of suicide risk on three methods of suicide-firearm, hanging and drug poisoning. Compared to women, men who completed suicide with firearms were more likely to be married and committed the act at home. Unmarried men were likelier to hang themselves than married men, but unmarried women were less likely to hang themselves than married women. Men with a history of depression were more likely to suicide by hanging, but women with depression were half as likely to hang themselves compared to the women without a history of depression. Men with a history of substance abuse were more likely to suicide by poisoning than men without such history, but substance abuse history had no influence on women's use of poisoning to suicide. For both sexes, the odds of suicide by poisoning were significantly higher for those on psychiatric medications.

Crystallization and preliminary X-ray diffraction studies of the ferredoxin reductase component in the Rieske nonhaem iron oxygenase system carbazole 1,9a-dioxygenase

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

Ashikawa, Yuji; Uchimura, Hiromasa; Fujimoto, Zui

2007-06-01

The NAD(P)H:ferredoxin oxidoreductase in carbazole 1,9a-dioxygenase from Janthinobacterium sp. J3 was crystallized and diffraction data were collected to 2.60 Å resolution. Carbazole 1,9a-dioxygenase (CARDO), which consists of an oxygenase component (CARDO-O) and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), catalyzes dihydroxylation at the C1 and C9a positions of carbazole. CARDO-R was crystallized at 277 K using the hanging-drop vapour-diffusion method with the precipitant PEG 8000. Two crystal types (types I and II) were obtained. The type I crystal diffracted to a maximum resolution of 2.80 Å and belonged to space group P4{sub 2}2{sub 1}2, with unit-cell parameters amore » = b = 158.7, c = 81.4 Å. The type II crystal was obtained in drops from which type I crystals had been removed; it diffracted to 2.60 Å resolution and belonged to the same space group, with unit-cell parameters a = b = 161.8, c = 79.5 Å.« less

Effect of Sediments on Rupture Dynamics of Shallow Subduction Zone Earthquakes and Tsunami Generation

NASA Astrophysics Data System (ADS)

Ma, S.

2011-12-01

Low-velocity fault zones have long been recognized for crustal earthquakes by using fault-zone trapped waves and geodetic observations on land. However, the most pronounced low-velocity fault zones are probably in the subduction zones where sediments on the seafloor are being continuously subducted. In this study I focus on shallow subduction zone earthquakes; these earthquakes pose a serious threat to human society in their ability in generating large tsunamis. Numerous observations indicate that these earthquakes have unusually long rupture durations, low rupture velocities, and/or small stress drops near the trench. However, the underlying physics is unclear. I will use dynamic rupture simulations with a finite-element method to investigate the dynamic stress evolution on faults induced by both sediments and free surface, and its relations with rupture velocity and slip. I will also explore the effect of off-fault yielding of sediments on the rupture characteristics and seafloor deformation. As shown in Ma and Beroza (2008), the more compliant hanging wall combined with free surface greatly increases the strength drop and slip near the trench. Sediments in the subduction zone likely have a significant role in the rupture dynamics of shallow subduction zone earthquakes and tsunami generation.

Crystallization and preliminary X-ray analysis of the atrial natriuretic peptide (ANP) receptor extracellular domain complex with ANP: use of ammonium sulfate as the cryosalt.

PubMed

Ogawa, Haruo; Zhang, Xiaolun; Qiu, Yue; Ogata, Craig M; Misono, Kunio S

2003-10-01

Atrial natriuretic peptide (ANP) plays a major role in blood pressure and volume regulation owing to its natriuretic and vasodilatory activities. The ANP receptor is a single-span transmembrane receptor coupled to its intrinsic guanylyl cyclase activity. The extracellular hormone-binding domain of rat ANP receptor (ANPR) was overexpressed by permanent transfection in CHO cells and purified. ANPR complexed with ANP was crystallized at 301 K by the hanging-drop vapor-diffusion method. The crystals were frozen in 3.4 M ammonium sulfate used as a cryoprotectant. The crystals diffracted to 3.1 A resolution using synchrotron radiation and belonged to the hexagonal space group P6(1), with unit-cell parameters a = b = 100.3, c = 258.6 A.

Crystallization and preliminary X-ray study of a (2R,3R)-2,3-butanediol dehydrogenase from Bacillus coagulans 2-6

PubMed Central

Miao, Xiangzhi; Huang, Xianhui; Zhang, Guofang; Zhao, Xiufang; Zhu, Xianming; Dong, Hui

2013-01-01

(2R,3R)-2,3-Butanediol dehydrogenase (R,R-BDH) from Bacillus coagulans 2-6 is a zinc-dependent medium-chain alcohol dehydrogenase. Recombinant R,R-BDH with a His6 tag at the C-terminus was expressed in Escherichia coli BL21 (DE3) cells and purified by Ni2+-chelating affinity and size-exclusion chromatography. Crystals were grown by the hanging-drop vapour-diffusion method at 289 K. The crystallization condition consisted of 8%(v/v) Tacsimate pH 4.6, 18%(w/v) polyethylene glycol 3350. The crystal diffracted to 2.8 Å resolution in the orthorhombic space group P212121, with unit-cell parameters a = 88.35, b = 128.73, c = 131.03 Å. PMID:24100567

Purification, crystallization and preliminary crystallographic study of low oxygen-affinity haemoglobin from cat (Felis silvestris catus) in two different crystal forms.

PubMed

Balasubramanian, M; Moorthy, Pon Sathya; Neelagandan, K; Ponnuswamy, M N

2009-03-01

Haemoglobin is a metalloprotein which plays a major role in the transportation of oxygen from the lungs to tissues and of carbon dioxide back to the lungs. The present work reports the preliminary crystallographic study of low oxygen-affinity haemoglobin from cat in different crystal forms. Cat blood was collected, purified by anion-exchange chromatography and crystallized in two different conditions by the hanging-drop vapour-diffusion method under unbuffered low-salt and buffered high-salt concentrations using PEG 3350 as a precipitant. Intensity data were collected using MAR345 and MAR345dtb image-plate detector systems. Cat haemoglobin crystallizes in monoclinic and orthorhombic crystal forms with one and two whole biological molecules (alpha(2)beta(2)), respectively, in the asymmetric unit.

Crystallization and preliminary crystallographic analysis of hygromycin B phosphotransferase from Escherichia coli.

PubMed

Iino, Daisuke; Takakura, Yasuaki; Kuroiwa, Mika; Kawakami, Ryouta; Sasaki, Yasuyuki; Hoshino, Takayuki; Ohsawa, Kanju; Nakamura, Akira; Yajima, Shunsuke

2007-08-01

Aminoglycoside antibiotics, such as hygromycin, kanamycin, neomycin, spectinomycin and streptomycin, inhibit protein synthesis by acting on bacterial and eukaryotic ribosomes. Hygromycin B phosphotransferase (Hph; EC 2.7.1.119) converts hygromycin B to 7''-O-phosphohygromycin using a phosphate moiety from ATP, resulting in the loss of its cell-killing activity. The Hph protein has been crystallized for the first time using a thermostable mutant and the hanging-drop vapour-diffusion method. The crystal provided diffraction data to a resolution of 2.1 A and belongs to space group P3(2)21, with unit-cell parameters a = b = 71.0, c = 125.0 A. Crystals of complexes of Hph with hygromycin B and AMP-PNP or ADP have also been obtained in the same crystal form as that of the apoprotein.

Crystallization and preliminary crystallographic analysis of hygromycin B phosphotransferase from Escherichia coli

PubMed Central

Iino, Daisuke; Takakura, Yasuaki; Kuroiwa, Mika; Kawakami, Ryouta; Sasaki, Yasuyuki; Hoshino, Takayuki; Ohsawa, Kanju; Nakamura, Akira; Yajima, Shunsuke

2007-01-01

Aminoglycoside antibiotics, such as hygromycin, kanamycin, neomycin, spectinomycin and streptomycin, inhibit protein synthesis by acting on bacterial and eukaryotic ribosomes. Hygromycin B phosphotransferase (Hph; EC 2.7.1.119) converts hygromycin B to 7′′-O-phosphohygromycin using a phosphate moiety from ATP, resulting in the loss of its cell-killing activity. The Hph protein has been crystallized for the first time using a thermostable mutant and the hanging-drop vapour-diffusion method. The crystal provided diffraction data to a resolution of 2.1 Å and belongs to space group P3221, with unit-cell parameters a = b = 71.0, c = 125.0 Å. Crystals of complexes of Hph with hygromycin B and AMP-PNP or ADP have also been obtained in the same crystal form as that of the apoprotein. PMID:17671368

Crystallization and preliminary X-ray study of a (2R,3R)-2,3-butanediol dehydrogenase from Bacillus coagulans 2-6.

PubMed

Miao, Xiangzhi; Huang, Xianhui; Zhang, Guofang; Zhao, Xiufang; Zhu, Xianming; Dong, Hui

2013-10-01

(2R,3R)-2,3-Butanediol dehydrogenase (R,R-BDH) from Bacillus coagulans 2-6 is a zinc-dependent medium-chain alcohol dehydrogenase. Recombinant R,R-BDH with a His6 tag at the C-terminus was expressed in Escherichia coli BL21 (DE3) cells and purified by Ni2+-chelating affinity and size-exclusion chromatography. Crystals were grown by the hanging-drop vapour-diffusion method at 289 K. The crystallization condition consisted of 8%(v/v) Tacsimate pH 4.6, 18%(w/v) polyethylene glycol 3350. The crystal diffracted to 2.8 Å resolution in the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a=88.35, b=128.73, c=131.03 Å.

Crystallization and Preliminary X-ray Diffraction Analysis of Hemextin A: A Unique Anticoagulant Protein from Hemachatus haemachatus Venom

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

Banerjee,Y.; Kumar, S.; Jobichen, C.

2007-01-01

Hemextin A was isolated and purified from African Ringhals cobra (Hemachatus haemachatus). It is a three-finger toxin that specifically inhibits blood coagulation factor VIIa and clot formation and that also interacts with hemextin B to form a unique anticoagulant complex. Hemextin A was crystallized by the hanging-drop vapor-diffusion method by equilibration against 0.2 M ammonium acetate, 0.1 M sodium acetate trihydrate pH 4.6 and 30% PEG 4000 as the precipitating agent. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 49.27, b = 49.51, c = 57.87 {angstrom} and two molecules in the asymmetricmore » unit. They diffracted to 1.5 {angstrom} resolution at beamline X25 at BNL.« less

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

Aoki, Ken-ichi; Tanaka, Nobutada, E-mail: ntanaka@pharm.showa-u.ac.jp; Ishikura, Shuhei

Pig heart carbonyl reductase has been crystallized in the presence of NADPH. Diffraction data have been collected using synchrotron radiation. Pig heart carbonyl reductase (PHCR), which belongs to the short-chain dehydrogenase/reductase (SDR) family, has been crystallized by the hanging-drop vapour-diffusion method. Two crystal forms (I and II) have been obtained in the presence of NADPH. Form I crystals belong to the tetragonal space group P4{sub 2}, with unit-cell parameters a = b = 109.61, c = 94.31 Å, and diffract to 1.5 Å resolution. Form II crystals belong to the tetragonal space group P4{sub 1}2{sub 1}2, with unit-cell parameters amore » = b = 120.10, c = 147.00 Å, and diffract to 2.2 Å resolution. Both crystal forms are suitable for X-ray structure analysis at high resolution.« less

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

Niemi, Merja, E-mail: merja.niemi@joensuu.fi; Jänis, Janne; Jylhä, Sirpa

The high-resolution mass-spectrometric characterization, crystallization and X-ray diffraction studies of a recombinant IgE Fab fragment in complex with bovine β-lactoglobulin are reported. A D1 Fab fragment containing the allergen-binding variable domains of the IgE antibody was characterized by ESI FT–ICR mass spectrometry and crystallized with bovine β-lactoglobulin (BLG) using the hanging-drop vapour-diffusion method at 293 K. X-ray data suitable for structure determination were collected to 2.8 Å resolution using synchrotron radiation. The crystal belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 67.0, b = 100.6, c = 168.1 Å. The three-dimensional structure ofmore » the D1 Fab fragment–BLG complex will provide the first insight into IgE antibody–allergen interactions at the molecular level.« less

Automation of Vapor-Diffusion Growth of Protein Crystals

NASA Technical Reports Server (NTRS)

Hamrick, David T.; Bray, Terry L.

2005-01-01

Some improvements have been made in a system of laboratory equipment developed previously for studying the crystallization of proteins from solution by use of dynamically controlled flows of dry gas. The improvements involve mainly (1) automation of dispensing of liquids for starting experiments, (2) automatic control of drying of protein solutions during the experiments, and (3) provision for automated acquisition of video images for monitoring experiments in progress and for post-experiment analysis. The automation of dispensing of liquids was effected by adding an automated liquid-handling robot that can aspirate source solutions and dispense them in either a hanging-drop or a sitting-drop configuration, whichever is specified, in each of 48 experiment chambers. A video camera of approximately the size and shape of a lipstick dispenser was added to a mobile stage that is part of the robot, in order to enable automated acquisition of images in each experiment chamber. The experiment chambers were redesigned to enable the use of sitting drops, enable backlighting of each specimen, and facilitate automation.

Bioinspired Fabrication of one dimensional graphene fiber with collection of droplets application.

PubMed

Song, Yun-Yun; Liu, Yan; Jiang, Hao-Bo; Li, Shu-Yi; Kaya, Cigdem; Stegmaier, Thomas; Han, Zhi-Wu; Ren, Lu-Quan

2017-09-21

We designed a kind of smart bioinspired fiber with multi-gradient and multi-scale spindle knots by combining polydimethylsiloxane (PDMS) and graphene oxide (GO). Multilayered graphene structures can produce obvious wettability change after laser etching due to increased roughness. We demonstrate that the cooperation between curvature and the controllable wettability play an important role in water gathering, which regulate effectively the motion of tiny water droplets. In addition, due to the effective cooperation of multi-gradient and multi-scale hydrophilic spindle knots, the length of the three-phase contact line (TCL) can be longer, which makes a great contribution to the improvement of collecting efficiency and water-hanging ability. This study offers a novel insight into the design of smart materials that may control the transport of tiny drops reversibly in directions, which could potentially be extended to the realms of in microfluidics, fog harvesting filtration and condensers designs, and further increase water collection efficiency and hanging ability.

Increase in Suicide Rates by Hanging in the Population of Tabasco, Mexico between 2003 and 2012

PubMed Central

Hernández-Alvarado, Mervyn Manuel; González-Castro, Thelma Beatriz; Tovilla-Zárate, Carlos Alfonso; Fresán, Ana; Juárez-Rojop, Isela E.; López-Narváez, María Lilia; Villar-Soto, Mario; Genis-Mendoza, Alma

2016-01-01

Background: Worldwide, the suicide rate is decreasing. To examine changes in the rates of completed suicide in the Mexican population from 2003 to 2012, we analyzed these changes according to: (i) the method of suicide; (ii) age group and (iii) gender. Methods: The data analyzed were obtained from governmental organizations from the State of Tabasco, Mexico. The data provided 1836 cases of subjects born and residing in Tabasco, who completed suicide in this state. Results: Suicide by hanging was a common choice of suicide method for Mexicans. The rate of suicide by hanging increased from 5.80 to 6.49 per 100,000 persons between 2003 and 2012, a rate percentage increase of 11.89%. Conclusions: Hanging was found to be the most common choice of suicide in the Mexican population, probably because the materials required are easily available and the method does not require complicated techniques, especially in the 55–64 age group. Strategies for prevention and intervention should be developed for the Mexican population considering suicide rates by age group and gender. PMID:27258292

Marmoset induced pluripotent stem cells: Robust neural differentiation following pretreatment with dimethyl sulfoxide.

PubMed

Qiu, Zhifang; Mishra, Anuja; Li, Miao; Farnsworth, Steven L; Guerra, Bernadette; Lanford, Robert E; Hornsby, Peter J

2015-07-01

The marmoset is an important nonhuman primate model for regenerative medicine. For experimental autologous cell therapy based on induced pluripotent (iPS) cells in the marmoset, cells must be able to undergo robust and reliable directed differentiation that will not require customization for each specific iPS cell clone. When marmoset iPS cells were aggregated in a hanging drop format for 3 days, followed by exposure to dual SMAD inhibitors and retinoic acid in monolayer culture for 3 days, we found substantial variability in the response of different iPS cell clones. However, when clones were pretreated with 0.05-2% dimethyl sulfoxide (DMSO) for 24 hours, all clones showed a very similar maximal response to the directed differentiation scheme. Peak responses were observed at 0.5% DMSO in two clones and at 1% DMSO in a third clone. When patterns of gene expression were examined by microarray analysis, hierarchical clustering showed very similar responses in all 3 clones when they were pretreated with optimal DMSO concentrations. The change in phenotype following exposure to DMSO and the 6 day hanging drop/monolayer treatment was confirmed by immunocytochemistry. Analysis of DNA content in DMSO-exposed cells indicated that it is unlikely that DMSO acts by causing cells to exit from the cell cycle. This approach should be generally valuable in the directed neural differentiation of pluripotent cells for experimental cell therapy. Copyright © 2015. Published by Elsevier B.V.

Electrochemical study of the interaction between dsDNA and copper(I) using carbon paste and hanging mercury drop electrode.

PubMed

Stanić, Z; Girousi, S

2008-06-30

The interaction of copper(I) with double-stranded (ds) calf thymus DNA was studied in solution and at the electrode surface by means of transfer voltammetry using a carbon paste electrode (CPE) as working electrode in 0.2 M acetate buffer solution (pH 5.0). As a result of the interaction of Cu(I) between the base pairs of the dsDNA, the characteristic peaks of dsDNA, due to the oxidation of guanine and adenine, increased and after a certain concentration of Cu(I) a new peak at +1.37 V appeared, probably due to the formation of a purine-Cu(I) complex (dsDNA-Cu(I) complex). Accordingly, the interaction of copper(I) with calf thymus dsDNA was studied in solution as well as at the electrode surface using hanging mercury drop electrode (HMDE) by means of alternating current voltammetry (AC voltammetry) in 0.3 M NaCl and 50 mM sodium phosphate buffer (pH 8.5) as supporting electrolyte. Its interaction with DNA is shown to be time dependent. Significant changes in the characteristic peaks of dsDNA were observed after addition of higher concentration of Cu(I) to a solution containing dsDNA, as a result of the interaction between Cu(I) and dsDNA. All the experimental results indicate that Cu(I) can bind to DNA by electrostatic binding and form an association complex.

Life cycle and population growth rate of Caenorhabditis elegans studied by a new method.

PubMed

Muschiol, Daniel; Schroeder, Fabian; Traunspurger, Walter

2009-05-16

The free-living nematode Caenorhabditis elegans is the predominant model organism in biological research, being used by a huge number of laboratories worldwide. Many researchers have evaluated life-history traits of C. elegans in investigations covering quite different aspects such as ecotoxicology, inbreeding depression and heterosis, dietary restriction/supplement, mutations, and ageing. Such traits include juvenile growth rates, age at sexual maturity, adult body size, age-specific fecundity/mortality, total reproduction, mean and maximum lifespan, and intrinsic population growth rates. However, we found that in life-cycle experiments care is needed regarding protocol design. Here, we test a recently developed method that overcomes some problems associated with traditional cultivation techniques. In this fast and yet precise approach, single individuals are maintained within hanging drops of semi-fluid culture medium, allowing the simultaneous investigation of various life-history traits at any desired degree of accuracy. Here, the life cycles of wild-type C. elegans strains N2 (Bristol, UK) and MY6 (Münster, Germany) were compared at 20 degrees C with 5 x 10(9) Escherichia coli ml-1 as food source. High-resolution life tables and fecundity schedules of the two strains are presented. Though isolated 700 km and 60 years apart from each other, the two strains barely differed in life-cycle parameters. For strain N2 (n = 69), the intrinsic rate of natural increase (r m d(-1)), calculated according to the Lotka equation, was 1.375, the net reproductive rate (R 0) 291, the mean generation time (T) 90 h, and the minimum generation time (T min) 73.0 h. The corresponding values for strain MY6 (n = 72) were r m = 1.460, R0 = 289, T = 84 h, and T min = 67.3 h. Peak egg-laying rates in both strains exceeded 140 eggs d(-1). Juvenile and early adulthood mortality was negligible. Strain N2 lived, on average, for 16.7 d, while strain MY6 died 2 days earlier; however, differences in survivorship curves were statistically non-significant. We found no evidence that adaptation to the laboratory altered the life history traits of C. elegans strain N2. Our results, discussed in the light of earlier studies on C. elegans, demonstrate certain advantages of the hanging drop method in investigations of nematode life cycles. Assuming that its reproducibility is validated in further studies, the method will reduce the inter-laboratory variability of life-history estimates and may ultimately prove to be more convenient than the current standard methods used by C. elegans researchers.

Life cycle and population growth rate of Caenorhabditis elegans studied by a new method

PubMed Central

Muschiol, Daniel; Schroeder, Fabian; Traunspurger, Walter

2009-01-01

Background The free-living nematode Caenorhabditis elegans is the predominant model organism in biological research, being used by a huge number of laboratories worldwide. Many researchers have evaluated life-history traits of C. elegans in investigations covering quite different aspects such as ecotoxicology, inbreeding depression and heterosis, dietary restriction/supplement, mutations, and ageing. Such traits include juvenile growth rates, age at sexual maturity, adult body size, age-specific fecundity/mortality, total reproduction, mean and maximum lifespan, and intrinsic population growth rates. However, we found that in life-cycle experiments care is needed regarding protocol design. Here, we test a recently developed method that overcomes some problems associated with traditional cultivation techniques. In this fast and yet precise approach, single individuals are maintained within hanging drops of semi-fluid culture medium, allowing the simultaneous investigation of various life-history traits at any desired degree of accuracy. Here, the life cycles of wild-type C. elegans strains N2 (Bristol, UK) and MY6 (Münster, Germany) were compared at 20°C with 5 × 109 Escherichia coli ml-1 as food source. Results High-resolution life tables and fecundity schedules of the two strains are presented. Though isolated 700 km and 60 years apart from each other, the two strains barely differed in life-cycle parameters. For strain N2 (n = 69), the intrinsic rate of natural increase (rmd-1), calculated according to the Lotka equation, was 1.375, the net reproductive rate (R0) 291, the mean generation time (T) 90 h, and the minimum generation time (Tmin) 73.0 h. The corresponding values for strain MY6 (n = 72) were rm = 1.460, R0 = 289, T = 84 h, and Tmin = 67.3 h. Peak egg-laying rates in both strains exceeded 140 eggs d-1. Juvenile and early adulthood mortality was negligible. Strain N2 lived, on average, for 16.7 d, while strain MY6 died 2 days earlier; however, differences in survivorship curves were statistically non-significant. Conclusion We found no evidence that adaptation to the laboratory altered the life history traits of C. elegans strain N2. Our results, discussed in the light of earlier studies on C. elegans, demonstrate certain advantages of the hanging drop method in investigations of nematode life cycles. Assuming that its reproducibility is validated in further studies, the method will reduce the inter-laboratory variability of life-history estimates and may ultimately prove to be more convenient than the current standard methods used by C. elegans researchers. PMID:19445697

Investigation of interaction between magnetic silica particles and lambda phage DNA fragment.

PubMed

Smerkova, Kristyna; Dostalova, Simona; Vaculovicova, Marketa; Kynicky, Jindrich; Trnkova, Libuse; Kralik, Miroslav; Adam, Vojtech; Hubalek, Jaromir; Provaznik, Ivo; Kizek, Rene

2013-12-01

Nucleic acids belong to the most important molecules and therefore the understanding of their properties, function and behavior is crucial. Even though a range of analytical and biochemical methods have been developed for this purpose, one common step is essential for all of them - isolation of the nucleic acid from the from complex sample matrix. The use of magnetic particles for the separation of nucleic acids has many advantages over other isolation methods. In this study, an isolation procedure for extraction of DNA was optimized. Each step of the isolation process including washing, immobilization and elution was optimized and therefore the efficiency was increased from 1.7% to 28.7% and the total time was shortened from 75 to 30min comparing to the previously described method. Quantification of the particular parameter influence was performed by square-wave voltammetry using hanging drop mercury electrode. Further, we compared the optimized method with standard chloroform extraction and applied on isolation of DNA from Staphylococcus aureus and Escherichia coli. Copyright © 2013 Elsevier B.V. All rights reserved.

Overcoming a hemihedral twinning problem in tetrahydrofolate-dependent O -demethylase crystals by the microseeding method

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

Harada, Ayaka; Sato, Yukari; Kamimura, Naofumi

2016-11-30

A tetrahydrofolate-dependentO-demethylase, LigM, fromSphingobiumsp. SYK-6 was crystallized by the hanging-drop vapour-diffusion method. However, the obtainedP3 121 orP3 221 crystals, which diffracted to 2.5–3.3 Å resolution, were hemihedrally twinned. To overcome the twinning problem, microseeding usingP3 121/P3 221 crystals as microseeds was performed with optimization of the reservoir conditions. As a result, another crystal form was obtained. The newly obtained crystal diffracted to 2.5–3.0 Å resolution and belonged to space groupP2 12 12, with unit-cell parametersa= 102.0,b= 117.3,c= 128.1 Å. TheP2 12 12 crystals diffracted to better than 2.0 Å resolution after optimizing the cryoconditions. Phasing using the single anomalous diffractionmore » method was successful at 3.0 Å resolution with a Pt-derivative crystal. This experience suggested that microseeding is an effective method to overcome the twinning problem, even when twinned crystals are utilized as microseeds.« less

Overcoming a hemihedral twinning problem in tetrahydrofolate-dependent O -demethylase crystals by the microseeding method

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

Harada, Ayaka; Sato, Yukari; Kamimura, Naofumi

2016-11-30

A tetrahydrofolate-dependentO-demethylase, LigM, from Sphingobiumsp. SYK-6 was crystallized by the hanging-drop vapour-diffusion method. However, the obtained P3 121 orP3 221 crystals, which diffracted to 2.5–3.3 Å resolution, were hemihedrally twinned. To overcome the twinning problem, microseeding using P3 121/P3 2 21 crystals as microseeds was performed with optimization of the reservoir conditions. As a result, another crystal form was obtained. The newly obtained crystal diffracted to 2.5–3.0 Å resolution and belonged to space group P2 12 12, with unit-cell parametersa= 102.0,b= 117.3,c = 128.1 Å. The P2 12 12 crystals diffracted to better than 2.0 Å resolution after optimizing themore » cryoconditions. Phasing using the single anomalous diffraction method was successful at 3.0 Å resolution with a Pt-derivative crystal. This experience suggested that microseeding is an effective method to overcome the twinning problem, even when twinned crystals are utilized as microseeds.« less

The trend in suicide methods in South Korea in 1997-2015.

PubMed

Sohn, Kitae

2017-01-01

South Korea exhibited the highest crude suicide rate in the world. To better understand this phenomenon, the author analyzed all suicides in South Korea from 1997 to 2015 and charted the trend in suicide methods by gender. Over time, both genders rapidly chose hanging for suicide at the expense of drug/pesticide poisoning. Gassing was hardly used in the beginning, but its recent gain in use is noteworthy. Including undetermined deaths did not change the main results. The author regressed hanging on demographics and found that hanging was particularly chosen by ever-married men of prime working age with a respectable level of education.

Purification, characterization and preliminary crystallographic studies of a cysteine protease from Pachyrrhizus erosus seeds.

PubMed

Chang, Shaojie; Song, Xiaomin; Yan, Ming; Zhou, Zhaocai; Wu, Fang; Gong, Weimin

2004-01-01

The proteins Spe31 and Spe32, named after their respective molecular weights of about 31 and 32 kDa, were purified simultaneously from the seeds of Pachyrrhizus erosus. They cannot be separated from each other by column chromatography. N-terminal sequence analysis indicated that they belonged to the papain family of cysteine proteases. An in-gel activity assay revealed that Spe31 possesses proteolytic activity while Spe32 only displays very weak activity for protein degradation. Both of them are glycoproteins as detected by the periodic acid and Schiff's reagent method. Crystals were obtained from the protein mixture by the hanging-drop vapour-diffusion method; they diffracted to a resolution of 2.61 A on an in-house X-ray source. The crystals belong to space group P4(1(3))2(1)2, with unit-cell parameters a = b = 61.96, c = 145.61 A. Gel electrophoresis under non-denaturing conditions showed that the protein crystallized was Spe31.

Crystallization and preliminary X-ray diffraction studies of NP24-I, an isoform of a thaumatin-like protein from ripe tomato fruits

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

Ghosh, Raka; Chakrabarti, Chandana, E-mail: chandana.chakrabarti@saha.ac.in

2005-08-01

A thaumatin-like antifungal protein, NP24-I, has been isolated from ripe tomato fruits. It was crystallized by the vapour-diffusion method and data were collected to 2.45 Å. The structure was solved by molecular replacement. NP24 is a 24 kDa (207-amino-acid) antifungal thaumatin-like protein (TLP) found in tomato fruits. An isoform of the protein, NP24-I, is reported to play a possible role in ripening of the fruit in addition to its antifungal properties. The protein has been isolated and purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the tetragonal space group P4{sub 3}, with unit-cell parameters a =more » b = 61.01, c = 62.90 Å and one molecule per asymmetric unit. X-ray diffraction data were processed to a resolution of 2.45 Å and the structure was solved by molecular replacement.« less

Measurement of thin liquid film drainage using a novel high-speed impedance analyzer

NASA Astrophysics Data System (ADS)

Hool, Kevin O.; Saunders, Robert C.; Ploehn, Harry J.

1998-09-01

This work describes the design and implementation of a new instrument, called the thin film impedance analyzer, which measures the rate of drainage of thin oil films. The instrument forms an oil film by elevating a planar oil-water interface into a water drop hanging from a stainless steel capillary tube immersed in the oil. The instrument measures the magnitude of the impedance of the matter between the capillary tube and a screen electrode immersed in the lower water phase. Under appropriate conditions, the capacitance of the oil film dominates the impedance. The instrument records the increase in the magnitude of the admittance associated with the draining and thinning of the oil film. The features of the drainage curves vary considerably with the type, amount, and location of surfactants in the oil and water phases, as well as with user-specified values of drop volume, drop equilibration time, and extent of drop compression. For this reason, the instrument has utility as a screening tool for selecting surfactants for emulsion formulations. Potential future uses include accelerated prediction of emulsion stability and extraction of oil-water interfacial rheological parameters.

Increase in Suicide Rates by Hanging in the Population of Tabasco, Mexico between 2003 and 2012.

PubMed

Hernández-Alvarado, Mervyn Manuel; González-Castro, Thelma Beatriz; Tovilla-Zárate, Carlos Alfonso; Fresán, Ana; Juárez-Rojop, Isela E; López-Narváez, María Lilia; Villar-Soto, Mario; Genis-Mendoza, Alma

2016-06-01

Worldwide, the suicide rate is decreasing. To examine changes in the rates of completed suicide in the Mexican population from 2003 to 2012, we analyzed these changes according to: (i) the method of suicide; (ii) age group and (iii) gender. The data analyzed were obtained from governmental organizations from the State of Tabasco, Mexico. The data provided 1836 cases of subjects born and residing in Tabasco, who completed suicide in this state. Suicide by hanging was a common choice of suicide method for Mexicans. The rate of suicide by hanging increased from 5.80 to 6.49 per 100,000 persons between 2003 and 2012, a rate percentage increase of 11.89%. Hanging was found to be the most common choice of suicide in the Mexican population, probably because the materials required are easily available and the method does not require complicated techniques, especially in the 55-64 age group. Strategies for prevention and intervention should be developed for the Mexican population considering suicide rates by age group and gender.

Fabrication of Intermetallic Titanium Alloy Based on Ti2AlNb by Rapid Quenching of Melt

NASA Astrophysics Data System (ADS)

Senkevich, K. S.; Serov, M. M.; Umarova, O. Z.

2017-11-01

The possibility of fabrication of rapidly quenched fibers from alloy Ti - 22Al - 27Nb by extracting a hanging melt drop is studied. The special features of the production of electrodes for spraying the fibers by sintering mechanically alloyed powdered components of the alloy, i.e., titanium hydride, niobium, and aluminum dust, are studied. The rapidly quenched fibers with homogeneous phase composition and fine-grained structure produced from alloy Ti - 22Al - 27Nb are suitable for manufacturing compact semiproducts by hot pressing.

Evaluation of Vitality in the Experimental Hanging Model of Rats by Using Immunohistochemical IL-1β Antibody Staining.

PubMed

Balandiz, Hüseyin; Pehlivan, Sultan; Çiçek, Ali Fuat; Tuğcu, Harun

2015-12-01

Hanging is the most common suicide method in the world, and the discrimination of antemortem-postmortem hanging must be done at autopsy. The aim of this experimental study was to examine the immunohistochemical expression of IL-1β antibody at the hanging mark skin samples of rats to discriminate antemortem and postmortem hangings. A total of 20 Wistar albino rats were used for this study. The groups were as follows: A-1, antemortem control group; A-2, antemortem second-hour hanging mark skin samples; A-3, antemortem 24th-hour hanging mark skin samples; A-4, antemortem 72nd-hour hanging mark skin samples; B-1, postmortem control group; and B-2, postmortem second-hour hanging mark skin samples. Interleukin-1β immunostaining was performed to all tissue samples. For epidermal cells, group A-1 samples did not show IL-1β immunostaining, group A-2 samples were severely immunostained, and groups A-3 and A-4 samples' staining were slightly decreased. There was no IL-1β antibody staining in groups B-1 and B-2 samples. For adnexal cells, groups A-1 and B-1 samples did not show IL-1β immunostaining, staining of group A-2 samples was mild to severe, and groups A-3 and A-4 samples' staining were slightly decreased. Half of the group B-2 samples did not show IL-1β immunostaining. For subepidermal cells, most of the samples of groups A-1 and B-1 showed slight immunostaining, groups A-2 and B-2 samples' staining were mild to severe, and there were slight immunostaining in groups A-3 and A-4 samples. The majority of vascular structure cells did not show IL-1β immunostaining. Interleukin-1β immunostaining of epidermal cells can discriminate antemortem-postmortem hangings, but vascular structure cells and subepidermal cells cannot discriminate vital hangings.

Preparation, crystallization and preliminary X-ray analysis of the methionine synthase (MetE) from Streptococcus mutans

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

Fu, Tian-Min; Zhang, Xiao-Yan; Li, Lan-Fen

2006-10-01

Methionine synthase (MetE) from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.2 Å resolution. The Streptococcus mutans metE gene encodes methionine synthase (MetE), which catalyzes the direct transfer of a methyl group from methyltetrahydrofolate to homocysteine in the last step of methionine synthesis. metE was cloned into pET28a and the gene product was expressed at high levels in the Escherichia coli strain BL21 (DE3). MetE was purified to homogeneity using Ni{sup 2+}-chelating chromatography followed by size-exclusion chromatography. Crystals of the protein were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.2 Å resolution.more » The crystal belongs to space group P2{sub 1}, with unit-cell parameters a = 52.85, b = 99.48, c = 77.88 Å, β = 94.55°.« less

Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants

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

Hu, Guan-Jing; Li, Lan-Fen; Li, Dan

2007-09-01

A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P2{sub 1}2{sub 1}2{sub 1}, withmore » unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å.« less

Preliminary X-ray crystallographic analysis of SMU.573, a putative sugar kinase from Streptococcus mutans

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

Zhou, Yan-Feng; Li, Lan-Fen; Yang, Cheng

2008-01-01

SMU.573 from S. mutans was expressed in E. coli and crystallized. The crystals belong to space group I4 and 2.5 Å resolution diffraction data were collected at an in-house chromium radiation source. SMU.573 from Streptococcus mutans is a structurally and functionally uncharacterized protein that was selected for structural biology studies. Native and SeMet-labelled proteins were expressed with an N-His tag in Escherichia coli BL21 (DE3) and purified by Ni{sup 2+}-chelating and size-exclusion chromatography. Crystals of the SeMet-labelled protein were obtained by the hanging-drop vapour-diffusion method and a 2.5 Å resolution diffraction data set was collected using an in-house chromium radiationmore » source. The crystals belong to space group I4, with unit-cell parameters a = b = 96.53, c = 56.26 Å, α = β = γ = 90°.« less

Purification, crystallization and preliminary X-ray crystallographic analysis of rice Bowman–Birk inhibitor from Oryza sativa

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

Lin, Yi-Hung; Li, Hsin-Tai; Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Hsinchu 30013,Taiwan

2006-06-01

Rice Bowman–Birk inhibitor was expressed and crystallized. Bowman–Birk inhibitors (BBIs) are cysteine-rich proteins with inhibitory activity against proteases that are widely distributed in monocot and dicot species. The expression of rice BBI from Oryza sativa is up-regulated and induced by pathogens or insects during germination of rice seeds. The rice BBI (RBTI) of molecular weight 15 kDa has been crystallized using the hanging-drop vapour-diffusion method. According to the diffraction of rice BBI crystals at a resolution of 2.07 Å, the unit cell belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 74.37, b = 96.69, cmore » = 100.36 Å. Preliminary analysis indicates four BBI molecules in an asymmetric unit, with a solvent content of 58.29%.« less

Three-dimensional structure of Erwinia carotovora L-asparaginase

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

Kislitsyn, Yu. A.; Kravchenko, O. V.; Nikonov, S. V.

2006-10-15

Three-dimensional structure of Erwinia carotovora L-asparaginase, which has antitumor activity and is used for the treatment of acute lymphoblastic leukemia, was solved at 3 A resolution and refined to R{sub cryst} = 20% and R{sub free} = 28%. Crystals of recombinant Erwinia carotovora L-asparaginase were grown by the hanging-drop vapor-diffusion method from protein solutions in a HEPES buffer (pH 6.5) and PEG MME 5000 solutions in a cacodylate buffer (pH 6.5) as the precipitant. Three-dimensional X-ray diffraction data were collected up to 3 A resolution from one crystal at room temperature. The structure was solved by the molecular replacement methodmore » using the coordinates of Erwinia chrysanthemi L-asparaginase as the starting model. The coordinates refined with the use of the CNS program package were deposited in the Protein Data Bank (PDB code 1ZCF)« less

Purification, identification and preliminary crystallographic studies of Pru du amandin, an allergenic protein from Prunus dulcis

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

Gaur, Vineet; Sethi, Dhruv K.; Salunke, Dinakar M., E-mail: dinakar@nii.res.in

The purification, identification, crystallization and preliminary crystallographic studies of an allergy-related protein, Pru du amandin, from P. dulcis nuts are reported. Food allergies appear to be one of the foremost causes of hypersensitivity reactions. Nut allergies account for most food allergies and are often permanent. The 360 kDa hexameric protein Pru du amandin, a known allergen, was purified from almonds (Prunus dulcis) by ammonium sulfate fractionation and ion-exchange chromatography. The protein was identified by a BLAST homology search against the nonredundant sequence database. Pru du amandin belongs to the 11S legumin family of seed storage proteins characterized by the presencemore » of a cupin motif. Crystals were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P4{sub 1} (or P4{sub 3}), with unit-cell parameters a = b = 150.7, c = 164.9 Å.« less

Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of great cormorant (Phalacrocorax carbo) haemoglobin.

PubMed

Jagadeesan, G; Malathy, P; Gunasekaran, K; Harikrishna Etti, S; Aravindhan, S

2014-11-01

Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins. Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350, NaCl and glycerol as precipitants. The crystals belonged to the trigonal system P3₁21, with unit-cell parameters a=b=55.64, c=153.38 Å, β=120.00°; a complete data set was collected to a resolution of 3.5 Å. Matthews coefficient analysis indicated that the crystals contained a half-tetramer in the asymmetric unit.

Bottom-Up Engineering of Well-Defined 3D Microtissues Using Microplatforms and Biomedical Applications.

PubMed

Lee, Geon Hui; Lee, Jae Seo; Wang, Xiaohong; Lee, Sang Hoon

2016-01-07

During the last decades, the engineering of well-defined 3D tissues has attracted great attention because it provides in vivo mimicking environment and can be a building block for the engineering of bioartificial organs. In this Review, diverse engineering methods of 3D tissues using microscale devices are introduced. Recent progress of microtechnologies has enabled the development of microplatforms for bottom-up assembly of diverse shaped 3D tissues consisting of various cells. Micro hanging-drop plates, microfluidic chips, and arrayed microwells are the typical examples. The encapsulation of cells in hydrogel microspheres and microfibers allows the engineering of 3D microtissues with diverse shapes. Applications of 3D microtissues in biomedical fields are described, and the future direction of microplatform-based engineering of 3D micro-tissues is discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Aravind, Penmatsa; Rajini, Bheemreddy; Sharma, Yogendra

The crystallization and preliminary X-ray diffraction analysis of AIM1g1, a βγ-crystallin domain of absent in melanoma (AIM1) protein from H. sapiens, is reported. AIM1g1 is a single βγ-crystallin domain from the protein absent in melanoma 1 (AIM1), which appears to play a role in the suppression of melanomas. This domain is known to bind calcium and its structure would help in identifying calcium-coordinating sites in vertebrate crystallins, which have hitherto been believed to have lost this ability during evolution. Crystallization of this domain was performed by the hanging-drop vapour-diffusion method. Crystals diffracted to a maximum resolution of 1.86 Å andmore » were found to belong to space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 54.98, c = 59.73 Å. Solvent-content analysis indicated the presence of one monomer per asymmetric unit.« less

Purification, characterization, and crystallization of monoamine oxidase from Escherichia coli K-12.

PubMed

Roh, J H; Suzuki, H; Azakami, H; Yamashita, M; Murooka, Y; Kumagai, H

1994-09-01

The gene for monoamine oxidase (MAO) was cloned from an Escherichia coli genomic library and MAO was overproduced in the periplasmic space. The enzyme was purified to homogeneity by preparation of a periplasmic fraction, followed by ammonium sulfate fractionation and DEAE-cellulose column chromatography. Crystals were obtained by the hanging drop method using sodium citrate as a precipitant. The enzyme was found to be a dimer of identical subunits with a molecular weight of 80,000, and showed the highest activity at pH 7.5 and 45 degrees C. The enzyme was inhibited by a MAO specific inhibitor, hydroxylamine, hydrazine, phenelzine, isoniazid, and tranycpromine. The enzyme oxidized tyramine, phenethylamine, and tryptamine at higher rates, but not oxidized diamine and polyamines such as putrescine and spermine. The antibody against E. coli MAO cross-reacted with purified MAO A from Klebsiella aerogenes.

Crystallization and preliminary crystallographic analysis of an acridone-producing novel multifunctional type III polyketide synthase from Huperzia serrata

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

Morita, Hiroyuki; Kondo, Shin; Kato, Ryohei

2007-07-01

An acridone-producing novel type III polyketide synthase from H. serrata has been overexpressed in E. coli, purified and crystallized. Diffraction data have been collected to 2.0 Å. Polyketide synthase 1 (PKS1) from Huperzia serrata is a plant-specific type III polyketide synthase that shows an unusually versatile catalytic potential, producing various aromatic tetraketides, including chalcones, benzophenones, phlorogulucinols and acridones. Recombinant H. serrata PKS1 expressed in Escherichia coli was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group I222 or I2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 73.3, b = 85.0, c = 137.7 Å, α =more » β = γ = 90.0°. Diffraction data were collected to 2.0 Å resolution using synchrotron radiation at BL24XU of SPring-8.« less

Crystallization and preliminary X-ray diffraction analysis of a cold-adapted catalase from Vibrio salmonicida

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

Riise, Ellen Kristin; Lorentzen, Marit Sjo; Helland, Ronny

2006-01-01

Monoclinic (P2{sub 1}) crystals of a His-tagged form of V. salmonicida catalase without cofactor diffract X-rays to 1.96 Å. Catalase (EC 1.11.1.6) catalyses the breakdown of hydrogen peroxide to water and molecular oxygen. Recombinant Vibrio salmonicida catalase (VSC) possesses typical cold-adapted features, with higher catalytic efficiency, lower thermal stability and a lower temperature optimum than its mesophilic counterpart from Proteus mirabilis. Crystals of VSC were produced by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 98.15, b = 217.76, c = 99.28 Å, βmore » = 110.48°. Data were collected to 1.96 Å and a molecular-replacement solution was found with eight molecules in the asymmetric unit.« less

Crystallization and preliminary X-ray data analysis of β-alanine synthase from Drosophila melanogaster

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

Lundgren, Stina; Andersen, Birgit; Piškur, Jure

2007-10-01

β-Alanine synthase catalyzes the last step in the reductive degradation pathway for uracil and thymine. Crystals of the recombinant enzyme from D. melanogaster belong to space group C2. Diffraction data to 3.3 Å resolution were collected and analyzed. β-Alanine synthase catalyzes the last step in the reductive degradation pathway for uracil and thymine, which represents the main clearance route for the widely used anticancer drug 5-fluorouracil. Crystals of the recombinant enzyme from Drosophila melanogaster, which is closely related to the human enzyme, were obtained by the hanging-drop vapour-diffusion method. They diffracted to 3.3 Å at a synchrotron-radiation source, belong tomore » space group C2 (unit-cell parameters a = 278.9, b = 95.0, c = 199.3 Å, β = 125.8°) and contain 8–10 molecules per asymmetric unit.« less

Crystallization and X-ray analysis of the salmon-egg lectin SEL24K.

PubMed

Murata, Kenji; Fisher, Andrew J; Hedrick, Jerry L

2007-05-01

The 24 kDa egg lectin of Chinook salmon (Oncorhynchus tshawytscha) is released from the egg during the cortical reaction. The lectin functions in blocking polyspermy during the fertilization process. The egg lectin was purified by affinity chromatography from salmon eggs and crystallized by the hanging-drop vapor-diffusion method using 15/4 EO/OH (pentaerythritol ethoxylate) as a precipitant. The crystal diffracted synchrotron-radiation X-rays to 1.63 A resolution. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 93.0, b = 73.6, c = 113.6 A, alpha = 90, beta = 92.82, gamma = 90 degrees. The crystal is likely to contain eight molecules in the asymmetric unit (V(M) = 2.3 A3 Da(-1)), corresponding to a solvent content of 45.5%. A self-rotation function suggests an arrangement with 222 point symmetry within the asymmetric unit.

Purification and crystallization of Bacillus subtilis NrnA, a novel enzyme involved in nanoRNA degradation

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

Nelersa, Claudiu M.; Schmier, Brad J.; Malhotra, Arun

2012-05-08

The final step in RNA degradation is the hydrolysis of RNA fragments five nucleotides or less in length (nanoRNA) to mononucleotides. In Escherichia coli this step is carried out by oligoribonuclease (Orn), a DEDD-family exoribonuclease that is conserved throughout eukaryotes. However, many bacteria lack Orn homologs, and an unrelated DHH-family phosphoesterase, NrnA, has recently been identified as one of the enzymes responsible for nanoRNA degradation in Bacillus subtilis. To understand its mechanism of action, B. subtilis NrnA was purified and crystallized at room temperature using the hanging-drop vapor-diffusion method with PEG 4000, PEG 3350 or PEG MME 2000 as precipitant.more » The crystals belonged to the primitive monoclinic space group P2{sub 1}, with unit-cell parameters a = 50.62, b = 121.3, c = 123.4 {angstrom}, {alpha} = 90, {beta} = 91.31, {gamma} = 90{sup o}.« less

Crystallization and preliminary X-ray diffraction studies of the ubiquitin-like (UbL) domain of the human homologue A of Rad23 (hHR23A) protein.

PubMed

Chen, Yu Wai; Tajima, Toshitaka; Rees, Martin; Garcia-Maya, Mitla

2009-09-01

Human homologue A of Rad23 (hHR23A) plays dual roles in DNA repair as well as serving as a shuttle vehicle targeting polyubiquitinated proteins for degradation. Its N-terminal ubiquitin-like (UbL) domain interacts with the 19S proteasomal cap and provides the docking mechanism for protein delivery. Pyramidal crystals of the UbL domain of hHR23A were obtained by the hanging-drop vapour-diffusion method with ammonium sulfate as the crystallizing agent. The crystals diffracted to beyond 2 A resolution and belonged to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 78.48, c = 63.57 A. The structure was solved by molecular replacement using the UbL domain of yeast Dsk2 as the search model.

Crystallization and preliminary X-ray diffraction studies of hyperthermophilic archaeal Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus P1

PubMed Central

Kounosu, Asako; Hasegawa, Kazuya; Iwasaki, Toshio; Kumasaka, Takashi

2010-01-01

The hyperthermophilic archaeal Rieske-type [2Fe–2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 contains a low-potential Rieske-type [2Fe–2S] cluster that has served as a tractable model for ligand-substitution studies on this protein family. Recombinant ARF harbouring a pET30a vector-derived N-­terminal extension region plus a hexahistidine tag has been heterologously overproduced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using 0.05 M sodium acetate, 0.05 M HEPES, 2 M ammonium sulfate pH 5.5. The crystals diffracted to 1.85 Å resolution and belonged to the tetragonal space group P43212, with unit-cell parameters a = 60.72, c = 83.31 Å. The asymmetric unit contains one protein molecule. PMID:20606288

Crystallization and preliminary X-ray diffraction studies of hyperthermophilic archaeal Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus P1.

PubMed

Kounosu, Asako; Hasegawa, Kazuya; Iwasaki, Toshio; Kumasaka, Takashi

2010-07-01

The hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 contains a low-potential Rieske-type [2Fe-2S] cluster that has served as a tractable model for ligand-substitution studies on this protein family. Recombinant ARF harbouring a pET30a vector-derived N-terminal extension region plus a hexahistidine tag has been heterologously overproduced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using 0.05 M sodium acetate, 0.05 M HEPES, 2 M ammonium sulfate pH 5.5. The crystals diffracted to 1.85 A resolution and belonged to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = 60.72, c = 83.31 A. The asymmetric unit contains one protein molecule.

An analysis of the predictors of mortality and morbidity in patients admitted after suicidal hanging to an Indian multidisciplinary Intensive Care Unit

PubMed Central

Renuka, MK; Kalaiselvan, MS; Arunkumar, AS

2017-01-01

Background and Aims: Hanging is a frequently used method to attempt suicide in India. There is a lack of data in the Indian population regarding clinical features and outcomes of suicidal hanging. The purpose of this study was to evaluate the factors affecting mortality and morbidity in patients admitted with suicidal hanging to the Intensive Care Unit (ICU). Methods: A 6-year retrospective study of adult patients admitted to the ICU with suicidal hanging was analysed for demographics, mode of hanging, lead time to emergency room (ER) admission, clinical presentation, Acute Physiology and Chronic Health Evaluation II (APACHE II) and Sequential Organ Failure Assessment (SOFA) scores, admission Glasgow coma scale (GCS) and neurological outcomes. The primary outcome was in-hospital mortality rate. Secondary outcomes were hospital length of stay (LOS), ICU-LOS, time for neurological recovery, organ support and duration of mechanical ventilation. Statistical analysis was performed using the Student's t-test for continuous variables and Chi-square test for categorical variables. Results: We analysed data of 106 patients. The median age was 27 years [Interquartile Range (IQR) (22–34)]. The median lead time to ER admission was 1 h [IQR (0.5–1.4)] with median ICU stay of 3 days [IQR (2–4)]. Vasopressors were administered to 27.4% patients. GCS was ≤7 in 65% patients, and 84.9% patients received mechanical ventilation. Mortality rate was 10.3%. Survivors recovered with normal organ function. Conclusion: Suicidal hanging is associated with significant mortality. Admission GCS, APACHE II and 48 h SOFA score were predictors of poor outcome. PMID:28794524

Over a century of neuron culture: from the hanging drop to microfluidic devices.

PubMed

Millet, Larry J; Gillette, Martha U

2012-12-01

The brain is the most intricate, energetically active, and plastic organ in the body. These features extend to its cellular elements, the neurons and glia. Understanding neurons, or nerve cells, at the cellular and molecular levels is the cornerstone of modern neuroscience. The complexities of neuron structure and function require unusual methods of culture to determine how aberrations in or between cells give rise to brain dysfunction and disease. Here we review the methods that have emerged over the past century for culturing neurons in vitro, from the landmark finding by Harrison (1910) - that neurons can be cultured outside the body - to studies utilizing culture vessels, micro-islands, Campenot and brain slice chambers, and microfluidic technologies. We conclude with future prospects for neuronal culture and considerations for advancement. We anticipate that continued innovation in culture methods will enhance design capabilities for temporal control of media and reagents (chemotemporal control) within sub-cellular environments of three-dimensional fluidic spaces (microfluidic devices) and materials (e.g., hydrogels). They will enable new insights into the complexities of neuronal development and pathology.

Over a Century of Neuron Culture: From the Hanging Drop to Microfluidic Devices

PubMed Central

Millet, Larry J.; Gillette, Martha U.

2012-01-01

The brain is the most intricate, energetically active, and plastic organ in the body. These features extend to its cellular elements, the neurons and glia. Understanding neurons, or nerve cells, at the cellular and molecular levels is the cornerstone of modern neuroscience. The complexities of neuron structure and function require unusual methods of culture to determine how aberrations in or between cells give rise to brain dysfunction and disease. Here we review the methods that have emerged over the past century for culturing neurons in vitro, from the landmark finding by Harrison (1910) — that neurons can be cultured outside the body — to studies utilizing culture vessels, micro-islands, Campenot and brain slice chambers, and microfluidic technologies. We conclude with future prospects for neuronal culture and considerations for advancement. We anticipate that continued innovation in culture methods will enhance design capabilities for temporal control of media and reagents (chemotemporal control) within sub-cellular environments of three-dimensional fluidic spaces (microfluidic devices) and materials (e.g., hydrogels). They will enable new insights into the complexities of neuronal development and pathology. PMID:23239951

Modified liver hanging maneuver focusing on outflow control in pure laparoscopic left-sided hepatectomy.

PubMed

Kim, Ji Hoon

2018-04-01

Outflow control during laparoscopic liver resection necessitates the use of technically demanding procedures since the hepatic veins are fragile and vulnerable to damage during parenchymal transection. The liver hanging maneuver reduces venous backflow bleeding during deep parenchymal transection. The present report describes surgical outcomes and a technique to achieve outflow control during application of the modified liver hanging maneuver in patients undergoing laparoscopic left-sided hepatectomy. A retrospective review was performed of clinical data from 29 patients who underwent laparoscopic left-sided hepatectomy using the modified liver hanging maneuver between February 2013 and March 2017. For this hanging technique, the upper end of the hanging tape was placed on the lateral aspect of the left hepatic vein. The tape was then aligned with the ligamentum venosum. The position of the lower end of the hanging tape was determined according to left-sided hepatectomy type. The hanging tape gradually encircled either the left hepatic vein or the common trunk of the left hepatic vein and middle hepatic vein. The surgical procedures comprised: left lateral sectionectomy (n = 10); left hepatectomy (n = 17); and extended left hepatectomy including the middle hepatic vein (n = 2). Median operative time was 210 min (range 90-350 min). Median intraoperative blood loss was 200 ml (range 60-600 ml). Two intraoperative major hepatic vein injuries occurred during left hepatectomy. Neither patient developed massive bleeding or air embolism. Postoperative major complications occurred in one patient (3.4%). Median postoperative hospital stay was 7 days (range 4-15 days). No postoperative mortality occurred. The present modified liver hanging maneuver is a safe and effective method of outflow control during laparoscopic left-sided hepatectomy.

Lepidoptera and associated parasitoids attacking Hass and non-Hass avocados in Guatemala.

PubMed

Hoddle, Mark S; Hoddle, Christina D

2008-08-01

A 5-mo survey for fruit feeding Lepidoptera attacking Hass and non-Hass avocados (Persea americana Miller [Lauraceae]) was conducted in Guatemala from 1 November 2006 to 1 April 2007. In total, 6,740 fruit were collected from 22 different areas in Guatemala. Eight species of Lepidoptera, of which at least two are species new to science, were reared from avocado fruit. Reared Lepidoptera were Amorbia santamaria Phillips and Powell, Cryptaspasma sp. nr. lugubris, Euxoa sorella Schaus, Histura n. sp., Holcocera n. sp., Micrathetis triplex Walker, Netechma pyrrhodelta (Meyrick), and Stenoma catenifer Walsingham. Hymenopteran parasitoids were reared from larvae of C. sp. nr. lugubris and S. catenifer. One species of parasitoid, Pseudophanerotoma sp., was reared from field collected C. sp. nr. lugubris larvae. The dominant parasitoid reared from S. catenifer was a gregarious Apanteles sp. Other parasitoid species reared from S. catenifer larvae were Brachycyrtus sp., Macrocentrus sp., and Pristomerus sp. The oviposition preference of C. sp. nr. lugubris for avocado fruit hanging in trees, dropped fruit on the ground, or exposed avocado seeds was investigated by studying the oviposition preferences of adult female moths and determining egg hatch times in the laboratory, and by investigating the longevity of avocado fruit on the ground under prevailing field conditions. Together, data from these studies suggested that C. sp. nr. lugubris may be an unrecognized pest of avocados that causes hanging fruit to drop to the ground prematurely. The influence of season and altitude on the phenology and distribution of avocado feeding Lepidoptera in Guatemala is discussed.

Surface-tension driven open microfluidic platform for hanging droplet culture

PubMed Central

de Groot, T. E.; Veserat, K. S.; Berthier, E.; Beebe, D. J.; Theberge, A. B.

2015-01-01

The hanging droplet technique for three-dimensional tissue culture has been used for decades in biology labs, with the core technology remaining relatively unchanged. Recently microscale approaches have expanded the capabilities of the hanging droplet method, making it more user-friendly. We present a spontaneously driven, open hanging droplet culture platform to address many limitations of current platforms. Our platform makes use of two interconnected hanging droplet wells, a larger well where cells are cultured and a smaller well for user interface via a pipette. The two-well system results in lower shear stress in the culture well during fluid exchange, enabling shear sensitive or non-adherent cells to be cultured in a droplet. The ability to perform fluid exchanges in-droplet enables long-term culture, treatment, and characterization without disruption of the culture. The open well format of the platform was utilized to perform time-dependent coculture, enabling culture configurations with bone tissue scaffolds and cells grown in suspension. The open nature of the system allowed the direct addition or removal of tissue over the course of an experiment, manipulations that would be impractical in other microfluidic or hanging droplet culture platforms. PMID:26660268

Electrochemistry of moexipril: experimental and computational approach and voltammetric determination.

PubMed

Taşdemir, Hüdai I; Kiliç, E

2014-09-01

The electrochemistry of moexipril (MOE) was studied by electrochemical methods with theoretical calculations performed at B3LYP/6-31 + G (d)//AM1. Cyclic voltammetric studies were carried out based on a reversible and adsorption-controlled reduction peak at -1.35 V on a hanging mercury drop electrode (HMDE). Concurrently irreversible diffusion-controlled oxidation peak at 1.15 V on glassy carbon electrode (GCE) was also employed. Potential values are according to Ag/AgCI, (3.0 M KCI) and measurements were performed in Britton-Robinson buffer of pH 5.5. Tentative electrode mechanisms were proposed according to experimental results and ab-initio calculations. Square-wave adsorptive stripping voltammetric methods have been developed and validated for quantification of MOE in pharmaceutical preparations. Linear working range was established as 0.03-1.35 microM for HMDE and 0.2-20.0 microM for GCE. Limit of quantification (LOQ) was calculated to be 0.032 and 0.47 microM for HMDE and GCE, respectively. Methods were successfully applied to assay the drug in tablets by calibration and standard addition methods with good recoveries between 97.1% and 106.2% having relative standard deviation less than 10%.

Control of solvent evaporation in hen egg white lysozyme crystallization

NASA Technical Reports Server (NTRS)

Wilson, L. J.; Suddath, F. L.

1992-01-01

An investigation of the role of solvent evaporation in tetragonal lysozyme crystallization was preformed with a device that employs N2(g) to control the evaporation of solvent from a micro-volume crystallization hanging drop. The number of crystals was found to vary with the rate at which the final supersaturation level was achieved. It was found that the more rapid the approach to supersaturation the larger the number of crystals. Accordingly, the crystals reached a smaller terminal size. Elongation of the (110) face parallel to the four-fold axis was observed with the slower evaporation rates.

Crystallization and preliminary X-ray crystallographic analysis of strictosidine synthase from Rauvolfia: the first member of a novel enzyme family.

PubMed

Ma, Xueyan; Koepke, Juergen; Fritzsch, Günter; Diem, Ralf; Kutchan, Toni M; Michel, Hartmut; Stöckigt, Joachim

2004-10-01

Strictosidine synthase is a central enzyme involved in the biosynthesis of almost all plant monoterpenoid indole alkaloids. Strictosidine synthase from Rauvolfia serpentina was heterologously expressed in Escherichia coli. Crystals of the purified recombinant enzyme have been obtained by the hanging-drop technique at 303 K with potassium sodium tartrate tetrahydrate as precipitant. The crystals belong to the space group R3 with cell dimensions of a=b=150.3 A and c=122.4 A. Under cryoconditions (120 K), the crystals diffract to about 2.95 A.

Why the water bridge does not collapse

NASA Astrophysics Data System (ADS)

Aerov, Artem A.

2011-09-01

In 2007 an interesting phenomenon was discovered [J. Phys. DJPAPBE0022-372710.1088/0022-3727/40/19/052 40, 6112 (2007)]: a horizontal thread of water, the so-called water bridge, hangs in a horizontal electrostatic field. A different explanation of the water bridge stability is proposed herein: the force supporting it is the surface tension of water, while the role of the electric field is to not allow the water bridge to reduce its surface energy by breaking into separate drops. It is proven that electrostatic field is not the origin of the tension holding the bridge.

Stripping voltammetry in environmental and food analysis.

PubMed

Brainina, K Z; Malakhova, N A; Stojko, N Y

2000-10-01

The review covers over 230 papers published mostly in the last 5 years. The goal of the review is to attract the attention of researchers and users to stripping voltammetry in particular, its application in environmental monitoring and analysis of foodstuffs. The sensors employed are impregnated graphite, carbon paste, thick film carbon/graphite and thin film metallic electrodes modified in-situ or beforehand. Hanging mercury drop electrodes and mercury coated glassy carbon electrodes are also mentioned. Strip and long-lived sensors for portable instruments and flow through systems are discussed as devices for future development and application of stripping voltammetry.

A new voltammetric strategy for sensitive and selective determination of gallium using cupferron as a complexing agent.

PubMed

Grabarczyk, Malgorzata; Wardak, Cecylia

2014-01-01

This article describes a differential pulse adsorptive stripping voltammetric method for the trace determination of gallium in environmental water samples. It is based on the adsorptive deposition of the complex Ga(III)-cupferron at the hanging mercury drop electrode (HMDE) at -0.4 V (versus Ag/AgCl) and its cathodic stripping during the potential scan. The method was optimized as concerns the main electrochemical parameters that affect the voltammetric determination (supporting electrolyte, pH, cupferron concentration, deposition potential and time). The calibration graph is linear from 5 × 10(-10) to 5 × 10(-7) mol L(-1) with a detection limit calculated as 1.3 × 10(-10) mol L(-1) for deposition time of 30 s. The influence of interfering substances such as surfactants and humic substances present in the matrices of natural water samples on the Ga(III) signal was examined and a satisfying minimization of these interferences was proposed. The procedure was applied to direct determination of gallium in environmental water samples.

3D spheroid cultures improve the metabolic gene expression profiles of HepaRG cells.

PubMed

Takahashi, Yu; Hori, Yuji; Yamamoto, Tomohisa; Urashima, Toshiki; Ohara, Yasunori; Tanaka, Hideo

2015-05-07

3D (three-dimensional) cultures are considered to be an effective method for toxicological studies; however, little evidence has been reported whether 3D cultures have an impact on hepatocellular physiology regarding lipid or glucose metabolism. In the present study, we conducted physiological characterization of hepatoma cell lines HepG2 and HepaRG cells cultured in 3D conditions using a hanging drop method to verify the effect of culture environment on cellular responses. Apo (Apolipoprotein)B as well as albumin secretion was augmented by 3D cultures. Expression of genes related to not only drug, but also glucose and lipid metabolism were significantly enhanced in 3D cultured HepaRG spheroids. Furthermore, mRNA levels of CYP (cytochrome P450) enzymes following exposure to corresponding inducers increased under the 3D condition. These data suggest that this simple 3D culture system without any special biomaterials can improve liver-specific characteristics including lipid metabolism. Considering that the system enables high-throughput assay, it may become a powerful tool for compound screening concerning hepatocellular responses in order to identify potential drugs. © 2015 Authors.

The Effect of Single or Repeated Home Visits on the Hanging and Use of Insecticide-Treated Mosquito Nets following a Mass Distribution Campaign - A Cluster Randomized, Controlled Trial

PubMed Central

Kilian, Albert; Balayo, Connie; Feldman, Mitra; Koenker, Hannah; Lokko, Kojo; Ashton, Ruth A.; Bruce, Jane; Lynch, Matthew; Boulay, Marc

2015-01-01

Background Study objective was to evaluate the effectiveness of commonly used post-campaign hang-up visits on the hanging and use of campaign nets. Methods A cluster-randomized trial was carried out in Uganda following an ITN distribution campaign. Five clusters (parishes, consisting of ∼11 villages each) were randomly selected for each of the three study arms with between 7,534 and 9,401 households per arm. Arm 1 received one hang-up visit, while Arm 2 received two visits by volunteers four and seven months after the campaign. Visits consisted of assistance hanging the net and education on net use. The control arm was only exposed to messages during the campaign itself. Three cross-sectional surveys with a two-stage cluster sampling design, representative of the study populations, were carried out to capture the two key outcome variables of net hanging and ITN use. Sample size was calculated to detect at least a 15 percentage-points change in net use, and was 1811 at endline. The analysis used an intention-to-treat approach. Findings Both hanging and use of ITN increased during follow-up in a similar way in all three study arms. The proportion of the population using an ITN the previous night was 64.0% (95% CI 60.8, 67.2), for one additional visit, 68.2% (63.8, 72.2) for two visits and 64.0% (59.4, 68.5) for the control. The proportion of households with all campaign nets hanging increased from 55.7% to 72.5% at endline (p<0.0005 for trend), with no difference between study arms. Financial cost per household visited was estimated as USD 2.33 for the first visit and USD 2.24 for the second. Conclusions Behavior change communication provided during the campaign or through other channels was sufficient to induce high levels of net hanging and use and additional “hang-up” activities were not cost-effective. PMID:25774676

Evaluation of a universal long-lasting insecticidal net (LLIN) distribution campaign in Ghana: cost effectiveness of distribution and hang-up activities

PubMed Central

2014-01-01

Background Between May 2010 and October 2012, approximately 12.5 million long-lasting insecticidal nets (LLINs) were distributed through a national universal mass distribution campaign in Ghana. The campaign included pre-registration of persons and sleeping places, door-to-door distribution of LLINs with ‘hang-up’ activities by volunteers and post-distribution ‘keep-up’ behaviour change communication activities. Hang-up activities were included to encourage high and sustained use. Methods The cost and cost-effectiveness of the LLIN Campaign were evaluated using a before-after design in three regions: Brong Ahafo, Central and Western. The incremental cost effectiveness of the ‘hang-up’ component was estimated using reported variation in the implementation of hang-up activities and LLIN use. Economic costs were estimated from a societal perspective assuming LLINs would be replaced after three years, and included the time of unpaid volunteers and household contributions given to volunteers. Results Across the three regions, 3.6 million campaign LLINs were distributed, and 45.5% of households reported the LLINs received were hung-up by a volunteer. The financial cost of the campaign was USD 6.51 per LLIN delivered. The average annual economic cost was USD 2.90 per LLIN delivered and USD 6,619 per additional child death averted by the campaign. The cost-effectiveness of the campaign was sensitive to the price, lifespan and protective efficacy of LLINs. Hang-up activities constituted 7% of the annual economic cost, though the additional financial cost was modest given the use of volunteers. LLIN use was greater in households in which one or more campaign LLINs were hung by a volunteer (OR = 1.57; 95% CI = 1.09, 2.27; p = 0.02). The additional economic cost of the hang-up activities was USD 0.23 per LLIN delivered, and achieved a net saving per LLIN used and per death averted. Conclusion In this campaign, hang-up activities were estimated to be net saving if hang-up increased LLIN use by 10% or more. This suggests hang-up activities can make a LLIN campaign more cost-effective. PMID:24581249

Personalized Medicine-Based Approach to Model Patterns of Chemoresistance and Tumor Recurrence Using Ovarian Cancer Stem Cell Spheroids.

PubMed

Raghavan, Shreya; Mehta, Pooja; Ward, Maria R; Bregenzer, Michael E; Fleck, Elyse M A; Tan, Lijun; McLean, Karen; Buckanovich, Ronald J; Mehta, Geeta

2017-11-15

Purpose: Chemoresistant ovarian cancers grow in suspension within the ascites fluid. To screen the effect of chemotherapeutics and biologics on resistant ovarian cancers with a personalized basis, we developed a 3D hanging drop spheroid platform. Experimental Design: We initiated spheroids with primary aldehyde dehydrogenase-positive (ALDH + ) CD133 + ovarian cancer stem cells (OvCSC) from different patient samples and demonstrated that stem cell progeny from harvested spheroids was similar to the primary tumor. OvCSC spheroids were utilized to initiate tumors in immunodeficient mice. Drug responses to cisplatin and ALDH-targeting compound or JAK2 inhibitor determined whether the OvCSC population within the spheroids could be targeted. Cells that escaped therapy were isolated and used to initiate new spheroids and model tumor reemergence in a personalized manner. Results: OvCSC spheroids from different patients exhibited varying and personalized responses to chemotherapeutics. Xenografts were established from OvCSC spheroids, even with a single spheroid. Distinct responses to therapy were observed in distinct primary tumor xenografts similar to those observed in spheroids. Spheroids resistant to cisplatin/ALDH inhibitor therapy had persistent, albeit lower ALDH expression and complete loss of CD133 expression, whereas those resistant to cisplatin/JAK2 inhibitor therapy were enriched for ALDH + cells. Conclusions: Our 3D hanging drop suspension platform can be used to propagate primary OvCSCs that represent individual patient tumors effectively by differentiating in vitro and initiating tumors in mice. Therefore, our platform can be used to study cancer stem cell biology and model tumor reemergence to identify new targeted therapeutics from an effective personalized medicine standpoint. Clin Cancer Res; 23(22); 6934-45. ©2017 AACR . ©2017 American Association for Cancer Research.

Identification of Hedgehog signaling outcomes in mouse testis development using a hanging drop-culture system.

PubMed

Szczepny, Anette; Hogarth, Cathryn A; Young, Julia; Loveland, Kate L

2009-02-01

The Hedgehog (Hh) signaling pathway affects fetal testis growth. Recently, we described the dynamic cellular production of Hh signaling pathway components in juvenile and adult rodent testes. The Hh signaling is understood to regulate cord formation in the fetal testis, but minimal knowledge exists regarding how Hh signaling impacts the postnatal testis. To investigate this, we employed hanging drop cultures, which are used routinely in embryoid body formation. This approach has the advantage of using small media volume, and we examined its suitability for short-term culture of both murine embryonic gonads and adult testis tubules. The effects of cyclopamine, a specific Hh signaling inhibitor, were examined following culture of Embryonic Day 11.5 urogenital ridges (as control) and adult seminiferous tubule fragments for 24-48 h using histological, cell proliferation, and gene expression analyses. Cultured embryonic testes displayed generally normal cord structure, anti-Müllerian hormone (Amh) expression, and cell proliferation; known Hh target gene expression (Gli1, osteopontin, official symbol Spp1, and Amh) was altered in response to cyclopamine. Cultured adult tubules exhibited some loss of seminiferous epithelium organization over 48 h. Spermatogonia continued to proliferate, however, and no significant loss of viability was noted overall. Addition of cyclopamine significantly affected levels of Gli1, Igfbp6, Ccnd2 (cyclin D2), Ccnb1 (cyclin B1), Spp1, Kit, and Amh mRNAs; these genes have been shown previously to be expressed in Sertoli and germ cells. These novel results identify Hh target genes in the testis and demonstrate this signaling pathway likely affects cell survival and differentiation in the context of normal adult testis.

Identification of Hedgehog Signaling Outcomes in Mouse Testis Development Using a Hanging Drop-Culture System1

PubMed Central

Szczepny, Anette; Hogarth, Cathryn A.; Young, Julia; Loveland, Kate L.

2008-01-01

The Hedgehog (Hh) signaling pathway affects fetal testis growth. Recently, we described the dynamic cellular production of Hh signaling pathway components in juvenile and adult rodent testes. The Hh signaling is understood to regulate cord formation in the fetal testis, but minimal knowledge exists regarding how Hh signaling impacts the postnatal testis. To investigate this, we employed hanging drop cultures, which are used routinely in embryoid body formation. This approach has the advantage of using small media volume, and we examined its suitability for short-term culture of both murine embryonic gonads and adult testis tubules. The effects of cyclopamine, a specific Hh signaling inhibitor, were examined following culture of Embryonic Day 11.5 urogenital ridges (as control) and adult seminiferous tubule fragments for 24–48 h using histological, cell proliferation, and gene expression analyses. Cultured embryonic testes displayed generally normal cord structure, anti-Müllerian hormone (Amh) expression, and cell proliferation; known Hh target gene expression (Gli1, osteopontin, official symbol Spp1, and Amh) was altered in response to cyclopamine. Cultured adult tubules exhibited some loss of seminiferous epithelium organization over 48 h. Spermatogonia continued to proliferate, however, and no significant loss of viability was noted overall. Addition of cyclopamine significantly affected levels of Gli1, Igfbp6, Ccnd2 (cyclin D2), Ccnb1 (cyclin B1), Spp1, Kit, and Amh mRNAs; these genes have been shown previously to be expressed in Sertoli and germ cells. These novel results identify Hh target genes in the testis and demonstrate this signaling pathway likely affects cell survival and differentiation in the context of normal adult testis. PMID:18843087

Arbitrary-level hanging nodes for adaptive hphp-FEM approximations in 3D

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

Pavel Kus; Pavel Solin; David Andrs

2014-11-01

In this paper we discuss constrained approximation with arbitrary-level hanging nodes in adaptive higher-order finite element methods (hphp-FEM) for three-dimensional problems. This technique enables using highly irregular meshes, and it greatly simplifies the design of adaptive algorithms as it prevents refinements from propagating recursively through the finite element mesh. The technique makes it possible to design efficient adaptive algorithms for purely hexahedral meshes. We present a detailed mathematical description of the method and illustrate it with numerical examples.

[Characteristics of Raman spectra of minerals in the veins of Wenchuan earthquake fault zone].

PubMed

Xie, Chao; Zhou, Ben-gang; Liu, Lei; Zhou, Xiao-cheng; Yi, Li; Chen, Zhi; Cui, Yue-ju; Li, Jing; Chen, Zheng-wei; Du, Jian-guo

2015-01-01

Quartz in the veins at the Shenxigou section of Wenchuan earthquake fault zone was investigated by micro-Raman spectroscopic measurement, and the distribution of compressive stress in the fault zone was estimated by the frequency shifts of the 464 cm-1 vibrational mode of quartz grains in the veins. It was showed that the 464 cm-1 peak arising from the quartz grains in the veins near the fault plane shifts by 3. 29 cm-1 , and the corresponding compressive stress is 368. 63 MPa, which is significantly lower than the stress accumulation on both sides due to multi-stage events. Stress accumulation increased with moving away from the fault plane in the footwall with the offset of the 464 cm-1 peak arising from the quartz grains in the veins increasing, which can reach 494. 77 MPa at a distance of 21 m with a high offset of 4. 40 cm-1 of the 464 cm-1 peak. The compressive stress gets the maximum value of 519.87 MPa at a distance of 10 m from the fault plane in the hanging wall with the offset of the 464 cm-1 peak arising from the quartz grains in the veins being 4. 62 cm-1, followed by a sudden drop in stress accumulation, and it drops to 359. 59 MPa at a distance of 17 m. Because of moving away from the foult plane at the edge of the foult zone, the stress drops to 359. 59 MPa with a small value of 464 cm-1 peak offset 3. 21 cm-1 at a distance of 27 m from the fault plane in the hanging wall due to the little effect by the fault activity. Therefore, the stress of Wenchuan earthquake fault zone is partially released, but the rest of the stress distribution is uneven, and there is also a high stress accumulation in somewhere in the fault zone, which reflects that the mechanical properties of the rocks in the fault zone have a characteristic of unevenness in space.

Purification, crystallization and preliminary X-ray diffraction analysis of a novel keto-deoxy-d-galactarate (KDG) dehydratase from Agrobacterium tumefaciens

PubMed Central

Taberman, Helena; Andberg, Martina; Parkkinen, Tarja; Richard, Peter; Hakulinen, Nina; Koivula, Anu; Rouvinen, Juha

2014-01-01

d-Galacturonic acid is the main component of pectin. It could be used to produce affordable renewable fuels, chemicals and materials through biotechnical conversion. Keto-deoxy-d-galactarate (KDG) dehydratase is an enzyme in the oxidative pathway of d-galacturonic acid in Agrobacterium tumefaciens (At). It converts 3-deoxy-2-keto-l-threo-hexarate to α-ketoglutaric semialdehyde. At KDG dehydratase was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 169.1, b = 117.8, c = 74.3 Å, β = 112.4° and an asymmetric unit of four monomers. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The three-dimensional structure of At KDG dehydratase will provide valuable information on the function of the enzyme and will allow it to be engineered for biorefinery-based applications. PMID:24419616

Purification, crystallization and preliminary X-ray analysis of the glucosamine-6-phosphate N-acetyltransferase from human liver

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

Wang, Juan; Zhou, Yan-Feng; Li, Lan-Fen

2006-11-01

Glucosamine-6-phosphate N-acetyltransferase from human liver was expressed, purified and crystallized. Diffraction data have been collected to 2.6 Å resolution. Glucosamine-6-phosphate N-acetyltransferase from human liver, which catalyzes the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to the primary amine of d-glucosamine 6-phosphate to form N-acetyl-d-glucosamine 6-phosphate, was expressed in a soluble form from Escherichia coli strain BL21 (DE3). The protein was purified to homogeneity using Ni{sup 2+}-chelating chromatography followed by size-exclusion chromatography. Crystals of the protein were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.6 Å resolution. The crystals belonged to space group P4{sub 1}2{sub 1}2more » or P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 50.08, c = 142.88 Å.« less

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

Gao, Xiong-Zhuo; National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871; Li, Lan-Fen

The SMU.961 protein from S. mutans was crystallized and preliminary characterization of the crystals, which diffracted to 2.9 Å resolution, shows them to belong to space group C2. The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni{sup 2+}-chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction weremore » obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 Å resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 Å, β = 98.82°.« less

Extracellular overproduction and preliminary crystallographic analysis of a family I.3 lipase

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

Angkawidjaja, Clement; You, Dong-Ju; Matsumura, Hiroyoshi

2007-03-01

A family I.3 lipase from Pseudomonas sp. MIS38 was secreted from Escherichia coli cells to the external medium, purified and crystallized and preliminary crystallographic studies were performed. A family I.3 lipase from Pseudomonas sp. MIS38 was secreted from Escherichia coli cells to the external medium, purified and crystallized and preliminary crystallographic studies were performed. The crystal was grown at 277 K by the hanging-drop vapour-diffusion method. Native X-ray diffraction data were collected to 1.7 Å resolution using synchrotron radiation at station BL38B1, SPring-8. The crystal belongs to space group P2{sub 1}, with unit-cell parameters a = 48.79, b = 84.06,more » c = 87.04 Å. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient V{sub M} was calculated to be 2.73 Å{sup 3} Da{sup −1} and the solvent content was 55%.« less

Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of great cormorant (Phalacrocorax carbo) haemoglobin

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

Jagadeesan, G.; Malathy, P.; Gunasekaran, K.

2014-10-25

The great cormorant hemoglobin has been isolated, purified and crystallized and the three dimensional structure is solved using molecular replacement technique. Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins. Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350, NaCl and glycerol as precipitants. The crystals belonged to themore » trigonal system P3{sub 1}21, with unit-cell parameters a = b = 55.64, c = 153.38 Å, β = 120.00°; a complete data set was collected to a resolution of 3.5 Å. Matthews coefficient analysis indicated that the crystals contained a half-tetramer in the asymmetric unit.« less

Biodynamic profiling of three-dimensional tissue growth techniques

NASA Astrophysics Data System (ADS)

Sun, Hao; Merrill, Dan; Turek, John; Nolte, David

2016-03-01

Three-dimensional tissue culture presents a more biologically relevant environment in which to perform drug development than conventional two-dimensional cell culture. However, obtaining high-content information from inside three dimensional tissue has presented an obstacle to rapid adoption of 3D tissue culture for pharmaceutical applications. Biodynamic imaging is a high-content three-dimensional optical imaging technology based on low-coherence interferometry and digital holography that uses intracellular dynamics as high-content image contrast. In this paper, we use biodynamic imaging to compare pharmaceutical responses to Taxol of three-dimensional multicellular spheroids grown by three different growth techniques: rotating bioreactor, hanging-drop and plate-grown spheroids. The three growth techniques have systematic variations among tissue cohesiveness and intracellular activity and consequently display different pharmacodynamics under identical drug dose conditions. The in vitro tissue cultures are also compared to ex vivo living biopsies. These results demonstrate that three-dimensional tissue cultures are not equivalent, and that drug-response studies must take into account the growth method.

Deer mouse hemoglobin exhibits a lowered oxygen affinity owing to mobility of the E helix.

PubMed

Inoguchi, Noriko; Oshlo, Jake R; Natarajan, Chandrasekhar; Weber, Roy E; Fago, Angela; Storz, Jay F; Moriyama, Hideaki

2013-04-01

The deer mouse, Peromyscus maniculatus, exhibits altitude-associated variation in hemoglobin oxygen affinity. To examine the structural basis of this functional variation, the structure of the hemoglobin was solved. Recombinant hemoglobin was expressed in Escherichia coli and was purified by ion-exchange chromatography. Recombinant hemoglobin was crystallized by the hanging-drop vapor-diffusion method using polyethylene glycol as a precipitant. The obtained orthorhombic crystal contained two subunits in the asymmetric unit. The refined structure was interpreted as the aquo-met form. Structural comparisons were performed among hemoglobins from deer mouse, house mouse and human. In contrast to human hemoglobin, deer mouse hemoglobin lacks the hydrogen bond between α1Trp14 in the A helix and α1Thr67 in the E helix owing to the Thr67Ala substitution. In addition, deer mouse hemoglobin has a unique hydrogen bond at the α1β1 interface between residues α1Cys34 and β1Ser128.

Deer mouse hemoglobin exhibits a lowered oxygen affinity owing to mobility of the E helix

PubMed Central

Inoguchi, Noriko; Oshlo, Jake R.; Natarajan, Chandrasekhar; Weber, Roy E.; Fago, Angela; Storz, Jay F.; Moriyama, Hideaki

2013-01-01

The deer mouse, Peromyscus maniculatus, exhibits altitude-associated variation in hemoglobin oxygen affinity. To examine the structural basis of this functional variation, the structure of the hemoglobin was solved. Recombinant hemoglobin was expressed in Escherichia coli and was purified by ion-exchange chromatography. Recombinant hemoglobin was crystallized by the hanging-drop vapor-diffusion method using polyethylene glycol as a precipitant. The obtained orthorhombic crystal contained two subunits in the asymmetric unit. The refined structure was interpreted as the aquo-met form. Structural comparisons were performed among hemoglobins from deer mouse, house mouse and human. In contrast to human hemoglobin, deer mouse hemoglobin lacks the hydrogen bond between α1Trp14 in the A helix and α1Thr67 in the E helix owing to the Thr67Ala substitution. In addition, deer mouse hemoglobin has a unique hydrogen bond at the α1β1 interface between residues α1Cys34 and β1Ser128. PMID:23545644

Purification, crystallization and preliminary crystallographic studies of haemoglobin from mongoose (Helogale parvula) in two different crystal forms induced by pH variation.

PubMed

Mohamed Abubakkar, M; Saraboji, K; Ponnuswamy, M N

2013-02-01

Haemoglobin (Hb) is a respiratory pigment; it is a tetrameric protein that ferries oxygen from the lungs to tissues and transports carbon dioxide on the return journey. The oxygen affinity of haemoglobin is regulated by the concentration of oxygen surrounding it and several efforts have revealed the shapes of Hb in different states and with different functions. However, study of the molecular basis of Hbs from low-oxygen-affinity species is critically needed in order to increase the understanding of the mechanism behind oxygen adaptation. The present study reports the preliminary crystallographic study of low-oxygen-affinity haemoglobin from mongoose, a burrowing mammal. Haemoglobin from mongoose was purified by anion-exchange chromatography, crystallized using the hanging-drop vapour-diffusion method and diffraction data sets were collected from monoclinic (2.3 Å resolution) and orthorhombic (2.9 Å resolution) crystal forms obtained by pH variation. The monoclinic and orthorhombic asymmetric units contained half and a whole biological molecule, respectively.

Expression, purification and preliminary X-ray analysis of the C-terminal domain of an arginine repressor protein from Mycobacterium tuberculosis

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

Lu, George J.; Garen, Craig R.; Cherney, Maia M.

2007-11-01

The C-terminal portion of the arginine repressor protein from M. tuberculosis H37Rv has been crystallized. The complete transcriptional factor regulates arginine biosynthesis by binding operator DNA when arginine is bound at the C-terminal domain. The gene product of an open reading frame Rv1657 from Mycobacterium tuberculosis is a putative arginine repressor protein (ArgR), a transcriptional factor that regulates the expression of arginine-biosynthetic enzymes. Rv1657 was expressed and purified and a C-terminal domain was crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 2.15 Å. The crystals belong to space group P1 and themore » Matthews coefficient suggests that the crystals contain six C-terminal domain molecules per unit cell. Previous structural and biochemical studies on the arginine repressor proteins from other organisms have likewise shown the presence of six molecules per unit cell.« less

Crystallization and preliminary crystallographic study of the yeast Malassezia sympodialis allergen Mala s 1

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

Vilhelmsson, Monica, E-mail: monica.vilhelmsson@medks.ki.se; Center for Infectious Medicine, Department of Medicine, Karolinska University Hospital, Huddinge, Stockholm; Hallberg, B. Martin

2006-02-01

Crystals of the M. sympodialis allergen Mala s 1 have been obtained using the hanging-drop vapour-diffusion method. A diffraction data set has been collected from native crystals to 1.35 Å resolution. The opportunistic yeast Malassezia sympodialis can act as an allergen and elicit specific IgE- and T-cell reactivity in patients with atopic eczema. The first identified major allergen from M. sympodialis, Mala s 1, is present on the cell surface of the yeast. Recombinant Mala s 1 was expressed in Escherichia coli, purified and refolded in a soluble form. Crystals of Mala s 1 were obtained in 25% PEG 8K,more » 0.2 M (NH{sub 4}){sub 2}SO{sub 4}. Crystals belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 44.4, b = 163.7, c = 50.6 Å, and diffract to 1.35 Å resolution.« less

Purification, crystallization and preliminary X-ray diffraction analysis of royal palm tree (Roystonea regia) peroxidase

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

Watanabe, Leandra; Nascimento, Alessandro S.; Zamorano, Laura S.

2007-09-01

The purification, crystallization, X-ray diffraction data acquisition and molecular-replacement results of royal palm tree (R. regia) peroxidase are described. Royal palm tree peroxidase (RPTP), which was isolated from Roystonea regia leaves, has an unusually high stability that makes it a promising candidate for diverse applications in industry and analytical chemistry [Caramyshev et al. (2005 ▶), Biomacromolecules, 6, 1360–1366]. Here, the purification and crystallization of this plant peroxidase and its X-ray diffraction data collection are described. RPTP crystals were obtained by the hanging-drop vapour-diffusion method and diffraction data were collected to a resolution of 2.8 Å. The crystals belong to themore » trigonal space group P3{sub 1}21, with unit-cell parameters a = b = 116.83, c = 92.24 Å, and contain one protein molecule per asymmetric unit. The V{sub M} value and solvent content are 4.07 Å{sup 3} Da{sup −1} and 69.8%, respectively.« less

Crystallization and preliminary crystallographic analysis of hygromycin B phosphotransferase from Escherichia coli

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

Iino, Daisuke; Takakura, Yasuaki; Kuroiwa, Mika

2007-08-01

The crystallization and preliminary X-ray studies of the aminoglycoside antibiotic-modifying enzyme hygromycin B phosphotransferase from E. coli are reported. Aminoglycoside antibiotics, such as hygromycin, kanamycin, neomycin, spectinomycin and streptomycin, inhibit protein synthesis by acting on bacterial and eukaryotic ribosomes. Hygromycin B phosphotransferase (Hph; EC 2.7.1.119) converts hygromycin B to 7′′-O-phosphohygromycin using a phosphate moiety from ATP, resulting in the loss of its cell-killing activity. The Hph protein has been crystallized for the first time using a thermostable mutant and the hanging-drop vapour-diffusion method. The crystal provided diffraction data to a resolution of 2.1 Å and belongs to space group P3{submore » 2}21, with unit-cell parameters a = b = 71.0, c = 125.0 Å. Crystals of complexes of Hph with hygromycin B and AMP-PNP or ADP have also been obtained in the same crystal form as that of the apoprotein.« less

Crystallization and preliminary X-ray crystallographic analysis of BxlA, an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520.

PubMed

Morioka, Hideaki; Miki, Yasuhiro; Saito, Kei; Tomoo, Koji; Ishida, Toshimasa; Hasegawa, Tomokazu; Yamano, Akihito; Takada, Chiaki; Miyamoto, Katsushiro; Tsujibo, Hiroshi

2010-07-01

BxlA from Streptomyces thermoviolaceus OPC-520, together with the extracellular BxlE and the integral membrane proteins BxlF and BxlG, constitutes a xylanolytic system that participates in the intracellular transport of xylan-degradation products and the production of xylose. To elucidate the mechanism of the hydrolytic degradation of xylooligosaccharides to xylose at the atomic level, X-ray structural analysis of BxlA was attempted. The recombinant BxlA protein (molecular weight 82 kDa) was crystallized by the hanging-drop vapour-diffusion method at 289 K. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 142.2, b = 129.5, c = 101.4 A, beta = 119.8 degrees , and contained two molecules per asymmetric unit (V(M) = 2.47 A(3) Da(-1)). Diffraction data were collected to a resolution to 2.50 A and provided a data set with an overall R(merge) of 8.3%.

Crystallization and preliminary X-ray crystallographic analysis of the GluR0 ligand-binding core from Nostoc punctiforme

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

Lee, Jun Hyuck; Park, Soo Jeong; Rho, Seong-Hwan

2005-11-01

The GluR0 ligand-binding core from N. punctiforme was expressed, purified and crystallized in the presence of l-glutamate. A diffraction data set was collected to a resolution of 2.1 Å. GluR0 from Nostoc punctiforme (NpGluR0) is a bacterial homologue of the ionotropic glutamate receptor. The ligand-binding core of NpGluR0 was crystallized at 294 K using the hanging-drop vapour-diffusion method. The l-glutamate-complexed crystal belongs to space group C222{sub 1}, with unit-cell parameters a = 78.0, b = 145.1, c = 132.1 Å. The crystals contain three subunits in the asymmetric unit, with a V{sub M} value of 2.49 Å{sup 3} Da{sup −1}.more » The diffraction limit of the l-glutamate complex data set was 2.1 Å using synchrotron X-ray radiation at beamline BL-4A of the Pohang Accelerator Laboratory (Pohang, Korea)« less

Purification, identification and preliminary crystallographic studies of a 2S albumin seed protein from Lens culinaris

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

Gupta, Pankaj; Gaur, Vineet; Salunke, Dinakar M., E-mail: dinakar@nii.res.in

2008-08-01

A 2S albumin from L. culinaris was purified and crystallized and preliminary crystallographic studies were carried out. Lens culinaris (lentil) is a widely consumed high-protein-content leguminous crop. A 2S albumin protein (26.5 kDa) has been identified using NH{sub 2}-terminal sequencing from a 90% ammonium sulfate saturation fraction of total L. culinaris seed protein extract. The NH{sub 2}-terminal sequence shows very high homology to PA2, an allergy-related protein from Pisum sativum. The 2S albumin protein was purified using a combination of size-exclusion and ion-exchange chromatography. Crystals of the 2S seed albumin obtained using the hanging-drop vapour-diffusion method diffracted to 2.5 Åmore » resolution and were indexed in space group P4{sub 1} (or P4{sub 3}), with unit-cell parameters a = b = 78.6, c = 135.2 Å.« less

Cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from Thalictrum flavum

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

Pasquo, Alessandra; Bonamore, Alessandra; Franceschini, Stefano

The cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from T. flavum, a protein which catalyzes the first committed step in the biosynthesis of benzylisoquinoline alkaloids, are reported. Norcoclaurine synthase (NCS) catalyzes the condensation of 3,4-dihydroxyphenylethylamine (dopamine) and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids in plants. The protein was cloned, expressed and purified. Crystals were obtained at 294 K by the hanging-drop vapour-diffusion method using ammonium sulfate and sodium chloride as precipitant agents and diffract to better than 3.0 Å resolution using a synchrotron-radiation source. The crystals belong to themore » trigonal space group P3{sub 1}21, with unit-cell parameters a = b = 86.31, c = 118.36 Å. A selenomethionine derivative was overexpressed, purified and crystallized in the same space group. A complete MAD data set was collected at 2.7 Å resolution. The model is under construction.« less

Development of a model system to analyze chondrogenic differentiation of mesenchymal stem cells

PubMed Central

Ruedel, Anke; Hofmeister, Simone; Bosserhoff, Anja-Katrin

2013-01-01

High-density cell culture is widely used for the analysis of cartilage development of human mesenchymal stem cells (HMSCs) in vitro. Several cell culture systems, as micromass, pellet culture and alginate culture, are applied by groups in the field to induce chondrogenic differentiation of HMSCs. A draw back of all model systems is the high amount of cells necessary for the experiments. Further, handling of large experimental approaches is difficult due to culturing e.g. in 15 ml tubes. Therefore, we aimed to develop a new model system based on “hanging drop” cultures using 10 to 100 fold less cells. Here, we demonstrate that differentiation of chondrogenic cells was induced as previously shown in other model systems. Real time RT-PCR analysis demonstrated that Collagen type II and MIA/CD-RAP were upregulated during culturing whereas for induction of hypertrophic markers like Collagen type X and AP-2 epsilon treatment with TGF beta was needed. To further test the system, siRNA against Sox9 was used and effects on chondrogenic gene expression were evaluated. In summary, the hanging drop culture system was determined to be a promising tool for in vitro chondrogenic studies. PMID:24294400

Three-dimensional crystals of ribosomes and their subunits from eu- and archaebacteria.

PubMed

Glotz, C; Müssig, J; Gewitz, H S; Makowski, I; Arad, T; Yonath, A; Wittmann, H G

1987-11-01

Ordered three-dimensional crystals of 70S ribosomes as well as of 30S and 50S ribosomal subunits from various bacteria (E. coli, Bacillus stearothermophilus, Thermus thermophilus and Halobacterium marismortui) have been grown by vapour diffusion in hanging drops using mono- and polyalcohols. A new compact crystal form of 50S subunits has been obtained, and it is suitable for crystallographic studies at medium resolution. In addition, from one crystal form large crystals could be grown in X-ray capillaries. In all cases the crystals were obtained from functionally active ribosomal particles, and the particles from dissolved crystals retained their integrity and biological activity.

Heterologous expression, purification, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis.

PubMed

Ruppert, Martin; Panjikar, Santosh; Barleben, Leif; Stöckigt, Joachim

2006-03-01

Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 A, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 A.

Nucleation and growth control in protein crystallization

NASA Technical Reports Server (NTRS)

Rosenberger, Franz; Nyce, Thomas A.; Meehan, Edward J.; Sowers, Jennifer W.; Monaco, Lisa A.

1990-01-01

The five topics summarized in this final report are as follows: (1) a technique for the expedient, semi-automated determination of protein solubilities as a function of temperature and application of this technique to proteins other than lysozyme; (2) a small solution cell with adjustable temperature gradients for the growth of proteins at a predetermined location through temperature programming; (3) a microscopy system with image storage and processing capability for high resolution optical studies of temperature controlled protein growth and etching kinetics; (4) growth experiments with lysozyme in thermosyphon flow ; and (5) a mathematical model for the evolution of evaporation/diffusion induced concentration gradients in the hanging drop protein crystallization technique.

Two approaches to the rapid screening of crystallization conditions

NASA Technical Reports Server (NTRS)

Mcpherson, Alexander

1992-01-01

A screening procedure is described for estimating conditions under which crystallization will proceed, thus providing a starting point for more careful experiments. The initial procedure uses the experimental setup of McPherson (1982) which supports 24 individual hanging drop experiments for screening variables such as the precipitant type, the pH, the temperature, and the effects of certain additives and which uses about 1 mg of protein. A second approach is proposed (which is rather hypothetical at this stage and needs a larger sample), based on the isoelectric focusing of protein samples on concentration gradients of common precipitating agents. Using this approach, crystals of concanavalin B and canavalin were obtained.

X-ray Experiments for Students at the SLS Optics Beamline

NASA Astrophysics Data System (ADS)

Flechsig, U.; Als-Nielsen, J.; Jaggi, A.; Krempaský, J.; Oberta, P.; Spielmann, S.; van der Veen, J. F.

2010-06-01

We present a X-ray training course for students. The course covers fundamental properties of synchrotron radiation and basic techniques like scattering and absorption. We prepared ten experiments together with a tutorial. The whole course takes about a week. A first student group from the University of Copenhagen passed the course in June 2009. The experiments were performed at the optics beamline of the Swiss Light Source which can be part-time allocated for training purposes. Two experiments are described in more detail: scattering from a hanging drop of water turning into ice and measurement of the power of a pink synchrotron beam using a simple calorimeter.

Suicide by pesticide poisoning remains a priority for suicide prevention in China: Analysis of national mortality trends 2006-2013.

PubMed

Page, Andrew; Liu, Shiwei; Gunnell, David; Astell-Burt, Thomas; Feng, Xiaoqi; Wang, Lijun; Zhou, Maigeng

2017-01-15

Despite recent declines, suicide remains a priority for China. Ease of availability of high-lethality suicide methods, such as pesticides and firearms, contributes to the overall incidence and is an important target for suicide prevention. This study investigates whether changes in the distribution of methods of suicide have contributed to the recent reduction in suicide in China. Suicide rates (2006-2013) were calculated using the Chinese Disease Surveillance Points system, stratified by gender, age group, and urban-rural residence, to investigate trends in suicide over the study period. Multilevel negative binomial regression models were used to investigate associations between socio-demographic factors and method-specific suicide. The most common method of suicide in China for both males and females was pesticide poisoning, followed by hanging. All methods declined over the study period, with the exception of suicide by jumping in males. Suicide rates for pesticide poisoning and for hanging increased exponentially with age in those aged over ≥45 years in both sexes. Pesticide poisoning declined from 55% to 49% of all suicides, while hanging increased from 27% to 31%. This was an ecological study of a time series of suicide rates, with risk factor adjustment being limited to population-level point estimates derived from a single census. Suicide by pesticide poisoning and hanging remain the leading methods of suicide in China. Changes to the safe use of pesticides and targeted prevention initiatives to restrict access, along with socio-economic development and urbanisation, are likely contributors to declines in suicide by pesticide poisoning. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

Association between the accessibility to lethal methods and method-specific suicide rates: An ecological study in Taiwan.

PubMed

Lin, Jin-Jia; Lu, Tsung-Hsueh

2006-07-01

To examine the association between availability of lethal methods of suicide and method-specific suicide rates at the city/ county level in Taiwan. Age-adjusted and age-specific suicide rates of 23 cities/counties in Taiwan for the years 1999 to 2003 were calculated. Partial correlation coefficients were used to examine cross-sectional associations between independent variables, i.e., proportion of agricultural population and proportion of households living on the sixth floor or above, and suicide rates by different methods (poisoning by solids/liquids, jumping, and hanging) after adjusting for unemployment rates and prevalence of depression. The partial correlation coefficient was 0.77 (p < .001) for proportion of agricultural population with solids/liquids poisoning suicide rates. It was 0.73 (p < .001) for the proportion of households living on the sixth floor or above with suicide rates by jumping. Correlations between hanging suicide rates and proportion of agricultural population or between hanging suicide rates and proportion of households living on the sixth floor or above were not significant. The results showed strong positive associations between access to lethal methods and method-specific suicide rates. Controlling the availability of pesticides and fencing high buildings or installing window guards may be effective measures for suicide prevention.

Suicide attempt by hanging in preadolescent children: a case series.

PubMed

Omigbodun, O O; Adejumo, O A; Babalola, O O

2008-10-01

Suicide is now among the five top causes of death in youth worldwide. However, during the preadolescent period, suicidal behaviour is rare and difficult to define because the cognitive level of young children limits their ability to plan and understand the consequences or the finality of suicide. There is virtually no information about preadolescent suicidal behaviour in Nigeria. To illustrate the presentation and psychosocial issues associated with preadolescent suicidal attempt using the 'hanging' method in Nigeria. Three case scenarios of suicide attempt by hanging in preadolescents seen at the University College Hospital, Ibadan between 2005 and 2006 were interviewed in detail along with mental state and physical examination. Family and individual therapies were embarked upon. Types of psychopathology found in the preadolescents include depressive symptoms, conduct and oppositional defiant disorder and impulse control problems. Stressful life events such as family disruption, physical abuse, and bullying at school were factors associated with suicidal behaviour. The influence of the media in providing information about 'hanging' as a method of suicide was evident. Therapy yield varying results. High risk parameters for suicide in children should be known to all health professionals. The importance of intervention strategies particularly media education, monitoring systems and further research on suicidal behaviour in this environment is apparent.

Design Tool Using a New Optimization Method Based on a Stochastic Process

NASA Astrophysics Data System (ADS)

Yoshida, Hiroaki; Yamaguchi, Katsuhito; Ishikawa, Yoshio

Conventional optimization methods are based on a deterministic approach since their purpose is to find out an exact solution. However, such methods have initial condition dependence and the risk of falling into local solution. In this paper, we propose a new optimization method based on the concept of path integrals used in quantum mechanics. The method obtains a solution as an expected value (stochastic average) using a stochastic process. The advantages of this method are that it is not affected by initial conditions and does not require techniques based on experiences. We applied the new optimization method to a hang glider design. In this problem, both the hang glider design and its flight trajectory were optimized. The numerical calculation results prove that performance of the method is sufficient for practical use.

Development of an in vitro culture method for stepwise differentiation of mouse embryonic stem cells and induced pluripotent stem cells into mature osteoclasts.

PubMed

Nishikawa, Keizo; Iwamoto, Yoriko; Ishii, Masaru

2014-05-01

The development of methods for differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cell (iPSCs) into functional cells have helped to analyze the mechanism regulating cellular processes and to explore cell-based assays for drug discovery. Although several reports have demonstrated methods for differentiation of mouse ESCs into osteoclast-like cells, it remains unclear whether these methods are applicable for differentiation of iPSCs to osteoclasts. In this study, we developed a simple method for stepwise differentiation of mouse ESCs and iPSCs into bone-resorbing osteoclasts based upon a monoculture approach consisting of three steps. First, based on conventional hanging-drop methods, embryoid bodies (EBs) were produced from mouse ESCs or iPSCs. Second, EBs were cultured in medium supplemented with macrophage colony-stimulating factor (M-CSF), and differentiated to osteoclast precursors, which expressed CD11b. Finally, ESC- or iPSC-derived osteoclast precursors stimulated with receptor activator of nuclear factor-B ligand (RANKL) and M-CSF formed large multinucleated osteoclast-like cells that expressed tartrate-resistant acid phosphatase and were capable of bone resorption. Molecular analysis showed that the expression of osteoclast marker genes such as Nfatc1, Ctsk, and Acp5 are increased in a RANKL-dependent manner. Thus, our procedure is simple and easy and would be helpful for stem cell-based bone research.

Cloning, purification, crystallization and preliminary structural studies of penicillin V acylase from Bacillus subtilis

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

Rathinaswamy, Priya; Pundle, Archana V.; Prabhune, Asmita A.

An unannotated protein reported from B. subtilis has been expressed in E. coli and identified as possessing penicillin V acylase activity. The crystallization and preliminary crystallographic analysis of this penicillin V acylase is presented. Penicillin acylase proteins are amidohydrolase enzymes that cleave penicillins at the amide bond connecting the side chain to their β-lactam nucleus. An unannotated protein from Bacillus subtilis has been expressed in Escherichia coli, purified and confirmed to possess penicillin V acylase activity. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 4 M sodium formate in 100 mM Tris–HCl buffer pH 8.2.more » Diffraction data were collected under cryogenic conditions to a spacing of 2.5 Å. The crystals belonged to the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 111.0, b = 308.0, c = 56.0 Å. The estimated Matthews coefficient was 3.23 Å{sup 3} Da{sup −1}, corresponding to 62% solvent content. The structure has been solved using molecular-replacement methods with B. sphaericus penicillin V acylase (PDB code 2pva) as the search model.« less

The Tool for Designing Engineering Systems Using a New Optimization Method Based on a Stochastic Process

NASA Astrophysics Data System (ADS)

Yoshida, Hiroaki; Yamaguchi, Katsuhito; Ishikawa, Yoshio

The conventional optimization methods were based on a deterministic approach, since their purpose is to find out an exact solution. However, these methods have initial condition dependence and risk of falling into local solution. In this paper, we propose a new optimization method based on a concept of path integral method used in quantum mechanics. The method obtains a solutions as an expected value (stochastic average) using a stochastic process. The advantages of this method are not to be affected by initial conditions and not to need techniques based on experiences. We applied the new optimization method to a design of the hang glider. In this problem, not only the hang glider design but also its flight trajectory were optimized. The numerical calculation results showed that the method has a sufficient performance.

Structural analysis of bacteriophage-encoded peptidoglycan hydrolase domain KMV36C: crystallization and preliminary X-ray diffraction

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

Van Hecke, Kristof, E-mail: kristof.vanhecke@chem.kuleuven.be; Briers, Yves; Derua, Rita

2008-04-01

Crystallization and X-ray data collection of the C-terminus of gp36 from bacteriophage ϕKMV (KMV36C) are reported. The C-terminus of gp36 of bacteriophage ϕKMV (KMV36C) functions as a particle-associated muramidase, presumably as part of the injection needle of the ϕKMV genome during infection. Crystals of KMV36C were obtained by hanging-drop vapour diffusion and diffracted to a resolution of 1.6 Å. The crystals belong to the cubic space group P432, with unit-cell parameters a = b = c = 102.52 Å. KMV36C shows 30% sequence identity to T4 lysozyme (PDB code)

Heterologous expression, purification, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis

PubMed Central

Ruppert, Martin; Panjikar, Santosh; Barleben, Leif; Stöckigt, Joachim

2006-01-01

Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 Å, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 Å. PMID:16511316

Overexpression, purification, crystallization and preliminary X-ray studies of Vibrio cholerae EpsG

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

Jens, Jason; Raghunathan, Kannan; Vago, Frank

2010-01-12

EpsG is the major pseudopilin protein of the Vibrio cholerae type II secretion system. An expression plasmid that encodes an N-terminally truncated form of EpsG with a C-terminal noncleavable His tag was constructed. Recombinant EpsG was expressed in Escherichia coli; the truncated protein was purified and crystallized by hanging-drop vapor diffusion against a reservoir containing 6 mM zinc sulfate, 60 mM MES pH 6.5, 15% PEG MME 550. The crystals diffracted X-rays to a resolution of 2.26 {angstrom} and belonged to space group P2{sub 1}, with unit-cell parameters a = 88.61, b = 70.02, c = 131.54 {angstrom}.

Characteristic Features of Hanging: A Study in Rural District of Central India.

PubMed

Ambade, Vipul Namdeorao; Kolpe, Dayanand; Tumram, Nilesh; Meshram, Satin; Pawar, Mohan; Kukde, Hemant

2015-09-01

The ligature mark is the most relevant feature of hanging. This study was undertaken with a view to determine the characteristic features of hanging and its association with ligature material or mode of suspension. Of a total medicolegal deaths reported at an Apex Medical Centre, hanging was noted in 4.1% cases, all suicidal with mortality rate of 1.5 per 100,000 population per year. The hanging was complete in 67.7% with nylon rope as the commonest type of ligature material used for ligation. The hanging mark was usually single, situated above thyroid cartilage, incomplete, prominent, and directed toward nape of neck. The mark of dribbling of saliva was seen in 11.8% cases. Facial congestion, petechial hemorrhage, and cyanosis were significantly seen in partial hanging. Though occasionally reported, the argent line was noted in 78.7% hanging deaths with neck muscle hemorrhage in 23.6% cases. Fracture of neck structure was predominant in complete hanging. © 2015 American Academy of Forensic Sciences.

Enhanced differentiation of mesenchymal stromal cells by three-dimensional culture and azacitidine

PubMed Central

Bae, Yoo-Jin; Kwon, Yong-Rim; Kim, Hye Joung; Lee, Seok

2017-01-01

Background Mesenchymal stromal cells (MSCs) are useful for cell therapy because of their potential for multilineage differentiation. However, MSCs that are expanded in traditional two-dimensional (2D) culture systems eventually lose their differentiation abilities. Therefore, we investigated whether azacitidine (AZA) supplementation and three-dimensional culture (3D) could improve the differentiation properties of MSCs. Methods 2D- or 3D-cultured MSCs which were prepared according to the conventional or hanging-drop culture method respectively, were treated with or without AZA (1 µM for 72 h), and their osteogenic and adipogenic differentiation potential were determined and compared. Results AZA treatment did not affect the cell apoptosis or viability in both 2D- and 3D-cultured MSCs. However, compared to conventionally cultured 2D-MSCs, AZA-treated 2D-MSCs showed marginally increased differentiation abilities. In contrast, 3D-MSCs showed significantly increased osteogenic and adipogenic differentiation ability. When 3D culture was performed in the presence of AZA, the osteogenic differentiation ability was further increased, whereas adipogenic differentiation was not affected. Conclusion 3D culture efficiently promoted the multilineage differentiation of MSCs, and in combination with AZA, it could help MSCs to acquire greater osteogenic differentiation ability. This optimized culture method can enhance the therapeutic potential of MSCs. PMID:28401097

Hang cleans and hang snatches produce similar improvements in female collegiate athletes

PubMed Central

Ayers, JL; DeBeliso, M; Sevene, TG

2016-01-01

Olympic weightlifting movements and their variations are believed to be among the most effective ways to improve power, strength, and speed in athletes. This study investigated the effects of two Olympic weightlifting variations (hang cleans and hang snatches), on power (vertical jump height), strength (1RM back squat), and speed (40-yard sprint) in female collegiate athletes. 23 NCAA Division I female athletes were randomly assigned to either a hang clean group or hang snatch group. Athletes participated in two workout sessions a week for six weeks, performing either hang cleans or hang snatches for five sets of three repetitions with a load of 80-85% 1RM, concurrent with their existing, season-specific, resistance training program. Vertical jump height, 1RM back squat, and 40-yard sprint all had a significant, positive improvement from pre-training to post-training in both groups (p≤0.01). However, when comparing the gain scores between groups, there was no significant difference between the hang clean and hang snatch groups for any of the three dependent variables (i.e., vertical jump height, p=0.46; 1RM back squat, p=0.20; and 40-yard sprint, p=0.46). Short-term training emphasizing hang cleans or hang snatches produced similar improvements in power, strength, and speed in female collegiate athletes. This provides strength and conditioning professionals with two viable programmatic options in athletic-based exercises to improve power, strength, and speed. PMID:27601779

Duodenoscope hang time does not correlate with risk of bacterial contamination.

PubMed

Heroux, Riley; Sheppard, Michelle; Wright, Sharon B; Sawhney, Mandeep; Hirsch, Elizabeth B; Kalaidjian, Robin; Snyder, Graham M

2017-04-01

Current professional guidelines recommend a maximum hang time for reprocessed duodenoscopes of 5-14 days. We sought to study the association between hang time and risk of duodenoscope contamination. We analyzed cultures of the elevator mechanism and working channel collected in a highly standardized fashion just before duodenoscope use. Hang time was calculated as the time from reprocessing to duodenoscope sampling. The relationship between hang time and duodenoscope contamination was estimated using a calculated correlation coefficient between hang time in days and degree of contamination on the elevator mechanism and working channel. The 18 study duodenoscopes were cultured 531 times, including 465 (87.6%) in the analysis dataset. Hang time ranged from 0.07-39.93 days, including 34 (7.3%) with hang time ≥7.00 days. Twelve cultures (2.6%) demonstrated elevator mechanism and/or working channel contamination. The correlation coefficients for hang time and degree of duodenoscope contamination were very small and not statistically significant (-0.0090 [P = .85] for elevator mechanism and -0.0002 [P = 1.00] for working channel). Odds ratios for hang time (dichotomized at ≥7.00 days) and elevator mechanism and/or working channel contamination were not significant. We did not find a significant association between hang time and risk of duodenoscope contamination. Future guidelines should consider a recommendation of no limit for hang time. Copyright © 2017 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Hang cleans and hang snatches produce similar improvements in female collegiate athletes.

PubMed

Ayers, J L; DeBeliso, M; Sevene, T G; Adams, K J

2016-09-01

Olympic weightlifting movements and their variations are believed to be among the most effective ways to improve power, strength, and speed in athletes. This study investigated the effects of two Olympic weightlifting variations (hang cleans and hang snatches), on power (vertical jump height), strength (1RM back squat), and speed (40-yard sprint) in female collegiate athletes. 23 NCAA Division I female athletes were randomly assigned to either a hang clean group or hang snatch group. Athletes participated in two workout sessions a week for six weeks, performing either hang cleans or hang snatches for five sets of three repetitions with a load of 80-85% 1RM, concurrent with their existing, season-specific, resistance training program. Vertical jump height, 1RM back squat, and 40-yard sprint all had a significant, positive improvement from pre-training to post-training in both groups (p≤0.01). However, when comparing the gain scores between groups, there was no significant difference between the hang clean and hang snatch groups for any of the three dependent variables (i.e., vertical jump height, p=0.46; 1RM back squat, p=0.20; and 40-yard sprint, p=0.46). Short-term training emphasizing hang cleans or hang snatches produced similar improvements in power, strength, and speed in female collegiate athletes. This provides strength and conditioning professionals with two viable programmatic options in athletic-based exercises to improve power, strength, and speed.

Changes in Suicide Rates by Hanging/Suffocation and Firearms among Young Persons Aged 10 to 24 Years in the United States: 1992–2006

PubMed Central

Bridge, Jeffrey A.; Greenhouse, Joel B.; Sheftall, Arielle H.; Fabio, Anthony; Campo, John V.; Kelleher, Kelly J.

2009-01-01

We examined changes in suicide rates among 10–24-year-olds in the US from 1992–2006. The overall suicide rate and the rate by firearms, poisoning, and other methods declined markedly, whereas the hanging/suffocation rate rose significantly from 1992–2006. This increase occurred across every major demographic subgroup, but was most dramatic for females. PMID:20413089

Raman Spectroscopy Adds Complementary Detail to the High-Resolution X-Ray Crystal Structure of Photosynthetic PsbP from Spinacia oleracea

PubMed Central

Lapkouski, Mikalai; Hofbauerova, Katerina; Sovova, Zofie; Ettrichova, Olga; González-Pérez, Sergio; Dulebo, Alexander; Kaftan, David; Kuta Smatanova, Ivana; Revuelta, Jose L.; Arellano, Juan B.; Carey, Jannette; Ettrich, Rüdiger

2012-01-01

Raman microscopy permits structural analysis of protein crystals in situ in hanging drops, allowing for comparison with Raman measurements in solution. Nevertheless, the two methods sometimes reveal subtle differences in structure that are often ascribed to the water layer surrounding the protein. The novel method of drop-coating deposition Raman spectropscopy (DCDR) exploits an intermediate phase that, although nominally “dry,” has been shown to preserve protein structural features present in solution. The potential of this new approach to bridge the structural gap between proteins in solution and in crystals is explored here with extrinsic protein PsbP of photosystem II from Spinacia oleracea. In the high-resolution (1.98 Å) x-ray crystal structure of PsbP reported here, several segments of the protein chain are present but unresolved. Analysis of the three kinds of Raman spectra of PsbP suggests that most of the subtle differences can indeed be attributed to the water envelope, which is shown here to have a similar Raman intensity in glassy and crystal states. Using molecular dynamics simulations cross-validated by Raman solution data, two unresolved segments of the PsbP crystal structure were modeled as loops, and the amino terminus was inferred to contain an additional beta segment. The complete PsbP structure was compared with that of the PsbP-like protein CyanoP, which plays a more peripheral role in photosystem II function. The comparison suggests possible interaction surfaces of PsbP with higher-plant photosystem II. This work provides the first complete structural picture of this key protein, and it represents the first systematic comparison of Raman data from solution, glassy, and crystalline states of a protein. PMID:23071614

Study of endolaryngeal structures by videolaryngoscopy after hanging: a new approach to understanding the physiopathogenesis.

PubMed

Duband, S; Timoshenko, A P; Mohammedi, R; Prades, J-M; Barral, F-G; Debout, M; Péoc'h, M

2009-11-20

To evaluate laryngoscopic findings in hanging cases and to compare them with magnetic resonance imaging (MRI) and forensic autopsy results. Postmortem nasolaryngofibroscopy and MRI of five people who died from hanging were performed. Three people who died from other causes than hanging were also examined with a flexible laryngofibroscope. The results were compared with injuries discovered during forensic autopsy. In all five hanging cases, laryngofibroscopic investigation showed a vocal fold position in complete adduction confirmed by MRI. This position did not seem to be influenced by the intensity of the forces applied to neck or postmortem delay and cadaveric phenomena. The vocal cords of the three non-hanging deceased were found in the intermediate position. These findings could suggest that pressure applied to the cervical nervous and cartilaginous structures or their elongation during hanging could lead to closure of the glottis with vocal cord adduction maintained after death. Laryngofibroscopic examination in hanging cases could be very useful in confirming the vital character of the hanging and understanding asphyxial phenomena in incomplete suspension without laryngeal crush.

Dynamic compaction of human mesenchymal stem/precursor cells (MSC) into spheres self-activates caspase-dependent IL1 signaling to enhance secretion of modulators of inflammation and immunity (PGE2, TSG6 and STC1)

PubMed Central

Bazhanov, Nikolay; Kuhlman, Jessica; Prockop, Darwin J.

2013-01-01

Human mesenchymal stem/precursor cells (MSC) are similar to some other stem/progenitor cells in that they compact into spheres when cultured in hanging drops or on non-adherent surfaces. Assembly of MSC into spheres alters many of their properties, including enhanced secretion of factors that mediate inflammatory and immune responses. Here we demonstrated that MSC spontaneously aggregated into sphere-like structures after injection into a subcutaneous air pouch or the peritoneum of mice. The structures were similar to MSC spheres formed in cultures demonstrated by the increased expression of genes for inflammation-modulating factors TSG6, STC1, and COX2, a key enzyme in production of PGE2. To identify the signaling pathways involved, hanging drop cultures were used to follow the time-dependent changes in the cells as they compacted into spheres. Among the genes up-regulated were genes for the stress-activated signaling pathway for IL1α/β, and the contact-dependent signaling pathway for Notch. An inhibitor of caspases reduced the up-regulation of IL1A/B expression, and inhibitors of IL1 signaling decreased production of PGE2, TSG6 and STC1. Also, inhibition of IL1A/B expression and secretion of PGE2 negated the anti-inflammatory effects of MSC spheres on stimulated macrophages. Experiments with γ-secretase inhibitors suggested that Notch signaling was also required for production of PGE2 but not TSG6 or STC1. The results indicated that assembly of MSC into spheres triggers caspase-dependent IL1 signaling and the secretion of modulators of inflammation and immunity. Similar aggregation in vivo may account for some of the effects observed with administration of the cells in animal models. PMID:23922312

Behaviour and quantification studies of terbacil and lenacil in environmental samples using cyclic and adsorptive stripping voltammetry at hanging mercury drop electrode.

PubMed

Thriveni, T; Rajesh Kumar, J; Sujatha, D; Sreedhar, N Y

2007-05-01

The cyclic voltammograms of terbacil and lenacil at the hanging mercury drop electrode showed a single well defined four electron irreversible peak in universal buffer of pH 4.0 for both compounds. The peak potentials were shifted to more negative values on the increase of pH of the medium, implying the involvement of protons in the electrode reaction and that the proton transfer reaction precedes the proper electrode process. The four electron single peak may be attributed to the simultaneous reduction of carbonyl groups present in 2 and 4 in pyrimidine ring of terbacil and lenacil to the corresponding hydroxy derivative. Based on the interfacial adsorptive character of the terbacil and lenacil onto the mercury electrode surface, a simple sensitive and low cost differential pulse adsorptive stripping voltammetric procedure was optimized for the analysis of terbacil and lenacil. The optimal operational conditions of the proposed procedure were accumulation potential E (acc) = -0.4 V, accumulation time t (acc) = 80 s, scan rate = 40 mV s(-1), pulse amplitude = 25 mV using a universal buffer pH 4.0 as a supporting electrolyte. The linear concentration range was found to be 1.5 x 10(-5) to 1.2 x 10(-9) mol/l and 1.5 x 10(-5) to 2.5 x 10(-8) mol/l with the lower detection limit of 1.22 x 10(-9) and 2.0 x 10(-8) mol/l. The correlation coefficient and relative standard deviation values are found to be 0.942, 0.996, 1.64% and 1.23%, respectively, for 10 replicants. The procedure was successfully applied for determination of terbacil and lenacil in formulations, mixed formulations, environmental samples such as fruit samples and spiked water samples.

Anti-cancer Effect of Xao Tam Phan Paramignya trimera Methanol Root Extract on Human Breast Cancer Cell Line MCF-7 in 3D Model.

PubMed

Nguyen-Thi, Lam-Huyen; Nguyen, Sinh Truong; Tran, Thao Phuong; Phan-Lu, Chinh-Nhan; The Van, Trung; Van Pham, Phuc

2018-04-24

Cancer is one of the leading causes of death in the world. A great deal of effort has been made to discover new agents for cancer treatment. Xao tam phan (Paramignya trimera) is a traditional medicine of Vietnam used in cancer treatment for a long time, yet there is not much scientific evidence proving its anticancer potency. The study aimed to evaluate the toxicity of Paramignya trimera extract (PTE) on multicellular tumor spheres (MCTS) of MCF-7 cells using hanging drop technique. Firstly, MCF-7 cells were seeded on hanging drop plates, spheroid size was tracked, and growth curve was measured by MTT assay and AlamarBlue ® assay. The necrotic core of MCTS was evaluated by propidium iodide (PI) staining. Toxicity of doxorubicin (DOX) and tirapazamine (TPZ) was then tested on 3D model compared to 2D culture condition. The results showed that the IC50 of DOX on 3D MCF-7 cells was nearly 50 times greater than monolayer MCF-7 cells. In contrast, TPZ (an agent which is specifically toxic under hypoxic conditions) had significantly lower IC50 in 3D condition than in 2D. The toxicity tests for PTE showed that PTE strongly inhibited MCF-7 cells in both 2D and 3D conditions. Interestingly, the IC50 of PTE in 3D model was remarkably lower than in 2D (IC50 value was 168.9 ± 11.65 μg/ml compared to 260.8 ± 16.54 μg/ml, respectively). The invasion assay showed that PTE completely inhibited invasion of MCF-7 cells at 250 μg/mL concentration. Also, flow cytometry results indicated that PTE effectively induced apoptosis in MCF-7 spheroids in 3D condition at 250 μg/mL concentration. The results from this study emphasize the promise of PTE in cancer therapy.

Celebrity Suicides and Their Differential Influence on Suicides in the General Population: A National Population-Based Study in Korea

PubMed Central

Myung, Woojae; Won, Hong-Hee; Fava, Maurizio; Mischoulon, David; Yeung, Albert; Lee, Dongsoo; Kim, Doh Kwan

2015-01-01

Objective Although evidence suggests that there is an increase in suicide rates in the general population following celebrity suicide, the rates are heterogeneous across celebrities and countries. It is unclear which is the more vulnerable population according to the effect sizes of celebrity suicides to general population. Methods All suicide victims in the general population verified by the Korea National Statistical Office and suicides of celebrity in South Korea were included for 7 years from 2005 to 2011. Effect sizes were estimated by comparing rates of suicide in the population one month before and after each celebrity suicide. The associations between suicide victims and celebrities were examined. Results Among 94,845 suicide victims, 17,209 completed suicide within one month after 13 celebrity suicides. Multivariate logistic regression analyses revealed that suicide victims who died after celebrity suicide were significantly likely to be of age 20-39, female, and to die by hanging. These qualities were more strongly associated among those who followed celebrity suicide with intermediate and high effect sizes than lower. Younger suicide victims were significantly associated with higher effect size, female gender, white collar employment, unmarried status, higher education, death by hanging, and night-time death. Characteristics of celebrities were significantly associated with those of general population in hanging method and gender. Conclusion Individuals who commit suicide after a celebrity suicide are likely to be younger, female, and prefer hanging as method of suicide, which are more strongly associated in higher effect sizes of celebrity suicide. PMID:25866521

Preliminary Crystallographic Study of Hemoglobin from Buffalo (Bubalus bubalis): A Low Oxygen Affinity Species.

PubMed

Balasubramanian, Moovarkumudalvan; Moorthy, Ponnuraj Sathya; Neelagandan, Kamariah; Ponnuswamy, Mondikalipudur Nanjappa Gounder

2009-01-01

Hemoglobin is a tetrameric, iron-containing metalloprotein, which plays a vital role in the transportation of oxygen from lungs to tissues and carbon dioxide back to lungs. Though good amount of work has already been done on hemoglobins, the scarcity of data on three dimensional structures pertaining to low oxygen affinity hemoglobins from mammalian species, motivated our group to work on this problem specifically. Herein, we report the preliminary crystallographic analysis of buffalo hemoglobin, which belongs to low oxygen affinity species. The buffalo blood was collected, purified by anion exchange chromatography and crystallized with PEG 3350 using 50mM phosphate buffer at pH 6.7 as a precipitant by hanging drop vapor diffusion method. Data collection was carried out using mar345dtb image plate detector system. Buffalo hemoglobin crystallizes in orthorhombic space group P2(1)2(1)2(1) with one whole biological molecule (alpha2beta2) in the asymmetric unit with cell dimensions a=63.064A, b=74.677A, c=110.224A.

Purification, crystallization and preliminary crystallographic studies of haemoglobin from mongoose (Helogale parvula) in two different crystal forms induced by pH variation

PubMed Central

Mohamed Abubakkar, M.; Saraboji, K.; Ponnuswamy, M. N.

2013-01-01

Haemoglobin (Hb) is a respiratory pigment; it is a tetrameric protein that ferries oxygen from the lungs to tissues and transports carbon dioxide on the return journey. The oxygen affinity of haemoglobin is regulated by the concentration of oxygen surrounding it and several efforts have revealed the shapes of Hb in different states and with different functions. However, study of the molecular basis of Hbs from low-oxygen-affinity species is critically needed in order to increase the understanding of the mechanism behind oxygen adaptation. The present study reports the preliminary crystallographic study of low-oxygen-affinity haemoglobin from mongoose, a burrowing mammal. Haemoglobin from mongoose was purified by anion-exchange chromatography, crystallized using the hanging-drop vapour-diffusion method and diffraction data sets were collected from monoclinic (2.3 Å resolution) and orthorhombic (2.9 Å resolution) crystal forms obtained by pH variation. The monoclinic and orthorhombic asymmetric units contained half and a whole biological molecule, respectively. PMID:23385751

Crystallization and preliminary X-ray diffraction analysis of mouse 3(17)α-hydroxysteroid dehydrogenase

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

El-Kabbani, Ossama, E-mail: ossama.el-kabbani@vcp.monash.edu.au; Ishikura, Syuhei; Wagner, Armin

2005-07-01

Orthorhombic crystals of mouse 3(17)α-hydroxysteroid dehydrogenase were obtained from buffered polyethylene glycol solutions. The crystals diffracted to a resolution of 1.8 Å at the Swiss Light Source beamline X06SA. The 3(17)α-hydroxysteroid dehydrogenase from mouse is involved in the metabolism of oestrogens, androgens, neurosteroids and xenobiotic compounds. The enzyme was crystallized by the hanging-drop vapour-diffusion method in space group P222{sub 1}, with unit-cell parameters a = 84.91, b = 84.90, c = 95.83 Å. The Matthews coefficient (V{sub M}) and the solvent content were 2.21 Å{sup 3} Da{sup −1} and 44.6%, respectively, assuming the presence of two molecules in the asymmetricmore » unit. Diffraction data were collected to a resolution of 1.8 Å at the Swiss Light Source beamline X06SA using a MAR CCD area detector and gave a data set with an overall R{sub merge} of 6.8% and a completeness of 91.1%.« less

Purification, crystallization and preliminary X-ray analysis of native and selenomethionine class I tagatose-1,6-bisphosphate aldolase from Streptococcus pyogenes.

PubMed

Liotard, Brigitte; Sygusch, Jurgen

2004-03-01

Tagatose-1,6-bisphosphate aldolase (EC 4.1.2.40) is situated at the branching of the tagatose-6-phosphate and Embden-Meyerhof-Parnas (glycolysis) metabolic pathways, where it catalyzes the reversible cleavage of tagatose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The recombinant protein from Streptococcus pyogenes was overexpressed in Escherichia coli in its native and selenomethionine-derivative forms and purified using ion-exchange and hydrophobic interaction chromatography. Orthorhombic crystals suitable for structural analysis were obtained by the hanging-drop vapour-diffusion method for both isoforms. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 63.7, b = 108.1, c = 238.7 A for the native form and a = 64.1, b = 108.3, c = 239.8 A for the selenomethionine derivative. The asymmetric unit contains four protomers, corresponding to a crystal volume per protein weight (V(M)) of 2.8 A(3) Da(-1) and a solvent content of 56% by volume.

Purification, partial characterization, crystallization and preliminary X-ray diffraction of a novel cardiotoxin-like basic protein from Naja naja atra (South Anhui) venom

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

Rong, Hui; Li, Yan; Lou, Xiao-hua

2007-02-01

A novel cardiotoxin-like basic protein from Naja naja atra was crystallized and diffraction data were collected to 2.35 Å resolution. A novel cardiotoxin-like basic protein was isolated from the venom of the Chinese cobra (Naja naja atra) from the south of Anhui in China. The protein inhibits the expression of vascular endothelial growth factor and basic fibroblast growth factor in human lung cancer cell line H1299 and induces the haemolysis of rabbit erythrocytes under low-lecithin conditions. After a two-step chromatographic purification, the resultant 7 kDa protein was crystallized by the hanging-drop vapour-diffusion method at room temperature. A complete data setmore » was collected to 2.35 Å resolution using an in-house X-ray diffraction system. The crystal belongs to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 43.2, c = 147.9 Å. There are two molecules in the crystallographic asymmetric unit.« less

Crystallization and preliminary X-ray crystallographic analysis of a carbonyl reductase from Candida parapsilosis

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

Zhang, Rongzhen; Xu, Yan, E-mail: biosean@yahoo.com.cn; Sun, Ying

2008-04-01

A novel short-chain NADPH-dependent (S)-1-phenyl-1,2-ethanediol dehydrogenase (SCR) has been crystallized. A novel short-chain NADPH-dependent (S)-1-phenyl-1,2-ethanediol dehydrogenase (SCR) has been crystallized. Two distinct but related crystal forms of SCR were obtained using the hanging-drop vapour-diffusion method and a reservoir solution consisting of 18%(w/v) polyethylene glycol 2000 monomethyl ether and 8%(v/v) 2-propanol as the precipitant. The crystals were rhomboid in shape with average dimensions of 0.3 × 0.3 × 0.4 mm and diffracted to a resolution of 2.7–3.0 Å. The crystal forms both belong to space group P2{sub 1}2{sub 1}2{sub 1} and have unit-cell parameters a = 104.7, b = 142.8, cmore » = 151.8 Å and a = 101.1, b = 146.0, c = 159.8 Å. The calculated values of V{sub M}, rotation-function and translation-function solutions and consideration of potential crystal packing suggest that there are eight protein subunits per asymmetric unit.« less

Purification, crystallization and preliminary X-ray crystallographic analysis of a cysteine-rich secretory protein (CRISP) from Naja atra venom.

PubMed

Wang, Yu-Ling; Goh, King-Xiang; Wu, Wen-guey; Chen, Chun-Jung

2004-10-01

Cysteine-rich secretory proteins (CRISPs) play an important role in the innate immune system and are transcriptionally regulated by androgens in several tissues. The proteins are mostly found in the epididymis and granules of mammals, whilst a number of snake venoms also contain CRISP-family proteins. The natrin protein from the venom of Naja atra (Taiwan cobra), which belongs to a family of CRISPs and has a cysteine-rich C-terminal amino-acid sequence, has been purified using a three-stage chromatography procedure and crystals suitable for X-ray analysis have been obtained using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.58 A resolution using synchrotron radiation; the crystals belong to space group C222(1), with unit-cell parameters a = 59.172, b = 65.038, c = 243.156 A. There are two protein molecules in the asymmetric unit and the Matthews coefficient is estimated to be 2.35 A3 Da(-1), corresponding to a solvent content of 47.60%.

Crystallization and preliminary X-ray analysis of pyruvate kinase from Bacillus stearothermophilus

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

Suzuki, Kenichiro; Ito, Sohei; Shimizu-Ibuka, Akiko

2005-08-01

This report describes the crystallization and X-ray diffraction data collection of three types (wild-type, W416F/V435W and C9S/C268S) of B. stearothermophilus. Crystals of C9S/C268S belonged to space group P6{sub 2}22 and diffracted to a resolution of 2.4 Å. Pyruvate kinase (PK) from a moderate thermophile, Bacillus stearothermophilus (BstPK), is an allosteric enzyme activated by AMP and ribose 5-phosphate but not by fructose 1,6-bisphosphate (FBP). However, almost all other PKs are activated by FBP. The wild-type and W416F/V435W mutant BstPKs were crystallized by the hanging-drop vapour-diffusion method. However, they were unsuitable for structural analysis because their data sets exhibited low completeness. Amore » crystal suitable for structural analysis was obtained using C9S/C268S enzyme. The crystal belonged to space group P6{sub 2}22, with unit-cell parameters a = b = 145.97, c = 118.03 Å.« less

Expression, purification and preliminary X-ray characterization of dl-2-haloacid dehalogenase from Methylobacterium sp. CPA1

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

Omi, Rie; Department of Chemistry, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585; Jitsumori, Keiji

A recombinant form of dl-2-haloacid dehalogenase from Methylobacterium sp. CPA1 has been expressed in E. coli, purified and crystallized. The crystal belongs to space group P6{sub 3}. Diffraction data have been collected to 1.75 Å resolution. dl-2-Haloacid dehalogenase from Methylobacterium sp. CPA1 (dl-DEX Mb) is a unique enzyme that catalyzes the dehalogenation reaction without the formation of an ester intermediate. A recombinant form of dl-DEX Mb has been expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the hexagonal space group P6{sub 3}, with unit-cell parameters a = b = 186.2, c =more » 114.4 Å. The crystals are likely to contain between four and eight monomers in the asymmetric unit, with a V{sub M} value of 4.20–2.10 Å{sup 3} Da{sup −1}. A self-rotation function revealed peaks on the χ = 180° section. X-ray data have been collected to 1.75 Å resolution.« less

Crystallization and preliminary X-ray analysis of a protease inhibitor from the latex of Carica papaya

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

Azarkan, Mohamed; Garcia-Pino, Abel; Dibiani, Rachid

2006-12-01

The Kunitz-type trypsin/chymotrypsin inhibitor isolated from C. papaya latex has been crystallized using the hanging-drop vapour-diffusion method. Two different crystal forms are observed, diffracting to 2.6 and 1.7 Å. A Kunitz-type protease inhibitor purified from the latex of green papaya (Carica papaya) fruits was crystallized in the presence and absence of divalent metal ions. Crystal form I, which is devoid of divalent cations, diffracts to a resolution of 2.6 Å and belongs to space group P3{sub 1} or P3{sub 2}. This crystal form is a merohedral twin with two molecules in the asymmetric unit and unit-cell parameters a = bmore » = 74.70, c = 78.97 Å. Crystal form II, which was grown in the presence of Co{sup 2+}, diffracts to a resolution of 1.7 Å and belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 44.26, b = 81.99, c = 140.89 Å.« less

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

Wang, Che-Yen; Karolinska Institute Structural Virology, F68 Karolinska University Hospital, SE-14186 Stockholm; Institute of Public Health, National Yang-Ming University, 112 Taipei,Taiwan

A recombinant virus-like particle that is a potential oral hepatitis E vaccine was crystallized. Diffraction data were collected to 8.3 Å resolution and the X-ray structure was phased with the aid of a low-resolution density map determined using cryo-electron microscopy data. Hepatitis E virus (HEV) accounts for the majority of enterically transmitted hepatitis infections worldwide. Currently, there is no specific treatment for or vaccine against HEV. The major structural protein is derived from open reading frame (ORF) 2 of the viral genome. A potential oral vaccine is provided by the virus-like particles formed by a protein construct of partial ORF3more » protein (residue 70–123) fused to the N-terminus of the ORF2 protein (residues 112–608). Single crystals obtained by the hanging-drop vapour-diffusion method at 293 K diffract X-rays to 8.3 Å resolution. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 337, b = 343, c = 346 Å, α = β = γ = 90°, and contain one particle per asymmetric unit.« less

Crystallization and preliminary X-ray diffraction analyses of the redox-controlled complex of terminal oxygenase and ferredoxin components in the Rieske nonhaem iron oxygenase carbazole 1,9a-dioxygenase

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

Matsuzawa, Jun; Aikawa, Hiroki; Umeda, Takashi

2014-09-25

A crystal was obtained of the complex between reduced terminal oxygenase and oxidized ferredoxin components of carbazole 1,9a-dioxygenase. The crystal belonged to space group P2{sub 1} and diffracted to 2.25 Å resolution. The initial reaction in bacterial carbazole degradation is catalyzed by carbazole 1,9a-dioxygenase, which consists of terminal oxygenase (Oxy), ferredoxin (Fd) and ferredoxin reductase components. The electron-transfer complex between reduced Oxy and oxidized Fd was crystallized at 293 K using the hanging-drop vapour-diffusion method with PEG 3350 as the precipitant under anaerobic conditions. The crystal diffracted to a maximum resolution of 2.25 Å and belonged to space group P2{submore » 1}, with unit-cell parameters a = 97.3, b = 81.6, c = 116.2 Å, α = γ = 90, β = 100.1°. The V{sub M} value is 2.85 Å{sup 3} Da{sup −1}, indicating a solvent content of 56.8%.« less

Crystallization and preliminary diffraction analysis of a DsbA homologue from Wolbachia pipientis

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

Kurz, M.; Iturbe-Ormaetxe, I.; Jarrott, R.

2008-02-01

The first crystallization of a W. pipientis protein, α-DsbA1, was achieved using hanging-drop and sitting-drop vapour diffusion. α-DsbA1 is one of two DsbA homologues encoded by the Gram-negative α-proteobacterium Wolbachia pipientis, an endosymbiont that can behave as a reproductive parasite in insects and as a mutualist in medically important filarial nematodes. The α-DsbA1 protein is thought to be important for the folding and secretion of Wolbachia proteins involved in the induction of reproductive distortions. Crystals of native and SeMet α-DsbA1 were grown by vapour diffusion and belong to the monoclinic space group C2, with unit-cell parameters a = 71.4, bmore » = 49.5, c = 69.3 Å, β = 107.0° and one molecule in the asymmetric unit (44% solvent content). X-ray data were recorded from native crystals to a resolution of 2.01 Å using a copper anode and data from SeMet α-DsbA1 crystals were recorded to 2.45 Å resolution using a chromium anode.« less

Outcomes of therapeutic hypothermia in unconscious patients after near-hanging.

PubMed

Lee, Byung Kook; Jeung, Kyung Woon; Lee, Hyoung Youn; Lim, Jae Hoon

2012-09-01

Hanging has been increasingly used to commit suicide. There is no specific treatment besides general intensive care after near-hanging. Therapeutic hypothermia (TH) has been used in unconscious patients after near-hanging. To describe the outcomes in unconscious patients after near-hanging in order to determine whether TH improves the outcome of near-hanging injury. Medical charts were reviewed of unconscious patients after near-hanging who presented to Chonnam National University Hospital between January 2006 and December 2010 and who were considered to be eligible for TH. According to local policy, unconscious survivors after near-hanging, whether or not they experienced cardiac arrest at the scene, were treated with TH if this was agreed by next-of-kin. There were 16 survivors of asphyxial cardiac arrest after near-hanging, of whom 13 received TH. Among them, only one (7.7%, 95% CI 1.4% to 33.3%) attained Cerebral Performance Category (CPC) 1; the other 15 patients had poor neurological outcomes (CPC 5 in seven patients and CPC 4 in eight patients). Nine of the patients did not experience cardiac arrest at the scene and of these, four received TH and five received normothermic treatment. All patients who did not have cardiac arrest recovered and were discharged with CPC 1. In this study, outcomes in unconscious near-hanging patients with cardiac arrest were poor despite treatment with TH. Before recommending TH in near-hanging patients, a prospective, randomised controlled study is required.

Hanging Out with Which Friends? Friendship-Level Predictors of Unstructured and Unsupervised Socializing in Adolescence

PubMed Central

Siennick, Sonja E.; Osgood, D. Wayne

2012-01-01

Companions are central to explanations of the risky nature of unstructured and unsupervised socializing, yet we know little about whom adolescents are with when hanging out. We examine predictors of how often friendship dyads hang out via multilevel analyses of longitudinal friendship-level data on over 5,000 middle schoolers. Adolescents hang out most with their most available friends and their most generally similar friends, not with their most at-risk or similarly at-risk friends. These findings vary little by gender and wave. Together, the findings suggest that the risks of hanging out stem from the nature of hanging out as an activity, not the nature of adolescents’ companions, and that hanging out is a context for friends’ mutual reinforcement of pre-existing characteristics. PMID:23204811

Hanging Out with Which Friends? Friendship-Level Predictors of Unstructured and Unsupervised Socializing in Adolescence.

PubMed

Siennick, Sonja E; Osgood, D Wayne

2012-12-01

Companions are central to explanations of the risky nature of unstructured and unsupervised socializing, yet we know little about whom adolescents are with when hanging out. We examine predictors of how often friendship dyads hang out via multilevel analyses of longitudinal friendship-level data on over 5,000 middle schoolers. Adolescents hang out most with their most available friends and their most generally similar friends, not with their most at-risk or similarly at-risk friends. These findings vary little by gender and wave. Together, the findings suggest that the risks of hanging out stem from the nature of hanging out as an activity, not the nature of adolescents' companions, and that hanging out is a context for friends' mutual reinforcement of pre-existing characteristics.

Accidental hanging by a T-shirt collar in a man with morphine intoxication: an unusual case.

PubMed

Kodikara, Sarathchandra; Alagiyawanna, Ramesh

2011-09-01

Accidental hanging is rare across all age groups, and it is even rarer in the adult population except in autoerotic asphyxia. Few cases have been reported in the literature, which describe unusual patterns of accidental hanging. This article focuses on an unusual pattern of accidental hanging of a 25-year-old man, who was in a state of morphine-induced central nervous system depression and found dead in a sitting position with the collar of his T-shirt hanging off a jutting-out root of a tree. The hanged collar acted as a ligature compressing the neck.

Retrospective analysis of 319 hanging and strangulation cases between 2001 and 2014 in Shanghai.

PubMed

Ma, Jianlong; Jing, Haojia; Zeng, Yan; Tao, Li; Yang, Yulei; Ma, Kaijun; Chen, Long

2016-08-01

In this study, we retrospectively analyzed 141 cases of hanging and 178 cases of ligature strangulation recorded in the Shanghai Municipal Public Security Bureau between January 2001 and December 2014 to explore the characteristics of hanging and ligature strangulation and to supply a scientific reference for forensic pathology. Several significant differences between hanging and ligature strangulation were found. Hanging cases were mostly suicide, with some accidental cases. Strangulation cases were mostly homicide, with a few cases of suicide or sexual asphyxia. Male hanging was more common than female hanging, with a ratio of 5:2. However, there were more female than male strangulation cases, with a ratio of 13:5. The ligature marks in hanging cases were almost all of a "U" type and above the hyoid bone. The ligature marks in strangulation cases were almost always a closed circle, but the position varied. The most common vital reactions were subcutaneous hemorrhage, exfoliation and blister, which are strong evidence of antemortem injury. Hemorrhagic spots were found on the temporalis, scalp, chest and back in strangulation cases, but were rare in hanging cases. Hemorrhagic manifestations were most common in the sternocleidomastoid muscle in hanging cases, and in the sternohyoid and sternothyroid muscles in strangulation cases. Fractures occurred in only ∼17% of victims. There are notable differences between hanging and ligature strangulation, which can help distinguish between these causes of death. These characteristics should be considered in forensic practice. Copyright © 2016 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Crystallization and preliminary X-ray analysis of native and selenomethionyl vinorine synthase from Rauvolfia serpentina.

PubMed

Ma, Xueyan; Koepke, Juergen; Bayer, Anja; Fritzsch, Günter; Michel, Hartmut; Stöckigt, Joachim

2005-06-01

Vinorine synthase (VS) is a central enzyme of the biosynthesis of the antiarrhythmic drug ajmaline and is a member of the BAHD superfamily of acyltransferases. So far, no three-dimensional structure with significant sequence homology with VS is known. Crystals of VS and selenomethionyl-labelled VS from the medicinal plant Rauvolfia serpentina have been obtained by the hanging-drop technique at 305 K with ammonium sulfate and PEG 400 as precipitants. VS crystals diffract to 2.8 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 82.3, b = 89.6, c = 136.2 A. The selenomethionyl VS crystal was nearly isomorphous with the VS crystal.

Overexpression, purification, crystallization and preliminary X-ray studies of Vibrio cholerae EpsG

PubMed Central

Jens, Jason; Raghunathan, Kannan; Vago, Frank; Arvidson, Dennis

2009-01-01

EpsG is the major pseudopilin protein of the Vibrio cholerae type II secretion system. An expression plasmid that encodes an N-terminally truncated form of EpsG with a C-terminal noncleavable His tag was constructed. Recombinant EpsG was expressed in Escherichia coli; the truncated protein was purified and crystallized by hanging-drop vapor diffusion against a reservoir containing 6 mM zinc sulfate, 60 mM MES pH 6.5, 15% PEG MME 550. The crystals diffracted X-rays to a resolution of 2.26 Å and belonged to space group P21, with unit-cell parameters a = 88.61, b = 70.02, c = 131.54 Å. PMID:19478449

Complex suicide with black powder muzzle loading derringer.

PubMed

Hejna, Petr; Šafr, Miroslav; Zátopková, Lenka; Straka, Luboš

2012-09-01

Planned complex suicide is defined as the combination of more than one method of suicide, previously planned by the victim, to prevent failure of the first method. Herein, we present a case of planned complex suicide, committed by a black powder muzzle loading handgun and hanging. A 39-year-old man was found dead in the bathroom of his flat, hanging by the neck with a huge atypical gunshot entrance in the right temporal region of his head with extensive backspatter. The skin defects, as well as soft tissues in the subcutaneous pocket undermining, were heavily burnt. Along the wound canal were multiple bone fragments, and at the end of the path at the left temple was an embedded lead ogival projectile with a cross shaped artificial incision at its tip. The hanging was incomplete. There were no fractures of the hyoid bone and laryngeal cartilages. Cervical muscles and vessels were intact. Simon's sign was negative. Signs of asphyxia were not present. This is the first reported case of complex suicide with a black powder derringer and manipulated projectile.

Celebrity suicides and their differential influence on suicides in the general population: a national population-based study in Korea.

PubMed

Myung, Woojae; Won, Hong-Hee; Fava, Maurizio; Mischoulon, David; Yeung, Albert; Lee, Dongsoo; Kim, Doh Kwan; Jeon, Hong Jin

2015-04-01

Although evidence suggests that there is an increase in suicide rates in the general population following celebrity suicide, the rates are heterogeneous across celebrities and countries. It is unclear which is the more vulnerable population according to the effect sizes of celebrity suicides to general population. All suicide victims in the general population verified by the Korea National Statistical Office and suicides of celebrity in South Korea were included for 7 years from 2005 to 2011. Effect sizes were estimated by comparing rates of suicide in the population one month before and after each celebrity suicide. The associations between suicide victims and celebrities were examined. Among 94,845 suicide victims, 17,209 completed suicide within one month after 13 celebrity suicides. Multivariate logistic regression analyses revealed that suicide victims who died after celebrity suicide were significantly likely to be of age 20-39, female, and to die by hanging. These qualities were more strongly associated among those who followed celebrity suicide with intermediate and high effect sizes than lower. Younger suicide victims were significantly associated with higher effect size, female gender, white collar employment, unmarried status, higher education, death by hanging, and night-time death. Characteristics of celebrities were significantly associated with those of general population in hanging method and gender. Individuals who commit suicide after a celebrity suicide are likely to be younger, female, and prefer hanging as method of suicide, which are more strongly associated in higher effect sizes of celebrity suicide.

Role of Marangoni stress during breakup of surfactant-covered liquid threads: Reduced rates of thinning and microthread cascades

NASA Astrophysics Data System (ADS)

Kamat, Pritish M.; Wagoner, Brayden W.; Thete, Sumeet S.; Basaran, Osman A.

2018-04-01

Adsorption onto and lowering of surface tension σ of fluid interfaces by surfactants is exploited in drop formation (e.g., inkjet printing) where a thinning liquid thread (radius h ) connects an about-to-form drop to the liquid that remains hanging from the nozzle when the former falls from it. Surfactants can affect thread pinch-off in two ways: first, by lowering σ , they lower capillary pressure (σ /h ), and second, as surfactant concentration along the interface can be nonuniform, they cause the interface to be subjected to a surface tension gradient or Marangoni stress. Recent studies show that the location where the thread breaks is devoid of surfactant, and others assert that the influence of Marangoni stress on pinch-off is negligible. We demonstrate by simulations and experiments that surfactants play a major role in drop formation and that Marangoni stresses acting near but not at the pinch point give rise to reduced rates of thread thinning and formation of multiple microthreads that distinguish pinch-off of surfactant-covered threads from surfactant-free ones. Thinning at finite Reynolds and Peclet numbers, Re and Pe, is shown to exhibit intermediate scaling regimes that have heretofore only been observed during pinch-off of threads undergoing creeping flow (Re=0 ) while convection of surfactant is weak compared to its diffusion (Pe<1 ).

Structural inheritance and selective reactivation in the central Andes: Cenozoic deformation guided by pre-Andean structures in southern Peru

NASA Astrophysics Data System (ADS)

Perez, Nicholas D.; Horton, Brian K.; Carlotto, Victor

2016-03-01

Structural, stratigraphic, and geochronologic constraints from the Eastern Cordillera in the central Andean plateau of southern Peru (14-15°S) demonstrate the existence and position of major pre-Andean structures that controlled the accumulation of Triassic synrift fill and guided subsequent Cenozoic deformation. The timing of initial clastic deposition of the Triassic Mitu Group is here constrained to ~ 242-233 Ma on the basis of detrital and volcanic zircon U-Pb geochronology. Regionally distinct provenance variations, as provided by U-Pb age populations from localized synrift accumulations, demonstrate Triassic erosion of multiple diagnostic sources from diverse rift-flank uplifts. Stratigraphic correlations suggest synchronous initiation of extensional basins containing the Mitu Group, in contrast with previous interpretations of southward rift propagation. Triassic motion along the NE-dipping San Anton normal fault accommodated up to 7 km of throw and hanging-wall deposition of a synrift Mitu succession > 2.5 km thick. The contrasting orientation of a non-reactivated Triassic normal fault suggests selective inversion of individual structures in the Eastern Cordillera was dependent on fault dip and strike. Selective preservation of a ~ 4 km thick succession of Carboniferous-Permian strata in the down-dropped San Anton hanging wall, beneath the synrift Mitu Group, suggests large-scale erosional removal in the uplifted footwall. Field and map observations identify additional pre-Andean thrust faults and folds attributed to poorly understood Paleozoic orogenic events preserved in the San Anton hanging wall. Selective thrust reactivation of normal and reverse faults during later compression largely guided Cenozoic deformation in the Eastern Cordillera. The resulting structural compartmentalization and across-strike variations in kinematics and deformation style highlight the influence of inherited Paleozoic structures and Triassic normal faults on the long-term history of convergent margin deformation in the Andes.

Splay fault slip in a subduction margin, a new model of evolution

NASA Astrophysics Data System (ADS)

Conin, Marianne; Henry, Pierre; Godard, Vincent; Bourlange, Sylvain

2012-08-01

In subduction zones, major thrusts called splay faults are thought to slip coseismically during large earthquakes affecting the main plate interface. We propose an analytical condition for the activation of a splay fault based on force balance calculations and suggest thrusting along the splay fault is generally conditioned by the growth of the accretionary wedge, or by the erosion of the hanging wall. In theory, normal slip on the splay fault may occur when the décollement has a very low friction coefficient seaward. Such a low friction also implies an unstable extensional state within the outer wedge. Finite element elasto-plastic calculations with a geometry based on the Nankai Kumano section were performed and confirm that this analytical condition is a valid approximation. Furthermore, localized extension at a shallow level in the splay hanging wall is observed in models for a wide range of friction coefficients (from ∼0 to the value of internal friction coefficient of the rock, here equals to 0.4). The timing of slip established for the splay fault branch drilled on Nankai Kumano transect suggests a phase of concurrent splay and accretionary wedge growth ≈2 Ma to ≈1.5 Ma, followed by a locking of the splay ≈1.3 Ma. Active extension is observed in the hanging wall. This evolution can be explained by the activation of a deeper and weaker décollement, followed by an interruption of accretion. Activation of a splay as a normal fault, as hypothesized in the case of the Tohoku 2011 earthquake, can be achieved only if the friction coefficient on the décollement drops to near zero. We conclude that the tectonic stress state largely determines long-term variations of tightly related splay fault and outer décollement activity and thus influences where and how coseismic rupture ends, but that occurrence of normal slip on a splay fault requires coseismic friction reduction.

SU-F-T-75: Dosimetry Considerations in the Use of Hanging-Eye Block for Lesions of the Conjunctiva

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

Grelewicz, Z; Lee, B; Cutright, D

Purpose: Superficial lesions in the conjunctiva are frequently treated with en face electrons, using a hanging block to spare dose to the lens of the eye. Dose to the tumor and lens depend on the design and setup of the block and supporting apparatus. We performed in phantom measurements in order to characterize the dose sparing effects of the block as well as the under-dosing effect under the supporting apparatus for 6 MeV treatment. Methods: The commercial hanging block studied uses a 1.2 cm diameter tungsten cylinder supported by a 3 mm diameter acrylic rod. Point dose measurements under themore » hanging block, under an unblocked part of the field, and under the acrylic rod were performed using MOSFET detectors. In addition, EBT3 film was used for both PDD and profile measurements at a depth in phantom of 3 mm for both 105 and 103 cm SSD. Results: MOSFET measurements reported a dose reduction of 95% under the tungsten block when using an SSD of 103 cm, and 86% when using an SSD of 105 cm at a depth in phantom of 3 mm. Film measurements showed that the area under the acrylic rod may be under-dosed by as much as 30% when using 103 SSD. MOSFET measurements confirmed that when using an SSD of 103 cm, the area under the acrylic rod is under-dosed by up to 30% at 3mm depth, compared to the unblocked part of the field. Conclusion: The effectiveness of the commercial hanging block apparatus depends on setup, with 95% lens sparing possible with an SSD of 103 cm. This short SSD is necessary for sharp penumbra. At this SSD, substantial under-dosing under the acrylic support rod is possible. This must be mitigated with either feathering, or using an alternative method of support for the tungsten block.« less

Measuring mechanodynamics in an unsupported epithelial monolayer grown at an air–water interface

PubMed Central

Gullekson, Corinne; Walker, Matthew; Harden, James L.; Pelling, Andrew E.

2017-01-01

Actomyosin contraction and relaxation in a monolayer is a fundamental biophysical process in development and homeostasis. Current methods used to characterize the mechanodynamics of monolayers often involve cells grown on solid supports such as glass or gels. The results of these studies are fundamentally influenced by these supporting structures. Here we describe a new method for measuring the mechanodynamics of epithelial monolayers by culturing cells at an air–liquid interface. These model monolayers are grown in the absence of any supporting structures, removing cell–substrate effects. This method’s potential was evaluated by observing and quantifying the generation and release of internal stresses upon actomyosin contraction (800 ± 100 Pa) and relaxation (600 ± 100 Pa) in response to chemical treatments. Although unsupported monolayers exhibited clear major and minor strain axes, they were not correlated with nuclear alignment as observed when the monolayers were grown on soft deformable gels. It was also observed that both gels and glass substrates led to the promotion of long-range cell nuclei alignment not seen in the hanging-drop model. This new approach provides us with a picture of basal actomyosin mechanodynamics in a simplified system, allowing us to infer how the presence of a substrate affects contractility and long-range multicellular organization and dynamics. PMID:28035043

Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module from family 64 (StX).

PubMed

Campos, Bruna Medeia; Liberato, Marcelo Vizona; Polikarpov, Igor; Zeri, Ana Carolina de Mattos; Squina, Fabio Marcio

2015-03-01

In recent years, biofuels have attracted great interest as a source of renewable energy owing to the growing global demand for energy, the dependence on fossil fuels, limited natural resources and environmental pollution. However, the cost-effective production of biofuels from plant biomass is still a challenge. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases to polysaccharides, is crucial for enzyme development. Aiming at the structural and functional characterization of novel CBMs involved in plant polysaccharide deconstruction, an analysis of the CAZy database was performed and CBM family 64 was chosen owing to its capacity to bind with high specificity to microcrystalline cellulose and to the fact that is found in thermophilic microorganisms. In this communication, the CBM-encoding module named StX was expressed, purified and crystallized, and X-ray diffraction data were collected from native and derivatized crystals to 1.8 and 2.0 Å resolution, respectively. The crystals, which were obtained by the hanging-drop vapour-diffusion method, belonged to space group P3121, with unit-cell parameters a = b = 43.42, c = 100.96 Å for the native form. The phases were found using the single-wavelength anomalous diffraction method.

Purification, crystallization and preliminary crystallographic study of an IDS-epimerase from Agrobacterium tumefaciens BY6

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

Bäuerle, Bettina; Sandalova, Tatyana; Schneider, Gunter

2006-08-01

This is the first report of the crystallization of an IDS-epimerase from A. tumefaciens BY6 and its l-selenomethionine derivative. The initial degradation of all stereoisomers of the complexing agent iminodisuccinate (IDS) is enabled by an epimerase in the bacterial strain Agrobacterium tumefaciens BY6. This protein was produced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method. Crystals of IDS-epimerase were obtained under several conditions. The best diffracting crystals were grown in 22% PEG 3350, 0.2 M (NH{sub 4}){sub 2}SO{sub 4} and 0.1 M bis-Tris propane pH 7.2 at 293 K. These crystals belong to the monoclinic space groupmore » P2{sub 1}, with unit-cell parameters a = 55.4, b = 104.2, c = 78.6 Å, β = 103.3°, and diffracted to 1.7 Å resolution. They contain two protein molecules per asymmetric unit. In order to solve the structure using the MAD phasing method, crystals of the l-selenomethionine-substituted epimerase were grown in the presence of 20% PEG 3350, 0.2 M Na{sub 2}SO{sub 4} and 0.1 M bis-Tris propane pH 8.5.« less

Using T-Z plots as a graphical method to infer lithological variations from growth strata

NASA Astrophysics Data System (ADS)

Castelltort, Sébastien; Pochat, Stéphane; Van Den Driessche, Jean

2004-08-01

The 'T-Z plot' method consists of plotting the throw of sedimentary horizons across a growth fault versus their depth in the hanging wall. This method has been initially developed for the analysis of growth fault kinematics from seismic data. A brief analytical examination of such plots shows that they can also provide valuable information about the evolution of fault topography. When growth is a continuous process, stages of topography creation (fault scarp) and filling (of the space available in the hanging-wall) are related to non-dynamic (draping, mud-prone pelagic settling) and dynamic (sand-prone, dynamically deposited) sedimentation, respectively. In this case, the T-Z plot analysis becomes a powerful tool to predict major lithological variations on seismic profiles in faulted settings.

Ingestion of safety razor blade and delayed hanging in a complex suicide.

PubMed

Chauhan, Mohit Singh; Behera, C; Naagar, Sunil; Sreenivas, M

2016-12-01

Ingestion of a foreign body is mostly accidental in children and intentional in prisoners to achieve hospitalization; however, use of this method of suicide is rare. We report a case where the victim first ingested a safety razor blade, but failed to die and then hanged himself, but failed again and finally succumbed to the complications on the sixth day. He had also attempted suicide by inflicting multiple incised wounds on his neck four days before the safety blade ingestion, but none were fatal. © The Author(s) 2016.

Overexpression, purification, crystallization and preliminary structural studies of p-coumaric acid decarboxylase from Lactobacillus plantarum

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

Rodríguez, Héctor; Rivas, Blanca de las; Muñoz, Rosario

2007-04-01

The enzyme p-coumaric acid decarboxylase (PDC) from L. plantarum has been recombinantly expressed, purified and crystallized. The structure has been solved at 2.04 Å resolution by the molecular-replacement method. The substrate-inducible p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum has been overexpressed in Escherichia coli, purified and confirmed to possess decarboxylase activity. The recombinant His{sub 6}-tagged enzyme was crystallized using the hanging-drop vapour-diffusion method from a solution containing 20%(w/v) PEG 4000, 12%(w/v) 2-propanol, 0.2 M sodium acetate, 0.1 M Tris–HCl pH 8.0 with 0.1 M barium chloride as an additive. Diffraction data were collected in-house to 2.04 Å resolution. Crystals belongedmore » to the tetragonal space group P4{sub 3}, with unit-cell parameters a = b = 43.15, c = 231.86 Å. The estimated Matthews coefficient was 2.36 Å{sup 3} Da{sup −1}, corresponding to 48% solvent content, which is consistent with the presence of two protein molecules in the asymmetric unit. The structure of PDC has been determined by the molecular-replacement method. Currently, the structure of PDC complexed with substrate analogues is in progress, with the aim of elucidating the structural basis of the catalytic mechanism.« less

Exposure assessment of ETBE in gas station workers and gasoline tanker truck drivers.

PubMed

Eitaki, Yoko; Kawai, Toshio; Omae, Kazuyuki

2011-01-01

In order to measure occupational exposure concentrations of ethyl tertiary-butyl ether (ETBE), we developed a diffusive sampling method for monitoring ETBE and performed an ETBE exposure assessment. The applicability of diffusive samplers was examined by exposing the samplers to ETBE vapor in test chambers. The personal exposure levels of workers and airborne concentrations were measured at 4 gas stations. The ETBE sampling rate for the diffusive samplers (VOC-SD, Sigma-Aldrich Japan) was 25.04 ml/min (25°C). Compared with the active sampling method, the diffusive samplers could be used for short-term measurements and in environments containing a mixture of organic solvents. The geometric mean (GM) of TWA-8h ETBE was 0.08 ppm (0.02-0.28 ppm) in 28 gas station workers and 0.04 ppm (0.01-0.21 ppm) in 2 gasoline tanker truck drivers. With regard to ETBE airborne concentrations, the GM was 4.12 ppm (0.93-8.71 ppm) at the handles of hanging pumps but dropped to less than 0.01 ppm (less than 0.01-0.01 ppm) at the side of a public road. The diffusive sampling method can be used for the measurement of occupational ETBE exposure. The threshold limit of TLV-TWA 5 ppm recommended by the ACGIH was not exceeded in any of the workers in this study.

[Phenotype-based primary screening for drugs promoting neuronal subtype differentiation in embryonic stem cells with light microscope].

PubMed

Gao, Yi-ning; Wang, Dan-ying; Pan, Zong-fu; Mei, Yu-qin; Wang, Zhi-qiang; Zhu, Dan-yan; Lou, Yi-jia

2012-07-01

To set up a platform for phenotype-based primary screening of drug candidates promoting neuronal subtype differentiation in embryonic stem cells (ES) with light microscope. Hanging drop culture 4-/4+ method was employed to harvest the cells around embryoid body (EB) at differentiation endpoint. Morphological evaluation for neuron-like cells was performed with light microscope. Axons for more than three times of the length of the cell body were considered as neuron-like cells. The compound(s) that promote neuron-like cells was further evaluated. Icariin (ICA, 10(-6)mol/L) and Isobavachin (IBA, 10(-7)mol/L) were selected to screen the differentiation-promoting activity on ES cells. Immunofluorescence staining with specific antibodies (ChAT, GABA) was used to evaluate the neuron subtypes. The cells treated with IBA showed neuron-like phenotype, but the cells treated with ICA did not exhibit the morphological changes. ES cells treated with IBA was further confirmed to be cholinergic and GABAergic neurons. Phenotypic screening with light microscope for molecules promoting neuronal differentiation is an effective method with advantages of less labor and material consuming and time saving, and false-positive results derived from immunofluorescence can be avoided. The method confirms that IBA is able to facilitate ES cells differentiating into neuronal cells, including cholinergic neurons and GABAergic neurons.

Hanging out with Which Friends? Friendship-Level Predictors of Unstructured and Unsupervised Socializing in Adolescence

ERIC Educational Resources Information Center

Siennick, Sonja E.; Osgood, D. Wayne

2012-01-01

Companions are central to explanations of the risky nature of unstructured and unsupervised socializing, yet we know little about whom adolescents are with when hanging out. We examine predictors of how often friendship dyads hang out via multilevel analyses of longitudinal friendship-level data on over 5,000 middle schoolers. Adolescents hang out…

Hands Tied with Bag Full of Books in Suicidal Hanging.

PubMed

Sikary, Asit Kumar; Behera, Chittaranjan; Murty, Om Prakash; Rautji, Ravi

2016-01-01

Hanging deaths associated with binding of limbs, masking of a face, and gagging are always suspicious. In suicidal hanging, the victim uses these added techniques to prevent him from backing out of his decision and to ensure death. However, binding of limbs and adding extra weight to the suspension in hanging are not reported. Herein, we report a case where the victim tied a bag containing books weighing 7 kg (15.4 lbs) to both his hands during hanging. The forensic specialist must be aware of the unusual presentation of suicidal hanging which may suggest foul play. The manner of death must be established after detailed analysis of circumstantial evidence, information obtained from the witnesses, complete autopsy, and toxicological examination. © 2015 American Academy of Forensic Sciences.

Vinorine synthase from Rauvolfia: the first example of crystallization and preliminary X-ray diffraction analysis of an enzyme of the BAHD superfamily.

PubMed

Ma, Xueyan; Koepke, Juergen; Bayer, Anja; Linhard, Verena; Fritzsch, Günter; Zhang, Bin; Michel, Hartmut; Stöckigt, Joachim

2004-09-01

Crystals of vinorine synthase (VS) from medicinal plant Rauvolfia serpentina expressed in Escherichia coli have been obtained by the hanging-drop technique at 305 K with ammonium sulfate and PEG 400 as precipitants. The enzyme is involved in the biosynthesis of the antiarrhythmic drug ajmaline and is a member of the BAHD superfamily of acyltransferases. So far, no three-dimensional structure of a member of this enzyme family is known. The crystals belong to the space group P2(1)2(1)2(1) with cell dimensions of a=82.3 A, b=89.6 A and c=136.2 A. Under cryoconditions (120 K), a complete data set up to 2.8 A was collected at a synchrotron source.

Crystallization and preliminary X-ray analysis of gamma-glutamyltranspeptidase from Escherichia coli K-12.

PubMed

Kumagai, H; Nohara, S; Suzuki, H; Hashimoto, W; Yamamoto, K; Sakai, H; Sakabe, K; Fukuyama, K; Sakabe, N

1993-12-20

gamma-Glutamyltranspeptidase (EC 2.3.2.2) from Escherichia coli K-12 has been purified and crystallized by means of vapor diffusion in hanging drops. Two kinds of crystals on cell dimensions were found for X-ray diffraction analysis, one from ammonium sulfate and the other from polyethylene glycol 6000 as precipitants. The crystals of the orthorhombic form grown in the presence of 15% polyethylene glycol and 20 mM sodium acetate buffer were chosen for further analysis. The crystals belonged to space group P2(1)2(1)2(1), with cell dimensions of a = 128.1, b = 129.9 and c = 79.2 A, and two molecules constitute an asymmetric unit. These crystals diffracted to 2.0 A resolution and were suitable for X-ray crystallographic studies.

Suicidal hanging within an automobile.

PubMed

Blanco Pampin, J M; López-Abajo Rodriguez, B A

2001-12-01

Accidental asphyxia related to cars has been described in different reports, but suicidal hanging in an automobile is very unusual. Two cases of suicidal hanging inside an automobile are described, illustrating an unusual form of hanging. In one case, the deceased used his belt as a ligature, and the point of attachment was the window of the car. The second victim used the safety belt of the passenger seat. In both cases, the automobile engine was turned off, all the windows were closed, and the door locks were blocked. The medicolegal cause of hanging was based on the scene of the investigation, police and witness reports, social history, autopsy findings, and toxicologic examinations.

Acute pulmonary emphysema in death by hanging: a morphometric digital study.

PubMed

Castiglioni, Claudia; Baumann, Pia; Fracasso, Tony

2016-09-01

Acute pulmonary emphysema (APE) has been described in cases of mechanical asphyxia such as ligature or manual strangulation but not in cases of hanging. In this study, we wanted to verify by morphometric digital analysis of lung tissue whether APE occurs in death by hanging.We investigated 16 cases of hanging (eight complete, eight incomplete), 10 cases of freshwater drowning (positive control group), and 10 cases of acute external bleeding (negative control group). Tissue sections were obtained from each pulmonary lobe. For each slide, five fields were randomly selected. The area of every alveolar space was measured by image analysis software. The mean alveolar area (MAA) was calculated for each group.In incomplete hanging, MAA was significantly higher than that observed in complete hanging and similar to the one observed in freshwater drowning.APE in cases of incomplete hanging can be considered as a sign of vitality. The high number of conditions that can cause alveolar distension (that were excluded in this study) limits the applicability of this vital sign in the routine forensic practice.

Agonal sequences in four filmed hangings: analysis of respiratory and movement responses to asphyxia by hanging.

PubMed

Sauvageau, Anny

2009-01-01

The human pathophysiology of asphyxia by hanging is still poorly understood, despite great advances in forensic science. In that context, filmed hangings may hold the key to answer questions regarding the sequence of events leading to death in human asphyxia. Four filmed hangings were analyzed. Rapid loss of consciousness was observed between 13 sec and 18 sec after onset of hanging, closely followed by convulsions (at 14-19 sec). A complex pattern of decerebration rigidity (19-21 sec in most cases), followed by a quick phase of decortication rigidity (1 min 00 sec-1 min 08 sec in most cases), an extended phase of decortication rigidity (1 min 04 sec-1 min 32 sec) and loss of muscle tone (1 min 38 sec-2 min 47 sec) was revealed. Very deep respiratory attempts started between 20 and 22 sec, the last respiratory attempt being detected between 2 min 00 sec and 2 min 04 sec. Despite differences in the types of hanging, this unique study reveals similarities that are further discussed.

Agonal sequences in a filmed suicidal hanging: analysis of respiratory and movement responses to asphyxia by hanging.

PubMed

Sauvageau, Anny; Racette, Stéphanie

2007-07-01

The forensic literature on the pathophysiology of human hanging is still limited. Therefore, forensic pathologists often feel uncomfortable when confronted with related questions. Here presented is the filmed suicidal hanging of a 37-year-old man. This recording allows a unique analysis of agonal movement sequences: loss of consciousness (13 sec), convulsions (15 sec), decortication rigidity (21 sec), decerebration rigidity (46 sec), second decortication rigidity (1 min 11 sec), loss of muscle tone, (1 min 38 sec) and last isolated muscle movement (4 min 10 sec). As for respiratory responses, very deep respiratory attempts started at 20 sec. Respiratory movements progressively decreased and completely stopped at 2 min. Despite the fact that extending the presented data on all cases of hanging asphyxia would be a mistake, this case gives a very interesting insight into movement and respiratory response to asphyxia by hanging.

Animations, games, and virtual reality for the Jing-Hang Grand Canal.

PubMed

Chen, Wenzhi; Zhang, Mingmin; Pan, Zhigeng; Liu, Gengdai; Shen, Huaqing; Chen, Shengnan; Liu, Yong

2010-01-01

Digital heritage, an effective method to preserve and present natural and cultural heritage, is engaging many heritage preservation specialists and computer scientists. In particular, computer graphics researchers have become involved, and digital heritage has employed many CG techniques. For example, Daniel Pletinckx and his colleagues employed VR in a real museum at Ename, Belgium, and Zhigeng Pan and his colleagues applied it to construct a virtual Olympics museum. Soo-Chang Pei and his colleagues focused on restoring ancient Chinese paintings. Here, we describe how we've applied animations, computer games, and VR to China's famous Jing-Hang Grand Canal.

Purification, crystallization and preliminary crystallographic study of haemoglobin from camel (Camelus dromedarius): a high oxygen-affinity lowland species.

PubMed

Balasubramanian, M; Moorthy, Pon Sathya; Neelagandan, K; Ponnuswamy, M N

2009-08-01

Haemoglobin is a prototypical allosteric protein that is mainly involved in the transportation of oxygen from the lungs to tissues and of carbon dioxide back to the lungs in an intrinsically coordinated manner to maintain the viability of cells. Haemoglobin from Camelus dromedarius provides an interesting case study of adaptation to life in deserts at extremely high temperatures. An ambition to unravel the integrated structural and functional aspects of the casual survival of this animal at high temperatures led us to specifically work on this problem. The present work reports the preliminary crystallographic study of camel haemoglobin. Camel blood was collected and the haemoglobin was purified by anion-exchange chromatography and crystallized using the hanging-drop vapour-diffusion method under buffered high salt concentration using PEG 3350 as a precipitant. Intensity data were collected using a MAR 345 dtb image-plate detector system. Camel haemoglobin crystallized in the monoclinic space group P2(1), with one whole biological molecule (alpha(2)beta(2)) in the asymmetric unit and unit-cell parameters a = 52.759, b = 116.782, c = 52.807 A, beta = 120.07 degrees .

Purification, crystallization and preliminary X-ray diffraction studies on goat (Capra hircus) hemoglobin - a low oxygen affinity species.

PubMed

Moorthy, Ponnuraj Sathya; Neelagandan, Kamariah; Balasubramanian, Moovarkumudalvan; Ponnuswamy, Mondikalipudur Nanjappa Gounder

2009-01-01

Hemoglobin is a vital protein present in almost all higher species. It is a transport protein involved in carrying oxygen from lungs to tissues and carbon dioxide back to lungs by an intrinsically coordinated manner. Even though a good amount of work has been carried out in this direction there exists scarcity of structural insight on low oxygen affinity species. Attempts are being made to unravel the structural insight of this low oxygen affinity species. Goat blood plasma was collected, treated with EDTA to avoid blood clotting and purification was accomplished using DEAE-anion chromatographic column. The goat hemoglobin was crystallized using 50mM of phosphate buffer at pH 6.7 with 1M NaCl and PEG 3350 as precipitant by hanging drop vapor diffusion method. Crystals obtained are screened and suitable crystals are taken for data collection using mar345dtb as image plate detector system. Goat hemoglobin crystal diffracted up to 2.61 A resolution. Goat hemoglobin crystallizes in orthorhombic space group P212(1)2(1) as a whole biological molecule in the asymmetric unit with cell dimensions a=53.568A, b=67.365A, c=154.183A.

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

Zhou, Dongwen; Sun, Jianping; Zhao, Wei

The CRD domain of GRP from H. sapiens has been expressed, purified and crystallized and X-ray diffraction data have been collected to a resolution of 2.0 Å. Galectins are a family of animal lectins which share similar carbohydrate-recognition domains (CRDs) and an affinity for β-galactosides. A novel human galectin-related protein named GRP (galectin-related protein; previously known as HSPC159) comprises only one conserved CRD with 38 additional N-terminal residues. The C-terminal fragment of human GRP (GRP-C; residues 38–172) containing the CRD has been expressed and purified. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 2% PEGmore » 400 and 2M ammonium sulfate in 100 mM Tris–HCl buffer pH 7.5. Diffraction data were collected to a resolution limit of 2.0 Å at beamline 3W1A of Beijing Synchrotron Radiation Facility at 100 K. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 123.07, b = 96.67, c = 61.56 Å, β = 118.72°. The estimated Matthews coefficient was 2.6 Å{sup 3} Da{sup −1}, corresponding to 51.8% solvent content.« less

A 3D magnetic tissue stretcher for remote mechanical control of embryonic stem cell differentiation.

PubMed

Du, Vicard; Luciani, Nathalie; Richard, Sophie; Mary, Gaëtan; Gay, Cyprien; Mazuel, François; Reffay, Myriam; Menasché, Philippe; Agbulut, Onnik; Wilhelm, Claire

2017-09-12

The ability to create a 3D tissue structure from individual cells and then to stimulate it at will is a major goal for both the biophysics and regenerative medicine communities. Here we show an integrated set of magnetic techniques that meet this challenge using embryonic stem cells (ESCs). We assessed the impact of magnetic nanoparticles internalization on ESCs viability, proliferation, pluripotency and differentiation profiles. We developed magnetic attractors capable of aggregating the cells remotely into a 3D embryoid body. This magnetic approach to embryoid body formation has no discernible impact on ESC differentiation pathways, as compared to the hanging drop method. It is also the base of the final magnetic device, composed of opposing magnetic attractors in order to form embryoid bodies in situ, then stretch them, and mechanically stimulate them at will. These stretched and cyclic purely mechanical stimulations were sufficient to drive ESCs differentiation towards the mesodermal cardiac pathway.The development of embryoid bodies that are responsive to external stimuli is of great interest in tissue engineering. Here, the authors culture embryonic stem cells with magnetic nanoparticles and show that the presence of magnetic fields could affect their aggregation and differentiation.

Differentiating Mouse Embryonic Stem Cells into Embryoid Bodies by Hanging-Drop Cultures.

PubMed

Behringer, Richard; Gertsenstein, Marina; Nagy, Kristina Vintersten; Nagy, Andras

2016-12-01

Embryonic stem (ES) cells can develop into many types of differentiated tissues if they are placed into a differentiating environment. This can occur in vivo when the ES cells are injected into or aggregated with an embryo, or in vitro if their culture conditions are modified to induce differentiation. There are an increasing number of differentiating culture conditions that can bias the differentiation of ES cells into desired cell types. Determining the mechanisms that control ES cell differentiation into therapeutically important cell types is a quickly growing area of research. Knowledge gained from these studies may eventually lead to the use of stem cells to repair specific damaged tissues. Many times ES cell differentiation proceeds through an intermediate stage called the embryoid body (EB). EBs are round structures composed of ES cells that have undergone some of the initial stages of differentiation. EBs can then be manipulated further to generate more specific cell types. This protocol describes a method to differentiate ES cells into EBs. It produces EBs of comparable size. This aspect is important because the differentiation processes taking place inside an EB are influenced by its size. © 2016 Cold Spring Harbor Laboratory Press.

Crystallization and preliminary X-ray diffraction analysis of hemextin A: a unique anticoagulant protein from Hemachatus haemachatus venom

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

Banerjee, Yajnavalka; Kumar, Sundramurthy; Jobichen, Chacko

2007-08-01

Crystals of hemextin A, a three-finger toxin isolated and purified from African Ringhals cobra (H. haemachatus), are orthorhombic, space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 49.27, b = 49.51, c = 57.87 Å, and diffract to 1.5 Å resolution. Hemextin A was isolated and purified from African Ringhals cobra (Hemachatus haemachatus). It is a three-finger toxin that specifically inhibits blood coagulation factor VIIa and clot formation and that also interacts with hemextin B to form a unique anticoagulant complex. Hemextin A was crystallized by the hanging-drop vapour-diffusion method by equilibration against 0.2 M ammonium acetate, 0.1more » M sodium acetate trihydrate pH 4.6 and 30% PEG 4000 as the precipitating agent. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 49.27, b = 49.51, c = 57.87 Å and two molecules in the asymmetric unit. They diffracted to 1.5 Å resolution at beamline X25 at BNL.« less

Purification, crystallization and preliminary X-ray analysis of a thermostable glycoside hydrolase family 43 β-xylosidase from Geobacillus thermoleovorans IT-08

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

Rohman, Ali; Oosterwijk, Niels van; Kralj, Slavko

2007-11-01

The β-xylosidase was crystallized using PEG 6000 as precipitant. 5% PEG 6000 yielded bipyramid-shaped tetragonal crystals diffracting to 1.55 Å resolution, and 13% PEG 6000 gave rectangular monoclinic crystals diffracting to 1.80 Å resolution. The main enzymes involved in xylan-backbone hydrolysis are endo-1,4-β-xylanase and β-xylosidase. β-Xylosidase converts the xylo-oligosaccharides produced by endo-1,4-β-xylanase into xylose monomers. The β-xylosidase from the thermophilic Geobacillus thermoleovorans IT-08, a member of glycoside hydrolase family 43, was crystallized at room temperature using the hanging-drop vapour-diffusion method. Two crystal forms were observed. Bipyramid-shaped crystals belonging to space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = bmore » = 62.53, c = 277.4 Å diffracted to 1.55 Å resolution. The rectangular crystals belonged to space group P2{sub 1}, with unit-cell parameters a = 57.94, b = 142.1, c = 153.9 Å, β = 90.5°, and diffracted to 1.80 Å resolution.« less

Dispersible oxygen microsensors map oxygen gradients in three-dimensional cell cultures.

PubMed

Lesher-Pérez, Sasha Cai; Kim, Ge-Ah; Kuo, Chuan-Hsien; Leung, Brendan M; Mong, Sanda; Kojima, Taisuke; Moraes, Christopher; Thouless, M D; Luker, Gary D; Takayama, Shuichi

2017-09-26

Phase fluorimetry, unlike the more commonly used intensity-based measurement, is not affected by differences in light paths from culture vessels or by optical attenuation through dense 3D cell cultures and hydrogels thereby minimizing dependence on signal intensity for accurate measurements. This work describes the use of phase fluorimetry on oxygen-sensor microbeads to perform oxygen measurements in different microtissue culture environments. In one example, cell spheroids were observed to deplete oxygen from the cell-culture medium filling the bottom of conventional microwells within minutes, whereas oxygen concentrations remained close to ambient levels for several days in hanging-drop cultures. By dispersing multiple oxygen microsensors in cell-laden hydrogels, we also mapped cell-generated oxygen gradients. The spatial oxygen mapping was sufficiently precise to enable the use of computational models of oxygen diffusion and uptake to give estimates of the cellular oxygen uptake rate and the half-saturation constant. The results show the importance of integrated design and analysis of 3D cell cultures from both biomaterial and oxygen supply aspects. While this paper specifically tests spheroids and cell-laden gel cultures, the described methods should be useful for measuring pericellular oxygen concentrations in a variety of biomaterials and culture formats.

Crystallization and preliminary crystallographic analysis of l-asparaginase from Erwinia carotovora

PubMed Central

Wikman, Linnea E. K.; Krasotkina, Julya; Kuchumova, Anastasia; Sokolov, Nikolay N.; Papageorgiou, Anastassios C.

2005-01-01

Bacterial l-asparaginases have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukaemia for over 30 y. However, their use is limited owing to the glutaminase activity of the administered enzymes, which results in serious side effects. In contrast, l-asparaginase from Erwinia carotovora exhibits low glutaminase activity at physiological concentrations of l-asparagine and l-glutamine in the blood. Recombinant Er. carotovora l-­asparaginase was crystallized in the presence of l-glutamate by the hanging-drop vapour-diffusion method using 10 mg ml−1 purified enzyme, 16–18%(w/v) PEG 3350 and 0.2 M NaF. X-ray diffraction data were collected to 2.6 Å at 293 K using an in-house rotating-anode generator. The crystals belong to the monoclinic P21 space group, with unit-cell parameters a = 78.0, b = 112.3, c = 78.7 Å, β = 101.9° and a homotetramer in the crystallographic asymmetric unit. A molecular-replacement solution has been found and refinement is currently in progress. The crystal structure may provide leads towards protein-engineering efforts aimed at safer asparaginase administration in leukaemia treatment. PMID:16511054

Trypanothione reductase from Leishmania infantum: cloning, expression, purification, crystallization and preliminary X-ray data analysis.

PubMed

Baiocco, Paola; Franceschini, Stefano; Ilari, Andrea; Colotti, Gianni

2009-01-01

The most promising targets for Leishmania-specific drug design are two key enzymes involved in the unique thiol-based metabolism, common to all parasites of the Trypanosomatidae family: trypanothione synthetase (TryS) and trypanothione reductase (TR). Recently, new inhibitors of TR have been identified such as polyamines and tricyclic compounds. The knowledge of the three-dimensional structure of Leishmania TR will shed light on the mechanism of interaction of these inhibitors with TR and will be the starting point to design novel lead candidates to facilitate the development of new effective and affordable drugs. Trypanothione reductase from Leishmania infantum has been cloned, expressed in E. coli and purified. Crystals were obtained at 294 K by the hanging drop vapour diffusion method using ammonium sulfate as precipitant agent and diffract to better than 2.95 A resolution using a synchrotron radiation source. The crystals exhibit an unusually high solvent content of 74 %, belong to the tetragonal space group P41 with units cell parameters a=b=103.45 A, c=192.62 A and two molecules in the asymmetric unit. The protein X-ray structure has been solved by Molecular Replacement and the model is under construction.

White River National Forest Hanging Lake Capacity Study

DOT National Transportation Integrated Search

2016-05-01

Due to overcrowding at Hanging Lake, the Volpe Center performed a capacity study for the White River National Forest. This capacity study examines the visitation and environmental constraints of Hanging Lake parking lot, trail, and site to develop th...

Craniocervical injuries in judicial hangings: an anthropologic analysis of six cases.

PubMed

Spence, M W; Shkrum, M J; Ariss, A; Regan, J

1999-12-01

Restoration projects and archaeologic excavations in two Canadian prisons resulted in the recovery of the skeletons of six felons executed by judicial hanging. Damage inflicted by hanging on various skeletal elements was observed. Among the injuries seen were fractures of the hyoid cornua, styloid processes, occipital bones, and cervical vertebral bodies (C2) and transverse processes (C1, C2, C3, and C5). Despite the general uniformity of the hanging technique, which involved a subaural knot, the trauma to the skeletal elements and the cause of death varied among individuals. Although some of this variation was probably due to minor differences in hanging practices, individual anatomic peculiarities of the victims likely also contributed.

Voltammetric method for the determination of sildenafil citrate (Viagra) in pure form and in pharmaceutical formulations.

PubMed

Tyszczuk, Katarzyna; Korolczuk, Mieczyslaw

2010-06-01

A highly sensitive and simple voltammetric method for the determination of sildenafil citrate (SC) was developed. The method is based on the accumulation by adsorption of SC on a lead film modified glassy carbon electrode (LF/GCE) and then the reduction of SC throughout the stripping step. During the determinations of SC at the lead film electrode three adsorptive stripping voltammetric peaks at -1.2, -1.33 and -1.45V were observed. The respective response selected for identification and quantification has been evaluated with respect to the composition and pH of the supporting electrolyte, the potential and the time of the lead film formation, the potential and the time of the SC accumulation and other variables. Experimental results indicate an excellent linear correlation between the peak current and concentration in the range of 2x10(-9)-1.5x10(-7)mol/L (for peaks 1 and 2) and 1x10(-8)-1.5x10(-7)mol/L (for the peak 3). The detection limits (LOD) for SC following 30s of accumulation time were equal to 9x10(-10)mol/L (for peaks 1 and 2) and 4.5x10(-9)mol/L (for the peak 3). The method was successfully applied to the determination of SC in the tablets (Viagra 25 and Viagra 50) and average the contents were in close agreement with those quoted by the manufacturer and with those obtained by the reported spectrophotometric method and voltammetric method using a hanging mercury drop electrode. Copyright 2009 Elsevier B.V. All rights reserved.

Graphene induces spontaneous cardiac differentiation in embryoid bodies

NASA Astrophysics Data System (ADS)

Ahadian, Samad; Zhou, Yuanshu; Yamada, Shukuyo; Estili, Mehdi; Liang, Xiaobin; Nakajima, Ken; Shiku, Hitoshi; Matsue, Tomokazu

2016-03-01

Graphene was embedded into the structure of mouse embryoid bodies (EBs) using the hanging drop technique. The inclusion of 0.2 mg per mL graphene in the EBs did not affect the viability of the stem cells. However, the graphene decreased the stem cell proliferation, probably by accelerating cell differentiation. The graphene also enhanced the mechanical properties and electrical conductivity of the EBs. Interestingly, the cardiac differentiation of the EB-graphene was significantly greater than that of the EBs at day 5 of culture, as confirmed by high-throughput gene analysis. Electrical stimulation (voltage, 4 V; frequency, 1 Hz; and duration, 10 ms for 2 continuous days) further enhanced the cardiac differentiation of the EBs, as demonstrated by analyses of the cardiac protein and gene expression and the beating activity of the EBs. Taken together, the results demonstrated that graphene played a major role in directing the cardiac differentiation of EBs, which has potential cell therapy and tissue regeneration applications.Graphene was embedded into the structure of mouse embryoid bodies (EBs) using the hanging drop technique. The inclusion of 0.2 mg per mL graphene in the EBs did not affect the viability of the stem cells. However, the graphene decreased the stem cell proliferation, probably by accelerating cell differentiation. The graphene also enhanced the mechanical properties and electrical conductivity of the EBs. Interestingly, the cardiac differentiation of the EB-graphene was significantly greater than that of the EBs at day 5 of culture, as confirmed by high-throughput gene analysis. Electrical stimulation (voltage, 4 V; frequency, 1 Hz; and duration, 10 ms for 2 continuous days) further enhanced the cardiac differentiation of the EBs, as demonstrated by analyses of the cardiac protein and gene expression and the beating activity of the EBs. Taken together, the results demonstrated that graphene played a major role in directing the cardiac differentiation of EBs, which has potential cell therapy and tissue regeneration applications. Electronic supplementary information (ESI) available: Fig. S1-S3, Tables S1-S4, and Movies S1-S4. See DOI: 10.1039/c5nr07059g

Postmortem Presence of Drugs and Method of Violent Suicide

PubMed Central

Sheehan, Connor M.; Rogers, Richard G.; Boardman, Jason D.

2016-01-01

The link between substance use and suicide is well established. However, little research analyzes how substance use is related to the method of suicide. This paper analyzes how specific drugs are associated with method of suicide, a critical topic because drug use bears on the etiology of suicide and may lead to policies aimed at deterring suicide. We use the COVDRS and logistic regression to examine postmortem presence of drugs among 3,389 hanging and firearm suicides in Colorado from 2004–2009. Net of demographic controls, we find that opiates are positively associated with firearms (OR: 1.92, 95% L: 1.27, 95% U: 2.86]) while antidepressants are positively associated with hanging (OR: 1.45, 95% L: 1.04, 95% U: 2.03). For cocaine and opiates, the association between drug use and violent method vary by educational attainment. Importantly, knowledge of the presence and type of specific drug is strongly associated with the method of suicide. PMID:27239069

The Discrete Hanging Cable

ERIC Educational Resources Information Center

Peters, James V.

2004-01-01

Using the methods of finite difference equations the discrete analogue of the parabolic and catenary cable are analysed. The fibonacci numbers and the golden ratio arise in the treatment of the catenary.

Factors Contributing to Pelvis Instability in Female Adolescent Athletes During Unilateral Repeated Partial Squat Activity

PubMed Central

Scarborough, Donna Moxley; Linderman, Shannon; Berkson, Eric M.; Oh, Luke S.

2017-01-01

Objectives: Unilateral partial squat tasks are often used to assess athletes’ lower extremity (LE) neuromuscular control. Single squat biomechanics such as lateral drop of the non-stance limb’s pelvis have been linked to knee injury risk. Yet, there are limited studies on the factors contributing to pelvic instability during the unilateral partial squat such as anatomical alignment of the knee and hip strength. The purpose of this study was 1) to assess the influence of leg dominance on pelvic drop among female athletes during the repeated unilateral partial squat activity and 2) to investigate the contributions that lower limb kinematics and hip strength have on pelvis drop. Methods: 42 female athletes (27= softball pitchers, 15=gymnasts, avg age=16.48 ± 2.54 years) underwent lower limb assessment. The quadriceps angle (Q angle) and the average of 3 trials for hip abduction and extension strength (handheld dynamometer measurements) were used for analyses. 3D biomechanical analysis of the repeated unilateral partial squat activity followed using a 20 motion capture camera system which created a 15 segment model of each subject. The subject stood on one leg at the lateral edge of a 17.78 cm box with hands placed on the hips and squatted so that the free hanging contralateral limb came as close to the ground without contact for 5 continuous repetitions. One trial for each limb was performed. Peak pelvic drop and ankle, knee and hip angles and torques (normalized by weight) at this time point were calculated using Visual 3D (C-Motion) biomechanical software. Paired T-test, Spearman correlations and multiple regression model statistical analyses were performed. Results: Peak pelvic drop during the unilateral partial squat did not differ significantly on the basis of limb dominance (p=0.831, Dom: -3.40 ± 5.10° , ND: -3.46 ± 4.44°). Peak pelvic drop displayed a Spearman correlation with the functional measure of hip abduction/adduction (ABD/ADD) angle (rs= 0.627, p< 0.001) (Figure 1). No association was noted between peak pelvic drop and anatomical measures of Q angle or isometric hip extension strength. A multiple regression was performed to predict pelvis drop angle from the following 6 variables: isometric hip ABD strength, hip ABD/ADD angle, hip internal/external rotation angle, ankle supination/pronation (S/P) angle, height and weight. These variables statistically predicted pelvis drop, F(6,73) = 17.848, p < .0005, R2 = 0.595. The strongest combined predictor variables for pelvic drop in the female athletes were hip abduction/ adduction angle and strength followed by subject’s weight and ankle S/P angle (Table 1). Conclusion: Peak pelvic drop during the repeated unilateral partial squat activity did not correlate significantly with Q angle and hip extension strength. Instead, peak pelvic drop appears more related to a combination of biomechanical limb positioning, hip ABD strength and subject demographics. The regression model run on the repeated unilateral partial squat demonstrates predictive power of this dynamic assessment tool based on kinematic measures across multiple joints. Results could guide clinician screening for excessive pelvic drop in female athletes and based on the predictive model make recommendations for corrective conditioning to help prevent knee injury and guide return to sport following LE surgery. Table 1: Multivariate linear regression model for pelvic drop, Isometric hip strength and lower extremity kinematics during repeated partial squat activity among female athletes. Variable P Isometric hip abduction strength 0.034* Hip Abduction/Adduction Angle <0.001* Hip Internal/External Rotation Angle 0.936 Ankle Internal/External Rotation Angle 0.072 Height 0.398 Weight 0.011* * Level of significance established at p<0.05

Nighttime behavioral study of flying foxes on the southern coast of West Java, Indonesia.

PubMed

Hengjan, Yupadee; Saputra, Vidi; Pramono, Didik; Basri, Chaerul; Ando, Takeshi; Ohmori, Yasushige; Agungpriyono, Srihadi; Hondo, Eiichi

2018-05-30

Flying foxes are important in the maintenance of forests and diversity. However, knowledge of their behavioral ecology, especially of movement and foraging patterns, which are essential for conservation and management of their populations, are not well known. Therefore, movement behavior of two individuals of Pteropus vampyrus were examined using an Argos telemetry system, and foraging pattern of Pteropus spp. was directly observed, at West Java province, Indonesia in October 2017. The maximum distance between the location at which bats were released and their furthest roost, recorded via satellite telemetry, was approximately 100 km. This reflects the long-distance flight ability of P. vampyrus. Daytime roosting sites and nighttime foraging places consisted of several types of habitats, such as intact forests, agricultural lands, and residential areas. This evidence indicated that there was habitat overlap between humans and bats in West Java province. According to direct observation of the behaviors of flying foxes at two locations within residental areas, various activities such as wing spreading, excretion, fighting, aggressive calls, movement, hanging relax, and hanging alert were found. The number of bat-visits to the trees varied among night hours, and had a positive correlation with the number of fruit dropping. The data obtained in this study have improved our understanding of nighttime behavior and habitat utilization of P. vampyrus, that can be used to support landscape management, species conservation, and disease prevention in regions of Southeast Asia.

Application of Thermoluminescence dating to Sambagawa metamorphic rocks for evaluation of the late Quaternary uprifting of Central Shikoku, Japan

NASA Astrophysics Data System (ADS)

Nishikawa, O.

2016-12-01

Thermoluminescence (TL) dating is one of the geochronometry with a low closure temperature, which covers a wide range of younger ages from 1k to 1m yrs, and used to be applied to young volcanics and archeological burnt materials. These materials experienced an instant temperature drop under the closure temperature just after they are generated. If crust is rapidly uplifting, it may possible to apply TL dating even for basement rocks to reconstruct a young history of orogeny. TL age applied to basement is not the age of rock itself, but the age since the rock rising from the deeper part crossed the depth of the closure temperature. Therefore TL age of basement rock is the function of both uplifting rate and geothermal gradient. In this study, in order to evaluation of the late Quaternary uplifting of the central Shikoku, Japan, TL dating of quartz grain derived from the Sambagawa metamorphic rocks has been performed. The ages are in 100-1000 kyr orders and much older than TL ages obtained from the hanging wall of Alpine fault, New Zealand (Nishikawa et al., 2015; AGU Fall meeting). This can be due to the difference of geothermal gradient and uplifting rate between two orogenic belts, and interpreted that the hanging wall of the Alpine fault has been rapidly lifted up from the shallower closure temperature depth, while the rocks in central Shikoku have been rising slowly from deeper part.

Can footwall unloading explain late Cenozoic uplift of the Sierra Nevada crest?

USGS Publications Warehouse

Thompson, G.A.; Parsons, T.

2009-01-01

Globally, normal-fault displacement bends and warps rift flanks upwards, as adjoining basins drop downwards. Perhaps the most evident manifestations are the flanks of the East African Rift, which cuts across the otherwise minimally deformed continent. Flank uplift was explained by Vening Meinesz (1950, Institut Royal Colonial Belge, Bulletin des Seances, v. 21, p. 539-552), who recognized that isostasy should cause uplift of a normal-faulted footwall and subsidence of its hanging wall. Uplift occurs because slip on a dipping normal fault creates a broader root of less-dense material beneath the footwall, and a narrowed one beneath the hanging wall. In this paper, we investigate the potential influence of this process on the latest stages of Sierra Nevada uplift. Through theoretical calculations and 3D finite element modelling, we find that cumulative slip of about 4km on range-front faults would have produced about 1.3km peak isostatic uplift at the ridge crest. Numerical models suggest that the zone of uplift is narrow, with the width controlled by bending resistance of the seismogenic crust. We conclude that footwall unloading cannot account for the entire elevation of the Sierran crest above sea level, but if range-front faulting initiated in an already elevated plateau like the adjacent Basin and Range Province, then a hybrid model of pre-existing regional uplift and localized footwall unloading can account for the older and newer uplift phases suggested by the geologic record.

Ground Motions Simulations and Site Effects in the Quito Basin (Ecuador)

NASA Astrophysics Data System (ADS)

Courboulex, F.; Castro-Cruz, D.; Laurendeau, A.; Bonilla, L. F.; Bertrand, E.; Mercerat, D.; Alvarado, A. P.

2017-12-01

The city of Quito (3M inhabitants), capital of Ecuador has been damaged several times in the past by large earthquakes. It is built on the hanging-wall of an active reverse fault, constituting a piggy-back basin. The deep structure of this basin and its seismic response remains badly known. We first use the recordings of 170 events on 18 accelerometers from the Quito permanent network and perform spectral ratio analysis. We find that the southern part of Quito shows strong site amplification at low frequency ( 0.35 Hz). Yet, high frequency ( 5 Hz) amplifications also exist, but exhibit a complex spatial variability. We then propose a new calibrated method based on empirical Green's functions (EGF) to simulate the ground motions due to a future earthquake in Quito. The idea is to use the results of a global database of source time functions (i.e., the SCARDEC database, Vallée and Douet, 2016; Courboulex et al., 2016) to define the average values and the variability of the stress-drop ratio parameter, which strongly affects the resulting simulations. We test the method on a Mw 7.8 event, similar in location and focal mechanism to the Pedernales earthquake that occurred on April 16th 2016 on the subduction zone. For this aim, we use the recordings of 6 aftershocks of magnitude 5.6 to 6.2 as EGF's. The predicted Fourier spectra, peak values and response spectra we obtain are in good agreement with real data from the 2016 event recorded on the Quito network. With the constraints we impose on stress-drop ratios, we expect that the simulated ground motions be representative of the variability of other Pedernales-type events that could occur in the future. Our results also well reproduce the low frequency site effects amplification in the south of the basin. This amplification could be particularly dangerous in the case of a mega subduction earthquake, like the one that struck Ecuador in 1906.

Liquid-liquid diffusion crystallization improves the X-ray diffraction of EndoS, an endo-β-N-acetylglucosaminidase from Streptococcus pyogenes with activity on human IgG.

PubMed

Trastoy, Beatriz; Lomino, Joseph V; Wang, Lai Xi; Sundberg, Eric J

2013-12-01

Endoglycosidase S (EndoS) is an enzyme secreted by Streptococcus pyogenes that specifically hydrolyzes the β-1,4-di-N-acetylchitobiose core glycan on immunoglobulin G (IgG) antibodies. One of the most common human pathogens and the cause of group A streptococcal infections, S. pyogenes secretes EndoS in order to evade the host immune system by rendering IgG effector mechanisms dysfunctional. On account of its specificity for IgG, EndoS has also been used extensively for chemoenzymatic synthesis of homogeneous IgG glycoprotein preparations and is being developed as a novel therapeutic for a wide range of autoimmune diseases. The structural basis of its enzymatic activity and substrate specificity, however, remains unknown. Here, the purification and crystallization of EndoS are reported. Using traditional hanging-drop and sitting-drop vapor-diffusion crystallization, crystals of EndoS were grown that diffracted to a maximum of 3.5 Å resolution but suffered from severe anisotropy, the data from which could only be reasonably processed to 7.5 Å resolution. When EndoS was crystallized by liquid-liquid diffusion, it was possible to grow crystals with a different space group to those obtained by vapor diffusion. Crystals of wild-type endoglycosidase and glycosynthase constructs of EndoS grown by liquid-liquid diffusion diffracted to 2.6 and 1.9 Å resolution, respectively, with a greatly diminished anisotropy. Despite extensive efforts, the failure to reproduce these liquid-liquid diffusion-grown crystals by vapor diffusion suggests that these crystallization methods each sample a distinct crystallization space.

KSC-05PD-1659

NASA Technical Reports Server (NTRS)

2005-01-01

KENNEDY SPACE CENTER, FLA. In the transfer aisle of the Vehicle Assembly Building at NASA Kennedy Space Center, In the transfer aisle of the Vehicle Assembly Building at NASA Kennedy Space Center, orbiter technicians are hooking a restraining cable around the orbiter lifting slings aft drop leg adjustment hand wheel. This is performed prior to disconnecting the aft spreader beam from the sling (the portion suspended by the 175 ton crane on the left side of the photo), and also prior to taking the final hang angle measurement of the orbiter prior to lifting it over the transom into the high bay. It will be mated with the Solid Rocket Boosters and External Tank already stacked there on the Mobile Launcher Platform. Atlantis is the designated orbiter for Return to Flight mission STS-121. The lighted planning window for launch extends from Sept. 9 to Sept. 25.

KSC-05PD-1660

NASA Technical Reports Server (NTRS)

2005-01-01

KENNEDY SPACE CENTER, FLA. In the transfer aisle of the Vehicle Assembly Building at NASA Kennedy Space Center, In the transfer aisle of the Vehicle Assembly Building at NASA Kennedy Space Center, orbiter technicians are hooking a restraining cable around the orbiter lifting slings aft drop leg adjustment hand wheel. This is performed prior to disconnecting the aft spreader beam from the sling (the portion suspended by the 175 ton crane on the left side of the photo), and also prior to taking the final hang angle measurement of the orbiter prior to lifting it over the transom into the high bay. It will be mated with the Solid Rocket Boosters and External Tank already stacked there on the Mobile Launcher Platform. Atlantis is the designated orbiter for Return to Flight mission STS-121. The lighted planning window for launch extends from Sept. 9 to Sept. 25.

Time lapse microscopy of temperature control during self-assembly of 3D DNA crystals

NASA Astrophysics Data System (ADS)

Conn, Fiona W.; Jong, Michael Alexander; Tan, Andre; Tseng, Robert; Park, Eunice; Ohayon, Yoel P.; Sha, Ruojie; Mao, Chengde; Seeman, Nadrian C.

2017-10-01

DNA nanostructures are created by exploiting the high fidelity base-pairing interactions of double-stranded branched DNA molecules. These structures present a convenient medium for the self-assembly of macroscopic 3D crystals. In some self-assemblies in this system, crystals can be formed by lowering the temperature, and they can be dissolved by raising it. The ability to monitor the formation and melting of these crystals yields information that can be used to monitor crystal formation and growth. Here, we describe the development of an inexpensive tool that enables direct observation of the crystal growth process as a function of both time and temperature. Using the hanging-drop crystallization of the well-characterized 2-turn DNA tensegrity triangle motif for our model system, its response to temperature has been characterized visually.

Crystallization and preliminary X-ray analysis of copper amine oxidase from Escherichia coli K-12.

PubMed

Roh, J H; Suzuki, H; Kumagai, H; Yamashita, M; Azakami, H; Murooka, Y; Mikami, B

1994-05-13

Copper-containing monoamine oxidase (MAO) from Escherichia coli was overproduced in the periplasmic space by expression of the cloned gene. The purified MAO has been crystallized by means of the hanging drop technique using sodium citrate as a precipitant. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit cell dimensions of a = 136.1 A, b = 168.4 A and c = 81.6 A. The asymmetric unit contains one molecule of MAO, with a crystal volume per protein mass (Vm) of 2.88 A3/Da and a solvent content of 58% by volume. The crystals diffract X-rays to a resolution limit of at least 2.7 A and are resistant to X-ray radiation damage. They appear to be suitable for X-ray structure analysis.

Crystallization and preliminary crystallographic analysis of porcine acylaminoacyl peptidase.

PubMed

Wright, Helena; Kiss, András L; Szeltner, Zoltán; Polgár, László; Fülöp, Vilmos

2005-10-01

Acylaminoacyl peptidase (also known as acylamino-acid-releasing enzyme or acylpeptide hydrolase; EC 3.4.19.1) is an unusual member of the prolyl oligopeptidase family catalysing the hydrolysis of an N-acylated peptide to an acylamino acid and a peptide with a free N-terminus. Acylaminoacyl peptidase purified from porcine liver has been crystallized in mother liquor containing 0.1 M Tris-HCl pH 7.0, 10%(w/v) polyethylene glycol 8000, 50 mM MgCl2 and 1%(w/v) CHAPS using the hanging-drop vapour-diffusion technique. A full data set to 3.4 A resolution was collected at ESRF beamline ID14-4 and space group C222 was assigned, with unit-cell parameters a = 84.8, b = 421.1, c = 212.0 A and four molecules in the asymmetric unit.

Crystallization and preliminary X-ray diffraction studies of the lipopolysaccharide core biosynthetic enzyme ADP-L-glycero-D-mannoheptose 6-epimerase from Escherichia coli K-12.

PubMed

Ding, L; Zhang, Y; Deacon, A M; Ealick, S E; Ni, Y; Sun, P; Coleman, W G

1999-03-01

ADP-L-glycero-D-mannoheptose 6-epimerase is a 240 kDa NAD-dependent nucleotide diphosphosugar epimerase from Escherichia coli K12 which catalyzes the interconversion of ADP-D-glycero-D-mannoheptose and ADP-L-glycero-D-mannoheptose. ADP-L-glycero-D-mannoheptose is a required intermediate for lipopolysaccharide inner-core and outer-membrane biosynthesis in several genera of pathogenic and non-pathogenic Gram-negative bacteria. ADP-L-glycero-D-mannoheptose 6-epimerase was overexpressed in E. coli and purified to apparent homogeneity by chromatographic methods. Three crystal forms of the epimerase were obtained by a hanging-drop vapor-diffusion method. A native data set for crystal form III was collected in-house on a Rigaku R-AXIS-IIC image plate at 3.0 A resolution. The form III crystals belong to the monoclinic space group P21. The unit-cell parameters are a = 98.94, b = 110.53, c = 180.68 A and beta = 90.94 degrees. Our recent results show that these crystals diffract to 2.0 A resolution at the Cornell High Energy Synchrotron Source. The crystal probably contains six 40 kDa monomers per asymmetric unit, with a corresponding volume per protein mass (Vm) of 4.11 A3 Da-1 and a solvent fraction of 70%.

75 FR 19895 - Final Flood Elevation Determinations

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-16

... +1529 Eastern Band of Cherokee Hanging Dog Creek. Indians, Unincorporated Areas of Cherokee County. Approximately 0.8 mile +1633 upstream of the confluence with Hanging Dog Creek. Big Witch Creek At the.... Indians. Approximately 1,050 feet +1904 upstream of Goose Creek Road. Hanging Dog Creek At the confluence...

78 FR 48657 - Procurement List; Additions

Federal Register 2010, 2011, 2012, 2013, 2014

2013-08-09

... Divider, 4 Sections, Light Blue NSN: 7530-00-NIB-1098--Folder, File, Hanging, Light Blue, Letter Size, 2-Dividers, 6 Sections NSN: 7530-00-NIB-1099--Folder, File, Hanging, Light Blue, Legal Size, 1-Divider, 4 Sections NSN: 7530-00-NIB-1100--Folder, File, Hanging, Light Blue, Legal Size, 2-Dividers, 6 Sections NPA...

75 FR 73074 - Duke Energy Hanging Rock II, LLC; Supplemental Notice That Initial Market-Based Rate Filing...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-11-29

... DEPARTMENT OF ENERGY Federal Energy Regulatory Commission [Docket No. ER11-2064-000] Duke Energy Hanging Rock II, LLC; Supplemental Notice That Initial Market-Based Rate Filing Includes Request for... proceeding, of Duke Energy Hanging Rock II, LLC's application for market-based rate authority, with an...

The Hanging Cord with a Real Tip Mass

ERIC Educational Resources Information Center

Deschaine, J. S.; Suits, B. H.

2008-01-01

Normal mode solutions for the perfectly flexible hanging cord problem have been known for over 200 years. More recently, theoretical results for a hanging cord with a point mass attached were presented. Here the theoretical results are tested experimentally using high-precision techniques which are accessible for use in an introductory laboratory.…

Verifying the Hanging Chain Model

ERIC Educational Resources Information Center

Karls, Michael A.

2013-01-01

The wave equation with variable tension is a classic partial differential equation that can be used to describe the horizontal displacements of a vertical hanging chain with one end fixed and the other end free to move. Using a web camera and TRACKER software to record displacement data from a vibrating hanging chain, we verify a modified version…

Hang-It-Up Artists

ERIC Educational Resources Information Center

Szekely, George

2009-01-01

The best lesson ideas often derive from childhood recollections. When brought to class, play memories and art mementos from the teacher's childhood paint a powerful homage to children's art. A survey of a child's room in the dark, or with lights on, discloses interesting hanging sites, means of attachment and unusual items drafted for hanging, all…

[Sensory illusions in hang-gliding].

PubMed

Bousquet, F; Bizeau, A; Resche-Rigon, P; Taillemite, J P; De Rotalier

1997-01-01

Sensory illusions in hang-gliding and para-gliding. Hang-gliding and para-gliding are at the moment booming sports. Sensory illusions are physiological phenomena sharing the wrong perception of the pilote's real position in space. These phenomena are very familiar to aeroplane pilotes, they can also be noticed on certain conditions with hang-gliding pilotes. There are many and various sensory illusions, but only illusions of vestibular origin will be dealt with in this article. Vestibular physiology is reminded with the working principle of a semicircular canal. Physiology and laws of physics explain several sensory illusions, especially when the pilote loses his visual landmarks: flying through a cloud, coriolis effect. Also some specific stages of hang-gliding foster those phenomena: spiraling downwards, self-rotation, following an asymetric closing of the parachute, spin on oneself. Therefore a previous briefing for the pilotes seems necessary.

Rapid Screening Method for Detecting Ethinyl Estradiol in Natural Water Employing Voltammetry

PubMed Central

2016-01-01

17α-Ethinyl estradiol (EE2), which is used worldwide in the treatment of some cancers and as a contraceptive, is often found in aquatic systems and is considered a pharmaceutically active compound (PhACs) in the environment. Current methods for the determination of this compound, such as chromatography, are expensive and lengthy and require large amounts of toxic organic solvents. In this work, a voltammetric procedure is developed and validated as a screening tool for detecting EE2 in water samples without prior extraction, clean-up, or derivatization steps. Application of the method we elaborate here to EE2 analysis is unprecedented. EE2 detection was carried out using differential pulse adsorptive cathodic stripping voltammetry (DP AdCSV) with a hanging mercury drop electrode (HMDE) in pH 7.0 Britton-Robinson buffer. The electrochemical process of EE2 reduction was investigated by cyclic voltammetry at different scan rates. Electroreduction of the hormone on a mercury electrode exhibited a peak at −1.16 ± 0.02 V versus Ag/AgCl. The experimental parameters were as follows: −0.7 V accumulation potential, 150 s accumulation time, and 60 mV s−1 scan rate. The limit of detection was 0.49 μg L−1 for a preconcentration time of 150 s. Relative standard deviations were less than 13%. The method was applied to the detection of EE2 in water samples with recoveries ranging from 93.7 to 102.5%. PMID:27738548

Multi-Section Sensing and Vibrotactile Perception for Walking Guide of Visually Impaired Person.

PubMed

Jeong, Gu-Young; Yu, Kee-Ho

2016-07-12

Electronic Travel Aids (ETAs) improve the mobility of visually-impaired persons, but it is not easy to develop an ETA satisfying all the factors needed for reliable object detection, effective notification, and actual usability. In this study, the authors developed an easy-to-use ETA having the function of reliable object detection and its successful feedback to the user by tactile stimulation. Seven ultrasonic sensors facing in different directions detect obstacles in the walking path, while vibrators in the tactile display stimulate the hand according to the distribution of obstacles. The detection of ground drop-offs activates the electromagnetic brakes linked to the rear wheels. To verify the feasibility of the developed ETA in the outdoor environment, walking tests by blind participants were performed, and the evaluation of safety to ground drop-offs was carried out. From the experiment, the feasibility of the developed ETA was shown to be sufficient if the sensor ranges for hanging obstacle detection is improved and learning time is provided for the ETA. Finally, the light-weight and low cost ETA designed and assembled based on the evaluation of the developed ETA is introduced to show the improvement of portability and usability, and is compared with the previously developed ETAs.

Multi-Section Sensing and Vibrotactile Perception for Walking Guide of Visually Impaired Person

PubMed Central

Jeong, Gu-Young; Yu, Kee-Ho

2016-01-01

Electronic Travel Aids (ETAs) improve the mobility of visually-impaired persons, but it is not easy to develop an ETA satisfying all the factors needed for reliable object detection, effective notification, and actual usability. In this study, the authors developed an easy-to-use ETA having the function of reliable object detection and its successful feedback to the user by tactile stimulation. Seven ultrasonic sensors facing in different directions detect obstacles in the walking path, while vibrators in the tactile display stimulate the hand according to the distribution of obstacles. The detection of ground drop-offs activates the electromagnetic brakes linked to the rear wheels. To verify the feasibility of the developed ETA in the outdoor environment, walking tests by blind participants were performed, and the evaluation of safety to ground drop-offs was carried out. From the experiment, the feasibility of the developed ETA was shown to be sufficient if the sensor ranges for hanging obstacle detection is improved and learning time is provided for the ETA. Finally, the light-weight and low cost ETA designed and assembled based on the evaluation of the developed ETA is introduced to show the improvement of portability and usability, and is compared with the previously developed ETAs. PMID:27420060

Distribution of hanging garden vegetation associations on the Colorado Plateau, USA

Treesearch

James F. Fowler; N. L. Stanton; Ronald L. Hartman

2007-01-01

Hanging gardens are island-like habitats dominated by mesophytic-hydrophytic plant communities, growing on seeps on the xeric canyon walls of the Colorado Plateau in the American West. We measured the abundance of species and physical microhabitat characteristics of 73 individual hanging gardens during the growing seasons of 1991-1993. Cluster analysis of a simplified...

Analysis and experimental studies of the control of hang gliders

NASA Technical Reports Server (NTRS)

Phillips, W. H.

1974-01-01

A theoretical analysis of the longitudinal and lateral characteristics of hang gliders in straight flight, pullups, and turns is presented. Some examples of the characteristics of a straight-wing configuration and a Rogallo-wing configuration are given. A means for improving the control of hang gliders while retaining the same basic control feel is proposed.

Expedition 54 plaque hanging ceremony

NASA Image and Video Library

2018-04-26

NASA astronauts Joe Acaba and Mark Vande Hei Acaba visited Marshall April 11 for their honorary Expedition 54 plaque hanging ceremony and to provide valuable feedback of their on-orbit science investigations with the Payload Operations and Integration Center team. Chris Buckley is awarded privilege of hanging Mission 54 placque, shown here with Astronaut Mark Vande Hei (L) and Astronaut Joe Acaba

Characteristics of Completed Suicides: Implications of Differences among Methods.

ERIC Educational Resources Information Center

Fischer, Ellen P.; And Others

1993-01-01

Compared characteristics of suicides by jumping to those of suicides by hanging, ingestion, or shooting. Method used was significantly associated with sociodemographics, occupation, and mental health status, even after adjustment for individual access to suicide means. Findings provide evidence for hypothesis that controlling access to agent of…

Water resources and effects of potential surface coal mining on dissolved solids in Hanging Woman Creek basin, southeastern Montana

USGS Publications Warehouse

Cannon, M.R.

1989-01-01

Groundwater resources of the Hanging Woman Creek basin, Montana include Holocene and Pleistocene alluvial aquifers and sandstone , coal, and clinker aquifers in the Paleocene Fort Union Formation. Surface water resources are composed of Hanging Woman Creek, its tributaries, and small stock ponds. Dissolved-solids concentrations in groundwater ranged from 200 to 11,00 mg/L. Generally, concentrations were largest in alluvial aquifers and smallest in clinker aquifers. Near its mouth, Hanging Woman Creek had a median concentration of about 1,800 mg/L. Mining of the 20-foot to 35-foot-thick Anderson coal bed and 3-foot to 16-foot thick Dietz coal bed could increase dissolved-solids concentrations in shallow aquifers and in Hanging Woman Creek because of leaching of soluble minerals from mine spoils. Analysis of saturated-paste extracts from 158 overburden samples indicated that water moving through mine spoils would have a median increase in dissolved-solids concentration of about 3,700 mg/L, resulting in an additional dissolved-solids load to Hanging Woman Creek of about 3.0 tons/day. Hanging Woman Creek near Birney could have an annual post-mining dissolved-solids load of 3,415 tons at median discharge, a 47% increase from pre-mining conditions load. Post-mining concentrations of dissolved solids, at median discharge, could range from 2,380 mg/L in March to 3,940 mg/L in August, compared to mean pre-mining concentrations that ranged from 1,700 mg/L in July, November, and December to 2,060 mg/L in May. Post-mining concentrations and loads in Hanging Woman Creek would be smaller if a smaller area were mined. (USGS)

Correlation of Coseismic Velocity and Static Volumetric Strain Changes Induced by the 2010 Mw6.3 Jiasian Earthquake under the Southern Taiwan Orogenic Belt

NASA Astrophysics Data System (ADS)

Wu, S. M.; Hung, S. H.

2015-12-01

Earthquake-induced temporal changes in seismic velocity of the earth's crust have been demonstrated to be monitored effectively by the time-lapse shifts of coda waves recently. Velocity drop during the coseismic rupture has been explicitly observed in proximity to the epicenters of large earthquakes with different styles of faulting. The origin of such sudden perturbation in crustal properties is closely related to the damage and/or volumetric strain change influenced by seismic slip distribution. In this study, we apply a coda wave interferometry method to investigate potential velocity change in both space and time related to the moderate-sized (Mw6.3) 2010 Jiasian earthquake, which nucleated deeply in the crust (~23 km), ruptured and terminated around the depth of 10 km along a previously unidentified blind thrust fault near the lithotectonic boundary of the southern Taiwan orogenic belt. To decipher the surface and crustal response to this relatively deep rupture, we first measure relative time-lapse changes of coda between different short-term time frames spanning one year covering the pre- and post-seismic stages by using the Moving Window Cross Spectral Method. Rather than determining temporal velocity variations based on a long-term reference stack, we conduct a Bayesian least-squares inversion to obtain the optimal estimates by minimizing the inconsistency between the relative time-lapse shifts of individual short-term stacks. The results show the statistically significant velocity reduction immediately after the mainshock, which is most pronounced at the pairs with the interstation paths traversing through the hanging-wall block of the ruptured fault. The sensitivity of surface wave coda arrivals mainly in the periods of 3-5 s to shear wave speed perturbation is confined within the depth of 10 km, where the crust mostly experienced extensional strain changes induced by the slip distribution from the finite-fault model. Compared with coseismic slip distribution from GPS data and finite-fault inversion, peak ground velocity, and static volumetric strain field following the earthquake, the velocity decrease observed in the hanging wall side of the shallow crust is most likely attributed to pervasive dilatational strain changes induced by the slip rupture on the underlying blind thrust.

Comparing hydraulic properties of soil-less substrates with natural soils: a more detailed look at hydraulic properties and their impact on plant water availability

NASA Astrophysics Data System (ADS)

Crawford, L.; Rivera, L. D.; van Iersel, M.

2013-12-01

Moisture release curves are often used when assessing plant-water relationships in soil-less substrates. However, differences between natural soils and soilless substrates make traditional assumptions about plant available water potentially invalid. If soil-less substrates are supposed to be treated like natural soils; why do plants begin wilting at very low water potentials (-10 to -30 kPa) and there is anywhere between 20 to 40 % water left (on a volumetric basis) in the soil (Abad et al., 2005; Arguedas et al., 2006; Ristvey et al, 2008) . We hypothesize that the fault lies in the methods used and the assumption that water potential is the only limiting factor in water availability to plants. Hydraulic properties, including the relationships that exist between plant available water, water content, and hydraulic conductivity of soil-less substrates have traditionally been characterized using instrumentation such as pressure plates, hanging water columns, and tempe cells. These approaches typically take a months and only provide data on select segments of the soil moisture release curve, and in the case of pressure plates and hanging water columns hydraulic conductivity is ignored and not very well understood. Using the Wind/Schindler Evaporation method more detailed measurements of these hydraulic properties can be measured in a less than a week. A more detailed look at the hydraulic properties of soil-less substrates and how they compare with natural soils may give us more insight into soil-plant-water-relations and what limits availability of water to plants. Soil moisture release curves and hydraulic conductivity curves of different soil-less substrates were compared with curves from typical agriculture soils to give insight into how these properties compare. Results of the soil moisture release curves showed that some soil-less substrates had comparable moisture release curves to agricultural soils while others had bi-modal curves indicating gap-gradation in the pore size distribution. These soils that showed this non-typical curve had hydraulic conductivities that dropped very low (500 times lower than agricultural soils) at low water potentials (around 10 kPa). This dramatically lower hydraulic conductivity could lead to zones of depletion around the roots hindering plant water uptake.

The Historical Method of Inquiry in a Teacher Training Program: Theory and Metatheory.

ERIC Educational Resources Information Center

Kimmons, Ron

A historical method of inquiry can be applied to an experimental teacher training program, specifically, the Ford Training and Preparation Program (FTPP). The historical method requires gathering a lot of loose ideas and events that have been part of the project and hanging them together in an integrated way. To achieve this, two organizing…

Methods of Suicide by Age: Sex and Race Differences among the Young and Old.

ERIC Educational Resources Information Center

McIntosh, John L.; Santos, John F.

1986-01-01

Annual official statistics for specific methods of suicide (firearms, hanging, poisons) by age for different sex and racial groups (Whites, Blacks, non-Whites excluding Black) were examined from 1960 to 1978. Comparisons among the age-sex-race groups, along with trends over time and differences in the methods employed, were noted. (Author/ABL)

Searching for new features of intravitality of hanging based on macro- and microscopic evaluation of the proximal attachment of the sternocleidomastoid muscle and the mastoid process of the temporal bone.

PubMed

Szleszkowski, Ł; Hałoń, A; Thannhäuser, A; Jurek, T

2015-01-01

Assessment of the usefulness of intravital lesions in the proximal attachment of the sternocleidomastoid muscle and the mastoid process of the temporal bone in medico-legal evaluation of death by hanging. The study material was obtained from the bodies of 35 people who died by hanging. The control group comprised specimens collected from 30 people who died of non-traumatic causes. The structures under study were examined macro- and microscopically. The basic change which could be recognized as a marker of intravitality of hanging was the presence of a macroscopically extensive blotchy area of abundant ecchymosis in the proximal muscle attachment, similar to that found in the distal attachment, and the presence of abundant diffuse intraosseous ecchymoses in the mastoid process. None of the cases revealed any ecchymoses in the proximal attachment of the muscle that would be similar to those present in the distal attachment. Discolourations within the mastoid processes, macroscopically suggestive of extensive intraosseous effusions arising from the mechanism of stretching, were not confirmed by microscopic evaluation and occurred at the same frequency as in the control group. Limitations of the study were related to the method which involved sample collection by means of bone chisels, decalcification and preparation of specimens, which had an effect, for example, on the measurable evaluation of the degree of congestion. The study has failed to provide convincing and unambiguous data on the usefulness of examining mastoid processes and proximal attachments of the sternocleidomastoid muscles during autopsy to determine the presence of intravitality features of hanging. A description of research methodology and its associated difficulties, e.g. with the interpretation of results, can also be useful for the planning of similar studies by other researchers.

String & Sticky Tape Experiments: Two-Dimensional Collisions Using Pendulums.

ERIC Educational Resources Information Center

Edge, R. D.

1989-01-01

Introduces a method for two-dimensional kinematics measurements by hanging marbles with long strings. Describes experimental procedures for conservation of momentum and obtaining the coefficient of restitution. Provides diagrams and mathematical expressions for the activities. (YP)

Identification and on-line monitoring of reduced sulphur species (RSS) by voltammetry in oxic waters.

PubMed

Superville, Pierre-Jean; Pižeta, Ivanka; Omanović, Dario; Billon, Gabriel

2013-08-15

Based on automatic on-line measurements on the Deûle River that showed daily variation of a peak around -0.56V (vs Ag|AgCl 3M), identification of Reduced Sulphur Species (RSS) in oxic waters was performed applying cathodic stripping voltammetry (CSV) with the hanging mercury drop electrode (HMDE). Pseudopolarographic studies accompanied with increasing concentrations of copper revealed the presence of elemental sulphur S(0), thioacetamide (TA) and reduced glutathione (GSH) as the main sulphur compounds in the Deûle River. In order to resolve these three species, a simple procedure was developed and integrated in an automatic on-line monitoring system. During one week monitoring with hourly measurements, GSH and S(0) exhibited daily cycles whereas no consequential pattern was observed for TA. Copyright © 2013 Elsevier B.V. All rights reserved.

In Situ μGISAXS: II. Thaumatin Crystal Growth Kinetic

PubMed Central

Gebhardt, Ronald; Pechkova, Eugenia; Riekel, Christian; Nicolini, Claudio

2010-01-01

The formation of thaumatin crystals by Langmuir-Blodgett (LB) film nanotemplates was studied by the hanging-drop technique in a flow-through cell by synchrotron radiation micrograzing-incidence small-angle x-ray scattering. The kinetics of crystallization was measured directly on the interface of the LB film crystallization nanotemplate. The evolution of the micrograzing-incidence small-angle x-ray scattering patterns suggests that the increase in intensity in the Yoneda region is due to protein incorporation into the LB film. The intensity variation suggests several steps, which were modeled by system dynamics based on first-order differential equations. The kinetic data can be described by two processes that take place on the LB film, a first, fast, process, attributed to the crystal growth and its detachment from the LB film, and a second, slower process, attributed to an unordered association and conversion of protein on the LB film. PMID:20713011

Expression, purification, crystallization and preliminary X-ray analysis of a C-terminal fragment of the Epstein–Barr virus ZEBRA protein

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

Morand, Patrice; Laboratoire de Virologie Moléculaire et Structurale, EA 2939, Université Joseph Fourier, Grenoble; Budayova-Spano, Monika

A C-terminal fragment of the Epstein–Barr virus lytic switch protein ZEBRA has been crystallized in complex with DNA. A C-terminal fragment of the Epstein–Barr virus immediate-early transcription factor ZEBRA has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. The fragment behaves as a dimer in solution, consistent with the presence of a basic region leucine-zipper (bZIP) domain. Crystals of the fragment in complex with a DNA duplex were grown by the hanging-drop vapour-diffusion technique using polyethylene glycol 4000 and magnesium acetate as crystallization agents. Crystals diffract to better than 2.5 Å resolution using synchrotron radiationmore » (λ = 0.976 Å). Crystals belong to space group C2, with unit-cell parameters a = 94.2, b = 26.5, c = 98.1 Å, β = 103.9°.« less

Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

PubMed Central

Rosenthal, Cindy; Mueller, Uwe; Panjikar, Santosh; Sun, Lianli; Ruppert, Martin; Zhao, Yu; Stöckigt, Joachim

2006-01-01

Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C2221 and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å. PMID:17142919

Magnetically levitated mesenchymal stem cell spheroids cultured with a collagen gel maintain phenotype and quiescence

PubMed Central

Lewis, Natasha S; Lewis, Emily EL; Mullin, Margaret; Wheadon, Helen; Dalby, Matthew J; Berry, Catherine C

2017-01-01

Multicellular spheroids are an established system for three-dimensional cell culture. Spheroids are typically generated using hanging drop or non-adherent culture; however, an emerging technique is to use magnetic levitation. Herein, mesenchymal stem cell spheroids were generated using magnetic nanoparticles and subsequently cultured within a type I collagen gel, with a view towards developing a bone marrow niche environment. Cells were loaded with magnetic nanoparticles, and suspended beneath an external magnet, inducing self-assembly of multicellular spheroids. Cells in spheroids were viable and compared to corresponding monolayer controls, maintained stem cell phenotype and were quiescent. Interestingly, core spheroid necrosis was not observed, even with increasing spheroid size, in contrast to other commonly used spheroid systems. This mesenchymal stem cell spheroid culture presents a potential platform for modelling in vitro bone marrow stem cell niches, elucidating interactions between cells, as well as a useful model for drug delivery studies. PMID:28616152

Preparation, crystallization and preliminary X-ray crystallographic studies of diadenosine tetraphosphate hydrolase from Shigella flexneri 2a.

PubMed

Hu, Wenxin; Wang, Qihai; Bi, Ruchang

2005-12-01

Diadenosine tetraphosphate (Ap4A) hydrolase (EC 3.6.1.41) hydrolyzes Ap4A symmetrically in prokaryotes. It plays a potential role in organisms by regulating the concentration of Ap4A in vivo. To date, no three-dimensional structures of proteins with significant sequence homology to this protein have been determined. The 31.3 kDa Ap4A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap4A hydrolase have been obtained by the hanging-drop technique at 291 K using PEG 550 MME as precipitant. Ap4A hydrolase crystals diffract X-rays to 3.26 A and belong to space group P2(1), with unit-cell parameters a = 118.9, b = 54.6, c = 128.5 A, beta = 95.7 degrees.

Droplets on bent fibers

NASA Astrophysics Data System (ADS)

Weyer, Floriane; Pan, Zhao; Pitt, William; Truscott, Tadd; Vandewalle, Nicolas

Droplets on fibers are part of our everyday lives. Many phenomena involve drops and fibers such as the formation of dew droplets on a spiderweb, the trapping of water droplets on cactus spines or the motion of droplets on wetted moss hairs. These topics have been widely studied. In particular, Lorenceau et al. determined the critical volume of a water droplet hanging on a horizontal fiber. Here, we address a similar question : we try to find out the maximum droplet size on bent fibers, which are able to hold significantly more water than horizontal fibers. Indeed, we noticed that, in nature, some specific plants can hold large rain droplets thanks to their Y-shaped leaves. We try to mimic these structures with nylon fibers, of different diameters, bent with various angles. For each set-up, the critical water volume is determined. Finally, we propose models of the physics involved in determining droplet size that could be implemented in future fiber-based microfluidic devices.

Coseismic source model of the 2003 Mw 6.8 Chengkung earthquake, Taiwan, determined from GPS measurements

USGS Publications Warehouse

Ching, K.-E.; Rau, R.-J.; Zeng, Y.

2007-01-01

A coseismic source model of the 2003 Mw 6.8 Chengkung, Taiwan, earthquake was well determined with 213 GPS stations, providing a unique opportunity to study the characteristics of coseismic displacements of a high-angle buried reverse fault. Horizontal coseismic displacements show fault-normal shortening across the fault trace. Displacements on the hanging wall reveal fault-parallel and fault-normal lengthening. The largest horizontal and vertical GPS displacements reached 153 and 302 mm, respectively, in the middle part of the network. Fault geometry and slip distribution were determined by inverting GPS data using a three-dimensional (3-D) layered-elastic dislocation model. The slip is mainly concentrated within a 44 ?? 14 km slip patch centered at 15 km depth with peak amplitude of 126.6 cm. Results from 3-D forward-elastic model tests indicate that the dome-shaped folding on the hanging wall is reproduced with fault dips greater than 40??. Compared with the rupture area and average slip from slow slip earthquakes and a compilation of finite source models of 18 earthquakes, the Chengkung earthquake generated a larger rupture area and a lower stress drop, suggesting lower than average friction. Hence the Chengkung earthquake seems to be a transitional example between regular and slow slip earthquakes. The coseismic source model of this event indicates that the Chihshang fault is divided into a creeping segment in the north and the locked segment in the south. An average recurrence interval of 50 years for a magnitude 6.8 earthquake was estimated for the southern fault segment. Copyright 2007 by the American Geophysical Union.

Complex rupture mechanism and topography control symmetry of mass-wasting pattern, 2010 Haiti earthquake

NASA Astrophysics Data System (ADS)

Gorum, Tolga; van Westen, Cees J.; Korup, Oliver; van der Meijde, Mark; Fan, Xuanmei; van der Meer, Freek D.

2013-02-01

The 12 January 2010 Mw 7.0 Haiti earthquake occurred in a complex deformation zone at the boundary between the North American and Caribbean plates. Combined geodetic, geological and seismological data posited that surface deformation was driven by rupture on the Léogâne blind thrust fault, while part of the rupture occurred as deep lateral slip on the Enriquillo-Plantain Garden Fault (EPGF). The earthquake triggered > 4490 landslides, mainly shallow, disrupted rock falls, debris-soil falls and slides, and a few lateral spreads, over an area of ~ 2150 km2. The regional distribution of these slope failures defies those of most similar earthquake-triggered landslide episodes reported previously. Most of the coseismic landslides did not proliferate in the hanging wall of the main rupture, but clustered instead at the junction of the blind Léogâne and EPGF ruptures, where topographic relief and hillslope steepness are above average. Also, low-relief areas subjected to high coseismic uplift were prone to lesser hanging wall slope instability than previous studies would suggest. We argue that a combined effect of complex rupture dynamics and topography primarily control this previously rarely documented landslide pattern. Compared to recent thrust fault-earthquakes of similar magnitudes elsewhere, we conclude that lower static stress drop, mean fault displacement, and blind ruptures of the 2010 Haiti earthquake resulted in fewer, smaller, and more symmetrically distributed landslides than previous studies would suggest. Our findings caution against overly relying on across-the-board models of slope stability response to seismic ground shaking.

Preparation of crystals for characterizing the Grb7 SH2 domain before and after complex formation with a bicyclic peptide antagonist.

PubMed

Ambaye, Nigus D; Gunzburg, Menachem J; Traore, Daouda A K; Del Borgo, Mark P; Perlmutter, Patrick; Wilce, Matthew C J; Wilce, Jacqueline A

2014-02-01

Human growth factor receptor-bound protein 7 (Grb7) is an adapter protein involved in cell growth, migration and proliferation. It is now recognized that Grb7 is an emerging therapeutic target in specific cancer subtypes. Recently, the discovery of a bicyclic peptide inhibitor that targets the Grb7 SH2 domain, named G7-B1, was reported. In an attempt to probe the foundation of its interaction with Grb7, the crystallization and preliminary data collection of both the apo and G7-B1-bound forms of the Grb7 SH2 domain are reported here. Diffraction-quality crystals were obtained using the hanging-drop vapour-diffusion method. After several rounds of microseeding, crystals of the apo Grb7 SH2 domain were obtained that diffracted to 1.8 Å resolution, while those of the G7-B1-Grb7 SH2 domain complex diffracted to 2.2 Å resolution. The apo Grb7 SH2 domain crystallized in the trigonal space group P63, whereas the G7-B1-Grb7 SH2 domain complex crystallized in the monoclinic space group P21. The experimental aspects of crystallization, crystal optimization and data collection and the preliminary data are reported.

Purification, crystallization and preliminary X-ray diffraction studies on avian haemoglobin from pigeon (Columba livia).

PubMed

Sathya Moorthy, Pon; Neelagandan, K; Balasubramanian, M; Ponnuswamy, M N

2009-02-01

Haemoglobin is a physiologically significant metalloprotein that is involved in the exchange of gases for sustaining life. The respiratory system of birds is unique and complex compared with that of mammals. Many investigations of avian haemoglobins have revealed the presence of inositol pentaphosphate (IP5), a principal allosteric effector that is involved in regulation of their function. Structural investigations of avian haemoglobins are presently not adequate to explain their function. Efforts have been made in this direction in order to understand the oxygen-binding affinity involved in adapting to hypoxia in avian haemoglobins. Fresh whole blood was collected from pigeon (Columba livia) and purified using a DEAE cellulose anion-exchange chromatographic column. Crystallization of pigeon haemoglobin was accomplished using the hanging-drop vapour-diffusion method using PEG 3350 as a precipitant in 50 mM sodium acetate buffer pH 5.5 with 1 M NaCl. Data collection was carried out using a MAR345 image-plate detector system. The crystals diffracted to 2 A resolution. Pigeon haemoglobin crystallizes in a triclinic space group, with two whole biological molecules in the asymmetric unit and with unit-cell parameters a = 55.005, b = 65.528, c = 104.370 A, alpha = 78.742, beta = 89.819, gamma = 65.320 degrees .

Crystallization and preliminary X-ray diffraction analysis of the arginine repressor of the hyperthermophile Thermotoga neapolitana

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

Massant, Jan, E-mail: jan.massant@vub.ac.be; Peeters, Eveline; Charlier, Daniel

2006-01-01

The arginine repressor of the hyperthermophile T. neapolitana was crystallized with and without its corepressor arginine. Both crystals diffracted to high resolution and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with similar unit-cell parameters. The arginine repressor of Thermotoga neapolitana (ArgRTnp) is a member of the family of multifunctional bacterial arginine repressors involved in the regulation of arginine metabolism. This hyperthermophilic repressor shows unique DNA-binding features that distinguish it from its homologues. ArgRTnp exists as a homotrimeric protein that assembles into hexamers at higher protein concentrations and/or in the presence of arginine. ArgRTnp was crystallized with andmore » without its corepressor arginine using the hanging-drop vapour-diffusion method. Crystals of the aporepressor diffracted to a resolution of 2.1 Å and belong to the orthorhombic P2{sub 1}2{sub 1}2{sub 1} space group, with unit-cell parameters a = 117.73, b = 134.15, c = 139.31 Å. Crystals of the repressor in the presence of its corepressor arginine diffracted to a resolution of 2.4 Å and belong to the same space group, with similar unit-cell parameters.« less

Physiologically Low Oxygen Enhances Biomolecule Production and Stemness of Mesenchymal Stem Cell Spheroids

PubMed Central

Shearier, Emily; Xing, Qi; Qian, Zichen

2016-01-01

Multicellular human mesenchymal stem cell (hMSC) spheroids have been demonstrated to be valuable in a variety of applications, including cartilage regeneration, wound healing, and neoangiogenesis. Physiological relevant low oxygen culture can significantly improve in vitro hMSC expansion by preventing cell differentiation. We hypothesize that hypoxia-cultured hMSC spheroids can better maintain the regenerative properties of hMSCs. In this study, hMSC spheroids were fabricated using hanging drop method and cultured under 2% O2 and 20% O2 for up to 96 h. Spheroid diameter and viability were examined, as well as extracellular matrix (ECM) components and growth factor levels between the two oxygen tensions at different time points. Stemness was measured among the spheroid culture conditions and compared to two-dimensional cell cultures. Spheroid viability and structural integrity were studied using different needle gauges to ensure no damage would occur when implemented in vivo. Spheroid attachment and integration within a tissue substitute were also demonstrated. The results showed that a three-dimensional hMSC spheroid cultured at low oxygen conditions can enhance the production of ECM proteins and growth factors, while maintaining the spheroids' stemness and ability to be injected, attached, and potentially be integrated within a tissue. PMID:26830500

Purification, crystallization and preliminary X-ray characterization of prunin-1, a major component of the almond (Prunus dulcis) allergen amandin.

PubMed

Albillos, Silvia M; Jin, Tengchuan; Howard, Andrew; Zhang, Yuzhu; Kothary, Mahendra H; Fu, Tong-Jen

2008-07-09

The 11S globulins from plant seeds account for a number of major food allergens. Because of the interest in the structural basis underlying the allergenicity of food allergens, we sought to crystallize the main 11S seed storage protein from almond ( Prunus dulcis). Prunin-1 (Pru1) was purified from defatted almond flour by water extraction, cryoprecipitation, followed by sequential anion exchange, hydrophobic interaction, and size exclusion chromatography. Single crystals of Pru1 were obtained in a screening with a crystal screen kit, using the hanging-drop vapor diffusion method. Diffraction quality crystals were grown after optimization. The Pru1 crystals diffracted to at least 3.0 A and belong to the tetragonal space group P4(1)22, with unit cell parameters of a = b = 150.912 A, c = 165.248 A. Self-rotation functions and molecular replacement calculations showed that there are three molecules in the asymmetry unit with water content of 51.41%. The three Pru1 protomers are related by a noncrystallographic 3-fold axis and they form a doughnut-shaped trimer. Two prunin trimers form a homohexamer. Elucidation of prunin structure will allow further characterization of the allergenic features of the 11S protein allergens at the molecular level.

Crystallization and preliminary X-ray analysis of cryptolepain, a novel glycosylated serine protease from Cryptolepis buchanani

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

Pande, Monu; Dubey, Vikash K.; Jagannadham, Medicherla V., E-mail: vdubey@iitg.ernet.in

2007-02-01

Cryptolepain is a stable glycosylated novel serine protease was crystallized by hanging-drop method. Crystal data was processed up to 2.25 Å with acceptable statistics and structure determination of the enzyme is under way. Cryptolepain is a stable glycosylated novel serine protease purified from the latex of the medicinally important plant Cryptolepis buchanani. The molecular weight of the enzyme is 50.5 kDa, as determined by mass spectrometry. The sequence of the first 15 N-terminal resides of the protease showed little homology with those of other plant serine proteases, suggesting it to be structurally unique. Thus, it is of interest to solvemore » the structure of the enzyme in order to better understand its structure–function relationship. X-ray diffraction data were collected from a crystal of cryptolepain and processed to 2.25 Å with acceptable statistics. The crystals belong to the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 81.78, b = 108.15, c = 119.86 Å. The Matthews coefficient was 2.62 Å{sup 3} Da{sup −1} with one molecule in the asymmetric unit. The solvent content was found to be 53%. Structure determination of the enzyme is under way.« less

A High Proliferation Rate is Critical for Reproducible and Standardized Embryoid Body Formation from Laminin-521-Based Human Pluripotent Stem Cell Cultures.

PubMed

Dziedzicka, Dominika; Markouli, Christina; Barbé, Lise; Spits, Claudia; Sermon, Karen; Geens, Mieke

2016-12-01

When aiming for homogenous embryoid body (EB) differentiation, the use of equal-sized EBs is required to avoid a size-induced differentiation bias. In this study we developed an efficient and standardized EB formation protocol for human pluripotent stem cells (hPSC) cultured in a laminin-521-based xeno-free system. As the cell proliferation rate of the cells growing on laminin-521 strongly affected the efficiency of aggregate formation, we found that recently passaged cells, as well as the addition of ROCK inhibitor, were essential for reproducible EB formation from hPSC single-cell suspensions. EBs could be obtained in a variety of differentiation media, in 96-well round-bottom plates and in hanging drops. Gene expression studies on differentially sized EBs from three individual human embryonic stem cell lines demonstrated that the medium used for differentiation influenced the differentiation outcome to a much greater extent than the number of cells used for the initial EB formation. Our findings give a new insight into factors that influence the EB formation and differentiation process. This optimized method allows us to easily manipulate EB formation and provide an excellent starting point for downstream EB-based differentiation protocols.

Biodynamic imaging for phenotypic profiling of three-dimensional tissue culture

PubMed Central

Sun, Hao; Merrill, Daniel; An, Ran; Turek, John; Matei, Daniela; Nolte, David D.

2017-01-01

Abstract. Three-dimensional (3-D) tissue culture represents a more biologically relevant environment for testing new drugs compared to conventional two-dimensional cancer cell culture models. Biodynamic imaging is a high-content 3-D optical imaging technology based on low-coherence interferometry and digital holography that uses dynamic speckle as high-content image contrast to probe deep inside 3-D tissue. Speckle contrast is shown to be a scaling function of the acquisition time relative to the persistence time of intracellular transport and hence provides a measure of cellular activity. Cellular responses of 3-D multicellular spheroids to paclitaxel are compared among three different growth techniques: rotating bioreactor (BR), hanging-drop (HD), and nonadherent (U-bottom, UB) plate spheroids, compared with ex vivo living tissues. HD spheroids have the most homogeneous tissue, whereas BR spheroids display large sample-to-sample variability as well as spatial heterogeneity. The responses of BR-grown tumor spheroids to paclitaxel are more similar to those of ex vivo biopsies than the responses of spheroids grown using HD or plate methods. The rate of mitosis inhibition by application of taxol is measured through tissue dynamics spectroscopic imaging, demonstrating the ability to monitor antimitotic chemotherapy. These results illustrate the potential use of low-coherence digital holography for 3-D pharmaceutical screening applications. PMID:28301634

Crystallization and preliminary X-ray analysis of binary and ternary complexes of Haloferax mediterranei glucose dehydrogenase

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

Esclapez, Julia; Britton, K. Linda; Baker, Patrick J.

2005-08-01

Single crystals of binary and ternary complexes of wild-type and D38C mutant H. mediterranei glucose dehydrogenase have been obtained by the hanging-drop vapour-diffusion method. Haloferax mediterranei glucose dehydrogenase (EC 1.1.1.47) belongs to the medium-chain alcohol dehydrogenase superfamily and requires zinc for catalysis. In the majority of these family members, the catalytic zinc is tetrahedrally coordinated by the side chains of a cysteine, a histidine, a cysteine or glutamate and a water molecule. In H. mediterranei glucose dehydrogenase, sequence analysis indicates that the zinc coordination is different, with the invariant cysteine replaced by an aspartate residue. In order to analyse themore » significance of this replacement and to contribute to an understanding of the role of the metal ion in catalysis, a range of binary and ternary complexes of the wild-type and a D38C mutant protein have been crystallized. For most of the complexes, crystals belonging to space group I222 were obtained using sodium/potassium citrate as a precipitant. However, for the binary and non-productive ternary complexes with NADPH/Zn, it was necessary to replace the citrate with 2-methyl-2,4-pentanediol. Despite the radical change in conditions, the crystals thus formed were isomorphous.« less

Biodynamic imaging for phenotypic profiling of three-dimensional tissue culture

NASA Astrophysics Data System (ADS)

Sun, Hao; Merrill, Daniel; An, Ran; Turek, John; Matei, Daniela; Nolte, David D.

2017-01-01

Three-dimensional (3-D) tissue culture represents a more biologically relevant environment for testing new drugs compared to conventional two-dimensional cancer cell culture models. Biodynamic imaging is a high-content 3-D optical imaging technology based on low-coherence interferometry and digital holography that uses dynamic speckle as high-content image contrast to probe deep inside 3-D tissue. Speckle contrast is shown to be a scaling function of the acquisition time relative to the persistence time of intracellular transport and hence provides a measure of cellular activity. Cellular responses of 3-D multicellular spheroids to paclitaxel are compared among three different growth techniques: rotating bioreactor (BR), hanging-drop (HD), and nonadherent (U-bottom, UB) plate spheroids, compared with ex vivo living tissues. HD spheroids have the most homogeneous tissue, whereas BR spheroids display large sample-to-sample variability as well as spatial heterogeneity. The responses of BR-grown tumor spheroids to paclitaxel are more similar to those of ex vivo biopsies than the responses of spheroids grown using HD or plate methods. The rate of mitosis inhibition by application of taxol is measured through tissue dynamics spectroscopic imaging, demonstrating the ability to monitor antimitotic chemotherapy. These results illustrate the potential use of low-coherence digital holography for 3-D pharmaceutical screening applications.

Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment

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

McKinstry, William J.; Polekhina, Galina; Diefenbach-Jagger, Hannelore

Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belongedmore » to the orthorhombic space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 {angstrom}, and diffracted to 2.0 {angstrom} resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.« less

Purification of a Multidrug Resistance Transporter for Crystallization Studies

PubMed Central

Alegre, Kamela O.; Law, Christopher J.

2015-01-01

Crystallization of integral membrane proteins is a challenging field and much effort has been invested in optimizing the overexpression and purification steps needed to obtain milligram amounts of pure, stable, monodisperse protein sample for crystallography studies. Our current work involves the structural and functional characterization of the Escherichia coli multidrug resistance transporter MdtM, a member of the major facilitator superfamily (MFS). Here we present a protocol for isolation of MdtM to increase yields of recombinant protein to the milligram quantities necessary for pursuit of structural studies using X-ray crystallography. Purification of MdtM was enhanced by introduction of an elongated His-tag, followed by identification and subsequent removal of chaperonin contamination. For crystallization trials of MdtM, detergent screening using size exclusion chromatography determined that decylmaltoside (DM) was the shortest-chain detergent that maintained the protein in a stable, monodispersed state. Crystallization trials of MdtM performed using the hanging-drop diffusion method with commercially available crystallization screens yielded 3D protein crystals under several different conditions. We contend that the purification protocol described here may be employed for production of high-quality protein of other multidrug efflux members of the MFS, a ubiquitous, physiologically and clinically important class of membrane transporters. PMID:27025617

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

G Deb; K Boeshanes; W Idler

Lipoxygenases are a family of nonheme iron-containing dioxygenases. An Escherichia coli expression system producing the bacterial chaperones GroES and GroEL was engineered and successfully used to produce large quantities of recombinant human 12R-LOX (LOXR; MW 80.34 kDa; 701 amino-acid residues). The co-overproduction of the two chaperones with 12R-LOX resulted in increased solubility of 12R-LOX and allowed the purification of milligram amounts of active enzyme for structural studies by X-ray diffraction. The lipoxygenase protein was purified on an affinity column and a gel-filtration column with chaperone protein (MW 57.16 kDa). The LOXR-chaperone complex was crystallized with ligand by the hanging-drop vapor-diffusionmore » method using 1.5 M ammonium hydrogen phosphate as precipitant. The crystals belonged to the monoclinic system, space group P2{sub 1}, with unit-cell parameters a = 138.97, b = 266.11, c = 152.26 {angstrom}, {beta} = 101.07{sup o}. Based on the calculated Matthews coefficient (3.1 {angstrom}3 Da{sup -1}), it is estimated that one molecule of LOXR complexed with two molecules of chaperone is present in the asymmetric unit of the crystal lattice. X-ray diffraction data were collected to 4 {angstrom} resolution using synchrotron radiation.« less

A square-wave adsorptive stripping voltammetric method for the determination of Amaranth, a food additive dye.

PubMed

Alghamdi, Ahmad H

2005-01-01

Square-wave adsorptive stripping voltammetric (AdSV) determinations of trace concentrations of the azo coloring agent Amaranth are described. The analytical methodology used was based on the adsorptive preconcentration of the dye on the hanging mercury drop electrode, followed by initiation of a negative sweep. In a pH 10 carbonate supporting electrolyte, Amaranth gave a well-defined and sensitive AdSV peak at -518 mV. The electroanalytical determination of this azo dye was found to be optimal in carbonate buffer (pH 10) under the following experimental conditions: accumulation time, 120 s; accumulation potential, 0.0 V; scan rate, 600 mV/s; pulse amplitude, 90 mV; and frequency, 50 Hz. Under these optimized conditions the AdSV peak current was proportional over the concentration range 1 x 10(-8)-1.1 x 10(-7) mol/L (r = 0.999) with a detection limit of 1.7 x 10(-9) mol/L (1.03 ppb). This analytical approach possessed enhanced sensitivity, compared with conventional liquid chromatography or spectrophotometry and it was simple and fast. The precision of the method, expressed as the relative standard deviation, was 0.23%, whereas the accuracy, expressed as the mean recovery, was 104%. Possible interferences by several substances usually present as food additive azo dyes (E110, E102), gelatin, natural and artificial sweeteners, preservatives, and antioxidants were also investigated. The developed electroanalyticals method was applied to the determination of Amaranth in soft drink samples, and the results were compared with those obtained by a reference spectrophotometric method. Statistical analysis (paired t-test) of these data showed that the results of the 2 methods compared favorably.

Calculation of the bending of electromechanical aircraft element made of the carbon fiber

NASA Astrophysics Data System (ADS)

Danilova-Volkovskaya, Galina; Chepurnenko, Anton; Begak, Aleksandr; Savchenko, Andrey

2017-10-01

We consider a method of calculation of an orthotropic plate with variable thickness. The solution is performed numerically by the finite element method. The calculation is made for the springs of a hang glider made of carbon fiber. The comparison of the results with Sofistik software complex is given.

HangOut: generating clean PSI-BLAST profiles for domains with long insertions.

PubMed

Kim, Bong-Hyun; Cong, Qian; Grishin, Nick V

2010-06-15

Profile-based similarity search is an essential step in structure-function studies of proteins. However, inclusion of non-homologous sequence segments into a profile causes its corruption and results in false positives. Profile corruption is common in multidomain proteins, and single domains with long insertions are a significant source of errors. We developed a procedure (HangOut) that, for a single domain with specified insertion position, cleans erroneously extended PSI-BLAST alignments to generate better profiles. HangOut is implemented in Python 2.3 and runs on all Unix-compatible platforms. The source code is available under the GNU GPL license at http://prodata.swmed.edu/HangOut/. Supplementary data are available at Bioinformatics online.

Suicide Methods in Singapore (2000-2004): Types and Associations

ERIC Educational Resources Information Center

Chia, Boon-Hock; Franzco, Audrey Chia; Ng, Wai-Yee; Tai, Bee-Choo

2011-01-01

The choice of suicide methods varies between countries. Common methods used in Singapore between 2000 and 2004 were jumping (72.4%), hanging (16.6%), and poisoning (5.9%). Those who jumped were more likely to be young, single, female, and to have had a major mental illness. By comparison, those who hung themselves were more likely to be older (OR…

[Hangings in the material of Department of Forensic Medicine, Nicolaus Copernicus University Collegium Medicum in Bydgoszcz, in the years 2000-2010].

PubMed

Sumińska-Ziemann, Barbara; Bloch-Bogusławska, Elzbieta

2013-01-01

This study presents an analysis of suicides based on autopsy protocols from the years 2000-2010. Out of all autopsies conducted at Department of Forensic Medicine, Nicolaus Copernicus University Collegium Medicum in Bydgoszcz, 477 cases resulted from hanging. During this period the majority of hangings were recorded in 2010 (61 cases). The parameters assessed in this study included age, sex, post-mortem blood ethanol concentration and place of death of the victims. The age range of the deceased was similar in the group of men and women. Males represented the majority of victims of hanging (89%); only 11% of all the victims were females. The authors observed differences in blood alcohol level of the victims in association with their sex. Not present ethanol was noted in approx. 42% of men and 71% of women. More than 65% cases of suicide hangings were encountered in urban areas, in living quarters and outbuildings.

Agonal sequences in eight filmed hangings: analysis of respiratory and movement responses to asphyxia by hanging.

PubMed

Sauvageau, Anny; LaHarpe, Romano; Geberth, Vernon J

2010-09-01

It has been proposed that filmed hangings may hold the key to a better understanding of human asphyxia, and The Working Group on Human Asphyxia was formed to systematically review and compare these video recordings. This study analyzed eight filmed hangings. Considering time 0 to represent the onset of the final hanging, rapid loss of consciousness was observed (at 8-18 sec), closely followed by convulsions (at 10-19 sec). A complex pattern of decerebrate rigidity and decorticate rigidity then followed. Between 1 min 38 sec and 2 min 15 sec, muscle tone seemed to be lost, the body becoming progressively flaccid. From then on, isolated body movements were observed from time to time, the last one occurring between 1 min 2 sec and 7 min 31 sec. As for the respiratory responses, all cases presented deep rhythmic abdominal respiratory movements (last one between 1 min 2 sec and 2 min 5 sec). © 2010 American Academy of Forensic Sciences.

Death by hanging while watching violent pornographic videos on the Internet - suicide or accidental autoerotic death?

PubMed

Vennemann, B; Pollak, S

2006-03-01

In deaths by hanging, it may sometimes be difficult to differentiate between autoerotic accident and suicide. Our report deals with a 30-year-old man who was found hanged in the living room of his flat. The deceased was wearing headphones connected to a PC. Within the deceased's view was a computer screen showing the last picture of a video file downloaded from the Internet with the head of an unclothed, allegedly hanged female. The deceased's left hand was inside his trousers in the genital region. The autopsy did not only show findings typical for hanging, but also advanced sarcoidosis, which was known to the victim. Although this basic illness could have been a possible motive for suicide, the circumstances in the presented case pointed more in the direction of an accidental autoerotic death. As far as we know, this is the first description of a death during autoerotic activity in which sexual stimulation was achieved by watching a video file downloaded from the Internet.

Rock Around the Clock. Hanging Rock State Park: An Environmental Education Learning Experience Designed for Grade 5.

ERIC Educational Resources Information Center

Dow, Jaye

This learning packet, one in a series of eight, was developed by the Hanging Roek State Park in North Carolina for grade 5 to teach about the geology of the park. Loose-leaf pages are presented in nine sections that contain: (1) introductions to the North Carolina State Park System, the Hanging Rock State Park, the park's activity packet, and to…

Geometric effects resulting from the asymmetry of dipping fault: Hanging wall/ footwall effects

NASA Astrophysics Data System (ADS)

Wang, Dong; Xie, Li-Li; Hu, Jin-Jun

2008-05-01

Root-mean-square distance D rms with characteristic of weighted-average is introduced in this article firstly. D rms can be used to capture the general proximity of a site to a dipping fault plane comparing with the rupture distance D rup and the seismogenic distance D seis. Then, using D rup, D seis and D rms, the hanging wall/footwall effects on the peak ground acceleration (PGA) during the 1999 Chi-Chi earthquake are evaluated by regression analysis. The logarithm residual shows that the PGA on hanging wall is much greater than that on footwall at the same D rup or D seis when the D rup or D seis is used as site-to-source distance measure. In contrast, there is no significant difference between the PGA on hanging wall and that on footwall at the same D rms when D rms is used. This result confirms that the hanging wall/footwall effect is mainly a geometric effect caused by the asymmetry of dipping fault. Therefore, the hanging wall/footwall effect on the near-fault ground motions can be ignored in the future attenuation analysis if the root-mean-square distance D rms is used as the site-to-source distance measure.

Life history trait analysis of the entomopathogenic nematode Steinernema feltiae provides the basis for prediction of dauer juvenile yields in monoxenic liquid culture.

PubMed

Addis, Temesgen; Teshome, Asmamaw; Strauch, Olaf; Ehlers, Ralf-Udo

2016-05-01

Entomopathogenic nematodes (Steinernema spp.) are used in integrated pest management to control insect pests in cryptic environments. The nematodes are mass produced in monoxenic liquid culture with their symbiotic bacteria Xenorhabdus spp. For a better understanding of nematode population dynamics, the life history traits (LHTs) of the entomopathogenic nematode Steinernema feltiae were assessed at 25 °C by observing single pairs of male and female nematodes using a hanging drop technique. To investigate the influence of different food supplies on nematode reproduction, the LHTs were assessed with a daily supply of 5 ×, 10 × and 20 × 10(9) cells ml(-1) of the nematode's bacterial symbiont Xenorhabdus bovienii in semi-solid nematode growth gelrite (NGG) medium. Increasing bacterial density had a significant positive influence on the average number of offspring produced, which ranged from 359 to 813 per female. The intrinsic rate of natural increase r m, which ranges from 1.10 to 1.19 day(-1), was neither influenced by the bacterial density, nor was the mean generation time T (5.12-5.25 days) and population doubling time (PDT) (0.64-0.59 days). The average lifespan of reproductive females, which ranged from 6.7 to 7.3 days, was positively correlated with bacterial density. A positive correlation between female body volume and bacterial density was recorded (R = 0.67) as well as a significant positive correlation between female body size and offspring production (R = 0.89) in hanging drops. Whether these data can be used to predict nematode yields in liquid culture was tested. The total female body volume calculated as the average female body volume × total number of parental females per millilitre 3 days after nematode inoculation was positively correlated (R = 0.72) with nematode yields. The total female body volume on process day 3 is thus a good indicator for the estimation of nematode yield at the end of the process (12-15 days post dauer juvenile (DJ) inoculation) in both Erlenmeyer flasks and bioreactors. With a mean deviation of 9467 DJs ml(-1), the error resembles approximately 5 % of the final DJ yields.

Sensitive detection of cyclophosphamide using DNA-modified carbon paste, pencil graphite and hanging mercury drop electrodes.

PubMed

Palaska, P; Aritzoglou, E; Girousi, S

2007-05-15

The interaction of cyclophosphamide (CP) with calf thymus double-stranded DNA (dsDNA) and thermally denatured single-stranded DNA (ssDNA) immobilized at the carbon paste (CPE) and pencil graphite electrodes (PGE), was studied electrochemically based on oxidation signals of guanine and adenine using differential pulse voltammetry (DPV). As a result of the interaction of CP with DNA, the voltammetric signals of guanine and adenine increased in the case of dsDNA while a slight increase was observed in ssDNA. The effect of experimental parameters such as the interaction time between CP and DNA forms and the concentration of CP, were studied using DPV with CPE and PGE. Additionally, reproducibility and detection limits were determined using both electrodes. A comparison of the analytical performance between CPE and PGE was done. Our results showed that these two different DNA biosensors could be used for the sensitive, rapid and cost effective detection of CP itself as well as of CP-DNA interaction. Furthermore, the interaction of CP with dsDNA and ssDNA was studied in solution and at the electrode surface by means of alternating current voltammetry (ACV) in 0.3M NaCl and 50mM sodium phosphate buffer (pH 8.5) supporting electrolyte, using a hanging mercury drop electrode (HMDE) as working electrode. The conclusions of this study were mainly based on tensammetric peaks I (at -1.183V) and II (-1.419V) of DNA. This study involved the interaction of CP with surface-confined and solution phase DNA where experimental parameters, such as the concentration of CP and the interaction time, were studied. By increasing the concentration of CP, an increase of peak II was observed in both ds and ssDNA, while an increase of peak I was observed only in the case of dsDNA. An overall conclusion of the study using HMDE was that the interaction of CP with surface-confined DNA significantly differed from that with solution phase DNA. The increase of peaks I and II was lower in the case of interaction of CP with surface-confined DNA, probably due to steric positioning of DNA at the electrode surface.

Neurological Outcomes Following Suicidal Hanging: A Prospective Study of 101 Patients

PubMed Central

Jawaid, Mohammed Turab; Amalnath, S. Deepak; Subrahmanyam, D. K. S.

2017-01-01

Context: Survivors of suicidal hanging can have variable neurological outcomes – from complete recovery to irreversible brain damage. Literature on the neurological outcomes in these patients is confined to retrospective studies and case series. Hence, this prospective study was carried out. Aims: The aim is to study the neurological outcomes in suicidal hanging. Settings and Design: This was a prospective observational study carried out from July 2014 to July 2016. Subjects and Methods: Consecutive patients admitted to the emergency and medicine wards were included in the study. Details of the clinical and radiological findings, course in hospital and at 1 month postdischarge were analyzed. Statistical Analysis Used: Statistical analysis was performed using IBM SPSS advanced statistics 20.0 (SPSS Inc., Chicago, USA). Univariate analysis was performed using Chi-square test for significance and Odd's ratio was calculated. Results: Of the 101 patients, 6 died and 4 had residual neuro deficits. Cervical spine injury was seen in 3 patients. Interestingly, 39 patients could not remember the act of hanging (retrograde amnesia). Hypotension, pulmonary edema, Glasgow coma scale (GCS) score <8 at admission, need for mechanical ventilation, and cerebral edema on plain computed tomography were more in those with amnesia as compared to those with normal memory and these findings were statistically significant. Conclusions: Majority of patients recovered without any sequelae. Routine imaging of cervical spine may not be warranted in all patients, even in those with poor GCS. Retrograde amnesia might be more common than previously believed and further studies are needed to analyze this peculiar feature. PMID:28584409

Diagnosis of Vitality in Skin Wounds in the Ligature Marks Resulting From Suicide Hanging.

PubMed

Legaz Pérez, Isabel; Falcón, Maria; Gimenez, M; Diaz, F Martínez; Pérez-Cárceles, M D; Osuna, E; Nuno-Vieira, D; Luna, A

2017-09-01

Ascertaining the vital origin of skin wounds is one of the most challenging problems in forensic pathology. The forensic literature describes biomarkers and methods for differentiating vital and postmortem wounds, although no clear conclusions have been reached. The aim of this study was to characterize human vital wounds by analyzing the concentrations of metallic ions and the expression of P-selectin and cathepsin D in skin wounds in the ligature marks in a cohort of suicidal hangings for which vitality was previously demonstrated.A total of 71 skin wounds were analyzed within a postmortem interval of 19 to 36 hours. The concentration of Fe, Zn, Mg, and Ca and the expression of P-selectin and cathepsin D were analyzed together and separately. The majority of autopsied suicidal hangings were men (86%) with complete hanging mode (60.7%) in which there was a high frequency of subcutaneous injuries (78.3%). High concentrations of Ca and Mg compared with Fe and Zn were found. Ca and Zn concentrations decreased, and Fe concentration increased with the seriousness of the injury. A high percentage of moderately negative expression of both proteins was correlated with subcutaneous injury and low or medium concentrations of Fe.In conclusion, the joint study of metallic ions and proteins allows to characterize and to differentiate an injured vital wound of noninjured skin, especially when the damage in the tissue affects to the majority of the structures of the skin, but these results will need to be complemented with other biomarkers in time-controlled samples to further help in the differentiation of vital and postmortem wounds.

Langmuir-Blodgett nanotemplates for protein crystallography.

PubMed

Pechkova, Eugenia; Nicolini, Claudio

2017-12-01

The new generation of synchrotrons and microfocused beamlines has enabled great progress in X-ray protein crystallography, resulting in new 3D atomic structures for proteins of high interest to the pharmaceutical industry and life sciences. It is, however, often still challenging to produce protein crystals of sufficient size and quality (order, intensity of diffraction, radiation stability). In this protocol, we provide instructions for performing the Langmuir-Blodgett (LB) nanotemplate method, a crystallization approach that can be used for any protein (including membrane proteins). We describe how to produce highly ordered 2D LB protein monolayers at the air-water interface and deposit them on glass slides. LB-film formation can be observed by surface-pressure measurements and Brewster angle microscopy (BAM), although its quality can be characterized by atomic force microscopy (AFM) and nanogravimetry. Such films are then used as a 2D template for triggering 3D protein crystal formation by hanging-drop vapor diffusion. The procedure for forming the 2D template takes a few minutes. Structural information about the protein reorganization in the LB film during the crystallization process on the nano level can be obtained using an in situ submicron GISAXS (grazing-incidence small-angle X-ray scattering) method. MicroGISAXS spectra, measured directly at the interface of the LB films and protein solution in real time, as described in this protocol, can be interpreted in terms of the buildup of layers, islands, or holes. In our experience, the obtained LB crystals take 1-10 d to prepare and they are more ordered and radiation stable as compared with those produced using other crystallization methods.

Amalgam Electrode-Based Electrochemical Detector for On-Site Direct Determination of Cadmium(II) and Lead(II) from Soils

PubMed Central

Nejdl, Lukas; Kynicky, Jindrich; Brtnicky, Martin; Vaculovicova, Marketa; Adam, Vojtech

2017-01-01

Toxic metal contamination of the environment is a global issue. In this paper, we present a low-cost and rapid production of amalgam electrodes used for determination of Cd(II) and Pb(II) in environmental samples (soils and wastewaters) by on-site analysis using difference pulse voltammetry. Changes in the electrochemical signals were recorded with a miniaturized potentiostat (width: 80 mm, depth: 54 mm, height: 23 mm) and a portable computer. The limit of detection (LOD) was calculated for the geometric surface of the working electrode 15 mm2 that can be varied as required for analysis. The LODs were 80 ng·mL−1 for Cd(II) and 50 ng·mL−1 for Pb(II), relative standard deviation, RSD ≤ 8% (n = 3). The area of interest (Dolni Rozinka, Czech Republic) was selected because there is a deposit of uranium ore and extreme anthropogenic activity. Environmental samples were taken directly on-site and immediately analysed. Duration of a single analysis was approximately two minutes. The average concentrations of Cd(II) and Pb(II) in this area were below the global average. The obtained values were verified (correlated) by standard electrochemical methods based on hanging drop electrodes and were in good agreement. The advantages of this method are its cost and time effectivity (approximately two minutes per one sample) with direct analysis of turbid samples (soil leach) in a 2 M HNO3 environment. This type of sample cannot be analyzed using the classical analytical methods without pretreatment. PMID:28792458

A square-wave adsorptive stripping voltammetric method for determination of fast green dye.

PubMed

Al-Ghamdi, Ali F

2009-01-01

Square-wave adsorptive stripping voltammetric (SW-AdSV) determinations of trace concentrations of the coloring agent fast green were described. The analytical methodology used was based on the adsorptive preconcentration of the dye on the hanging mercury drop electrode, and then a negative sweep was initiated. In pH 10 carbonate supporting electrolyte, fast green gave a well-defined and sensitive SW-AdSV peak at -1220 mV. The electroanalytical determination of this dye was found to be optimized in carbonate buffer (pH 10) with the following experimental conditions: accumulation time (120 s); accumulation potential (-0.8 V); scan rate (800 mV/s); pulse amplitude (90 mV); frequency (90 Hz); surface area of the working electrode (0.6 mm2); and the convection rate (2000 rpm). Under these optimized conditions, the AdSV peak current was proportional over the concentration range 2 x 10(-8) -6 x 10(-7) M (r = 0.999), with an LOD of 1.63 x 10(-10) M (0.132 ppb). This analytical approach possessed more enhanced sensitivity than conventional chromatography or spectrophotometry, and was simple and quick. The precision of the method in terms of RSD was 0.17%, whereas the accuracy was evaluated via the mean recovery of 99.6%. Possible interferences by several substances usually present as food additive azo dyes (E110, E102, E123, and E129), natural and artificial sweeteners, and antioxidants were also investigated. Applicability of the developed electroanalysis method was illustrated via the determination of fast green in ice cream and soft drink samples.

Development of transferrin targeted NCL-240 micelles and their evaluation using in-vitro 3D cancer cell culture (spheroid) models

NASA Astrophysics Data System (ADS)

Nagelli, Srikar Goud

The main objective of this project was to develop targeted micellar delivery systems of a novel cytotoxic drug (NCL-240; a second generation DM-PIT-1 analog) and to evaluate their efficacy using optimized 3D cell culture spheroid models. Spheroids were optimized for several cancer cell lines using a range of techniques such as non-adhesive liquid overlay method, hanging drop method, and co-culturing. Transferrin (Tf)-conjugated NCL-240 micelles were prepared with varying Tf amounts and their cytotoxicities were evaluated using the optimized spheroid models. The uptake and penetration of the formulations were also studied using confocal microscopy. The results indicated that the concentration of NCL-240 micelles required to achieve the same cytotoxicity was relatively higher in spheroids compared to the monolayers. Also, In NCI-ADR-RES, Tf-targeted NCL-240 micelles were shown to have a significant increase in cytotoxicity compared to untargeted NCL-240 micelles. Even the penetration and uptake studies indicated that targeting improves the uptake and penetration of formulations. However, in U87-MG spheroids, there was a significant difference in cell viability among micelles compared to free drug but no significant benefit due to targeting was observed. The same formulations penetrated lesser in U87-MG spheroids compared to NCI-ADR-RES spheroids. This study thereby emphasizes the importance of drug screening in spheroid models as the penetration dynamics are varying from cell line to cell line because of the 3D structure.

Structural analysis using thrust-fault hanging-wall sequence diagrams: Ogden duplex, Wasatch Range, Utah

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

Schirmer, T.W.

1988-05-01

Detailed mapping and cross-section traverses provide the control for structural analysis and geometric modeling of the Ogden duplex, a complex thrust system exposed in the Wasatch Mountains, east of Ogden, Utah. The structures consist of east-dipping folded thrust faults, basement-cored horses, lateral ramps and folds, and tear faults. The sequence of thrusting determined by means of lateral overlap of horses, thrust-splay relationships, and a top-to-bottom piggyback development is Willard thrust, Ogden thrust, Weber thrust, and Taylor thrust. Major decollement zones occur in the Cambrian shales and limestones. The Tintic Quartzite is the marker for determining gross geometries of horses. Thismore » exposed duplex serves as a good model to illustrate the method of constructing a hanging-wall sequence diagram - a series of longitudinal cross sections that move forward in time and space, and show how a thrust system formed as it moved updip over various footwall ramps. A hanging wall sequence diagram also shows the complex lateral variations in a thrust system and helps to locate lateral ramps, lateral folds, tear faults, and other features not shown on dip-oriented cross sections. 8 figures.« less

Liver hanging maneuver for right hemiliver in situ donation – anatomical considerations

PubMed Central

Gadžijev, E.M.; Ravnik, D.; Hribernik, M.

2006-01-01

Background. An anatomical study was carried out to evaluate the safety of the liver hanging maneuver for the right hemiliver in living donor and in situ splitting transplantation. During this procedure a 4–6 cm blind dissection is performed between the inferior vena cava and the liver. Short subhepatic veins entering the inferior vena cava from segments 1 and 9 could be torn with consequent hemorrhage. Materials and methods. One hundred corrosive casts of livers were evaluated to establish the position and diameter of short subhepatic veins and the inferior right hepatic vein. Results. The average distance from the right border of the inferior vena cava to the opening of segment 1 veins was 16.7±3.4 mm and to the entrance of segment 9 veins was 5.0±0.5 mm. The width of the narrowest point on the route of blind dissection was determined, with the average value being 8.7±2.3 mm (range 2–15 mm). Discussion. The results show that the liver hanging maneuver is a safe procedure. A proposed route of dissection minimizes the risk of disrupting short subhepatic veins (7%). PMID:18333236

Self-harm and overcrowding among prisoners in Geneva, Switzerland.

PubMed

Wolff, Hans; Casillas, Alejandra; Perneger, Thomas; Heller, Patrick; Golay, Diane; Mouton, Elisabeth; Bodenmann, Patrick; Getaz, Laurent

2016-01-01

Prison institutional conditions affect risk for self-harm among detainees. In particular, prison overcrowding may increase the likelihood of self-harm by creating competition for resources, space, and enhancing a "deprivation state." The purpose of this paper is to examine the association between overcrowding and prisoner acts of self-harm. This cross-sectional study took place at Geneva's pre-trial prison (capacity:376) between 2006 and 2014. Outcomes were acts of self-harm that required medical attention, and self-strangulation/hanging events (combined into one group, as these are difficult to differentiate). Dichotomous predictors were overcrowding index- annual mean daily population divided by capacity ( > 200 percent vs < 200 percent), and year group (2006-2009 vs 2011-2014). Self-harm and self-strangulations/hangings increased in 2011-2014 compared to 2006-2010 (p < 0.001). Overcrowding in excess of 200 percent was associated with self-strangulation/hangings (p < 0.001) but not with all self-harm events. In terms of pertinent demographics that would affect self-harm, there was no prison change in gender, area of origin, foreign residency, religion, or psychiatric treatment. The present study is limited by the definition and identification of self-harm. The distinction between self-strangulation and self-hanging, and the precise classification of an intent to die is difficult to make in practice, especially with limited prison data records available. The relevant literature addresses the complexity of the association between non-suicidal and suicidal behavior. Despite this, the combined category self-strangulations/hangings gives some indication of severe self-harm events, especially since the methodology of categorization employed was consistent throughout the entire period of the study. Other limitations include the small sample size and the lack of individual patient data and prison data to help control for confounding factors. Despite these drawbacks, pertinent data (socio-demographics and number of prisoners treated for mental health and drug abuse) remained stable over the years. Thus, there are no apparent changes in the inmate population that could be linked to an increase in self-harm. High-security placements and mean prisoner stay have increased over time, with a decrease in staff to prisoner ratio - and these must be looked into further as contributors. Additionally, qualitative methods such as semi-structured interviews and focus groups could delineate the impact of overcrowding on prisoner well-being and self-harm potential. The authors observed a significant increase in self-harm and self-strangulation/hangings over time, and overcrowding was significantly associated with self-strangulation/hangings (but not with all self-harm events). Overcrowding can impose destructive effects on the psychological and behavioral well being of inmates in prison, influencing a myriad of emotional and livelihood factors that predispose to harmful behavior. This report should alert public health and prison authorities to this issue, and garner resources to address such an alarming rise. The findings from this short report demonstrate the need for a further examination of the mechanisms affecting self-harm among prisoners in this population, particularly the relationship between self-strangulations/hangings and overcrowding.

Ex vivo culture of human fetal gonads: manipulation of meiosis signalling by retinoic acid treatment disrupts testis development.

PubMed

Jørgensen, A; Nielsen, J E; Perlman, S; Lundvall, L; Mitchell, R T; Juul, A; Rajpert-De Meyts, E

2015-10-01

What are the effects of experimentally manipulating meiosis signalling by addition of retinoic acid (RA) in cultured human fetal gonads? RA-treatment accelerated meiotic entry in cultured fetal ovary samples, while addition of RA resulted in a dysgenetic gonadal phenotype in fetal testis cultures. One of the first manifestations of sex differentiation is the initiation of meiosis in fetal ovaries. In contrast, meiotic entry is actively prevented in the fetal testis at this developmental time-point. It has previously been shown that RA-treatment mediates initiation of meiosis in human fetal ovary ex vivo. This was a controlled ex vivo study of human fetal gonads treated with RA in 'hanging-drop' tissue cultures. The applied experimental set-up preserves germ cell-somatic niche interactions and the investigated outcomes included tissue integrity and morphology, cell proliferation and survival and the expression of markers of meiosis and sex differentiation. Tissue from 24 first trimester human fetuses was included in this study, all from elective terminations at gestational week (GW) 7-12. Gonads were cultured for 2 weeks with and without addition of 1 µM RA. Samples were subsequently formalin-fixed and investigated by immunohistochemistry and cell counting. Proteins investigated and quantified included; octamer-binding transcription factor 4 (OCT4), transcription factor AP-2 gamma (AP2γ) (embryonic germ cell markers), SRY (sex determining region Y)-box 9 (SOX9), anti-Müllerian hormone (AMH) (immature Sertoli cell markers), COUP transcription factor 2 (COUP-TFII) (marker of interstitial cells), forkhead box L2 (FOXL2) (granulosa cell marker), H2A histone family, member X (γH2AX) (meiosis marker), doublesex and mab-3 related transcription factor 1 (DMRT1) (meiosis regulator), cleaved poly ADP ribose polymerase (PARP), cleaved Caspase 3 (apoptosis markers) and Ki-67 antigen (Ki-67) (proliferation marker). Also, proliferation was determined using a 5'-bromo-2'-deoxyuridine (BrdU) incorporation assay. A novel ex vivo 'hanging-drop' culture model for human fetal gonads was successfully established. Continued proliferation of cells without signs of increased apoptosis was observed after 2 weeks of culture. In cultured fetal ovaries treated with RA, an increased number of meiotic germ cells (P < 0.05) and DMRT1-positive oogonia initiating meiosis (P < 0.05) was observed, which is in agreement with a previous study. In fetal testes, RA-treatment resulted in a decreased number of gonocytes (P < 0.05), a reduced percentage of proliferating gonocytes (P < 0.05), altered expression pattern of the somatic cell markers AMH and COUP-TFII, as well as disrupted seminiferous cord structure and testis morphology. The number of samples included in this study was relatively small due to the limited availability of human fetal tissue. The hanging-drop culture, similarly to other organ culture approaches, allows studies of germ cell-somatic niche interactions and determination of effects after manipulating specific signalling pathways. Our novel finding of disrupted fetal testis development after treatment with RA indicates that abnormal meiosis regulation can potentially cause gonadal dysgenesis. Further studies will elucidate the exact mechanisms and timing of observed effects. This work was supported in part by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S to A.Jø. Additional funding for this project was obtained from The Research Council of the Capital Region of Denmark (E.R.-D.M.), The Research Fund at Rigshospitalet (A.Ju. and J.E.N.), Familien Erichssens Fund (A.Jø.), Dagmar Marshalls Fund (A.Jø.) and Aase & Ejnar Danielsens Fund (A.Jø.). The authors have no conflicts of interest. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Swimming Behaviour of the upside-down swimming Catfish (Synodontis nigriventris) at high-quality Microgravity - a Drop-Tower Experiment

NASA Astrophysics Data System (ADS)

Knie, M.; Hilbig, R.; Anken, R.

The catfish Synodontis nigriventris often shows a unique swimming behaviour in being oriented upside down In the course of a parabolic aircraft flight PF experiment conducted by Ohnishi et al Abstract COSPAR04-A-00961 2004 www cosis net specimens of this species were subjected to diminished gravity and the dorsal light response DLR was tested Usually the DLR is more clearly exhibited by fish in a low-gravity environment since they then need to use visual input as the major or even the sole cue for postural control It was shown by Ohnishi et al 2004 however that S nigriventris did not reveal a DLR during the PF-phases of diminished gravity and it was concluded that the species has a novel balance sensation which does not induce a DLR In the course of an earlier drop-tower ZARM Bremen experiment we had analysed the swimming behaviour of cichlid fish Oreochromis mossambicus at various levels of diminished gravity ranging from 0 009g until 0 3g the animals were housed within a centrifuge during the drop-tower flights with the finding that the residual level of gravity which is usually gained aboard PFs i e 0 03-0 05g is sufficient for most fish of a given batch to maintain a normal postural control Anken Medicine and Mobility 7 18 2005 The vestibular organ of S nigriventris moreover is assumed to be more sensitive than that of O mossambicus due to hanging utricular otoliths in the upside-down posture Thus we hypothesized that the residual gravity aboard PFs might well be sufficient for the catfish to be perceived and

Escapes from herbivory in relation to the structure of mangrove island macroalgal communities.

PubMed

Taylor, Phillip R; Littler, Mark M; Littler, Diane S

1986-07-01

Some shallow habitats that surround mangrove islands exhibit abruptly discontinuous macrophyte boundaries; in other regions, plant distributional patterns are less defined. Where distinct boundaries do occur, fleshy algae predominate on the roots of the red mangrove, Rhizophora mangle, which do not contact the bottom sediments ("hanging roots"), while calcifying algae dominate on the substratum-penetrating roots and banks (=embedded-root habitat) surrounding the mangrove thickets. Considerable natural-history and floristic information reveals that the fleshy hanging-root species are not specialists, for that type of habitat. Experimental transplants showed that on banks and embedded roots where there typically are abundant macroherbivores (particularly sea urchins), most fleshy algae are eliminated.The dominants of the hanging-root habitat (e.g, Acanthophora spicifera, Spyridia filamentosa, Caulerpa racemosa var. Occidentalis) are 6-20 times more susceptible to herbivores than the dominants of the embedded-root habitat (e.g., Halimeda opuntia f. triloba, H. monile). Consequently, we suggest the former are relegated to the spatial refugia from herbivores (=non-coexistence escapes) provided by the hanging roots. Factors associated with these palatability differences include higher average calorific values (6.5 times) of the fleshy hanging-root dominants, greater proportions of organic content (2.6 times) and the general absence of calcification. The dominants of the embedded-root habitat show reduced edibility as a probable consequence of low calorific values, heavy calcification and potential herbivore-detering secondary metabolites. Correlative evidence and preliminary experimental results tentatively indicate that, in the absence of macroherbivores, the hanging-root dominants, which exhibit production rates 4.7 times greater than the dominants of the embedded-root habitat, are better competitors for space.We suggest that variations in herbivory are responsible, in part, for maintaining greater algal diversity in mangrove systems. At a study site with abundant sea urchins, five algal species were found only in the embedded-root habitat three species were confined to the hanging roots, while three others occurred in both. At an urchin-free site, no macrophytes were found only on embedded-root substrata, while one (in trace amounts) was found only on hanging-root habitat and eight occurred in both. We predict that in the absence of herbivores, the species assemblage characteristic of the hanging-roots would exclude many of the dominants from the embedded-root habitat.

Flat-ramp vs. convex-concave thrust geometries in a deformable hanging wall: new insights from analogue modeling experiments

NASA Astrophysics Data System (ADS)

Almeida, Pedro; Tomas, Ricardo; Rosas, Filipe; Duarte, Joao; Terrinha, Pedro

2015-04-01

Different modes of strain accommodation affecting a deformable hanging-wall in a flat-ramp-flat thrust system were previously addressed through several (sandbox) analog modeling studies, focusing on the influence of different variables, such as: a) thrust ramp dip angle and friction (Bonini et al, 2000); b) prescribed thickness of the hanging-wall (Koy and Maillot, 2007); and c) sin-thrust erosion (compensating for topographic thrust edification, e.g. Persson and Sokoutis, 2002). In the present work we reproduce the same experimental procedure to investigate the influence of two different parameters on hanging-wall deformation: 1) the geometry of the thrusting surface; and 2) the absence of a velocity discontinuity (VD) that is always present in previous similar analogue modeling studies. Considering the first variable we use two end member ramp geometries, flat-ramp-flat and convex-concave, to understand the control exerted by the abrupt ramp edges in the hanging-wall stress-strain distribution, comparing the obtain results with the situation in which such edge singularities are absent (convex-concave thrust ramp). Considering the second investigated parameter, our motivation was the recognition that the VD found in the different analogue modeling settings simply does not exist in nature, despite the fact that it has a major influence on strain accommodation in the deformable hanging-wall. We thus eliminate such apparatus artifact from our models and compare the obtained results with the previous ones. Our preliminary results suggest that both investigated variables play a non-negligible role on the structural style characterizing the hanging-wall deformation of convergent tectonic settings were such thrust-ramp systems were recognized. Acknowledgments This work was sponsored by the Fundação para a Ciência e a Tecnologia (FCT) through project MODELINK EXPL/GEO-GEO/0714/2013. Pedro Almeida wants to thank to FCT for the Ph.D. grant (SFRH/BD/52556/2014) under the Doctoral Program EarthSystems in IDL/UL. References Bonini, M., Sokoutis, D., Mulugeta, G., Katrivanos, E. (2000) - Modelling hanging wall accommodation above rigid thrust ramps. Journal of Structural Geology, 22, pp. 1165-1179. Persson, K. & Sokoutis, D (2002) - Analogue models of orogenic wedges controlled by erosion. Tectonophysics, 356, pp. 323- 336. Koy, H. & Bertrand, M. (2007) - Tectonic thickening of hanging-wall units over a ramp.Journal of Structural Geology, 29, pp. 924-932.

Properties of Dental Pulp-derived Mesenchymal Stem Cells and the Effects of Culture Conditions.

PubMed

Kawashima, Nobuyuki; Noda, Sonoko; Yamamoto, Mioko; Okiji, Takashi

2017-09-01

Dental pulp mesenchymal stem cells (DPMSCs) highly express mesenchymal stem cell markers and possess the potential to differentiate into neural cells, osteoblasts, adipocytes, and chondrocytes. Thus, DPMSCs are considered suitable for tissue regeneration. The colony isolation method has commonly been used to collect relatively large amounts of heterogeneous DPMSCs. Homogenous DPMSCs can be isolated by fluorescence-activated cell sorting using antibodies against mesenchymal stem cell markers, although this method yields a limited number of cells. Both quality and quantity of DPMSCs are critical to regenerative therapy, and cell culture methods need to be improved. We thus investigated the properties of DPMSCs cultured with different methods. DPMSCs in a three-dimensional spheroid culture system, which is similar to the hanging drop culture for differentiation of embryonic stem cells, showed upregulation of odonto-/osteoblastic markers and mineralized nodule formation. This suggests that this three-dimensional spheroid culturing system for DPMSCs may be suitable for inducing hard tissues. We further examined the effect of cell culture density on the properties of DPMSCs because the properties of stem cells can be altered depending on the cell density. DPMSCs cultured under the confluent cell density condition showed slight downregulation of some mesenchymal stem cell markers compared with those under the sparse condition. The ability of DPMSCs to differentiate into hard tissue-forming cells was found to be enhanced in the confluent condition, suggesting that the confluent culture condition may not be suitable for maintaining the stemness of DPMSCs. When DPMSCs are to be used for hard tissue regeneration, dense followed by sparse cell culture conditions may be a better alternative strategy. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Geologic history of the Yosemite Valley

USGS Publications Warehouse

Matthes, Francois E.

1930-01-01

Projection of the longitudinal profiles of these hanging valleys forward to the axis of the Merced Canyon shows that they are closely accordant in height. Their profiles indicate a series of points on a former profile of the Merced with respect to which the side streams had graded their courses prior to the last uplift. This old profile can be extended upward into the glaciated part of the Merced Canyon above El Portal and even into the profoundly glaciated Yosemite Valley, accordant points being furnished by a number of hanging side valleys (due allowance being made for glacial erosion suffered by those valleys). However, not all the hanging valleys of the Yosemite region are accordant with this set. Several of them, including the upland valley of Yosemite Creek, constitute a separate set indicating another old profile of the Merced at a level 600 to 1,000 feet higher than the first. Others, including the hanging gulch of lower Bridalveil Creek, point to an old profile of the Merced about 1,200 feet lower than the first. There are thus three distinct sets of hanging valleys produced in three cycles of stream erosion. The valleys of the upper set, like those of the middle set, were left hanging as a result of rapid trenching by the Merced induced by an uplift of the range, there having been two such uplifts. Only the valleys of the lower set hang because of glacial deepening and widening of the Yosemite Valley, the cycle in which they were cut having been interrupted by the advent of the Pleistocene glaciers. They consequently indicate the preglacial depth of the Yosemite Valley. That depth, measured from the brow of El Capitan, was about 2,400 feet; measured from the rim at Glacier Point it was about 2,000 feet.

Thermodynamics of adsorption of dithiocarbamates at the hanging mercury drop.

PubMed

Giannakopoulos, Evangelos; Deligiannakis, Yiannis

2007-02-27

Two dimethyldithiocarbamate (DMDTC) pesticides, thiram and ziram, are adsorbed onto a Hg drop via an entropically driven process. The adsorption isotherms are described by the Frumkin equation. For both molecules, the adsorption is characterized by a nonlinear pseudosigmoid temperature dependence of the Gibbs free energy. For the temperature range of 273-313 K, DeltaGADS varies between -43.4 and -56.71 kJ/mol for thiram and -42.60 and -55.67 kJ/mol for ziram. This variation of DeltaGADS reveals that the adsorption strength is increased at higher temperatures. During the adsorption of either molecule, strong lateral interactions are developed between neighboring adsorbates, which are severely weakened as the temperature increases. A unified reaction scheme is suggested for both ziram and thiram that predicts the formation and adsorption of a surface complex, (DMDTC)2Hg. In the case of thiram, two DMDTC molecules are formed by the cleavage of the disulfide S-S bond near the Hg electrode. The thermodynamic and structural parameters reveal that there are two limiting thermodynamic regimes for the adsorbed (DMDTC)2Hg species that originate from two limiting adsorption conformations of the adsorbates on the Hg surface. A transition occurs between these two conformations at temperatures in the region of 285-295 K. This transition is accompanied by large entropic and enthalpic changes.

Frictional heterogeneities on carbonate-bearing normal faults: Insights from the Monte Maggio Fault, Italy

NASA Astrophysics Data System (ADS)

Carpenter, B. M.; Scuderi, M. M.; Collettini, C.; Marone, C.

2014-12-01

Observations of heterogeneous and complex fault slip are often attributed to the complexity of fault structure and/or spatial heterogeneity of fault frictional behavior. Such complex slip patterns have been observed for earthquakes on normal faults throughout central Italy, where many of the Mw 6 to 7 earthquakes in the Apennines nucleate at depths where the lithology is dominated by carbonate rocks. To explore the relationship between fault structure and heterogeneous frictional properties, we studied the exhumed Monte Maggio Fault, located in the northern Apennines. We collected intact specimens of the fault zone, including the principal slip surface and hanging wall cataclasite, and performed experiments at a normal stress of 10 MPa under saturated conditions. Experiments designed to reactivate slip between the cemented principal slip surface and cataclasite show a 3 MPa stress drop as the fault surface fails, then velocity-neutral frictional behavior and significant frictional healing. Overall, our results suggest that (1) earthquakes may readily nucleate in areas of the fault where the slip surface separates massive limestone and are likely to propagate in areas where fault gouge is in contact with the slip surface; (2) postseismic slip is more likely to occur in areas of the fault where gouge is present; and (3) high rates of frictional healing and low creep relaxation observed between solid fault surfaces could lead to significant aftershocks in areas of low stress drop.

Long-term trends of suicide by choice of method in Norway: a joinpoint regression analysis of data from 1969 to 2012.

PubMed

Puzo, Quirino; Qin, Ping; Mehlum, Lars

2016-03-11

Suicide mortality and the rates by specific methods in a population may change over time in response to concurrent changes in relevant factors in society. This study aimed to identify significant changing points in method-specific suicide mortality from 1969 to 2012 in Norway. Data on suicide mortality by specific methods and by sex and age were retrieved from the Norwegian Cause-of-Death Register. Long-term trends in age-standardized rates of suicide mortality were analyzed by using joinpoint regression analysis. The most frequently used suicide method in the total population was hanging, followed by poisoning and firearms. Men chose suicide by firearms more often than women, whereas poisoning and drowning were more frequently used by women. The joinpoint analysis revealed that the overall trend of suicide mortality significantly changed twice along the period of 1969 to 2012 for both sexes. The male age-standardized suicide rate increased by 3.1% per year until 1989, and decreased by 1.2% per year between 1994 and 2012. Among females the long-term suicide rate increased by 4.0% per year until 1988, decreased by 5.5% through 1995, and then stabilized. Both sexes experienced an upward trend for suicide by hanging during the 44-year observation period, with a particularly significant increase in 15-24 year old males. The most distinct change among men was seen for firearms after 1988 with a significant decrease through 2012 of around 5% per year. For women, significant reductions since 1985-88 were observed for suicide by drowning and poisoning. The present study demonstrates different time trends for different suicide methods with significant reductions in suicide by firearms, drowning and poisoning after the peak in the suicide rate in the late 1980s. Suicide by means of hanging continuously increased, but did not fully compensate for the reduced use of other methods. This lends some support for the effectiveness of method-specific suicide preventive measures, such as restrictions to the access to firearms, which had been implemented in Norway during the relevant time period.

INTERIOR OF COLD STORAGE ROOM, SHOWING MOVABLE HANGING RACKS. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

INTERIOR OF COLD STORAGE ROOM, SHOWING MOVABLE HANGING RACKS. - Naval Air Station Barbers Point, Aircraft Storehouse, Between Midway & Card Streets at Enterprise Avenue intersection, Ewa, Honolulu County, HI

Electrical Properties of the Hanging Wall of the Alpine Fault, New Zealand, from DFDP-2 Wireline Data

NASA Astrophysics Data System (ADS)

Remaud, L.; Doan, M. L.; Pezard, P. A.; Celerier, B. P.; Townend, J.; Sutherland, R.; Toy, V.

2015-12-01

The DFDP-2B borehole drilled at Whataroa, New Zealand, provides a first-hand rare opportunity to investigate the damage pattern next to a major active fault. It was drilled along more than 893 m (820 m TVD) within hanging-wall protomylonites and mylonites. The interval between 264 m and 886 m (measured depth) was intensively investigated by wireline logging. Notably, electrical laterolog data were recorded over almost 3 km of cumulative logs, providing a homogeneous, uniformly sampled recording of the electrical properties of the borehole wall. The laterolog tool measures resistivity with two different electrode configurations, and hence achieves two different depths of penetration. Numerical simulations of the tool's response show that the true resistivity of the rock is close to the deep resistivity measurement, which in DFDP-2 varies between 300 Ω.m and 700 Ω.m. The shallow resistivity is about 75% of this value, as it is more sensitive to the presence of conductive borehole fluid. However, the large borehole diameter (averaging 8.5 inch = or 21.59 cm) only partially explains this value. The strong anisotropy suggested by laboratory measurements on outcrop samples also contributes to the separation between deep and shallow resistivity. The shallow and deep resistivities exhibit many significant drops that are coincident with the presence of fractures detected in borehole televiewer data. More than 700 electrical anomalies have been manually picked. The major ones are correlated with attenuation of the sonic data and sometimes with anomalies in fluid conductivity (temperature and conductivity). Their frequency gradually increases with depth, reaching a plateau below 700 m. This increase with depth may be related to closer proximity to the Alpine Fault.

A weak Galerkin least-squares finite element method for div-curl systems

NASA Astrophysics Data System (ADS)

Li, Jichun; Ye, Xiu; Zhang, Shangyou

2018-06-01

In this paper, we introduce a weak Galerkin least-squares method for solving div-curl problem. This finite element method leads to a symmetric positive definite system and has the flexibility to work with general meshes such as hybrid mesh, polytopal mesh and mesh with hanging nodes. Error estimates of the finite element solution are derived. The numerical examples demonstrate the robustness and flexibility of the proposed method.

MUTAGENIC CHARACTERISTICS OF RIVER WATERS FLOWING THROUGH LARGE METROPOLITAN AREAS IN NORTH AMERICA

EPA Science Inventory

Mutagenic characteristics of river waters flowing through large metropolitan areas in North America

The hanging technique using blue rayon, which specifically adsorbs mutagens with multicyclic planar structures, has the advantages over most conventional methods of not havi...

MUTAGENIC PROPERTIES OF RIVER WATERS FLOWING THROUGH BIG CITY AREAS IN NORTH AMERICA

EPA Science Inventory

The hanging technique using Blue rayon, which specifically adsorbs mutagens with multicyclic planer structures, has advantages over the conventional method of bringing-large volumes of water back to the laboratory for extraction. Therefore, the analysis of many samples from remot...

EAST Multicenter Trial on Targeted Temperature Management for Hanging-Induced Cardiac Arrest.

PubMed

Hsu, Cindy H; Haac, Bryce E; Drake, Mack; Bernard, Andrew C; Aiolfi, Alberto; Inaba, Kenji; Hinson, Holly E; Agarwal, Chinar; Galante, Joseph; Tibbits, Emily M; Johnson, Nicholas J; Carlbom, David; Mirhoseini, Mina F; Patel, Mayur B; OʼBosky, Karen R; Chan, Christian; Udekwu, Pascal O; Farrell, Megan; Wild, Jeffrey L; Young, Katelyn A; Cullinane, Daniel C; Gojmerac, Deborah J; Weissman, Alexandra; Callaway, Clifton; Perman, Sarah M; Guerrero, Mariana; Aisiku, Imoigele P; Seethala, Raghu R; Co, Ivan N; Madhok, Debbie Y; Darger, Bryan; Kim, Dennis Y; Spence, Lara; Scalea, Thomas M; Stein, Deborah M

2018-04-19

We sought to determine the outcome of suicidal hanging and the impact of targeted temperature management (TTM) on hanging-induced cardiac arrest (CA) through an Eastern Association for the Surgery of Trauma (EAST) multicenter retrospective study. We analyzed hanging patient data and TTM variables from January 1992 to December 2015. Cerebral performance category (CPC) score of 1 or 2 was considered good neurologic outcome, while CPC of 3 or 4 was considered poor outcome. Classification and Regression Trees (CART) recursive partitioning was used to develop multivariate predictive models for survival and neurological outcome. Total of 692 hanging patients from 17 centers were analyzed for this study. Their overall survival rate was 77%, and the CA survival rate was 28.6%. The CA patients had significantly higher severity of illness and worse outcome than the non-CA patients. Of the 175 CA patients who survived to hospital admission, 81 patients (46.3%) received post-cardiac arrest TTM. The unadjusted survival of TTM CA patients (24.7% vs 39.4%, p<0.05) and good neurologic outcome (19.8% vs 37.2%, p<0.05) were worse than non-TTM CA patients. However, when subgroup analyses were performed between those with admission GCS of 3-8, the differences between TTM and non-TTM CA survival (23.8% vs 30.0%, p=0.37) and good neurologic outcome (18.8% vs 28.7%, p=0.14) were not significant. TTM implementation and post-cardiac arrest management varied between the participating centers. CART models identified variables predictive of favorable and poor outcome for hanging and TTM patients with excellent accuracy. CA hanging patients had worse outcome than non-CA patients. TTM CA patients had worse unadjusted survival and neurologic outcome than non-TTM patients. These findings may be explained by their higher severity of illness, variable TTM implementation, and differences in post-cardiac arrest management. Future prospective studies are necessary to ascertain the effect of TTM on hanging outcome and to validate our CART models. Therapeutic study, level III; prognostic study, level III.

Toxicology findings in cases of hanging in the City and County of San Francisco over the 3-year period from 2011 to 2013.

PubMed

San Nicolas, A C; Lemos, N P

2015-10-01

In postmortem cases where the cause of death is hanging, toxicological analyses may be considered unnecessary by some medical examiners, toxicologists, and other persons involved in medico-legal investigations because the cause of death seems "obvious." To ascertain if toxicological analyses are necessary when the cause of death is hanging, all 102 hanging cases (25 females; 77 males) from 2011 to 2013 that came under the jurisdiction of the San Francisco Office of the Chief Medical Examiner were examined from a total of 3912 sudden, unexpected, or violent death cases in the same period. Suicide was the manner of death in 99 of these cases, with two accidental and one undetermined death. The average age of decedents was 43.9 years (median 41), the youngest was an 11-year old male and the oldest was an 86-year old female. Of the 102 cases, 33 had negative toxicology while 69 cases had at least one positive toxicology result. Females were equally likely to have negative or positive results (12 and 13 cases respectively), but males were 37.5% more likely to have positive toxicology (n=56) rather than negative toxicology (n=21). For females, alcohol, mirtazapine, venlafaxine, and trazodone were the top psychoactive substances in peripheral blood while THC, cocaine, hydrocodone, bupropion, olanzapine, doxylamine, quetiapine and dextromethorphan were also reported. For males, alcohol, THC, cocaine, amphetamine, methamphetamine, bupropion, and diphenhydramine were the top psychoactive substances in blood, but several other drugs were also found in individual cases. Our study of hanging cases over a 3-year period support the idea that complete postmortem toxicology investigation of hangings should be performed, even when the "obvious" cause of death is asphyxia due to hanging. Many of these cases involved psychoactive substances (most often alcohol and cannabis), and having such knowledge provides a better understanding of the circumstances surrounding the decedent's death, their possible state of impairment, including the possibility of a staged suicide if the decedent was too impaired to perform a self-hanging. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation.

PubMed

Chuaychu-Noo, Napapach; Thananurak, Pachara; Chankitisakul, Vibantita; Vongpralub, Thevin

2017-02-01

Little is known about the effects of Cholesterol-Loaded Cyclodextrin (CLC) on post-thaw semen quality in chicken. The aim of the present study is to investigate the efficacy of CLC levels (0, 1, 2 and 3 mg/mL Schramm diluent) on post-thawed semen quality and fertility in two breeds of chicken Pradu Hang Dum (native chicken) and Rhode Island Red. Semen samples of each breed were pooled, divided into 4 aliquots and diluted with Schramm diluents, cooled to 5 °C when DMF was added (6% of final volume). Semen straws were subjected to cryopreservation using the liquid nitrogen vapor method. Post-thawed sperm motility, viability, acrosome integrity, mitochondrial function, and the Malondialdehyde (MDA) level were determined. The fertility of frozen semen was tested by inseminating laying hens. Post-thaw motility between Pradu Hang Dum and Rhode Island Red was no different; but Rhode Island Red had a higher semen viability and live cell intact acrosomes than Pradu Hang Dum (P < 0.05). The percentage of high functioning mitochondria in the Pradu Hang Dum was higher than the Rhode Island Red. CLC at 2 and 3 mg/mL supplementation was associated with improved viability of frozen semen; that is, acrosome integrity and mitochondrial function (P < 0.01), albeit having no effect on MDA levels. The sperm with 1 mg/mL CLC yielded a significantly better fertility (P < 0.01). CLC (1 mg/mL) improved the quality of frozen rooster semen. There was no interaction among breeds and CLC on post-thaw semen quality and fertility. Copyright © 2017 Elsevier Inc. All rights reserved.

37. PATTERNS HANGING FROM CEILING AND OFFICE WALL, NOTE CRAFTSMANSHIP ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

37. PATTERNS HANGING FROM CEILING AND OFFICE WALL, NOTE CRAFTSMANSHIP OF CURVE-LOOKING NORTHWEST. - W. A. Young & Sons Foundry & Machine Shop, On Water Street along Monongahela River, Rices Landing, Greene County, PA

Variations in suicide method and in suicide occurrence by season and day of the week in Russia and the Nenets Autonomous Okrug, Northwestern Russia: a retrospective population-based mortality study.

PubMed

Sumarokov, Yury A; Brenn, Tormod; Kudryavtsev, Alexander V; Nilssen, Odd

2015-09-23

Suicide is an important world health issue, especially in territories inhabited by indigenous people. This investigated differences in suicide rates, suicide methods, and suicide occurrence by month and day of the week among the indigenous and non-indigenous populations of the Nenets Autonomous Okrug (NAO) and to compare the findings from the NAO with national Russian statistics. In this retrospective population-based mortality study we investigated all suicides that occurred in the NAO in 2002-2012 (N = 252). Suicide method and the month and day of the week suicide occurred was taken from autopsy reports and disaggregated by ethnic group (indigenous and non-indigenous) and sex. Data from the NAO were then compared with national data from the Russian Federal Statistics Service (Rosstat). Hanging was the most common suicide method in the NAO in both indigenous and non-indigenous populations. The proportion of suicides by hanging among males was lower in the NAO than in national data (69.3 vs 86.2 %), but the inverse was true for females (86.5 vs 74.9 %). Suicide by firearm and by cutting was significantly higher among the indigenous population in the NAO when compared with national data. Peaks in suicide occurrence were observed in May and September in the NAO, whereas national data showed only one peak in May. Suicide occurrence in the indigenous population of the NAO was highest in April, while the non-indigenous population showed peaks in May and September. Suicide occurrence in the NAO was highest on Fridays; in national data this occurrence was highest on Mondays. We showed different relative frequencies of suicide by hanging, cutting, and firearm, as well as different suicide occurrence by month and day of the week in the NAO compared with Russia as a whole. These results can be used to plan suicide prevention activities in the Russian Arctic.

Purification, crystallization and preliminary X-ray diffraction studies on avian haemoglobin from pigeon (Columba livia)

PubMed Central

Sathya Moorthy, Pon.; Neelagandan, K.; Balasubramanian, M.; Ponnuswamy, M. N.

2009-01-01

Haemoglobin is a physiologically significant metalloprotein that is involved in the exchange of gases for sustaining life. The respiratory system of birds is unique and complex compared with that of mammals. Many investigations of avian haemoglobins have revealed the presence of inositol pentaphosphate (IP5), a principal allosteric effector that is involved in regulation of their function. Structural investigations of avian haemoglobins are presently not adequate to explain their function. Efforts have been made in this direction in order to understand the oxygen-binding affinity involved in adapting to hypoxia in avian haemoglobins. Fresh whole blood was collected from pigeon (Columba livia) and purified using a DEAE cellulose anion-exchange chromatographic column. Crystallization of pigeon haemoglobin was accomplished using the hanging-drop vapour-diffusion method using PEG 3350 as a precipitant in 50 mM sodium acetate buffer pH 5.5 with 1 M NaCl. Data collection was carried out using a MAR345 image-plate detector system. The crystals diffracted to 2 Å resolution. Pigeon haemoglobin crystallizes in a triclinic space group, with two whole biological molecules in the asymmetric unit and with unit-cell parameters a = 55.005, b = 65.528, c = 104.370 Å, α = 78.742, β = 89.819, γ = 65.320°. PMID:19194000

Structural studies of haemoglobin from pisces species shortfin mako shark (Isurus oxyrinchus) at 1.9 Å resolution.

PubMed

Ramesh, Pandian; Sundaresan, S S; Sathya Moorthy, Pon; Balasubramanian, M; Ponnuswamy, M N

2013-11-01

Haemoglobin (Hb) is a tetrameric iron-containing protein that carries oxygen from the lungs to tissues and carbon dioxide from tissues back to the lungs. Pisces are the advanced aquatic vertebrates capable of surviving at wide depth ranges. The shortfin mako shark (SMS) is the pelagic, largest, fastest and most sophisticated species of the shark kingdom with well developed eyes. Mostly the pisces species are cold blooded in nature. Distinctly, the SMSs are warm-blooded animals with an advanced circulatory system. SMSs are capable of maintaining elevated muscle temperatures up to 33 K above the ambient water temperatures at a depth of 150-500 m. SMSs have a diverged air-breathing mechanism compared with other vertebrates. The haemoglobin molecule consists of four polypeptide chains, namely two α chains, each with 140 amino acids and two β chains each having 136 amino acids. The SMS Hb was found to crystallize in monoclinic space group P21 using the hanging-drop vapour-diffusion method at room temperature. The crystal packing parameters for the SMS Hb structure contain one whole biological molecule in the asymmetric unit with a solvent content of 47%. The SMS Hb quaternary structural features interface-interface interactions and heme binding sites are discussed with different state Hbs and the results reveal that SMS Hb adopts an unliganded deoxy T state conformation.

Purification, crystallization and preliminary X-ray analysis of haemoglobin from ostrich (Struthio camelus).

PubMed

Sundaresan, S S; Ramesh, P; Sivakumar, K; Ponnuswamy, M N

2009-07-01

Haemoglobin is a tetrameric protein that carries oxygen from the lungs to tissues and carbon dioxide from tissues back to the lungs. The oxygen-binding properties of haemoglobin are regulated through the binding of allosteric effectors. The respiratory system of avian species is unique and complex in nature when compared with that of mammals. In avian species, inositol pentaphosphate (inositol-P(5)) is present in the erythrocytes of the adult and is thought to be the major factor responsible for the relatively high oxygen affinity of the whole blood. The ostrich (Struthio camelus) is a large flightless bird which contains inositol tetrakisphosphate (inositol-P(4)) in its erythrocytes and its whole blood oxygen affinity is higher. Efforts have been made to explore the structure-function relationship of ostrich haemoglobin. Ostrich haemoglobin was purified using ion-exchange chromatography. Haemoglobin crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant in 50 mM phosphate buffer pH 7.2. Data were collected using a MAR345 image-plate detector system. The crystals of ostrich haemoglobin diffracted to 2.2 A resolution. They belonged to the orthorhombic space group P2(1)2(1)2(1) with one whole biological molecule in the asymmetric unit; the unit-cell parameters were a = 80.93, b = 81.68, c = 102.05 A.

Purification, Crystallization and Preliminary X-ray Characterization of Prunin-1, a Major Component of the Almond (Prunus dulcis) Allergen Amandin

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

Albillos, Silvia M.; Jin, Tengchuan; Howard, Andrew

2008-08-04

The 11S globulins from plant seeds account for a number of major food allergens. Because of the interest in the structural basis underlying the allergenicity of food allergens, we sought to crystallize the main 11S seed storage protein from almond (Prunus dulcis). Prunin-1 (Pru1) was purified from defatted almond flour by water extraction, cryoprecipitation, followed by sequential anion exchange, hydrophobic interaction, and size exclusion chromatography. Single crystals of Pru1 were obtained in a screening with a crystal screen kit, using the hanging-drop vapor diffusion method. Diffraction quality crystals were grown after optimization. The Pru1 crystals diffracted to at least 3.0more » {angstrom} and belong to the tetragonal space group P4{sub 1}22, with unit cell parameters of a = b = 150.912 {angstrom}, c = 165.248 {angstrom}. Self-rotation functions and molecular replacement calculations showed that there are three molecules in the asymmetry unit with water content of 51.41%. The three Pru1 protomers are related by a noncrystallographic 3-fold axis and they form a doughnut-shaped trimer. Two prunin trimers form a homohexamer. Elucidation of prunin structure will allow further characterization of the allergenic features of the 11S protein allergens at the molecular level.« less

Purification, crystallization and preliminary crystallographic studies of plant S-adenosyl-l-homocysteine hydrolase (Lupinus luteus)

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

Brzezinski, Krzysztof; Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan; Bujacz, Grzegorz

2008-07-01

Single crystals of recombinant S-adenosyl-l-homocysteine hydrolase from L. luteus in complex with adenosine diffract X-rays to 1.17 Å resolution at 100 K. The crystals are tetragonal, space group P4{sub 3}2{sub 1}2, and contain one copy of the dimeric enzyme in the asymmetric unit. By degrading S-adenosyl-l-homocysteine, which is a byproduct of S-adenosyl-l-methionine-dependent methylation reactions, S-adenosyl-l-homocysteine hydrolase (SAHase) acts as a regulator of cellular methylation processes. S-Adenosyl-l-homocysteine hydrolase from the leguminose plant yellow lupin (Lupinus luteus), LlSAHase, which is composed of 485 amino acids and has a molecular weight of 55 kDa, has been cloned, expressed in Escherichia coli and purified.more » Crystals of LlSAHase in complex with adenosine were obtained by the hanging-drop vapour-diffusion method using 20%(w/v) PEG 4000 and 10%(v/v) 2-propanol as precipitants in 0.1 M Tris–HCl buffer pH 8.0. The crystals were tetragonal, space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = 122.4, c = 126.5 Å and contained two protein molecules in the asymmetric unit, corresponding to the functional dimeric form of the enzyme. Atomic resolution (1.17 Å) X-ray diffraction data have been collected using synchrotron radiation.« less

Purification, crystallization and preliminary X-ray diffraction analysis of saxthrombin, a thrombin-like enzyme from Gloydius saxatilis venom

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

Wei, Wenqing; Zhao, Wei; Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027

2007-08-01

The thrombin-like enzyme saxthrombin has been purified from G. saxatilis snake venom. Crystallization conditions were found and a data set was obtained to 1.43 Å. The snake-venom thrombin-like enzymes (SVTLEs) are a class of serine proteinases that show fibrinogen-clotting and esterolytic activities. Most TLEs convert fibrinogen to fibrin by releasing either fibrinopeptide A or fibrinopeptide B and cannot activate factor XIII. The enzymes hydrolyze fibrinogen to produce non-cross-linked fibrins, which are susceptible to the lytic action of plasmin. Because of these physiological properties, TLEs have important medical applications in myocardial infarction, ischaemic stroke and thrombotic diseases. Here, a three-step chromatographymore » procedure was used to purify saxthrombin (AAP20638) from Gloydius saxatilis venom to homogeneity. Its molecular weight is about 30 kDa as estimated by SDS–PAGE. A saxthrombin crystal was obtained using the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 1.43 Å. The crystal belongs to space group C2, with unit-cell parameters a = 97.23, b = 52.21, c = 50.10 Å, β = 96.72°, and the Matthews coefficient (V{sub M}) was calculated to be 2.13 Å{sup 3} Da{sup −1} with one molecule in the asymmetric unit.« less