Integrated development of up- and downstream processes supported by the Cherry-Tag™ for real-time tracking of stability and solubility of proteins.

PubMed

Baumann, Pascal; Bluthardt, Nicolai; Renner, Sarah; Burghardt, Hannah; Osberghaus, Anna; Hubbuch, Jürgen

2015-04-20

Product analytics is the bottleneck of most processes in bioprocess engineering, as it is rather time-consuming. Real-time and in-line product tracing without sample pre-treatment is only possible for few products. The Cherry-Tag™ (Delphi Genetics, Belgium) which can be fused to any target protein allows for straightforward product analytics by VIS absorption measurements. When the fused protein becomes unstable or insoluble, the chromophore function of the group is lost, which makes this technology an ideal screening tool for solubility and stability in up- and downstream process development. The Cherry-Tag™ technology will be presented for the tagged enzyme glutathione-S-transferase (GST) from Escherichia coli in a combined up- and downstream process development study. High-throughput cultivations were carried out in a 48-well format in a BioLector system (m2p-Labs, Germany). The best cultivation setup of highest product titer was scaled up to a 2.5L shake flask culture, followed by a selective affinity chromatography product capturing step. In upstream applications the tag was capable of identifying conditions where insoluble and non-native inclusion bodies were formed. In downstream applications the red-colored product was found to be bound effectively to a GST affinity column. Thus, it was identified to be a native and active protein, as the binding mechanism relies on catalytic activity of the enzyme. The Cherry-Tag™ was found to be a reliable and quantitative tool for real-time tracking of stable and soluble proteins in up- and downstream processing applications. Denaturation and aggregation of the product can be detected in-line at any stage of the process. Critical stages can be identified and subsequently changed or replaced. Copyright © 2015 Elsevier B.V. All rights reserved.

The Effect of Backward-Facing Step Height on Instability Growth and Breakdown in Swept Wing Boundary-Layer Transition

NASA Technical Reports Server (NTRS)

Eppink, Jenna L.; Wlezien, Richard W.; King, Rudolph A.; Choudhari, Meelan

2015-01-01

A low-speed experiment was performed on a swept at plate model with an imposed pressure gradient to determine the effect of a backward-facing step on transition in a stationary-cross flow dominated flow. Detailed hot-wire boundary-layer measurements were performed for three backward-facing step heights of approximately 36, 45, and 49% of the boundary-layer thickness at the step. These step heights correspond to a subcritical, nearly-critical, and critical case. Three leading-edge roughness configurations were tested to determine the effect of stationary-cross flow amplitude on transition. The step caused a local increase in amplitude of the stationary cross flow for the two larger step height cases, but farther downstream the amplitude decreased and remained below the baseline amplitude. The smallest step caused a slight local decrease in amplitude of the primary stationary cross flow mode, but the amplitude collapsed back to the baseline case far downstream of the step. The effect of the step on the amplitude of the primary cross flow mode increased with step height, however, the stationary cross flow amplitudes remained low and thus, stationary cross flow was not solely responsible for transition. Unsteady disturbances were present downstream of the step for all three step heights, and the amplitudes increased with increasing step height. The only exception is that the lower frequency (traveling crossflow-like) disturbance was not present in the lowest step height case. Positive and negative spikes in instantaneous velocity began to occur for the two larger step height cases and then grew in number and amplitude downstream of reattachment, eventually leading to transition. The number and amplitude of spikes varied depending on the step height and cross flow amplitude. Despite the low amplitude of the disturbances in the intermediate step height case, breakdown began to occur intermittently and the flow underwent a long transition region.

Flue gas desulfurization

DOEpatents

Im, K.H.; Ahluwalia, R.K.

1984-05-01

The invention involves a combustion process in which combustion gas containing sulfur oxide is directed past a series of heat exchangers to a stack and in which a sodium compound is added to the combustion gas in a temparature zone of above about 1400 K to form Na/sub 2/SO/sub 4/. Preferably, the temperature is above about 1800 K and the sodium compound is present as a vapor to provide a gas-gas reaction to form Na/sub 2/SO/sub 4/ as a liquid. Since liquid Na/sub 2/SO/sub 4/ may cause fouling of heat exchanger surfaces downstream from the combustion zone, the process advantageously includes the step of injecting a cooling gas downstream of the injection of the sodium compound yet upstream of one or more heat exchangers to cool the combustion gas to below about 1150 K and form solid Na/sub 2/SO/sub 4/. The cooling gas is preferably a portion of the combustion gas downstream which may be recycled for cooling. It is further advantageous to utilize an electrostatic precipitator downstream of the heat exchangers to recover the Na/sub 2/SO/sub 4/. It is also advantageous in the process to remove a portion of the combustion gas cleaned in the electrostatic precipitator and recycle that portion upstream to use as the cooling gas. 3 figures.

A flow-through chromatography process for influenza A and B virus purification.

PubMed

Weigel, Thomas; Solomaier, Thomas; Peuker, Alessa; Pathapati, Trinath; Wolff, Michael W; Reichl, Udo

2014-10-01

Vaccination is still the most efficient measure to protect against influenza virus infections. Besides the seasonal wave of influenza, pandemic outbreaks of bird or swine flu represent a high threat to human population. With the establishment of cell culture-based processes, there is a growing demand for robust, economic and efficient downstream processes for influenza virus purification. This study focused on the development of an economic flow-through chromatographic process avoiding virus strain sensitive capture steps. Therefore, a three-step process consisting of anion exchange chromatography (AEC), Benzonase(®) treatment, and size exclusion chromatography with a ligand-activated core (LCC) was established, and tested for purification of two influenza A virus strains and one influenza B virus strain. The process resulted in high virus yields (≥68%) with protein contamination levels fulfilling requirements of the European Pharmacopeia for production of influenza vaccines for human use. DNA was depleted by ≥98.7% for all strains. The measured DNA concentrations per dose were close to the required limits of 10ng DNA per dose set by the European Pharmacopeia. In addition, the added Benzonase(®) could be successfully removed from the product fraction. Overall, the presented downstream process could potentially represent a simple, robust and economic platform technology for production of cell culture-derived influenza vaccines. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel cell autolysis system for cost-competitive downstream processing.

PubMed

Hajnal, Ivan; Chen, Xiangbin; Chen, Guo-Qiang

2016-11-01

The industrial production of low value-added biological products poses significant challenges due to cost pressures. In recent years, it has been argued that synthetic biology approaches will lead to breakthroughs that eliminate price bottlenecks for the production of a wide range of biological products including bioplastics and biofuels. One significant bottleneck lies in the necessity to break the tough cell walls of microbes in order to release intracellular products. We here report the implementation of the first synthetic biology standard part based on the lambda phage SRRz genes and a synthetic ribosome binding site (RBS) that works in Escherichia coli and Halomonas campaniensis, which enables the producer strains to induce lysis after the addition of small amounts (1-5 %) of solvents or to spontaneously lyse during the stresses of downstream processing, and thus has the potential to eliminate the mechanical cell disruption step as both an efficiency bottleneck and a significant capex barrier when implementing downstream bioprocesses.

System and method for reducing combustion dynamics and NO.sub.x in a combustor

DOEpatents

Uhm, Jong H.; Johnson, Thomas Edward

2015-11-20

A system for reducing combustion dynamics and NO.sub.x in a combustor includes a tube bundle that extends radially across at least a portion of the combustor, wherein the tube bundle comprises an upstream surface axially separated from a downstream surface. A shroud circumferentially surrounds the upstream and downstream surfaces. A plurality of tubes extends through the tube bundle from the upstream surface through the downstream surface, wherein the downstream surface is stepped to produce tubes having different lengths through the tube bundle. A method for reducing combustion dynamics and NO.sub.x in a combustor includes flowing a working fluid through a plurality of tubes radially arranged between an upstream surface and a downstream surface of an end cap that extends radially across at least a portion of the combustor, wherein the downstream surface is stepped.

Continuous bind-and-elute protein A capture chromatography: Optimization under process scale column constraints and comparison to batch operation.

PubMed

Kaltenbrunner, Oliver; Diaz, Luis; Hu, Xiaochun; Shearer, Michael

2016-07-08

Recently, continuous downstream processing has become a topic of discussion and analysis at conferences while no industrial applications of continuous downstream processing for biopharmaceutical manufacturing have been reported. There is significant potential to increase the productivity of a Protein A capture step by converting the operation to simulated moving bed (SMB) mode. In this mode, shorter columns are operated at higher process flow and corresponding short residence times. The ability to significantly shorten the product residence time during loading without appreciable capacity loss can dramatically increase productivity of the capture step and consequently reduce the amount of Protein A resin required in the process. Previous studies have not considered the physical limitations of how short columns can be packed and the flow rate limitations due to pressure drop of stacked columns. In this study, we are evaluating the process behavior of a continuous Protein A capture column cycling operation under the known pressure drop constraints of a compressible media. The results are compared to the same resin operated under traditional batch operating conditions. We analyze the optimum system design point for a range of feed concentrations, bed heights, and load residence times and determine achievable productivity for any feed concentration and any column bed height. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:938-948, 2016. © 2016 American Institute of Chemical Engineers.

Disposable world-to-chip interface for digital microfluidics

DOEpatents

Van Dam, R. Michael; Shah, Gaurav; Keng, Pei-Yuin

2017-05-16

The present disclosure sets forth incorporating microfluidic chips interfaces for use with digital microfluidic processes. Methods and devices according to the present disclosure utilize compact, integrated platforms that interface with a chip upstream and downstream of the reaction, as well as between intermediate reaction steps if needed. In some embodiments these interfaces are automated, including automation of a multiple reagent process. Various reagent delivery systems and methods are also disclosed.

Mixed-mode resins: taking shortcut in downstream processing of raw-starch digesting α-amylases

PubMed Central

Lončar, Nikola; Slavić, Marinela Šokarda; Vujčić, Zoran; Božić, Nataša

2015-01-01

Bacillus licheniformis 9945a α-amylase is known as a potent enzyme for raw starch hydrolysis. In this paper, a mixed mode Nuvia cPrime™ resin is examined with the aim to improve the downstream processing of raw starch digesting amylases and exploit the hydrophobic patches on their surface. This resin combines hydrophobic interactions with cation exchange groups and as such the presence of salt facilitates hydrophobic interactions while the ion-exchange groups enable proper selectivity. α-Amylase was produced using an optimized fed-batch approach in a defined media and significant overexpression of 1.2 g L−1 was achieved. This single step procedure enables simultaneous concentration, pigment removal as well as purification of amylase with yields of 96% directly from the fermentation broth. PMID:26492875

Transient Fluvial Response to Alpine Deglaciation, Mount Rainier, WA: Geomorphic Process Domains and Proglacial Flux Controls on Channel Evolution.

NASA Astrophysics Data System (ADS)

Beyeler, J. D.; Montgomery, D.; Kennard, P. M.

2016-12-01

Downwasting of all glaciers on the flanks of Mount Rainier, WA, in recent decades has debuttressed Little Ice Age glaciogenic sediments driving proglacial responses to regionally warming climate. Rivers draining the deglaciating edifice are responding to paraglacial sedimentation processes through transient storage of retreat-liberated sediments in aggrading (e.g., >5m) fluvial networks with widening channel corridors (i.e., 50-150%) post-LIA (ca., 1880-1910 locally). We hypothesize that the downstream transmission of proglacial fluxes (i.e., sediment and water) through deglaciating alpine terrain is a two-step geomorphic process. The ice-proximal portion of the proglacial system is dominated by the delivery of high sediment-to-water ratio flows (i.e., hyperconcentrated and debris slurries) and sediment retention by in-channel accumulation (e.g., confined debris fans within channel margins of valley segments) exacerbated by recruitment and accumulation of large wood (e.g., late seral stage conifers), whereas ice-distal fluvial reworking of transient sediment accumulations generates downstream aggradation. Historical Carbon River observations show restricted ice-proximal proglacial aggradation until a mainstem avulsion in 2009 initiated incision into sediment accumulations formed in recent decades, which is translating into aggradation farther down the network. Surficial morphology mapped with GPS, exposed subsurface sedimentology, and preliminary dating of buried trees suggest a transitional geomorphic process zone has persisted along the proglacial Carbon River through recent centuries and prior to the ultimate LIA glaciation. Structure-from-motion DEM differencing through the 2016 water year shows discrete zones of proglacial evolution through channel-spanning bed aggradation forced by interactions between large wood and sediment-rich flows that transition to fluvial process dominance as sediment is transported downstream. Long-term DEM differencing suggests these are persistent geomorphic processes as rivers respond to alpine deglaciation. This process-based study implies downstream river flooding in deglaciating alpine terrain globally is driven by glaciogenic sediment release and downstream channel aggradation irrespective of changes in discharge.

A sandpile model of grain blocking and consequences for sediment dynamics in step-pool streams

NASA Astrophysics Data System (ADS)

Molnar, P.

2012-04-01

Coarse grains (cobbles to boulders) are set in motion in steep mountain streams by floods with sufficient energy to erode the particles locally and transport them downstream. During transport, grains are often blocked and form width-spannings structures called steps, separated by pools. The step-pool system is a transient, self-organizing and self-sustaining structure. The temporary storage of sediment in steps and the release of that sediment in avalanche-like pulses when steps collapse, leads to a complex nonlinear threshold-driven dynamics in sediment transport which has been observed in laboratory experiments (e.g., Zimmermann et al., 2010) and in the field (e.g., Turowski et al., 2011). The basic question in this paper is if the emergent statistical properties of sediment transport in step-pool systems may be linked to the transient state of the bed, i.e. sediment storage and morphology, and to the dynamics in sediment input. The hypothesis is that this state, in which sediment transporting events due to the collapse and rebuilding of steps of all sizes occur, is analogous to a critical state in self-organized open dissipative dynamical systems (Bak et al., 1988). To exlore the process of self-organization, a cellular automaton sandpile model is used to simulate the processes of grain blocking and hydraulically-driven step collapse in a 1-d channel. Particles are injected at the top of the channel and are allowed to travel downstream based on various local threshold rules, with the travel distance drawn from a chosen probability distribution. In sandpile modelling this is a simple 1-d limited non-local model, however it has been shown to have nontrivial dynamical behaviour (Kadanoff et al., 1989), and it captures the essence of stochastic sediment transport in step-pool systems. The numerical simulations are used to illustrate the differences between input and output sediment transport rates, mainly focussing on the magnification of intermittency and variability in the system response by the processes of grain blocking and step collapse. The temporal correlation in input and output rates and the number of grains stored in the system at any given time are quantified by spectral analysis and statistics of long-range dependence. Although the model is only conceptually conceived to represent the real processes of step formation and collapse, connections will be made between the modelling results and some field and laboratory data on step-pool systems. The main focus in the discussion will be to demonstrate how even in such a simple model the processes of grain blocking and step collapse may impact the sediment transport rates to the point that certain changes in input are not visible anymore, along the lines of "shredding the signals" proposed by Jerolmack and Paola (2010). The consequences are that the notions of stability and equilibrium, the attribution of cause and effect, and the timescales of process and form in step-pool systems, and perhaps in many other fluvial systems, may have very limited applicability.

Sequential lignin depolymerization by combination of biocatalytic and formic acid/formate treatment steps.

PubMed

Gasser, Christoph A; Čvančarová, Monika; Ammann, Erik M; Schäffer, Andreas; Shahgaldian, Patrick; Corvini, Philippe F-X

2017-03-01

Lignin, a complex three-dimensional amorphous polymer, is considered to be a potential natural renewable resource for the production of low-molecular-weight aromatic compounds. In the present study, a novel sequential lignin treatment method consisting of a biocatalytic oxidation step followed by a formic acid-induced lignin depolymerization step was developed and optimized using response surface methodology. The biocatalytic step employed a laccase mediator system using the redox mediator 1-hydroxybenzotriazole. Laccases were immobilized on superparamagnetic nanoparticles using a sorption-assisted surface conjugation method allowing easy separation and reuse of the biocatalysts after treatment. Under optimized conditions, as much as 45 wt% of lignin could be solubilized either in aqueous solution after the first treatment or in ethyl acetate after the second (chemical) treatment. The solubilized products were found to be mainly low-molecular-weight aromatic monomers and oligomers. The process might be used for the production of low-molecular-weight soluble aromatic products that can be purified and/or upgraded applying further downstream processes.

Development of a 3-step straight-through purification strategy combining membrane adsorbers and resins.

PubMed

Hughson, Michael D; Cruz, Thayana A; Carvalho, Rimenys J; Castilho, Leda R

2017-07-01

The pressures to efficiently produce complex biopharmaceuticals at reduced costs are driving the development of novel techniques, such as in downstream processing with straight-through processing (STP). This method involves directly and sequentially purifying a particular target with minimal holding steps. This work developed and compared six different 3-step STP strategies, combining membrane adsorbers, monoliths, and resins, to purify a large, complex, and labile glycoprotein from Chinese hamster ovary cell culture supernatant. The best performing pathway was cation exchange chromatography to hydrophobic interaction chromatography to affinity chromatography with an overall product recovery of up to 88% across the process and significant clearance of DNA and protein impurities. This work establishes a platform and considerations for the development of STP of biopharmaceutical products and highlights its suitability for integration with single-use technologies and continuous production methods. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:931-940, 2017. © 2017 American Institute of Chemical Engineers.

Methods of downstream processing for the production of biodiesel from microalgae.

PubMed

Kim, Jungmin; Yoo, Gursong; Lee, Hansol; Lim, Juntaek; Kim, Kyochan; Kim, Chul Woong; Park, Min S; Yang, Ji-Won

2013-11-01

Despite receiving increasing attention during the last few decades, the production of microalgal biofuels is not yet sufficiently cost-effective to compete with that of petroleum-based conventional fuels. Among the steps required for the production of microalgal biofuels, the harvest of the microalgal biomass and the extraction of lipids from microalgae are two of the most expensive. In this review article, we surveyed a substantial amount of previous work in microalgal harvesting and lipid extraction to highlight recent progress in these areas. We also discuss new developments in the biodiesel conversion technology due to the importance of the connectivity of this step with the lipid extraction process. Furthermore, we propose possible future directions for technological or process improvements that will directly affect the final production costs of microalgal biomass-based biofuels. Copyright © 2013 Elsevier Inc. All rights reserved.

Integrating Sediment Connectivity into Water Resources Management Trough a Graph Theoretic, Stochastic Modeling Framework.

NASA Astrophysics Data System (ADS)

Schmitt, R. J. P.; Castelletti, A.; Bizzi, S.

2014-12-01

Understanding sediment transport processes at the river basin scale, their temporal spectra and spatial patterns is key to identify and minimize morphologic risks associated to channel adjustments processes. This work contributes a stochastic framework for modeling bed-load connectivity based on recent advances in the field (e.g., Bizzi & Lerner, 2013; Czubas & Foufoulas-Georgiu, 2014). It presents river managers with novel indicators from reach scale vulnerability to channel adjustment in large river networks with sparse hydrologic and sediment observations. The framework comprises three steps. First, based on a distributed hydrological model and remotely sensed information, the framework identifies a representative grain size class for each reach. Second, sediment residence time distributions are calculated for each reach in a Monte-Carlo approach applying standard sediment transport equations driven by local hydraulic conditions. Third, a network analysis defines the up- and downstream connectivity for various travel times resulting in characteristic up/downstream connectivity signatures for each reach. Channel vulnerability indicators quantify the imbalance between up/downstream connectivity for each travel time domain, representing process dependent latency of morphologic response. Last, based on the stochastic core of the model, a sensitivity analysis identifies drivers of change and major sources of uncertainty in order to target key detrimental processes and to guide effective gathering of additional data. The application, limitation and integration into a decision analytic framework is demonstrated for a major part of the Red River Basin in Northern Vietnam (179.000 km2). Here, a plethora of anthropic alterations ranging from large reservoir construction to land-use changes results in major downstream deterioration and calls for deriving concerted sediment management strategies to mitigate current and limit future morphologic alterations.

Framework for Integrating Science Data Processing Algorithms Into Process Control Systems

NASA Technical Reports Server (NTRS)

Mattmann, Chris A.; Crichton, Daniel J.; Chang, Albert Y.; Foster, Brian M.; Freeborn, Dana J.; Woollard, David M.; Ramirez, Paul M.

2011-01-01

A software framework called PCS Task Wrapper is responsible for standardizing the setup, process initiation, execution, and file management tasks surrounding the execution of science data algorithms, which are referred to by NASA as Product Generation Executives (PGEs). PGEs codify a scientific algorithm, some step in the overall scientific process involved in a mission science workflow. The PCS Task Wrapper provides a stable operating environment to the underlying PGE during its execution lifecycle. If the PGE requires a file, or metadata regarding the file, the PCS Task Wrapper is responsible for delivering that information to the PGE in a manner that meets its requirements. If the PGE requires knowledge of upstream or downstream PGEs in a sequence of executions, that information is also made available. Finally, if information regarding disk space, or node information such as CPU availability, etc., is required, the PCS Task Wrapper provides this information to the underlying PGE. After this information is collected, the PGE is executed, and its output Product file and Metadata generation is managed via the PCS Task Wrapper framework. The innovation is responsible for marshalling output Products and Metadata back to a PCS File Management component for use in downstream data processing and pedigree. In support of this, the PCS Task Wrapper leverages the PCS Crawler Framework to ingest (during pipeline processing) the output Product files and Metadata produced by the PGE. The architectural components of the PCS Task Wrapper framework include PGE Task Instance, PGE Config File Builder, Config File Property Adder, Science PGE Config File Writer, and PCS Met file Writer. This innovative framework is really the unifying bridge between the execution of a step in the overall processing pipeline, and the available PCS component services as well as the information that they collectively manage.

Development and validation of an affinity chromatography step using a peptide ligand for cGMP production of factor VIII.

PubMed

Kelley, Brian D; Tannatt, Molly; Magnusson, Robert; Hagelberg, Sigrid; Booth, James

2004-08-05

An affinity chromatography step was developed for purification of recombinant B-Domain Deleted Factor VIII (BDDrFVIII) using a peptide ligand selected from a phage display library. The peptide library had variegated residues, contained both within a disulfide bond-constrained ring and flanking the ring. The peptide ligand binds to BDDrFVIII with a dissociation constant of approximately 1 microM both in free solution and when immobilized on a chromatographic resin. The peptide is chemically synthesized and the affinity resin is produced by coupling the peptide to an agarose matrix preactivated with N-hydroxysuccinimide. Coupling conditions were optimized to give consistent and complete ligand incorporation and validated with a robustness study that tested various combinations of processing limits. The peptide affinity chromatographic operation employs conditions very similar to an immunoaffinity chromatography step currently in use for BDDrFVIII manufacture. The process step provides excellent recovery of BDDrFVIII from a complex feed stream and reduces host cell protein and DNA by 3-4 logs. Process validation studies established resin reuse over 26 cycles without changes in product recovery or purity. A robustness study using a factorial design was performed and showed that the step was insensitive to small changes in process conditions that represent normal variation in commercial manufacturing. A scaled-down model of the process step was qualified and used for virus removal studies. A validation package addressing the safety of the leached peptide included leaching rate measurements under process conditions, testing of peptide levels in product pools, demonstration of robust removal downstream by spiking studies, end product testing, and toxicological profiling of the ligand. The peptide ligand affinity step was scaled up for cGMP production of BDDrFVIII for clinical trials.

Channel formation by flow stripping: large-scale scour features along the Monterey East Channel and their relation to sediment waves

USGS Publications Warehouse

Fildani, A.; Normark, W.R.; Kostic, S.; Parker, G.

2006-01-01

The Monterey East system is formed by large-scale sediment waves deposited as a result of flows stripped from the deeply incised Monterey fan valley (Monterey Channel) at the apex of the Shepard Meander. The system is dissected by a linear series of steps that take the form of scour-shaped depressions ranging from 3·5 to 4·5 km in width, 3 to 6 km in length and from 80 to 200 m in depth. These giant scours are aligned downstream from a breech in the levee on the southern side of the Shepard Meander. The floor of the breech is only 150 m above the floor of the Monterey fan valley but more than 100 m below the levee crests resulting in significant flow stripping. Numerical modeling suggests that the steps in the Monterey East system were created by Froude-supercritical turbidity currents stripped from the main flow in the Monterey channel itself. Froude-supercritical flow over an erodible bed can be subject to an instability that gives rise to the formation of cyclic steps, i.e. trains of upstream-migrating steps bounded upstream and downstream by hydraulic jumps in the flow above them. The flow that creates these steps may be net-erosional or net-depositional. In the former case it gives rise to trains of scours such as those in the Monterey East system, and in the latter case it gives rise to the familiar trains of upstream-migrating sediment waves commonly seen on submarine levees. The Monterey East system provides a unique opportunity to introduce the concept of cyclic steps in the submarine environment to study processes that might result in channel initiation on modern submarine fans.

Perfusion mammalian cell culture for recombinant protein manufacturing - A critical review.

PubMed

Bielser, Jean-Marc; Wolf, Moritz; Souquet, Jonathan; Broly, Hervé; Morbidelli, Massimo

The manufacturing of recombinant protein is traditionally divided in two main steps: upstream (cell culture and synthesis of the target protein) and downstream (purification and formulation of the protein into a drug substance or drug product). Today, cost pressure, market uncertainty and market growth, challenge the existing manufacturing technologies. Leaders in the field are active in designing the process of the future and continuous manufacturing is recurrently mentioned as a potential solution to address some of the current limitations. This review focuses on the application of continuous processing to the first step of the manufacturing process. Enabling technologies and operation modes are described in the first part. In the second part, recent advances in the field that have the potential to support its successful future development are critically discussed. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Successive membrane separation processes simplify concentration of lipases produced by Aspergillus niger by solid-state fermentation.

PubMed

Reinehr, Christian Oliveira; Treichel, Helen; Tres, Marcus Vinicius; Steffens, Juliana; Brião, Vandré Barbosa; Colla, Luciane Maria

2017-06-01

In this study, we developed a simplified method for producing, separating, and concentrating lipases derived from solid-state fermentation of agro-industrial residues by filamentous fungi. First, we used Aspergillus niger to produce lipases with hydrolytic activity. We analyzed the separation and concentration of enzymes using membrane separation processes. The sequential use of microfiltration and ultrafiltration processes made it possible to obtain concentrates with enzymatic activities much higher than those in the initial extract. The permeate flux was higher than 60 L/m 2 h during microfiltration using 20- and 0.45-µm membranes and during ultrafiltration using 100- and 50-kDa membranes, where fouling was reversible during the filtration steps, thereby indicating that the fouling may be removed by cleaning processes. These results demonstrate the feasibility of lipase production using A. niger by solid-state fermentation of agro-industrial residues, followed by successive tangential filtration with membranes, which simplify the separation and concentration steps that are typically required in downstream processes.

Effect of Transport and Aging Processes on Metal Speciation in Iron Oxyhydroxide Aggregates, Tar Creek Superfund Site, Oklahoma

NASA Astrophysics Data System (ADS)

Estes, E. R.; Schaider, L. A.; Shine, J. P.; Brabander, D. J.

2010-12-01

Following the cessation of mining activity in the late 20th century, Tar Creek Superfund Site was left highly contaminated by Pb, Zn, and Cd. Tar Creek, which flows through the site and into the Neosho River, has been studied extensively because of its potential to transport metals from the mining site to downstream communities. Previous research identified aggregated iron oxyhydroxide material, which forms when mine seepage mixes with Tar Creek surface water, as a major transport vector of metals. Frequent flooding in Tar Creek deposits aggregates on downstream floodplains, where wetting and drying processes alter the speciation of iron and other metals. This study seeks to better quantify those changes and to determine how transport and aging affects the human and ecological health risk. Sequential extractions of aggregate samples collected from the creek demonstrate that Fe is present in both amorphous (10-35% of Fe extracted) and more crystalline (8-23% of Fe extracted) phases. Substantial portions of heavy metals sorb to amorphous iron oxyhydroxide phases (accounting for 10-30% of Pb and Zn extracted) but are not associated with more crystalline iron oxide phases (representing only 1% or less of the Pb and Zn extracted). Samples have a high organic matter content (18-25% mass loss on ignition), but only Fe was significantly extracted by the oxidizing step targeting organic matter (1-2% of Pb and Zn extracted, but 10-26% of Fe extracted). The majority of metals were extracted by the soluble or residual steps. If metals and organic matter inhibit transformation of amorphous iron oxyhydroxide material to nano and crystalline iron oxides, then a steady-state volume of amorphous iron oxyhydroxide material with a high total sorption capacity may exist within Tar Creek, enhancing the metal flux accommodated by this transport mechanism. Once transported downstream and deposited on floodplains, however, it is hypothesized that repeated changes in soil matrix composition and thermodynamic conditions could facilitate a transformation to more crystalline iron phases and increase metal bioavailability. While preliminary data from in-creek aggregates show no clear trend in mineralogical composition with downstream transport, only the furthest downstream samples have 2-line ferrihydrite in amounts detectable by XRD.

Quantifying the Impact of geographically isolated wetlands on the downstream hydrology of a Canadian Prairie watershed

NASA Astrophysics Data System (ADS)

Muhammad, A.; Evenson, G. R.; Boluwade, A.; Jha, S. K.; Rasmussen, P. F.

2016-12-01

Hydrological processes are highly complex and strongly nonlinear and cannot be represented through simple means. Models are built to replicate these processes. However, models due to various sources of uncertainty including their structural capability often lead to inaccurate results. The aim of this study is to setup the soil water assessment tool (SWAT) for a watershed that is dominated by potholes in the Prairie region of Canada. The potholes not connected to the stream, also known as geographically isolated wetlands (GIWs), are dynamic in nature leading to a fill and spill situation due to varying surface runoff conditions. Significant land use changes have resulted in almost 70% of wetlands being lost and have posed threat of flooding to downstream areas. While some studies were devoted to identify the presence of potholes only few have explored the impacts of wetlands on the downstream hydrology. In this study, we follow Evenson et al., (2016) approach of modifying SWAT model. The modification enhances structural capability of SWAT while depicting the dynamics of wetlands at HRUs level. Redefining the formation of HRUs in such way effectively captures the spatial presence of potholes. We then routed the potholes' fill and spill hydrology to direct the flow to the potholes immediately downstream. The model was calibrated for 2005-2008 and verified over 2009-2011 at a daily time step. We tested our model with three land use change scenarios by varying the presence of potholes and evaluated its impact on the downstream hydrograph. We foresee a significant improvement in replicating stream flow using this novel approach. We believe that it will effectively improve the predictive power of SWAT for this highly complex sub basin (Upper Assiniboine catchment at Kamsack) located in Canadian Prairie.

Utilization of extracellular information before ligand-receptor binding reaches equilibrium expands and shifts the input dynamic range

PubMed Central

Ventura, Alejandra C.; Bush, Alan; Vasen, Gustavo; Goldín, Matías A.; Burkinshaw, Brianne; Bhattacharjee, Nirveek; Folch, Albert; Brent, Roger; Chernomoretz, Ariel; Colman-Lerner, Alejandro

2014-01-01

Cell signaling systems sense and respond to ligands that bind cell surface receptors. These systems often respond to changes in the concentration of extracellular ligand more rapidly than the ligand equilibrates with its receptor. We demonstrate, by modeling and experiment, a general “systems level” mechanism cells use to take advantage of the information present in the early signal, before receptor binding reaches a new steady state. This mechanism, pre-equilibrium sensing and signaling (PRESS), operates in signaling systems in which the kinetics of ligand-receptor binding are slower than the downstream signaling steps, and it typically involves transient activation of a downstream step. In the systems where it operates, PRESS expands and shifts the input dynamic range, allowing cells to make different responses to ligand concentrations so high as to be otherwise indistinguishable. Specifically, we show that PRESS applies to the yeast directional polarization in response to pheromone gradients. Consideration of preexisting kinetic data for ligand-receptor interactions suggests that PRESS operates in many cell signaling systems throughout biology. The same mechanism may also operate at other levels in signaling systems in which a slow activation step couples to a faster downstream step. PMID:25172920

Diatom Milking: A Review and New Approaches

PubMed Central

Vinayak, Vandana; Manoylov, Kalina M.; Gateau, Hélène; Blanckaert, Vincent; Hérault, Josiane; Pencréac’h, Gaëlle; Marchand, Justine; Gordon, Richard; Schoefs, Benoît

2015-01-01

The rise of human populations and the growth of cities contribute to the depletion of natural resources, increase their cost, and create potential climatic changes. To overcome difficulties in supplying populations and reducing the resource cost, a search for alternative pharmaceutical, nanotechnology, and energy sources has begun. Among the alternative sources, microalgae are the most promising because they use carbon dioxide (CO2) to produce biomass and/or valuable compounds. Once produced, the biomass is ordinarily harvested and processed (downstream program). Drying, grinding, and extraction steps are destructive to the microalgal biomass that then needs to be renewed. The extraction and purification processes generate organic wastes and require substantial energy inputs. Altogether, it is urgent to develop alternative downstream processes. Among the possibilities, milking invokes the concept that the extraction should not kill the algal cells. Therefore, it does not require growing the algae anew. In this review, we discuss research on milking of diatoms. The main themes are (a) development of alternative methods to extract and harvest high added value compounds; (b) design of photobioreactors; (c) biodiversity and (d) stress physiology, illustrated with original results dealing with oleaginous diatoms. PMID:25939034

Design and scaleup of downstream processing of monoclonal antibodies for cancer therapy: from research to clinical proof of principle.

PubMed

Horenstein, Alberto L; Crivellin, Federico; Funaro, Ada; Said, Marcela; Malavasi, Fabio

2003-04-01

Murine monoclonal antibodies (mAb) from cell culture supernatants have been purified in order to acquire clinical grade for in vivo cancer treatment. The starting material was purified by high performance liquid chromatography (HPLC) systems ranging from the analytical scale process to a scaleup to 1 g per batch. Three columns (Protein A affinity chromatography with single-step elution, hydroxyapatite (HA) chromatography followed by linear gradient elution and endotoxin removing-gel chromatography), exploiting different properties of the mAb were applied. The final batches of antibody were subjected to a large panel of tests for the purpose of evaluating the efficacy of the downstream processing. The resulting data have allowed us to determine the maximum number of times the column can be used and to precisely and thoroughly characterize antibody integrity, specificity, and potency according to in-house reference standards. The optimized bioprocessing is rapid, efficient, and reproducible. Not less importantly, all the techniques applied are characterized by costs which are affordable to medium-sized laboratories. They represent the basis for implementing immunotherapeutic protocols transferable to clinical medicine.

Deconstruction of Lignin Model Compounds and Biomass-Derived Lignin using Layered Double Hydroxide Catalysts

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

Chmely, S. C.; McKinney, K. A.; Lawrence, K. R.

2013-01-01

Lignin is an underutilized value stream in current biomass conversion technologies because there exist no economic and technically feasible routes for lignin depolymerization and upgrading. Base-catalyzed deconstruction (BCD) has been applied for lignin depolymerization (e.g., the Kraft process) in the pulp and paper industry for more than a century using aqueous-phase media. However, these efforts require treatment to neutralize the resulting streams, which adds significantly to the cost of lignin deconstruction. To circumvent the need for downstream treatment, here we report recent advances in the synthesis of layered double hydroxide and metal oxide catalysts to be applied to the BCDmore » of lignin. These catalysts may prove more cost-effective than liquid-phase, non-recyclable base, and their use obviates downstream processing steps such as neutralization. Synthetic procedures for various transition-metal containing catalysts, detailed kinetics measurements using lignin model compounds, and results of the application of these catalysts to biomass-derived lignin will be presented.« less

Recent Advances and Challenges towards Sustainable Polyhydroxyalkanoate (PHA) Production.

PubMed

Kourmentza, Constantina; Plácido, Jersson; Venetsaneas, Nikolaos; Burniol-Figols, Anna; Varrone, Cristiano; Gavala, Hariklia N; Reis, Maria A M

2017-06-11

Sustainable biofuels, biomaterials, and fine chemicals production is a critical matter that research teams around the globe are focusing on nowadays. Polyhydroxyalkanoates represent one of the biomaterials of the future due to their physicochemical properties, biodegradability, and biocompatibility. Designing efficient and economic bioprocesses, combined with the respective social and environmental benefits, has brought together scientists from different backgrounds highlighting the multidisciplinary character of such a venture. In the current review, challenges and opportunities regarding polyhydroxyalkanoate production are presented and discussed, covering key steps of their overall production process by applying pure and mixed culture biotechnology, from raw bioprocess development to downstream processing.

An Experimental Study of a Separated/Reattached Flow Behind a Backward-Facing Step. Re(sub h) = 37,000

NASA Technical Reports Server (NTRS)

Jovic, Srba

1996-01-01

An experimental study was carried out to investigate turbulent structure of a two-dimensional incompressible separating/reattaching boundary layer behind a backward-facing step. Hot-wire measurement technique was used to measure three Reynolds stresses and higher-order mean products of velocity fluctuations. The Reynolds number, Re(sub h), based on the step height, h, and the reference velocity, U(sub 0), was 37,000. The upstream oncoming flow was fully developed turbulent boundary layer with the Re(sub theta) = 3600. All turbulent properties, such as Reynolds stresses, increase dramatically downstream of the step within an internally developing mixing layer. Distributions of dimensionless mean velocity, turbulent quantities and antisymmetric distribution of triple velocity products in the separated free shear layer suggest that the shear layer above the recirculating region strongly resembles free-shear mixing layer structure. In the reattachment region close to the wall, turbulent diffusion term balances the rate of dissipation since advection and production terms appear to be negligibly small. Further downstream, production and dissipation begin to dominate other transport processes near the wall indicating the growth of an internal turbulent boundary layer. In the outer region, however, the flow still has a memory of the upstream disturbance even at the last measuring station of 51 step-heights. The data show that the structure of the inner layer recovers at a much faster rate than the outer layer structure. The inner layer structure resembles the near-wall structure of a plane zero pressure-gradient turbulent boundary layer (plane TBL) by 25h to 30h, while the outer layer structure takes presumably over 100h.

Escherichia coli as a fatty acid and biodiesel factory: current challenges and future directions.

PubMed

Rahman, Ziaur; Rashid, Naim; Nawab, Javed; Ilyas, Muhammad; Sung, Bong Hyun; Kim, Sun Chang

2016-06-01

Biodiesel has received widespread attention as a sustainable, environment-friendly, and alternative source of energy. It can be derived from plant, animal, and microbial organisms in the form of vegetable oil, fats, and lipids, respectively. However, biodiesel production from such sources is not economically feasible due to extensive downstream processes, such as trans-esterification and purification. To obtain cost-effective biodiesel, these bottlenecks need to be overcome. Escherichia coli, a model microorganism, has the potential to produce biodiesel directly from ligno-cellulosic sugars, bypassing trans-esterification. In this process, E. coli is engineered to produce biodiesel using metabolic engineering technology. The entire process of biodiesel production is carried out in a single microbial cell, bypassing the expensive downstream processing steps. This review focuses mainly on production of fatty acid and biodiesel in E. coli using metabolic engineering approaches. In the first part, we describe fatty acid biosynthesis in E. coli. In the second half, we discuss bottlenecks and strategies to enhance the production yield. A complete understanding of current developments in E. coli-based biodiesel production and pathway optimization strategies would reduce production costs for biofuels and plant-derived chemicals.

Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris.

PubMed

Sreenivas, Suma; Krishnaiah, Sateesh M; Govindappa, Nagaraja; Basavaraju, Yogesh; Kanojia, Komal; Mallikarjun, Niveditha; Natarajan, Jayaprakash; Chatterjee, Amarnath; Sastry, Kedarnath N

2015-01-01

Glargine is an analog of Insulin currently being produced by recombinant DNA technology using two different hosts namely Escherichia coli and Pichia pastoris. Production from E. coli involves the steps of extraction of inclusion bodies by cell lysis, refolding, proteolytic cleavage and purification. In P. pastoris, a single-chain precursor with appropriate disulfide bonding is secreted to the medium. Downstream processing currently involves use of trypsin which converts the precursor into two-chain final product. The use of trypsin in the process generates additional impurities due to presence of Lys and Arg residues in the Glargine molecule. In this study, we describe an alternate approach involving over-expression of endogenous Kex2 proprotein convertase, taking advantage of dibasic amino acid sequence (Arg-Arg) at the end of B-chain of Glargine. KEX2 gene over-expression in Pichia was accomplished by using promoters of varying strengths to ensure production of greater levels of fully functional two-chain Glargine product, confirmed by HPLC and mass analysis. In conclusion, this new production process involving Kex2 protease over-expression improves the downstream process efficiency, reduces the levels of impurities generated and decreases the use of raw materials.

Stereo Particle Image Velocimetry Measurements of Transition Downstream of a Backward-Facing Step in a Swept-Wing Boundary Layer

NASA Technical Reports Server (NTRS)

Eppink, Jenna L.; Yao, Chung-Sheng

2017-01-01

Stereo particle image velocimetry measurements were performed downstream of a backward-facing step in a stationary-cross flow dominated flow. The PIV measurements exhibit excellent quantitative and qualitative agreement with the previously acquired hotwire data. Instantaneous PIV snapshots reveal new information about the nature and cause of the \\spikes" that occurred prior to breakdown in both the hotwire and PIV data. The PIV snapshots show that the events occur simultaneously across multiple stationary cross flow wavelengths, indicating that this is not simply a local event, but is likely caused by the 2D Tollmien-Schlichting instability that is introduced by the step. While the TS instability is a 2D instability, it is also modulated in the spanwise direction due to interactions with the stationary cross flow, as are the other unsteady disturbances present. Because of this modulation, the "spike" events cause an instantaneous increase of the spanwise modulation of the streamwise and spanwise velocity initially caused by the stationary cross flow. Breakdown appears to be caused by this instantaneous modulation, possibly due to a high-frequency secondary instability similar to a traveling-cross flow breakdown scenario. These results further illuminate the respective roles of the stationary cross flow and unsteady disturbances in transition downstream of a backward-facing step.

Very large scale monoclonal antibody purification: the case for conventional unit operations.

PubMed

Kelley, Brian

2007-01-01

Technology development initiatives targeted for monoclonal antibody purification may be motivated by manufacturing limitations and are often aimed at solving current and future process bottlenecks. A subject under debate in many biotechnology companies is whether conventional unit operations such as chromatography will eventually become limiting for the production of recombinant protein therapeutics. An evaluation of the potential limitations of process chromatography and filtration using today's commercially available resins and membranes was conducted for a conceptual process scaled to produce 10 tons of monoclonal antibody per year from a single manufacturing plant, a scale representing one of the world's largest single-plant capacities for cGMP protein production. The process employs a simple, efficient purification train using only two chromatographic and two ultrafiltration steps, modeled after a platform antibody purification train that has generated 10 kg batches in clinical production. Based on analyses of cost of goods and the production capacity of this very large scale purification process, it is unlikely that non-conventional downstream unit operations would be needed to replace conventional chromatographic and filtration separation steps, at least for recombinant antibodies.

recount workflow: Accessing over 70,000 human RNA-seq samples with Bioconductor

PubMed Central

Collado-Torres, Leonardo; Nellore, Abhinav; Jaffe, Andrew E.

2017-01-01

The recount2 resource is composed of over 70,000 uniformly processed human RNA-seq samples spanning TCGA and SRA, including GTEx. The processed data can be accessed via the recount2 website and the recountBioconductor package. This workflow explains in detail how to use the recountpackage and how to integrate it with other Bioconductor packages for several analyses that can be carried out with the recount2 resource. In particular, we describe how the coverage count matrices were computed in recount2 as well as different ways of obtaining public metadata, which can facilitate downstream analyses. Step-by-step directions show how to do a gene-level differential expression analysis, visualize base-level genome coverage data, and perform an analyses at multiple feature levels. This workflow thus provides further information to understand the data in recount2 and a compendium of R code to use the data. PMID:29043067

Overview of several applications of chemical downstream etching (CDE) for IC manufacturing: advantages and drawbacks versus WET processes

NASA Astrophysics Data System (ADS)

de Buttet, Côme; Prevost, Emilie; Campo, Alain; Garnier, Philippe; Zoll, Stephane; Vallier, Laurent; Cunge, Gilles; Maury, Patrick; Massin, Thomas; Chhun, Sonarith

2017-03-01

Today the IC manufacturing faces lots of problematics linked to the continuous down scaling of printed structures. Some of those issues are related to wet processing, which are often used in the IC manufacturing flow for wafer cleaning, material etching and surface preparation. In the current work we summarize the limitations for the next nodes of wet processing such as metallic contaminations, wafer charging, corrosion and pattern collapse. As a replacement, we promoted the isotropic chemical dry etching (CDE) which is supposed to fix all the above drawbacks. Etching steps of SI3N4 layers were evaluated in order to prove the interest of such technique.

The Importance of Splat Events to the Spatiotemporal Structure of Near-Bed Fluid Velocity and Bed Load Motion Over Bed Forms: Laboratory Experiments Downstream of a Backward Facing Step

NASA Astrophysics Data System (ADS)

Leary, K. C. P.; Schmeeckle, M. W.

2017-12-01

Flow separation/reattachment on the lee side of alluvial bed forms is known to produce a complex turbulence field, but the spatiotemporal details of the associated patterns of bed load sediment transported remain largely unknown. Here we report turbulence-resolving, simultaneous measurements of bed load motion and near-bed fluid velocity downstream of a backward facing step in a laboratory flume. Two synchronized high-speed video cameras simultaneously observed bed load motion and the motion of neutrally buoyant particles in a laser light sheet 6 mm above the bed at 250 frames/s downstream of a 3.8 cm backward facing step. Particle Imaging Velocimetry (PIV) and Acoustic Doppler Velocimetry (ADV) were used to characterize fluid turbulent patterns, while manual particle tracking techniques were used to characterize bed load transport. Octant analysis, conducted using ADV data, coupled with Markovian sequence probability analysis highlights differences in the flow near reattachment versus farther downstream. Near reattachment, three distinct flow patterns are apparent. Farther downstream we see the development of a dominant flow sequence. Localized, intermittent, high-magnitude transport events are more apparent near flow reattachment. These events are composed of streamwise and cross-stream fluxes of comparable magnitudes. Transport pattern and fluid velocity data are consistent with the existence of permeable "splat events," wherein a volume of fluid moves toward and impinges on the bed (sweep) causing a radial movement of fluid in all directions around the point of impingement (outward interaction). This is congruent with flow patterns, identified with octant analysis, proximal to flow reattachment.

Has1 regulates consecutive maturation and processing steps for assembly of 60S ribosomal subunits

PubMed Central

Dembowski, Jill A.; Kuo, Benjamin; Woolford, John L.

2013-01-01

Ribosome biogenesis requires ∼200 assembly factors in Saccharomyces cerevisiae. The pre-ribosomal RNA (rRNA) processing defects associated with depletion of most of these factors have been characterized. However, how assembly factors drive the construction of ribonucleoprotein neighborhoods and how structural rearrangements are coupled to pre-rRNA processing are not understood. Here, we reveal ATP-independent and ATP-dependent roles of the Has1 DEAD-box RNA helicase in consecutive pre-rRNA processing and maturation steps for construction of 60S ribosomal subunits. Has1 associates with pre-60S ribosomes in an ATP-independent manner. Has1 binding triggers exonucleolytic trimming of 27SA3 pre-rRNA to generate the 5′ end of 5.8S rRNA and drives incorporation of ribosomal protein L17 with domain I of 5.8S/25S rRNA. ATP-dependent activity of Has1 promotes stable association of additional domain I ribosomal proteins that surround the polypeptide exit tunnel, which are required for downstream processing of 27SB pre-rRNA. Furthermore, in the absence of Has1, aberrant 27S pre-rRNAs are targeted for irreversible turnover. Thus, our data support a model in which Has1 helps to establish domain I architecture to prevent pre-rRNA turnover and couples domain I folding with consecutive pre-rRNA processing steps. PMID:23788678

Declines in the dissolved organic carbon (DOC) concentration and flux from the UK

NASA Astrophysics Data System (ADS)

Worrall, Fred; Howden, Nicholas J. K.; Burt, Tim P.; Bartlett, Rebecca

2018-01-01

Increased concentrations of dissolved organic carbon (DOC) have been reported for many catchments across the northern hemisphere. Hypotheses to explain the increase have varied (eg. increasing air temperature or recovery from acidification) but one test of alternative hypotheses is the trend over the recent decade, with the competing hypotheses predicting: continuing increase; the rate of increase declining with time; and even decrease in concentration. In this study, records of DOC concentration in non-tidal rivers across the UK were examined for the period 2003-2012. The study found that: Of the 62 decade-long concentration trends that could be examined, 3 showed a significant increase, 17 experienced no significant change and 42 showed a significant decrease; in 28 of the 42 significant decreases, a significant step change was apparent with step changes being a decrease in concentration in every case. Of the 118 sites where annual flux and concentration records were available from 1974, 28 showed a significant step change down in flux and 52 showed a step down in concentration. The modal year of the step changes was 2000 with no step changes observed before 1982. At the UK national scale, DOC flux peaked in 2005 at 1354 ktonnes C/yr (5.55 tonnes C/km2/yr) but has declined since. The study suggests that there is a disconnection between DOC records from large catchments at their tidal limits and complementary records from headwater catchments, which means that mechanisms believed to be driving increases in DOC concentrations in headwaters will not necessarily be those controlling trends in DOC concentration further downstream. We propose that the changes identified here have been driven by changes in in-stream processing and changes brought about by the Urban Waste Water Treatment Directive. Therefore, signals identified in headwater catchments may bear little relation to those observed in large rivers much further downstream and vice versa.

Zeolite based microconcentrators for volatile organic compounds sensing at trace-level: fabrication and performance

NASA Astrophysics Data System (ADS)

Almazán, Fernando; Pellejero, Ismael; Morales, Alberto; Urbiztondo, Miguel A.; Sesé, Javier; Pina, M. Pilar; Santamaría, Jesús

2016-08-01

A novel 6-step microfabrication process is proposed in this work to prepare microfluidic devices with integrated zeolite layers. In particular, microfabricated preconcentrators designed for volatile organic compounds (VOC) sensing applications are fully described. The main novelty of this work is the integration of the pure siliceous MFI type zeolite (silicalite-1) polycrystalline layer, i.e. 4.0  ±  0.5 μm thick, as active phase, within the microfabrication process just before the anodic bonding step. Following this new procedure, Si microdevices with an excellent distribution of the adsorbent material, integrated resistive heaters and Pyrex caps have been obtained. Firstly, the microconcentrator performance has been assessed by means of the normal hexane breakthrough curves as a function of sampling and desorption flowrates, temperature and micropreconcentrator design. In a step further, the best preconcentrator device has been tested in combination with downstream Si based microcantilevers deployed as VOC detectors. Thus, a preliminar evaluation of the improvement on detection sensitivity by silicalite-1 based microconcentrators is presented.

Achieving Continuous Manufacturing for Final Dosage Formation: Challenges and How to Meet Them May 20-21 2014 Continuous Manufacturing Symposium.

PubMed

Byrn, Stephen; Futran, Maricio; Thomas, Hayden; Jayjock, Eric; Maron, Nicola; Meyer, Robert F; Myerson, Allan S; Thien, Michael P; Trout, Bernhardt L

2015-03-01

We describe the key issues and possibilities for continuous final dosage formation, otherwise known as downstream processing or drug product manufacturing. A distinction is made between heterogeneous processing and homogeneous processing, the latter of which is expected to add more value to continuous manufacturing. We also give the key motivations for moving to continuous manufacturing, some of the exciting new technologies, and the barriers to implementation of continuous manufacturing. Continuous processing of heterogeneous blends is the natural first step in converting existing batch processes to continuous. In heterogeneous processing, there are discrete particles that can segregate, versus in homogeneous processing, components are blended and homogenized such that they do not segregate. Heterogeneous processing can incorporate technologies that are closer to existing technologies, where homogeneous processing necessitates the development and incorporation of new technologies. Homogeneous processing has the greatest potential for reaping the full rewards of continuous manufacturing, but it takes long-term vision and a more significant change in process development than heterogeneous processing. Heterogeneous processing has the detriment that, as the technologies are adopted rather than developed, there is a strong tendency to incorporate correction steps, what we call below "The Rube Goldberg Problem." Thus, although heterogeneous processing will likely play a major role in the near-term transformation of heterogeneous to continuous processing, it is expected that homogeneous processing is the next step that will follow. Specific action items for industry leaders are. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Recent Advances and Challenges towards Sustainable Polyhydroxyalkanoate (PHA) Production

PubMed Central

Kourmentza, Constantina; Plácido, Jersson; Venetsaneas, Nikolaos; Burniol-Figols, Anna; Varrone, Cristiano; Gavala, Hariklia N.; Reis, Maria A. M.

2017-01-01

Sustainable biofuels, biomaterials, and fine chemicals production is a critical matter that research teams around the globe are focusing on nowadays. Polyhydroxyalkanoates represent one of the biomaterials of the future due to their physicochemical properties, biodegradability, and biocompatibility. Designing efficient and economic bioprocesses, combined with the respective social and environmental benefits, has brought together scientists from different backgrounds highlighting the multidisciplinary character of such a venture. In the current review, challenges and opportunities regarding polyhydroxyalkanoate production are presented and discussed, covering key steps of their overall production process by applying pure and mixed culture biotechnology, from raw bioprocess development to downstream processing. PMID:28952534

Blue and red light-induced germination of resting spores in the red-tide diatom Leptocylindrus danicus.

PubMed

Shikata, Tomoyuki; Iseki, Mineo; Matsunaga, Shigeru; Higashi, Sho-ichi; Kamei, Yasuhiro; Watanabe, Masakatsu

2011-01-01

Photophysiological and pharmacological approaches were used to examine light-induced germination of resting spores in the red-tide diatom Leptocylindrus danicus. The equal-quantum action spectrum for photogermination had peaks at about 440 nm (blue light) and 680 nm (red light), which matched the absorption spectrum of the resting spore chloroplast, as well as photosynthetic action spectra reported for other diatoms. DCMU, an inhibitor of photosynthetic electron flow near photosystem II, completely blocked photogermination. These results suggest that the photosynthetic system is involved in the photoreception process of light-induced germination. Results of pharmacological studies of the downstream signal transduction pathway suggested that Ca(2+) influx is the closest downstream neighbor, followed by steps involving calmodulin, nitric oxide synthase, guanylyl cyclase, protein-tyrosine-phosphatase, protein kinase C and actin polymerization and translation. © 2011 The Authors. Photochemistry and Photobiology © 2011 The American Society of Photobiology.

Temperature change rate actuated bubble mixing for homogeneous rehydration of dry pre-stored reagents in centrifugal microfluidics.

PubMed

Hin, S; Paust, N; Keller, M; Rombach, M; Strohmeier, O; Zengerle, R; Mitsakakis, K

2018-01-16

In centrifugal microfluidics, dead volumes in valves downstream of mixing chambers can hardly be avoided. These dead volumes are excluded from mixing processes and hence cause a concentration gradient. Here we present a new bubble mixing concept which avoids such dead volumes. The mixing concept employs heating to create a temperature change rate (TCR) induced overpressure in the air volume downstream of mixing chambers. The main feature is an air vent with a high fluidic resistance, representing a low pass filter with respect to pressure changes. Fast temperature increase causes rapid pressure increase in downstream structures pushing the liquid from downstream channels into the mixing chamber. As air further penetrates into the mixing chamber, bubbles form, ascend due to buoyancy and mix the liquid. Slow temperature/pressure changes equilibrate through the high fluidic resistance air vent enabling sequential heating/cooling cycles to repeat the mixing process. After mixing, a complete transfer of the reaction volume into the downstream fluidic structure is possible by a rapid cooling step triggering TCR actuated valving. The new mixing concept is applied to rehydrate reagents for loop-mediated isothermal amplification (LAMP). After mixing, the reaction mix is aliquoted into several reaction chambers for geometric multiplexing. As a measure for mixing efficiency, the mean coefficient of variation (C[combining macron]V[combining macron], n = 4 LabDisks) of the time to positivity (t p ) of the LAMP reactions (n = 11 replicates per LabDisk) is taken. The C[combining macron]V[combining macron] of the t p is reduced from 18.5% (when using standard shake mode mixing) to 3.3% (when applying TCR actuated bubble mixing). The bubble mixer has been implemented in a monolithic fashion without the need for any additional actuation besides rotation and temperature control, which are needed anyhow for the assay workflow.

Evaluation of target efficiencies for solid-liquid separation steps in biofuels production.

PubMed

Kochergin, Vadim; Miller, Keith

2011-01-01

Development of liquid biofuels has entered a new phase of large scale pilot demonstration. A number of plants that are in operation or under construction face the task of addressing the engineering challenges of creating a viable plant design, scaling up and optimizing various unit operations. It is well-known that separation technologies account for 50-70% of both capital and operating cost. Additionally, reduction of environmental impact creates technological challenges that increase project cost without adding to the bottom line. Different technologies vary in terms of selection of unit operations; however, solid-liquid separations are likely to be a major contributor to the overall project cost. Despite the differences in pretreatment approaches, similar challenges arise for solid-liquid separation unit operations. A typical process for ethanol production from biomass includes several solid-liquid separation steps, depending on which particular stream is targeted for downstream processing. The nature of biomass-derived materials makes it either difficult or uneconomical to accomplish complete separation in a single step. Therefore, setting realistic efficiency targets for solid-liquid separations is an important task that influences overall process recovery and economics. Experimental data will be presented showing typical characteristics for pretreated cane bagasse at various stages of processing into cellulosic ethanol. Results of generic material balance calculations will be presented to illustrate the influence of separation target efficiencies on overall process recoveries and characteristics of waste streams.

Magnetically Enhanced Solid-Liquid Separation

NASA Astrophysics Data System (ADS)

Rey, C. M.; Keller, K.; Fuchs, B.

2005-07-01

DuPont is developing an entirely new method of solid-liquid filtration involving the use of magnetic fields and magnetic field gradients. The new hybrid process, entitled Magnetically Enhanced Solid-Liquid Separation (MESLS), is designed to improve the de-watering kinetics and reduce the residual moisture content of solid particulates mechanically separated from liquid slurries. Gravitation, pressure, temperature, centrifugation, and fluid dynamics have dictated traditional solid-liquid separation for the past 50 years. The introduction of an external field (i.e. the magnetic field) offers the promise to manipulate particle behavior in an entirely new manner, which leads to increased process efficiency. Traditional solid-liquid separation typically consists of two primary steps. The first is a mechanical step in which the solid particulate is separated from the liquid using e.g. gas pressure through a filter membrane, centrifugation, etc. The second step is a thermal drying process, which is required due to imperfect mechanical separation. The thermal drying process is over 100-200 times less energy efficient than the mechanical step. Since enormous volumes of materials are processed each year, more efficient mechanical solid-liquid separations can be leveraged into dramatic reductions in overall energy consumption by reducing downstream drying requirements have a tremendous impact on energy consumption. Using DuPont's MESLS process, initial test results showed four very important effects of the magnetic field on the solid-liquid filtration process: 1) reduction of the time to reach gas breakthrough, 2) less loss of solid into the filtrate, 3) reduction of the (solids) residual moisture content, and 4) acceleration of the de-watering kinetics. These test results and their potential impact on future commercial solid-liquid filtration is discussed. New applications can be found in mining, chemical and bioprocesses.

PSR Injection Line Upgrade

NASA Astrophysics Data System (ADS)

Blind, Barbara; Jason, Andrew J.

1997-05-01

We describe the new injection line to be implemented for the Los Alamos Proton Storage Ring in the change from a two-step process to direct H- injection. While obeying all geometrical constraints imposed by the existing structures, the new line has properties not found in the present injection line. In particular, it features decoupled transverse phase spaces downstream of the skew bend and a high degree of tunability of the beam at the injection foil. A comprehensive set of error studies has dictated the component tolerances imposed and has indicated the expected performance of the system.

LysM receptor-like kinases to improve plant defense response against fungal pathogens

DOEpatents

Wan, Jinrong [Columbia, MO; Stacey, Gary [Columbia, MO; Stacey, Minviluz [Columbia, MO; Zhang, Xuecheng [Columbia, MO

2012-01-17

Perception of chitin fragments (chitooligosaccharides) is an important first step in plant defense response against fungal pathogen. LysM receptor-like kinases (LysM RLKs) are instrumental in this perception process. LysM RLKs also play a role in activating transcription of chitin-responsive genes (CRGs) in plants. Mutations in the LysM kinase receptor genes or the downstream CRGs may affect the fungal susceptibility of a plant. Mutations in LysM RLKs or transgenes carrying the same may be beneficial in imparting resistance against fungal pathogens.

LysM receptor-like kinases to improve plant defense response against fungal pathogens

DOEpatents

Wan, Jinrong; Stacey, Gary; Stacey, Minviluz; Zhang, Xuecheng

2013-10-15

Perception of chitin fragments (chitooligosaccharides) is an important first step in plant defense response against fungal pathogen. LysM receptor-like kinases (LysM RLKs) are instrumental in this perception process. LysM RLKs also play a role in activating transcription of chitin-responsive genes (CRGs) in plants. Mutations in the LysM kinase receptor genes or the downstream CRGs may affect the fungal susceptibility of a plant. Mutations in LysM RLKs or transgenes carrying the same may be beneficial in imparting resistance against fungal pathogens.

Ecogeomorphic feedbacks in regrowth of travertine step-pool morphology after dam decommissioning, Fossil Creek, Arizona

NASA Astrophysics Data System (ADS)

Fuller, Brian M.; Sklar, Leonard S.; Compson, Zacchaeus G.; Adams, Kenneth J.; Marks, Jane C.; Wilcox, Andrew C.

2011-03-01

The linkages between fluvial geomorphology and aquatic ecosystems are commonly conceptualized as a one-way causal chain in which geomorphic processes create the physical template for ecological dynamics. In streams with a travertine step-pool morphology, however, biotic processes strongly influence the formation and growth of travertine dams, creating the potential for numerous feedbacks. Here we take advantage of the decommissioning of a hydroelectric project on Fossil Creek, Arizona, where restoration of CaCO 3-rich baseflow has triggered rapid regrowth of travertine dams, to explore the interactions between biotic and abiotic factors in travertine morphodynamics. We consider three conceptual frameworks, where biotic factors independently modulate the rate of physical and chemical processes that produce travertine dams; combine with abiotic factors in a set of feedback loops; and work in opposition to abiotic processes, such that the travertine step-pool morphology reflects a dynamic balance between dominantly-biotic constructive processes and dominantly-abiotic destructive processes. We consider separately three phases of an idealized life cycle of travertine dams: dam formation, growth, and destruction by erosive floods. Dam formation is catalyzed by abiotic factors (e.g. channel constrictions, and bedrock steps) and biotic factors (e.g. woody debris, and emergent vegetation). From measurements of changes over time in travertine thickness on a bedrock step, we find evidence for a positive feedback between flow hydraulics and travertine accrual. Measurements of organic content in travertine samples from this step show that algal growth contributes substantially to travertine accumulation and suggest that growth is most rapid during seasonal algal blooms. To document vertical growth of travertine dams, we embedded 252 magnets into nascent travertine dams, along a 10 km stretch of river. Growth rates are calculated from changes over time in the magnetic field intensity at the dam surface. At each magnet we record a range of hydraulic and travertine composition variables to characterize the dominant mechanism of growth: abiotic precipitation, algal growth, trapping of organic material, or in situ plant growth. We find: (1) rapid growth of travertine dams following flow restoration, averaging more than 2 cm/year; (2) growth rates decline downstream, consistent with loss of dissolved constituents because of upstream travertine deposition, but also parallel to a decline in organic content in dam surface material and a downstream shift in dominant biotic mechanism; (3) biotic mechanisms are associated with faster growth rates; and (4) correlations between hydraulic attributes and growth rates are more consistent with biotic than abiotic controls. We conclude that the strong influence of living organisms on rates of travertine growth, coupled with the beneficial effects of travertine on ecosystem dynamics, demonstrate a positive feedback between biology and geomorphology. During our two-year study period, erosive flood flows occurred causing widespread removal of travertine. The temporal distribution of travertine growth and erosion over the study period is consistent with a bimodal magnitude-frequency relation in which growth dominates except when large, infrequent storms occur. This model may be useful in other systems where biology exerts strong controls on geomorphic processes.

First Steps Toward Incorporating Image Based Diagnostics Into Particle Accelerator Control Systems Using Convolutional Neural Networks

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

Edelen, A. L.; Biedron, S. G.; Milton, S. V.

At present, a variety of image-based diagnostics are used in particle accelerator systems. Often times, these are viewed by a human operator who then makes appropriate adjustments to the machine. Given recent advances in using convolutional neural networks (CNNs) for image processing, it should be possible to use image diagnostics directly in control routines (NN-based or otherwise). This is especially appealing for non-intercepting diagnostics that could run continuously during beam operation. Here, we show results of a first step toward implementing such a controller: our trained CNN can predict multiple simulated downstream beam parameters at the Fermilab Accelerator Science andmore » Technology (FAST) facility's low energy beamline using simulated virtual cathode laser images, gun phases, and solenoid strengths.« less

Municipal wastewater treatment by biofiltration: comparisons of various treatment layouts. Part 1: assessment of carbon and nitrogen removal.

PubMed

Rocher, Vincent; Paffoni, Catherine; Gonçalves, Alexandre; Guérin, Sabrina; Azimi, Sam; Gasperi, Johnny; Moilleron, Régis; Pauss, André

2012-01-01

One of the largest wastewater treatment plants in the Paris conurbation (240,000 m(3)/d) has been studied over several years in order to provide technical and economical information about biological treatment by biofiltration. Biofiltration systems are processes in which carbon and nitrogen pollution of wastewater are treated by ascendant flow through immersed fixed cultures. This paper, focused on technical information, aims: (1) to compare performances of the three biological treatment layouts currently used in biofiltration systems: upstream denitrification (UD), downstream denitrification (DD) and combined upstream-downstream denitrification (U-DD) layouts; and (2) to describe in detail each treatment step. Our study has shown that more than 90% of the carbon and ammoniacal pollution is removed during biological treatment, whatever the layout used. Nitrate, produced during nitrification, is then reduced to atmospheric nitrogen. This reduction is more extensive when the denitrification stage occurs downstream from the treatment (DD layout with methanol addition), whereas it is only partial when it is inserted upstream from the treatment (UD layout - use of endogenous carbonaceous substrate). So, the UD layout leads to a nitrate concentration that exceeds the regulatory threshold in the effluent, and the treatment must be supplemented with a post-denitrification step (U-DD layout). Our work has also shown that the optimal ammonium-loading rate is about 1.1-1.2 kg N-NH(4)(+) per m(3) media (polystyrene) and day. For denitrification, the optimal nitrate-loading rate is about 2.5 kg N per m(3) media (expanded clay) and day in the case of DD with methanol, and is about 0.25 kg N-NO(3)(-) per m(3) media and day in the case of UD with exogenous carbonaceous substrate.

The influence of combustion liner holes on noise production by ducted burners

NASA Technical Reports Server (NTRS)

Mahan, J. R.; Jones, J. D.

1984-01-01

The thermoacoustic energy conversion process in a turbulent flame is not yet sufficiently well understood to allow accurate prediction of the sound pressure field of even the simplest of laboratory burners. The present contribution is intended to be a step toward fuller understanding of this process. In particular, the possibility is explored that the source structure, in the form of the thermoacoustic efficiency spectrum, might be influenced by the acoustic response of the burner itself. Experimental results are presented which seem to establish that, at least for the gas-fueled laboratory burner studied, source activity is not affected by the addition of downstream combustion liner holes which otherwise alter the acoustic response of the burner.

Dynamics of Leading-strand Lesion Skipping by the Replisome

PubMed Central

Yeeles, Joseph T.P.; Marians, Kenneth J.

2013-01-01

SUMMARY The E. coli replisome stalls transiently when it encounters a lesion in the leading-strand template, skipping over the damage by reinitiating replication at a new primer synthesized downstream by the primase. We report here that template unwinding and lagging-strand synthesis continue downstream of the lesion at a reduced rate after replisome stalling, that one replisome is capable of skipping multiple lesions, and that the rate limiting steps of replication restart involve the synthesis and activation of the new primer downstream. We also find little support for the concept that polymerase uncoupling, where extensive lagging-strand synthesis proceeds downstream in the absence of leading-strand synthesis, involves physical separation of the leading-strand polymerase from the replisome. Instead, our data indicate that extensive uncoupled replication likely results from a failure of the leading-strand polymerase still associated with the DNA helicase and the lagging-strand polymerase that are proceeding downstream to reinitiate synthesis. PMID:24268579

A Framework for Propagation of Uncertainties in the Kepler Data Analysis Pipeline

NASA Technical Reports Server (NTRS)

Clarke, Bruce D.; Allen, Christopher; Bryson, Stephen T.; Caldwell, Douglas A.; Chandrasekaran, Hema; Cote, Miles T.; Girouard, Forrest; Jenkins, Jon M.; Klaus, Todd C.; Li, Jie;

Sensing fluid pressure during plucking events in a natural bedrock channel and experimental flume

NASA Astrophysics Data System (ADS)

Wilkinson, C.; Harbor, D. J.; Keel, D.; Levy, S.; Kuehner, J. P.

2016-12-01

River channel erosion by plucking is believed to be the dominant erosional process in channels with fractured or jointed bedrock. However, despite its significance as an erosional mechanism, plucking is poorly studied in both laboratory and natural channels. In previous flume studies, model bedrock was plucked by fluid forces alone in nonuniform flow near jumps and waves even where blocks do not protrude into the flow. Here we develop sensor systems to test the hypothesis that bed fluid pressure gradients lift "pluckable" bedrock blocks in a natural field setting and a hydraulic flume. The field setting closely mimics the previous flume setup; the instrumented block is downstream of a roughly 1m step and exhibits no protrusion into the flow. The presence of the step promotes nonuniform flow which changes pressure in the bedrock crack network; slabs of bedrock that have slid downstream and sediment that has been pushed upstream 3-4 m under the bed and in the cracks suggest the influence of pressure differences throughout the crack network and below the bed. In this initial deployment, we evaluate a sensor that monitors movement and simultaneous pressure above and below the block. Sensors are emplaced in a 26kg, 45-cm-long, 20-cm-wide block broken from a 4.5-m-long, 11-cm-thick sandstone bed with a dense network of cracks nearly parallel to flow direction and include a tri-axial accelerometer/gyroscope and two fluid pressure sensors. The electronics are housed in a custom-designed 3D-printed ABS waterproof capsule that is mounted in a vertical hole through the rock. A concurrent flume study develops the sensors necessary to investigate the longitudinal pressure difference below a step using multiple analog sensors (0-1 psi gauge pressure) mounted flush to a false floor under the center of a 30x14-cm test zone. The 15-mm-wide sensors are aligned along the flow centerline and are placed under 25 1-cm-thick "pluckable" bedrock blocks constructed with a proprietary plaster cement. Measured mean pressure and transmission of pressure pulses under the test bed are compared to the visual record of plucking. In addition, conducting runs with blocks removed permits simulation of the mean and varying pressure conditions above the modeled "pluckable" layer as a hydraulic jump is moved downstream through the step.

COLD-PCR enriches low-level variant DNA sequences and increases the sensitivity of genetic testing.

PubMed

Castellanos-Rizaldos, Elena; Milbury, Coren A; Guha, Minakshi; Makrigiorgos, G Mike

2014-01-01

Detection of low-level mutations is important for cancer biomarker and therapy targets discovery, but reliable detection remains a technical challenge. The newly developed method of CO-amplification at Lower Denaturation temperature PCR (COLD-PCR) helps to circumvent this issue. This PCR-based technology preferentially enriches minor known or unknown variants present in samples with a high background of wild type DNA which often hampers the accurate identification of these minority alleles. This is a simple process that consists of lowering the temperature at the denaturation step during the PCR-cycling protocol (critical denaturation temperature, T c) and inducing DNA heteroduplexing during an intermediate step. COLD-PCR in its simplest forms does not need additional reagents or specific instrumentation and thus, can easily replace conventional PCR and at the same time improve the mutation detection sensitivity limit of downstream technologies. COLD-PCR can be applied in two basic formats: fast-COLD-PCR that can enrich T m-reducing mutations and full-COLD-PCR that can enrich all mutations, though it requires an intermediate cross-hybridization step that lengthens the thermocycling program. An improved version of full-COLD-PCR (improved and complete enrichment, ice-COLD-PCR) has also been described. Finally, most recently, we developed yet another form of COLD-PCR, temperature-tolerant-COLD-PCR, which gradually increases the denaturation temperature during the COLD-PCR reaction, enriching diverse targets using a single cycling program. This report describes practical considerations for application of fast-, full-, ice-, and temperature-tolerant-COLD-PCR for enrichment of mutations prior to downstream screening.

Exploring continuous and integrated strategies for the up- and downstream processing of human mesenchymal stem cells.

PubMed

Cunha, Bárbara; Aguiar, Tiago; Silva, Marta M; Silva, Ricardo J S; Sousa, Marcos F Q; Pineda, Earl; Peixoto, Cristina; Carrondo, Manuel J T; Serra, Margarida; Alves, Paula M

2015-11-10

The integration of up- and downstream unit operations can result in the elimination of hold steps, thus decreasing the footprint, and ultimately can create robust closed system operations. This type of design is desirable for the bioprocess of human mesenchymal stem cells (hMSC), where high numbers of pure cells, at low volumes, need to be delivered for therapy applications. This study reports a proof of concept of the integration of a continuous perfusion culture in bioreactors with a tangential flow filtration (TFF) system for the concentration and washing of hMSC. Moreover, we have also explored a continuous alternative for concentrating hMSC. Results show that expanding cells in a continuous perfusion operation mode provided a higher expansion ratio, and led to a shift in cells' metabolism. TFF operated either in continuous or discontinuous allowed to concentrate cells, with high cell recovery (>80%) and viability (>95%); furthermore, continuous TFF permitted to operate longer with higher cell concentrations. Continuous diafiltration led to higher protein clearance (98%) with lower cell death, when comparing to discontinuous diafiltration. Overall, an integrated process allowed for a shorter process time, recovering 70% of viable hMSC (>95%), with no changes in terms of morphology, immunophenotype, proliferation capacity and multipotent differentiation potential. Copyright © 2015 Elsevier B.V. All rights reserved.

Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

PubMed

Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

2016-10-01

Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a cost-effective, rapid, and reliable avenue for the purification of recombinant proteins in heterologous hosts. Copyright © 2016 Elsevier Inc. All rights reserved.

Biophysical Interactions within Step-Pool Mountain Streams Following Wildfire

NASA Astrophysics Data System (ADS)

Parker, A.; Chin, A.; O'Dowd, A. P.

2014-12-01

Recovery of riverine ecosystems following disturbance is driven by a variety of interacting processes. Wildfires pose increasing disturbances to riverine landscapes, with rising frequencies and magnitudes owing to warming climates and increased fuel loads. The effects of wildfire include loss of vegetation, elevated runoff and flash floods, erosion and deposition, and changing biological habitats and communities. Understanding process interactions in post-fire landscapes is increasingly urgent for successful management and restoration of affected ecosystems. In steep channels, steps and pools provide prominent habitats for organisms and structural integrity in high energy environments. Step-pools are typically stable, responding to extreme events with recurrence intervals often exceeding 50 years. Once wildfire occurs, however, intensification of post-fire flood events can potentially overpower the inherent stability of these systems, with significant consequences for aquatic life and human well-being downstream. This study examined the short-term response of step-pool streams following the 2012 Waldo Canyon Fire in Colorado. We explored interacting feedbacks among geomorphology, hydrology, and ecology in the post-fire environment. At selected sites with varying burn severity, we established baseline conditions immediately after the fire with channel surveys, biological assessment using benthic macroinvertebrates, sediment analysis including pebble counts, and precipitation gauging. Repeat measurements after major storm events over several years enabled analysis of the interacting feedbacks among post-fire processes. We found that channels able to retain the step-pool structure changed less and facilitated recovery more readily. Step habitats maintained higher percentages of sensitive macroinvertebrate taxa compared to pools through post-fire floods. Sites burned with high severity experienced greater reduction in the percentage of sensitive taxa. The decimation of macroinvertebrates closely coincides with the physical destruction of the step-pool morphology. The role that step-pools play in enhancing the ecological quality of fluvial systems, therefore, provides a key focus for effective management and restoration of aquatic resources following wildfires.

Extraction and downstream processing of plant-derived recombinant proteins.

PubMed

Buyel, J F; Twyman, R M; Fischer, R

2015-11-01

Plants offer the tantalizing prospect of low-cost automated manufacturing processes for biopharmaceutical proteins, but several challenges must be addressed before such goals are realized and the most significant hurdles are found during downstream processing (DSP). In contrast to the standardized microbial and mammalian cell platforms embraced by the biopharmaceutical industry, there are many different plant-based expression systems vying for attention, and those with the greatest potential to provide inexpensive biopharmaceuticals are also the ones with the most significant drawbacks in terms of DSP. This is because the most scalable plant systems are based on the expression of intracellular proteins in whole plants. The plant tissue must therefore be disrupted to extract the product, challenging the initial DSP steps with an unusually high load of both particulate and soluble contaminants. DSP platform technologies can accelerate and simplify process development, including centrifugation, filtration, flocculation, and integrated methods that combine solid-liquid separation, purification and concentration, such as aqueous two-phase separation systems. Protein tags can also facilitate these DSP steps, but they are difficult to transfer to a commercial environment and more generic, flexible and scalable strategies to separate target and host cell proteins are preferable, such as membrane technologies and heat/pH precipitation. In this context, clarified plant extracts behave similarly to the feed stream from microbes or mammalian cells and the corresponding purification methods can be applied, as long as they are adapted for plant-specific soluble contaminants such as the superabundant protein RuBisCO. Plant-derived pharmaceutical proteins cannot yet compete directly with established platforms but they are beginning to penetrate niche markets that allow the beneficial properties of plants to be exploited, such as the ability to produce 'biobetters' with tailored glycans, the ability to scale up production rapidly for emergency responses and the ability to produce commodity recombinant proteins on an agricultural scale. Copyright © 2015 Elsevier Inc. All rights reserved.

A comparison of protein A chromatographic stationary phases: performance characteristics for monoclonal antibody purification.

PubMed

Liu, Zhuo; Mostafa, Sigma S; Shukla, Abhinav A

2015-01-01

Protein A chromatography remains the dominant capture step used during the downstream purification of monoclonal antibodies (mAbs). With the recent expiry of the Repligen patent on recombinant Protein A, a variety of new Protein A resins have been introduced in the market. Given productivity limitations during downstream processing that have come into sharper focus with the recent increase in cell culture titers for mAbs, the selection of an appropriate Protein A resin has direct implications on the overall process economics of mAb production. The performance of seven different Protein A chromatographic resins was compared with respect to static binding capacity and dynamic binding capacity as a function of flow rate. This data was translated into a comparison of productivity (g mAb purified per unit resin volume per unit time) for the seven stationary phases. In addition, elution pH and host cell protein impurity levels after product capture on each of these resins were determined. The current article provides an effective methodology and dataset for the selection of the optimal Protein A chromatographic resin. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

GABA-CREB signalling regulates maturation and survival of newly generated neurons in the adult hippocampus

PubMed Central

Jagasia, Ravi; Steib, Kathrin; Englberger, Elisabeth; Herold, Sabine; Faus-Kessler, Theresa; Saxe, Michael; Gage, Fred H.; Song, Hongjun; Lie, D. Chichung

2009-01-01

Survival and integration of new neurons in the hippocampal circuit are rate-limiting steps in adult hippocampal neurogenesis. Neuronal network activity is a major regulator of these processes, yet little is known about the respective downstream signalling pathways. Here, we investigate the role of CREB signalling in adult hippocampal neurogenesis. CREB is activated in new granule neurons during a distinct developmental period. Loss of CREB function in a cell-autonomous fashion impairs dendritic development, decreases the expression of the neurogenic transcription factor NeuroD and of the neuronal microtubule associated protein, DCX, and compromises the survival of newborn neurons. In addition, GABA-mediated excitation regulates CREB activation at early developmental stages. Importantly, developmental defects following loss of GABA-mediated excitation can be compensated by enhanced CREB signalling. These results indicate that CREB signalling is a central pathway in adult hippocampal neurogenesis, regulating the development and survival of new hippocampal neurons downstream of GABA-mediated excitation. PMID:19553437

Ubiquitinated Sirtuin 1 (SIRT1) Function Is Modulated during DNA Damage-induced Cell Death and Survival*

PubMed Central

Peng, Lirong; Yuan, Zhigang; Li, Yixuan; Ling, Hongbo; Izumi, Victoria; Fang, Bin; Fukasawa, Kenji; Koomen, John; Chen, Jiandong; Seto, Edward

2015-01-01

Downstream signaling of physiological and pathological cell responses depends on post-translational modification such as ubiquitination. The mechanisms regulating downstream DNA damage response (DDR) signaling are not completely elucidated. Sirtuin 1 (SIRT1), the founding member of Class III histone deacetylases, regulates multiple steps in DDR and is closely associated with many physiological and pathological processes. However, the role of post-translational modification or ubiquitination of SIRT1 during DDR is unclear. We show that SIRT1 is dynamically and distinctly ubiquitinated in response to DNA damage. SIRT1 was ubiquitinated by the MDM2 E3 ligase in vitro and in vivo. SIRT1 ubiquitination under normal conditions had no effect on its enzymatic activity or rate of degradation; hypo-ubiquitination, however, reduced SIRT1 nuclear localization. Ubiquitination of SIRT1 affected its function in cell death and survival in response to DNA damage. Our results suggest that ubiquitination is required for SIRT1 function during DDR. PMID:25670865

Application of coupled affinity-sizing chromatography for the detection of proteolyzed HSA-tagged proteins.

PubMed

London, Anne Serdakowski; Patel, Kunal; Quinn, Lisa; Lemmerer, Martin

2015-04-01

Coupled affinity liquid chromatography and size exclusion chromatography (ALC-SEC) is a technique that has been shown to successfully report product quality of proteins during cell expression and prior to the commencement of downstream processing chromatography steps. This method was applied to monitoring the degradation and subsequent partial remediation of a HSA-tagged protein which showed proteolysis, allowing for rapid cell line development to address this product quality dilemma. This paper outlines the novel application of this method for measuring and addressing protease-induced proteolysis. Copyright © 2014 Elsevier Inc. All rights reserved.

Ontogenetic behavior and migration of Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus, and shortnose sturgeon, A. brevirostrum, with notes on social behavior

USGS Publications Warehouse

Kynard, B.; Horgan, M.

2002-01-01

Ontogenetic behavior of Hudson River Atlantic sturgeon and Connecticut River shortnose sturgeon early life intervals were similar during laboratory observations. After hatching, free embryos were photonegative and sought cover. When embryos developed into larvae, fish left cover, were photopositive, and initiated downstream migration. Free embryos may remain at the spawning site instead of migrating downstream because the risk of predation at spawning sites is low. The two species are sympatric, but not closely related, so the similarities in innate behaviors suggest common adaptations, not phylogenetlc relationship. Atlantic sturgeon migrated downstream for 12 days (peak, first 6 days), shortnose sturgeon migrated for 3 days, and year-0 juveniles of both species did not resume downstream migration. Short or long migrations of larvae may reflect different styles related to the total migratory distance from spawning sites to juvenile rearing areas. Atlantic sturgeon need to move a short distance to reach rearing areas and they had a long 1-step migration of 6-12 days. In contrast, shortnose sturgeon need to move a long distance to reach all rearing areas. This may be accomplished by a 2-step migration, of which the brief migration of larvae is only the first step. Early migrant Atlantic sturgeon were nocturnal, while late migrants were diurnal, and shortnose sturgeon were diurnal. These diel differences may also be adaptations for long (Atlantic sturgeon) or short (shortnose sturgeon) migrations. Cultured shortnose sturgeon, and possibly Atlantic sturgeon, have a dominance hierarchy with large fish dominant when competing for limited foraging space. Social behavior may be more important in the life history of wild sturgeons than is generally recognized.

Determination of in vivo regulation kinetics of small non-coding RNA in bacteria

NASA Astrophysics Data System (ADS)

Fei, Jingyi

Small RNAs (sRNAs) play important roles in regulating gene expression through a variety of mechanisms. As one of the most common strategies, sRNA induced target messenger RNA (mRNA) includes two major steps: target search by base-pairing interactions with the and downstream execution by modulating translation or the stability of the mRNA. Here we describe a new imaging and analysis platform based on super-resolution fluorescence microscopy, which enabled the first in vivo kinetic measurement of sRNA-mediated gene regulation. Specifically, this platform was used to investigate a sugar-phosphate stress-induced bacterial sRNA that induces the degradation of target mRNAs. The data reveal that the sRNA binds to a primary target mRNA in a reversible and dynamic fashion, and that formation of the sRNA-mRNA complexes is the rate-limiting step, dictating the overall efficiency of regulation in vivo; whereas the downstream co-degradation of sRNA-mRNA complex can kinetically compete with the fast complex disassembly. Examination of a secondary target of this sRNA indicated that differences in the target search kinetics contribute to setting the regulation priority among different target mRNAs. This super-resolution imaging and analysis approach provides a conceptual framework that can be generalized to other sRNA systems and other target search processes.

Optimization and revision of the production process of the Necator americanus glutathione S-transferase 1 (Na-GST-1), the lead hookworm vaccine recombinant protein candidate

PubMed Central

Curti, Elena; Seid, Christopher A; Hudspeth, Elissa; Center, Lori; Rezende, Wanderson; Pollet, Jeroen; Kwityn, Cliff; Hammond, Molly; Matsunami, Rise K; Engler, David A; Hotez, Peter J; Elena Bottazzi, Maria

2014-01-01

Infection by the human hookworm Necator americanus is a leading cause of anemia and disability in the developing countries of Africa, Asia, and the Americas. In order to prevent childhood hookworm disease in resource poor settings, a recombinant vaccine is under development by the Sabin Vaccine Institute and Texas Children’s Hospital Center for Vaccine Development, a Product Development Partnership (PDP). Previously, we reported on the expression and purification of a highly promising hookworm vaccine candidate, Na-GST-1, an N. americanus glutathione s-transferase expressed in Pichia pastoris (yeast), which led to production of 1.5 g of 95% pure recombinant protein at a 20L scale.1, 2, 3 This yield and purity of Na-GST-1 was sufficient for early pilot manufacturing and initial phase 1 clinical testing. However, based on the number of doses which would be required to allow mass vaccination and a potential goal to deliver a vaccine as inexpensively as possible, a higher yield of expression of the recombinant antigen at the lowest possible cost is highly desirable. Here we report on modifications to the fermentation (upstream process) of the antigen expressed in P. pastoris, and to the purification (downstream process) of the recombinant protein that allowed for a 2–3-fold improvement in the final yield of Na-GST-1 purified protein. The major improvements included upstream process changes such as the addition of a sorbitol pulse and co-feed during methanol induction as well as an extension of the induction stage to approximately 96 hours; downstream process changes included modifying the UFDF to flat sheet with a 10 kDa Molecular Weight cut-off (MWCO), adjusting the capacity of an ion-exchange chromatography step utilizing a gradient elution as opposed to the original step elution, and altering the hydrophobic interaction chromatography conditions. The full process, as well as the purity and stability profiles of the target Na-GST-1, and its formulation on Alhydrogel®, is described. PMID:25424799

Minding One's Reach (To Eat): The Promise of Computer Mouse-Tracking to Study Self-Regulation of Eating.

PubMed

Lopez, Richard B; Stillman, Paul E; Heatherton, Todd F; Freeman, Jonathan B

2018-01-01

In this review, we present the case for using computer mouse-tracking techniques to examine psychological processes that support (and hinder) self-regulation of eating. We first argue that computer mouse-tracking is suitable for studying the simultaneous engagement of-and dynamic interactions between-multiple perceptual and cognitive processes as they unfold and interact over a fine temporal scale (i.e., hundreds of milliseconds). Next, we review recent work that implemented mouse-tracking techniques by measuring mouse movements as participants chose between various food items (of varying nutritional content). Lastly, we propose next steps for future investigations to link behavioral features from mouse-tracking paradigms, corresponding neural correlates, and downstream eating behaviors.

Clarification of vaccines: An overview of filter based technology trends and best practices.

PubMed

Besnard, Lise; Fabre, Virginie; Fettig, Michael; Gousseinov, Elina; Kawakami, Yasuhiro; Laroudie, Nicolas; Scanlan, Claire; Pattnaik, Priyabrata

2016-01-01

Vaccines are derived from a variety of sources including tissue extracts, bacterial cells, virus particles, recombinant mammalian, yeast and insect cell produced proteins and nucleic acids. The most common method of vaccine production is based on an initial fermentation process followed by purification. Production of vaccines is a complex process involving many different steps and processes. Selection of the appropriate purification method is critical to achieving desired purity of the final product. Clarification of vaccines is a critical step that strongly impacts product recovery and subsequent downstream purification. There are several technologies that can be applied for vaccine clarification. Selection of a harvesting method and equipment depends on the type of cells, product being harvested, and properties of the process fluids. These techniques include membrane filtration (microfiltration, tangential-flow filtration), centrifugation, and depth filtration (normal flow filtration). Historically vaccine harvest clarification was usually achieved by centrifugation followed by depth filtration. Recently membrane based technologies have gained prominence in vaccine clarification. The increasing use of single-use technologies in upstream processes necessitated a shift in harvest strategies. This review offers a comprehensive view on different membrane based technologies and their application in vaccine clarification, outlines the challenges involved and presents the current state of best practices in the clarification of vaccines. Copyright © 2015 Elsevier Inc. All rights reserved.

Process Intensification for Cellulosic Biorefineries.

PubMed

Sadula, Sunitha; Athaley, Abhay; Zheng, Weiqing; Ierapetritou, Marianthi; Saha, Basudeb

2017-06-22

Utilization of renewable carbon source, especially non-food biomass is critical to address the climate change and future energy challenge. Current chemical and enzymatic processes for producing cellulosic sugars are multistep, and energy- and water-intensive. Techno-economic analysis (TEA) suggests that upstream lignocellulose processing is a major hurdle to the economic viability of the cellulosic biorefineries. Process intensification, which integrates processes and uses less water and energy, has the potential to overcome the aforementioned challenges. Here, we demonstrate a one-pot depolymerization and saccharification process of woody biomass, energy crops, and agricultural residues to produce soluble sugars with high yields. Lignin is separated as a solid for selective upgrading. Further integration of our upstream process with a reactive extraction step makes energy-efficient separation of sugars in the form of furans. TEA reveals that the process efficiency and integration enable, for the first time, economic production of feed streams that could profoundly improve process economics for downstream cellulosic bioproducts. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of adsorptive hybrid filters to enable two-step purification of biologics

PubMed Central

Peck, Michael; Voloshin, Alexei M.; Moreno, Angela M.; Tan, Zhijun; Hester, Jonathan; Borys, Michael C.; Li, Zheng Jian

2017-01-01

ABSTRACT Recent progress in mammalian cell culture process has resulted in significantly increased product titers, but also a substantial increase in process- and product-related impurities. Due to the diverse physicochemical properties of these impurities, there is constant need for new technologies that offer higher productivity and improved economics without sacrificing the process robustness required to meet final drug substance specifications. Here, we examined the use of new synthetic adsorptive hybrid filters (AHF) modified with the high binding capacity of quaternary amine (Emphaze™ AEX) and salt-tolerant biomimetic (Emphaze™ ST-AEX) ligands for clearance of process-related impurities like host cell protein (HCP), residual DNA, and virus. The potential to remove soluble aggregates was also examined. Our aim was to develop a mechanistic understanding of the interactions governing adsorptive removal of impurities during filtration by evaluating the effect of various filter types, feed streams, and process conditions on impurity removal. The ionic capacity of these filters was measured and correlated with their ability to remove impurities for multiple molecules. The ionic capacity of AHF significantly exceeded that of traditional adsorptive depth filters (ADF) by 40% for the Emphaze™ AEX and by 700% for the Emphaze™ ST-AEX, providing substantially higher reduction of soluble anionic impurities, including DNA, HCPs and model virus. Nevertheless, we determined that ADF with filter aid provided additional hydrophobic functionality that resulted in removal of higher molecular weight species than AHF. Implementing AHF demonstrated improved process-related impurity removal and viral clearance after Protein A chromatography and enabled a two-step purification process. The consequences of enhanced process performance are far reaching because it allows the downstream polishing train to be restructured and simplified, and chromatographic purity standards to be met with a reduced number of chromatographic steps. PMID:27929735

Utilization of xylitol dehydrogenase in a combined microbial/enzymatic process for production of xylitol from D-glucose.

PubMed

Mayer, Gerhard; Kulbe, Klaus D; Nidetzky, Bernd

2002-01-01

The production of xylitol from D-glucose occurs through a three-step process in which D-arabitol and D-xylulose are formed as the first and second intermediate product, respectively, and both are obtained via microbial bioconversion reactions. Catalytic hydrogenation of D-xylulose yields xylitol; however, it is contaminated with D-arabitol. The aim of this study was to increase the stereoselectivity of the D-xylulose reduction step by using enzymatic catalysis. Recombinant xylitol dehydrogenase from the yeast Galactocandida mastotermitis was employed to catalyze xylitol formation from D-xylulose in an NADH-dependent reaction, and coenzyme regeneration was achieved by means of formate dehydrogenase-catalyzed oxidation of formate into carbon dioxide. The xylitol yield from D-xylulose was close to 100%. Optimal productivity was found for initial coenzyme concentrations of between 0.5 and 0.75 mM. In the presence of 0.30 M (45 g/L) D-xylulose and 2000 U/L of both dehydrogenases, exhaustive substrate turnover was achieved typically in a 4-h reaction time. The enzymes were recovered after the reaction in yields of approx 90% by means of ultrafiltration and could be reused for up to six cycles of D-xylulose reduction. The advantages of incorporating the enzyme-catalyzed step in a process for producing xylitol from D-glucose are discussed, and strategies for downstream processing are proposed by which the observed coenzyme turnover number of approx 600 could be increased significantly.

Process engineering economics of bioethanol production.

PubMed

Galbe, Mats; Sassner, Per; Wingren, Anders; Zacchi, Guido

2007-01-01

This work presents a review of studies on the process economics of ethanol production from lignocellulosic materials published since 1996. Our objective was to identify the most costly process steps and the impact of various parameters on the final production cost, e.g. plant capacity, raw material cost, and overall product yield, as well as process configuration. The variation in estimated ethanol production cost is considerable, ranging from about 0.13 to 0.81 US$ per liter ethanol. This can be explained to a large extent by actual process differences and variations in the assumptions underlying the techno-economic evaluations. The most important parameters for the economic outcome are the feedstock cost, which varied between 30 and 90 US$ per metric ton in the papers studied, and the plant capacity, which influences the capital cost. To reduce the ethanol production cost it is necessary to reach high ethanol yields, as well as a high ethanol concentration during fermentation, to be able to decrease the energy required for distillation and other downstream process steps. Improved pretreatment methods, enhanced enzymatic hydrolysis with cheaper and more effective enzymes, as well as improved fermentation systems present major research challenges if we are to make lignocellulose-based ethanol production competitive with sugar- and starch-based ethanol. Process integration, either internally or externally with other types of plants, e.g. heat and power plants, also offers a way of reducing the final ethanol production cost.

Manufacturing human mesenchymal stem cells at clinical scale: process and regulatory challenges.

PubMed

Jossen, Valentin; van den Bos, Christian; Eibl, Regine; Eibl, Dieter

2018-05-01

Human mesenchymal stem cell (hMSC)-based therapies are of increasing interest in the field of regenerative medicine. As economic considerations have shown, allogeneic therapy seems to be the most cost-effective method. Standardized procedures based on instrumented single-use bioreactors have been shown to provide billion of cells with consistent product quality and to be superior to traditional expansions in planar cultivation systems. Furthermore, under consideration of the complex nature and requirements of allogeneic hMSC-therapeutics, a new equipment for downstream processing (DSP) was successfully evaluated. This mini-review summarizes both the current state of the hMSC production process and the challenges which have to be taken into account when efficiently producing hMSCs for the clinical scale. Special emphasis is placed on the upstream processing (USP) and DSP operations which cover expansion, harvesting, detachment, separation, washing and concentration steps, and the regulatory demands.

PI3K{gamma} activation by CXCL12 regulates tumor cell adhesion and invasion

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

Monterrubio, Maria; Mellado, Mario; Carrera, Ana C.

Tumor dissemination is a complex process, in which certain steps resemble those in leukocyte homing. Specific chemokine/chemokine receptor pairs have important roles in both processes. CXCL12/CXCR4 is the most commonly expressed chemokine/chemokine receptor pair in human cancers, in which it regulates cell adhesion, extravasation, metastatic colonization, angiogenesis, and proliferation. All of these processes require activation of signaling pathways that include G proteins, phosphatidylinositol-3 kinase (PI3K), JAK kinases, Rho GTPases, and focal adhesion-associated proteins. We analyzed these pathways in a human melanoma cell line in response to CXCL12 stimulation, and found that PI3K{gamma} regulates tumor cell adhesion through mechanisms different frommore » those involved in cell invasion. Our data indicate that, following CXCR4 activation after CXCL12 binding, the invasion and adhesion processes are regulated differently by distinct downstream events in these signaling cascades.« less

Downstream processing and chromatography based analytical methods for production of vaccines, gene therapy vectors, and bacteriophages.

PubMed

Kramberger, Petra; Urbas, Lidija; Štrancar, Aleš

2015-01-01

Downstream processing of nanoplexes (viruses, virus-like particles, bacteriophages) is characterized by complexity of the starting material, number of purification methods to choose from, regulations that are setting the frame for the final product and analytical methods for upstream and downstream monitoring. This review gives an overview on the nanoplex downstream challenges and chromatography based analytical methods for efficient monitoring of the nanoplex production.

Downstream processing and chromatography based analytical methods for production of vaccines, gene therapy vectors, and bacteriophages

PubMed Central

Kramberger, Petra; Urbas, Lidija; Štrancar, Aleš

2015-01-01

Downstream processing of nanoplexes (viruses, virus-like particles, bacteriophages) is characterized by complexity of the starting material, number of purification methods to choose from, regulations that are setting the frame for the final product and analytical methods for upstream and downstream monitoring. This review gives an overview on the nanoplex downstream challenges and chromatography based analytical methods for efficient monitoring of the nanoplex production. PMID:25751122

Colloid particle sizes in the Mississippi River and some of its tributaries, from Minneapolis to below New Orleans

USGS Publications Warehouse

Rostad, C.E.; Rees, T.F.; Daniel, S.R.

1998-01-01

An on-board technique was developed that combined discharge-weighted pumping to a high-speed continuous-flow centrifuge for isolation of the particulate-sized material with ultrafiltration for isolation of colloid-sized material. In order to address whether these processes changed the particle sizes during isolation, samples of particles in suspension were collected at various steps in the isolation process to evaluate changes in particle size. Particle sizes were determined using laser light-scattering photon correlation spectroscopy and indicated no change in size during the colloid isolation process. Mississippi River colloid particle sizes from twelve sites from Minneapolis to below New Orleans were compared with sizes from four tributaries and three seasons, and from predominantly autochthonous sources upstream to more allochthonous sources downstream. ?? 1998 John Wiley Sons, Ltd.

Experimental study of flow reattachment in a single-sided sudden expansion

NASA Technical Reports Server (NTRS)

Westphal, R. V.; Johnston, J. P.; Eaton, J. K.

1984-01-01

The reattachment of a fully turbulent, two dimensional, separated shear layer downstream of a single-sided sudden expansion in a planar duct flow was examined experimentally. The importance of changing the structure of the separated shear layer on the reattachment process itself was examined. For all cases, the Reynolds number based on step height was greater than 20,000, the expansion ratio was 5/3, and the inlet boundary layer was less than one-half step height in thickness. A crucially important phase was the development of a pulsed wall probe for measurement of skin friction in the reattachment region, thus providing an unambiguous definition of the reattachment length. Quantitative features of reattachment - including streamwise development of the mean and fluctuating velocity field, pressure rise, and skin friction - were found to be similar for all cases studied when scaled by the reattachment length. A definition of the reattachment zone is proposed.

Gasification of high ash, high ash fusion temperature bituminous coals

DOEpatents

Liu, Guohai; Vimalchand, Pannalal; Peng, WanWang

2015-11-13

This invention relates to gasification of high ash bituminous coals that have high ash fusion temperatures. The ash content can be in 15 to 45 weight percent range and ash fusion temperatures can be in 1150.degree. C. to 1500.degree. C. range as well as in excess of 1500.degree. C. In a preferred embodiment, such coals are dealt with a two stage gasification process--a relatively low temperature primary gasification step in a circulating fluidized bed transport gasifier followed by a high temperature partial oxidation step of residual char carbon and small quantities of tar. The system to process such coals further includes an internally circulating fluidized bed to effectively cool the high temperature syngas with the aid of an inert media and without the syngas contacting the heat transfer surfaces. A cyclone downstream of the syngas cooler, operating at relatively low temperatures, effectively reduces loading to a dust filtration unit. Nearly dust- and tar-free syngas for chemicals production or power generation and with over 90%, and preferably over about 98%, overall carbon conversion can be achieved with the preferred process, apparatus and methods outlined in this invention.

Components of the Engulfment Machinery Have Distinct Roles in Corpse Processing

PubMed Central

Meehan, Tracy L.; Joudi, Tony F.; Timmons, Allison K.; Taylor, Jeffrey D.; Habib, Corey S.; Peterson, Jeanne S.; Emmanuel, Shanan; Franc, Nathalie C.; McCall, Kimberly

2016-01-01

Billions of cells die in our bodies on a daily basis and are engulfed by phagocytes. Engulfment, or phagocytosis, can be broken down into five basic steps: attraction of the phagocyte, recognition of the dying cell, internalization, phagosome maturation, and acidification. In this study, we focus on the last two steps, which can collectively be considered corpse processing, in which the engulfed material is degraded. We use the Drosophila ovarian follicle cells as a model for engulfment of apoptotic cells by epithelial cells. We show that engulfed material is processed using the canonical corpse processing pathway involving the small GTPases Rab5 and Rab7. The phagocytic receptor Draper is present on the phagocytic cup and on nascent, phosphatidylinositol 3-phosphate (PI(3)P)- and Rab7-positive phagosomes, whereas integrins are maintained on the cell surface during engulfment. Due to the difference in subcellular localization, we investigated the role of Draper, integrins, and downstream signaling components in corpse processing. We found that some proteins were required for internalization only, while others had defects in corpse processing as well. This suggests that several of the core engulfment proteins are required for distinct steps of engulfment. We also performed double mutant analysis and found that combined loss of draper and αPS3 still resulted in a small number of engulfed vesicles. Therefore, we investigated another known engulfment receptor, Crq. We found that loss of all three receptors did not inhibit engulfment any further, suggesting that Crq does not play a role in engulfment by the follicle cells. A more complete understanding of how the engulfment and corpse processing machinery interact may enable better understanding and treatment of diseases associated with defects in engulfment by epithelial cells. PMID:27347682

Microwave-Accelerated Method for Ultra-Rapid Extraction of Neisseria gonorrhoeae DNA for Downstream Detection

PubMed Central

Melendez, Johan H.; Santaus, Tonya M.; Brinsley, Gregory; Kiang, Daniel; Mali, Buddha; Hardick, Justin; Gaydos, Charlotte A.; Geddes, Chris D.

2016-01-01

Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by the detection of the genomic target often involving PCR-based approaches. In an effort to improve on current detection approaches, we have developed a unique two-step microwave-accelerated approach for rapid extraction and detection of Neisseria gonorrhoeae (GC) DNA. Our approach is based on the use of highly-focused microwave radiation to rapidly lyse bacterial cells, release, and subsequently fragment microbial DNA. The DNA target is then detected by a process known as microwave-accelerated metal-enhanced fluorescence (MAMEF), an ultra-sensitive direct DNA detection analytical technique. In the present study, we show that highly focused microwaves at 2.45 GHz, using 12.3 mm gold film equilateral triangles, are able to rapidly lyse both bacteria cells and fragment DNA in a time- and microwave power-dependent manner. Detection of the extracted DNA can be performed by MAMEF, without the need for DNA amplification in less than 10 minutes total time or by other PCR-based approaches. Collectively, the use of a microwave-accelerated method for the release and detection of DNA represents a significant step forward towards the development of a point-of-care (POC) platform for detection of gonorrhea infections. PMID:27325503

Forces on stationary particles in near-bed turbulent flows

NASA Astrophysics Data System (ADS)

Schmeeckle, Mark W.; Nelson, Jonathan M.; Shreve, Ronald L.

2007-06-01

In natural flows, bed sediment particles are entrained and moved by the fluctuating forces, such as lift and drag, exerted by the overlying flow on the particles. To develop a better understanding of these forces and the relation of the forces to the local flow, the downstream and vertical components of force on near-bed fixed particles and of fluid velocity above or in front of them were measured synchronously at turbulence-resolving frequencies (200 or 500 Hz) in a laboratory flume. Measurements were made for a spherical test particle fixed at various heights above a smooth bed, above a smooth bed downstream of a downstream-facing step, and in a gravel bed of similarly sized particles as well as for a cubical test particle and 7 natural particles above a smooth bed. Horizontal force was well correlated with downstream velocity and not correlated with vertical velocity or vertical momentum flux. The standard drag formula worked well to predict the horizontal force, but the required value of the drag coefficient was significantly higher than generally used to model bed load motion. For the spheres, cubes, and natural particles, average drag coefficients were found to be 0.76, 1.36, and 0.91, respectively. For comparison, the drag coefficient for a sphere settling in still water at similar particle Reynolds numbers is only about 0.4. The variability of the horizontal force relative to its mean was strongly increased by the presence of the step and the gravel bed. Peak deviations were about 30% of the mean force for the sphere over the smooth bed, about twice the mean with the step, and 4 times it for the sphere protruding roughly half its diameter above the gravel bed. Vertical force correlated poorly with downstream velocity, vertical velocity, and vertical momentum flux whether measured over or ahead of the test particle. Typical formulas for shear-induced lift based on Bernoulli's principle poorly predict the vertical forces on near-bed particles. The measurements suggest that particle-scale pressure variations associated with turbulence are significant in the particle momentum balance.

Forces on stationary particles in near-bed turbulent flows

USGS Publications Warehouse

Schmeeckle, M.W.; Nelson, J.M.; Shreve, R.L.

2007-01-01

In natural flows, bed sediment particles are entrained and moved by the fluctuating forces, such as lift and drag, exerted by the overlying flow on the particles. To develop a better understanding of these forces and the relation of the forces to the local flow, the downstream and vertical components of force on near-bed fixed particles and of fluid velocity above or in front of them were measured synchronously at turbulence-resolving frequencies (200 or 500 Hz) in a laboratory flume. Measurements were made for a spherical test particle fixed at various heights above a smooth bed, above a smooth bed downstream of a downstream-facing step, and in a gravel bed of similarly sized particles as well as for a cubical test particle and 7 natural particles above a smooth bed. Horizontal force was well correlated with downstream velocity and not correlated with vertical velocity or vertical momentum flux. The standard drag formula worked well to predict the horizontal force, but the required value of the drag coefficient was significantly higher than generally used to model bed load motion. For the spheres, cubes, and natural particles, average drag coefficients were found to be 0.76, 1.36, and 0.91, respectively. For comparison, the drag coefficient for a sphere settling in still water at similar particle Reynolds numbers is only about 0.4. The variability of the horizontal force relative to its mean was strongly increased by the presence of the step and the gravel bed. Peak deviations were about 30% of the mean force for the sphere over the smooth bed, about twice the mean with the step, and 4 times it for the sphere protruding roughly half its diameter above the gravel bed. Vertical force correlated poorly with downstream velocity, vertical velocity, and vertical momentum flux whether measured over or ahead of the test particle. Typical formulas for shear-induced lift based on Bernoulli's principle poorly predict the vertical forces on near-bed particles. The measurements suggest that particle-scale pressure variations associated with turbulence are significant in the particle momentum balance. Copyright 2007 by the American Geophysical Union.

Low-Cost alpha Alane for Hydrogen Storage

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

Fabian, Tibor; Petrie, Mark; Crouch-Baker, Steven

This project was directed towards the further development of the Savannah River National Laboratory (SRNL) lab-scale electrochemical synthesis of the hydrogen storage material alpha-alane and Ardica Technologies-SRI International (SRI) chemical downstream processes that are necessary to meet DoE cost metrics and transition alpha-alane synthesis to an industrial scale. Ardica has demonstrated the use of alpha-alane in a fuel-cell system for the U.S. Army WFC20 20W soldier power system that has successfully passed initial field trials with individual soldiers. While alpha-alane has been clearly identified as a desirable hydrogen storage material, cost-effective means for its production and regeneration on a scalemore » of use applicable to the industry have yet to be established. We focused on three, principal development areas: 1. The construction of a comprehensive engineering techno-economic model to establish the production costs of alpha-alane by both electrochemical and chemical routes at scale. 2. The identification of critical, cost-saving design elements of the electrochemical cell and the quantification of the product yields of the primary electrochemical process. A moving particle-bed reactor design was constructed and operated. 3. The experimental quantification of the product yields of candidate downstream chemical processes necessary to produce alpha-alane to complete the most cost-effective overall manufacturing process. Our techno-economic model shows that under key assumptions most 2015 and 2020 DOE hydrogen storage system cost targets for low and medium power can be achieved using the electrochemical alane synthesis process. To meet the most aggressive 2020 storage system cost target, $1/g, our model indicates that 420 metric tons per year (MT/y) production of alpha-alane is required. Laboratory-scale experimental work demonstrated that the yields of two of the three critical component steps within the overall “electrochemical process” were sufficiently high to meet this production target. In the case of the yield of the third step, the crystallization of alpha-alane from the primary alane-related product of the electrochemical reaction, further development is required.« less

Investigation of the Evolution of Crystal Size and Shape during Temperature Cycling and in the Presence of a Polymeric Additive Using Combined Process Analytical Technologies

PubMed Central

2017-01-01

Crystal size and shape can be manipulated to enhance the qualities of the final product. In this work the steady-state shape and size of succinic acid crystals, with and without a polymeric additive (Pluronic P123) at 350 mL, scale is reported. The effect of the amplitude of cycles as well as the heating/cooling rates is described, and convergent cycling (direct nucleation control) is compared to static cycling. The results show that the shape of succinic acid crystals changes from plate- to diamond-like after multiple cycling steps, and that the time required for this morphology change to occur is strongly related to the type of cycling. Addition of the polymer is shown to affect both the final shape of the crystals and the time needed to reach size and shape steady-state conditions. It is shown how this phenomenon can be used to improve the design of the crystallization step in order to achieve more efficient downstream operations and, in general, to help optimize the whole manufacturing process. PMID:28867966

Bioprocessing of plant-derived virus-like particles of Norwalk virus capsid protein under current Good Manufacture Practice regulations

PubMed Central

Lai, Huafang; Chen, Qiang

2012-01-01

Despite the success in expressing a variety of subunit vaccine proteins in plants and the recent stride in improving vaccine accumulation levels by transient expression systems, there is still no plant-derived vaccine that has been licensed for human use. The lack of commercial success of plant-made vaccines lies in several technical and regulatory barriers that remain to be overcome. These challenges include the lack of scalable downstream processing procedures, the uncertainty of regulatory compliance of production processes, and the lack of demonstration of plant-derived products that meet the required standards of regulatory agencies in identity, purity, potency and safety. In this study, we addressed these remaining challenges and successfully demonstrate the ability of using plants to produce a pharmaceutical grade Norwalk virus (NV) vaccine under current Good Manufacture Practice (cGMP) guidelines at multiple gram scales. Our results demonstrate that an efficient and scalable extraction and purification scheme can established for processing virus-like particles (VLP) of NV capsid protein (NVCP). We successfully operated the upstream and downstream NVCP production processes under cGMP regulations. Furthermore, plant-derived NVCP VLP demonstrates the identity, purity, potency and safety that meet the preset release specifications. This material is being tested in a Phase I human clinical trial. This research provides the first report of producing a plant-derived vaccine at scale under cGMP regulations in an academic setting and an important step for plant-produced vaccines to become a commercial reality. PMID:22134876

Chromatographic matrix based on hydrogel-coated reticulated polyurethane foams, prepared by gamma irradiation

NASA Astrophysics Data System (ADS)

Sánchez, Mirna L.; Giménez, Claudia Y.; Delgado, Juan F.; Martínez, Leandro J.; Grasselli, Mariano

2017-12-01

Novel chromatographic materials for protein purification with high adsorption capacity and fouling resistance are highly demanded to improve downstream processes. Here, we describe a novel adsorptive material based on reticulated polyurethane foam (rPUF) coated with a functional hydrogel layer. rPUF provides physical rigidity through its macroscopic structure, whereas the hydrogel layer provides capacity to adsorb proteins by specific interactions. The hydrogel coating process was performed by the dip-coating method, using a polyvinyl alcohol (PVA) solution. The PVA hydrogel was linked to the rPUF material by using a radiation-induced crosslinking process in aqueous ethanol solution. The ethanol in the solvent mixture allowed a balance between PVA swelling and PVA dissolution during the irradiation step. The resulting material showed higher thermal stability than the non-irradiated one. In addition, a simultaneous radiation-induced grafting polymerization (SRIGP) was done by simple addition of glycidyl methacrylate monomer into the irradiation solution. In a further step, sulfonic ligands were included specifically in the hydrogel layer, which contained around 200% of PVA respect to the original rPUF. Materials were characterized by FT-IR, thermogravimetric analysis, SEM microscopy and EDX analysis. The cation-exchange rPUF material was functionally characterized by the Langmuir isotherm and a dynamic adsorption experiment to analyze the chromatographic properties for protein purification processes.

Assessment and management of the performance risk of a pilot reclaimed water disinfection process.

PubMed

Zhou, Guangyu; Zhao, Xinhua; Zhang, Lei; Wu, Qing

2013-10-01

Chlorination disinfection has been widely used in reclaimed water treatment plants to ensure water quality. In order to assess the downstream quality risk of a running reclaimed water disinfection process, a set of dynamic equations was developed to simulate reactions in the disinfection process concerning variables of bacteria, chemical oxygen demand (COD), ammonia and monochloramine. The model was calibrated by the observations obtained from a pilot disinfection process which was designed to simulate the actual process in a reclaimed water treatment plant. A Monte Carlo algorithm was applied to calculate the predictive effluent quality distributions that were used in the established hierarchical assessment system for the downstream quality risk, and the key factors affecting the downstream quality risk were defined using the Regional Sensitivity Analysis method. The results showed that the seasonal upstream quality variation caused considerable downstream quality risk; the effluent ammonia was significantly influenced by its upstream concentration; the upstream COD was a key factor determining the process effluent risk of bacterial, COD and residual disinfectant indexes; and lower COD and ammonia concentrations in the influent would mean better downstream quality.

Sexy splicing: regulatory interplays governing sex determination from Drosophila to mammals.

PubMed

Lalli, Enzo; Ohe, Kenji; Latorre, Elisa; Bianchi, Marco E; Sassone-Corsi, Paolo

2003-02-01

A remarkable array of strategies is used to produce sexual differentiation in different species. Complex gene hierarchies govern sex determination pathways, as exemplified by the classic D. melanogaster paradigm, where an interplay of transcriptional, splicing and translational mechanisms operate. Molecular studies support the hypothesis that genetic sex determination pathways evolved in reverse order, from downstream to upstream genes, in the cascade. The recent identification of a role for the key regulatory factors SRY and WT1(+KTS) in pre-mRNA splicing indicates that important steps in the mammalian sex determination process are likely to operate at the post-transcriptional level.

High-throughput process development: I. Process chromatography.

PubMed

Rathore, Anurag S; Bhambure, Rahul

2014-01-01

Chromatographic separation serves as "a workhorse" for downstream process development and plays a key role in removal of product-related, host cell-related, and process-related impurities. Complex and poorly characterized raw materials and feed material, low feed concentration, product instability, and poor mechanistic understanding of the processes are some of the critical challenges that are faced during development of a chromatographic step. Traditional process development is performed as trial-and-error-based evaluation and often leads to a suboptimal process. High-throughput process development (HTPD) platform involves an integration of miniaturization, automation, and parallelization and provides a systematic approach for time- and resource-efficient chromatography process development. Creation of such platforms requires integration of mechanistic knowledge of the process with various statistical tools for data analysis. The relevance of such a platform is high in view of the constraints with respect to time and resources that the biopharma industry faces today. This protocol describes the steps involved in performing HTPD of process chromatography step. It described operation of a commercially available device (PreDictor™ plates from GE Healthcare). This device is available in 96-well format with 2 or 6 μL well size. We also discuss the challenges that one faces when performing such experiments as well as possible solutions to alleviate them. Besides describing the operation of the device, the protocol also presents an approach for statistical analysis of the data that is gathered from such a platform. A case study involving use of the protocol for examining ion-exchange chromatography of granulocyte colony-stimulating factor (GCSF), a therapeutic product, is briefly discussed. This is intended to demonstrate the usefulness of this protocol in generating data that is representative of the data obtained at the traditional lab scale. The agreement in the data is indeed very significant (regression coefficient 0.93). We think that this protocol will be of significant value to those involved in performing high-throughput process development of process chromatography.

A Statistical Selection Strategy for Normalization Procedures in LC-MS Proteomics Experiments through Dataset Dependent Ranking of Normalization Scaling Factors

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

Webb-Robertson, Bobbie-Jo M.; Matzke, Melissa M.; Jacobs, Jon M.

2011-12-01

Quantification of LC-MS peak intensities assigned during peptide identification in a typical comparative proteomics experiment will deviate from run-to-run of the instrument due to both technical and biological variation. Thus, normalization of peak intensities across a LC-MS proteomics dataset is a fundamental step in pre-processing. However, the downstream analysis of LC-MS proteomics data can be dramatically affected by the normalization method selected . Current normalization procedures for LC-MS proteomics data are presented in the context of normalization values derived from subsets of the full collection of identified peptides. The distribution of these normalization values is unknown a priori. If theymore » are not independent from the biological factors associated with the experiment the normalization process can introduce bias into the data, which will affect downstream statistical biomarker discovery. We present a novel approach to evaluate normalization strategies, where a normalization strategy includes the peptide selection component associated with the derivation of normalization values. Our approach evaluates the effect of normalization on the between-group variance structure in order to identify candidate normalization strategies that improve the structure of the data without introducing bias into the normalized peak intensities.« less

Selection of peptides interfering with protein-protein interaction.

PubMed

Gaida, Annette; Hagemann, Urs B; Mattay, Dinah; Räuber, Christina; Müller, Kristian M; Arndt, Katja M

2009-01-01

Cell physiology depends on a fine-tuned network of protein-protein interactions, and misguided interactions are often associated with various diseases. Consequently, peptides, which are able to specifically interfere with such adventitious interactions, are of high interest for analytical as well as medical purposes. One of the most abundant protein interaction domains is the coiled-coil motif, and thus provides a premier target. Coiled coils, which consist of two or more alpha-helices wrapped around each other, have one of the simplest interaction interfaces, yet they are able to confer highly specific homo- and heterotypic interactions involved in virtually any cellular process. While there are several ways to generate interfering peptides, the combination of library design with a powerful selection system seems to be one of the most effective and promising approaches. This chapter guides through all steps of such a process, starting with library options and cloning, detailing suitable selection techniques and ending with purification for further down-stream characterization. Such generated peptides will function as versatile tools to interfere with the natural function of their targets thereby illuminating their down-stream signaling and, in general, promoting understanding of factors leading to specificity and stability in protein-protein interactions. Furthermore, peptides interfering with medically relevant proteins might become important diagnostics and therapeutics.

Quantitative Evaluation of E1 Endoglucanase Recovery from Tobacco Leaves Using the Vacuum Infiltration-Centrifugation Method

PubMed Central

Kingsbury, Nathaniel J.; McDonald, Karen A.

2014-01-01

As a production platform for recombinant proteins, plant leaf tissue has many advantages, but commercialization of this technology has been hindered by high recovery and purification costs. Vacuum infiltration-centrifugation (VI-C) is a technique to obtain extracellularly-targeted products from the apoplast wash fluid (AWF). Because of its selective recovery of secreted proteins without homogenizing the whole tissue, VI-C can potentially reduce downstream production costs. Lab scale experiments were conducted to quantitatively evaluate the VI-C method and compared to homogenization techniques in terms of product purity, concentration, and other desirable characteristics. From agroinfiltrated Nicotiana benthamiana leaves, up to 81% of a truncated version of E1 endoglucanase from Acidothermus cellulolyticus was recovered with VI-C versus homogenate extraction, and average purity and concentration increases of 4.2-fold and 3.1-fold, respectively, were observed. Formulas were developed to predict recovery yields of secreted protein obtained by performing multiple rounds of VI-C on the same leaf tissue. From this, it was determined that three rounds of VI-C recovered 97% of the total active recombinant protein accessible to the VI-C procedure. The results suggest that AWF recovery is an efficient process that could reduce downstream processing steps and costs for plant-made recombinant proteins. PMID:24971334

Dam Removal Provides Fish Passage, Water Quality Benefits

EPA Pesticide Factsheets

The Bishopville Pond Dam in Maryland has been replaced with a series of pools, runs and step-like structures, improving water quality downstream and providing access for key fish species to spawn upstream.

Time Resolved Stereo Particle Image Velocimetry Measurements of the Instabilities Downstream of a Backward-Facing Step in a Swept-Wing Boundary Layer

NASA Technical Reports Server (NTRS)

Eppink, Jenna L.; Yao, Chung-Sheng

2017-01-01

Time-resolved particle image velocimetry (TRPIV) measurements are performed down-stream of a swept backward-facing step, with a height of 49% of the boundary-layer thickness. The results agree well qualitatively with previously reported hotwire measurements, though the amplitudes of the fluctuating components measured using TRPIV are higher. Nonetheless, the low-amplitude instabilities in the flow are fairly well resolved using TR- PIV. Proper orthogonal decomposition is used to study the development of the traveling cross flow and Tollmien-Schlichting (TS) instabilities downstream of the step and to study how they interact to form the large velocity spikes that ultimately lead to transition. A secondary mode within the traveling cross flow frequency band develops with a wavelength close to that of the stationary cross flow instability, so that at a certain point in the phase, it causes an increase in the spanwise modulation initially caused by the stationary cross flow mode. This increased modulation leads to an increase in the amplitude of the TS mode, which, itself, is highly modulated through interactions with the stationary cross flow. When the traveling cross flow and TS modes align in time and space, the large velocity spikes occur. Thus, these three instabilities, which are individually of low amplitude when the spikes start to occur (U'rms/Ue <0.03), interact and combine to cause a large flow disturbance that eventually leads to transition.

Recovery of succinic acid produced by fermentation of a metabolically engineered Mannheimia succiniciproducens strain.

PubMed

Song, Hyohak; Huh, Yun Suk; Lee, Sang Yup; Hong, Won Hi; Hong, Yeon Ki

2007-12-01

There have recently been much advances in the production of succinic acid, an important four-carbon dicarboxylic acid for many industrial applications, by fermentation of several natural and engineered bacterial strains. Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is able to produce succinic acid with high efficiency, but also produces acetic, formic and lactic acids just like other anaerobic succinic acid producers. We recently reported the development of an engineered M. succiniciproducens LPK7 strain which produces succinic acid as a major fermentation product while producing much reduced by-products. Having an improved succinic acid producer developed, it is equally important to develop a cost-effective downstream process for the recovery of succinic acid. In this paper, we report the development of a simpler and more efficient method for the recovery of succinic acid. For the recovery of succinic acid from the fermentation broth of LPK7 strain, a simple process composed of a single reactive extraction, vacuum distillation, and crystallization yielded highly purified succinic acid (greater than 99.5% purity, wt%) with a high yield of 67.05wt%. When the same recovery process or even multiple reactive extraction steps were applied to the fermentation broth of MBEL55E, lower purity and yield of succinic acid were obtained. These results suggest that succinic acid can be purified in a cost-effective manner by using the fermentation broth of engineered LPK7 strain, showing the importance of integrating the strain development, fermentation and downstream process for optimizing the whole processes for succinic acid production.

Integrated viral clearance strategies-reflecting on the present, projecting to the future.

PubMed

Roush, David J

2018-01-20

Viral clearance and inactivation are critical steps in ensuring the safety of biological products derived from mammalian cell culture and are a component of an adventitious agent control strategy which spans both upstream and downstream processes. Although these approaches have been sufficient to support the development of biologics to date, the empirical and semi-quantitative nature of the approach leaves some potential gaps. For example, the concept of performing a quantitative risk assessment for the downstream components of virus safety was introduced in ICH Q5A for XMuLV. An ideal future state would be to perform a similar quantitative risk assessment for a range of viruses based on an assessment of potential virus risk in both upstream and downstream processes. This assessment combined with an integrated control strategy (including monitoring) would be extremely beneficial in minimizing potential adventitious agent risks. Significant progress has been achieved towards this goal in the last several years including recent advances in quantification of virus sequences in cell banks (ADVTIG), development of truly modular or generic viral clearance claims for specific unit operations, enhanced controls of upstream media (HTST/nanofiltration) and the use of RVLP for in-process monitoring. The recent shift towards continuous processing has the potential to enhance the criticality of in-line monitoring and the complexity of viral clearance and inactivation (owing to a wide range of potential 'worst case' viral clearance scenarios). However, gaps exist in, firstly, the ability to quantify potential virus risk levels in process streams in real-time, secondly, mechanistic understanding of virus/chromatography media interactions, and thirdly, mechanistic understanding of virus/filter interactions. Some new technologies may also need to be developed to allow for real-time confirmation of virus inactivation and clearance to support process development (both batch and continuous) and assessment of the impact of process deviations during manufacturing. This review paper provides an overview of the current state of an overall integrated control strategy for upstream and downstream processing and highlights the investments that could be pursued to achieve the future state of a quantitative virus risk assessment for a range of viruses. One potential approach to address these gaps is the use of data mining from large, comprehensive and diverse data sets to establish heuristic rules for virus detection, clearance and inactivation followed by specific hypothesis-driven experiments for cases that fall outside of the normal paradigm. Once this approach reaches a mature state suitable for implementation, there is an opportunity to update regulatory guidance (e.g. ICH Q5A) accordingly. Copyright © 2018 Elsevier Ltd. All rights reserved.

Proteolytic Cascade for the Activation of the Insect Toll Pathway Induced by the Fungal Cell Wall Component

PubMed Central

Roh, Kyung-Baeg; Kim, Chan-Hee; Lee, Hanna; Kwon, Hyun-Mi; Park, Ji-Won; Ryu, Ji-Hwan; Kurokawa, Kenji; Ha, Nam-Chul; Lee, Won-Jae; Lemaitre, Bruno; Söderhäll, Kenneth; Lee, Bok-Luel

2009-01-01

The insect Toll signaling pathway is activated upon recognition of Gram-positive bacteria and fungi, resulting in the expression of antimicrobial peptides via NF-κB-like transcription factor. This activation is mediated by a serine protease cascade leading to the processing of Spätzle, which generates the functional ligand of the Toll receptor. Recently, we identified three serine proteases mediating Toll pathway activation induced by lysine-type peptidoglycan of Gram-positive bacteria. However, the identities of the downstream serine protease components of Gram-negative-binding protein 3 (GNBP3), a receptor for a major cell wall component β-1,3-glucan of fungi, and their order of activation have not been characterized yet. Here, we identified three serine proteases that are required for Toll activation by β-1,3-glucan in the larvae of a large beetle, Tenebrio molitor. The first one is a modular serine protease functioning immediately downstream of GNBP3 that proteolytically activates the second one, a Spätzle-processing enzyme-activating enzyme that in turn activates the third serine protease, a Spätzle-processing enzyme. The active form of Spätzle-processing enzyme then cleaves Spätzle into the processed Spätzle as Toll ligand. In addition, we show that injection of β-1,3-glucan into Tenebrio larvae induces production of two antimicrobial peptides, Tenecin 1 and Tenecin 2, which are also inducible by injection of the active form of Spätzle-processing enzyme-activating enzyme or processed Spätzle. These results demonstrate a three-step proteolytic cascade essential for the Toll pathway activation by fungal β-1,3-glucan in Tenebrio larvae, which is shared with lysine-type peptidoglycan-induced Toll pathway activation. PMID:19473968

Broad application and optimization of a single wash-step for integrated endotoxin depletion during protein purification.

PubMed

Koziel, David; Michaelis, Uwe; Kruse, Tobias

2018-08-01

Endotoxins contaminate proteins that are produced in E. coli. High levels of endotoxins can influence cellular assays and cause severe adverse effects when administered to humans. Thus, endotoxin removal is important in protein purification for academic research and in GMP manufacturing of biopharmaceuticals. Several methods exist to remove endotoxin, but often require additional downstream-processing steps, decrease protein yield and are costly. These disadvantages can be avoided by using an integrated endotoxin depletion (iED) wash-step that utilizes Triton X-114 (TX114). In this paper, we show that the iED wash-step is broadly applicable in most commonly used chromatographies: it reduces endotoxin by a factor of 10 3 to 10 6 during NiNTA-, MBP-, SAC-, GST-, Protein A and CEX-chromatography but not during AEX or HIC-chromatography. We characterized the iED wash-step using Design of Experiments (DoE) and identified optimal experimental conditions for application scenarios that are relevant to academic research or industrial GMP manufacturing. A single iED wash-step with 0.75% (v/v) TX114 added to the feed and wash buffer can reduce endotoxin levels to below 2 EU/ml or deplete most endotoxin while keeping the manufacturing costs as low as possible. The comprehensive characterization enables academia and industry to widely adopt the iED wash-step for a routine, efficient and cost-effective depletion of endotoxin during protein purification at any scale. Copyright © 2018. Published by Elsevier B.V.

Genome engineering for improved recombinant protein expression in Escherichia coli.

PubMed

Mahalik, Shubhashree; Sharma, Ashish K; Mukherjee, Krishna J

2014-12-19

A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review.

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

PubMed

Bova, G Steven; Eltoum, Isam A; Kiernan, John A; Siegal, Gene P; Frost, Andra R; Best, Carolyn J M; Gillespie, John W; Emmert-Buck, Michael R

2005-01-01

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of pancreatic malignancy and other biological phenomena. This chapter reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed-over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification. High-quality tissue microdissection does not necessarily mean high-quality samples to analyze. The quality of biomaterials obtained for analysis is highly dependent on steps upstream and downstream from tissue microdissection. We provide protocols for each of these steps, and encourage you to improve upon these. It is worth the effort of every laboratory to optimize and document its technique at each stage of the process, and we provide a starting point for those willing to spend the time to optimize. In our view, poor documentation of tissue and cell type of origin and the use of nonoptimized protocols is a source of inefficiency in current life science research. Even incremental improvement in this area will increase productivity significantly.

Potential GLOF Hazards and Initiatives taken to minimize its Impacts on Downstream Communities and Infrastructures in Nepal Himalaya

NASA Astrophysics Data System (ADS)

Regmi, D.; Kargel, J. S.; Leonard, G. J.; Haritashya, U. K.; Karki, A.; Poudyal, S.

2017-12-01

With long-term temperature increases due to climate change, glacier lakes in several parts of the world are a fast-developing threat to infrastructure and downstream communities. There are more than 2000 glacier lakes in Nepal; while most pose no significant hazard to people, a comparative few are very dangerous, such as Tso Rolpa, Imja, Barun and Thulagi glacier lakes. The objectives of this study are to present 1) a review of prior glacier lake studies that have been carried out in the Nepal Himalaya; 2) recent research results, including bathymetric studies of the lakes; 3) a summary of possible infrastructure damages, especially multi-million-dollar hydropower projects, that are under threat of glacier lake outburst floods (GLOFs); 4) to present the outcome of the recently completed Imja lake lowering project, which is the highest altitude lake ever controlled by lowering the water level. This project is being undertaken as a response to a scientific ground-based bathymetric and geophysical survey funded by the United Nations Development Program and a satellite-based study of the long-term development of the lake (funded by NASA's SERVIR program, J. Kargel, PI). The objective of the Imja Lake GLOF mitigation project is to lower the water level by three meters to reduce the lake volume, increase the freeboard, and improve the safety of tourism, downstream communities, and the infrastructure of Nepal's Everest region. This GLOF mitigation step taken by Nepal's government to reduce the risk of an outburst flood is a good step to reduce the chances of a GLOF, and to reduce the magnitude of a disaster if a GLOF nonetheless occurs despite our best efforts. We will also present the prospects for the future of Imja Lake, including an outline of possible steps that could further reduce the hazards faced by downstream communities and infrastructure. Key words: Glacier Lakes; GLOF; Hydropower; Imja lake; lake lowering

Double air-fuel ratio sensor system having double-skip function

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

Katsuno, T.

1988-01-26

A method for controlling the air-fuel ratio in an internal combustion engine is described having a catalyst converter for removing pollutants in the exhaust gas thereof, and upstream-side and downstream-side air-fuel ratio sensors disposed upstream and downstream, respectively, of the catalyst converter for detecting a concentration of a specific component in an exhaust gas, comprising the steps of: comparing the output of the upstream-side air-fuel ratio sensor with a first predetermined value; gradually changing a first air-fuel ratio correction amount in accordance with a result of the comparison of the output of the upstream-side air-fuel ratio sensor with the predeterminedmore » value; shifting the first air-fuel ratio correction amount by a first skip amount during a predetermined time period after the result of the comparison of the upstream-side air-fuel ratio sensor is changed; shifting the first air-fuel ratio correction amount by a second skip amount smaller than the first skip amount after the predetermined time period has passed; comparing the output of the downstream-side air-fuel ratio with a second predetermined value, calculating a second air-fuel ratio correction amount in accordance with the comparison result of the output of the downstream-side air-fuel ratio sensor with the second predetermined value; and adjusting the actual air-fuel ratio in accordance with the first and second air-fuel ratio correction amounts; wherein the gradually-changing step comprises the steps of: gradually decreasing the first air-fuel ratio correction amount when the output of the upstream-side air-fuel sensor is on the rich side with respect to the first predetermined value; and gradually increasing the first air-fuel ratio correction amount when the output of the upstream-side air-fuel sensor is on the lean side with respect to the first predetermined value.« less

Separated and Recovering Turbulent Boundary Layer Flow Behind a Backward Facing Step For Different Reynolds Numbers

NASA Technical Reports Server (NTRS)

Jovic, Srba; Kutler, Paul F. (Technical Monitor)

1994-01-01

Experimental results for a two-dimensional separated turbulent boundary layer behind a backward facing step for five different Reynolds numbers are reported. Results are presented in the form of tables, graphs and a floppy disk for an easy access of the data. Reynolds number based on the step height was varied by changing the reference velocity upstream of the step, U(sub o), and the step height, h. Hot-wire measurement techniques were used to measure three Reynolds stresses and four triple-velocity correlations. In addition, surface pressure and skin friction coefficients were measured. All hot-wire measurements were acquired in a measuring domain which excluded recirculating flow region due to the directional insensitivity of hot-wires. The downstream extent of the domain from the step was 51 h for the largest and I 14h for the smallest step height. This significant downstream length permitted extensive study of the flow recovery. Prediction of perturbed flows and their recovery is particularly attractive for popular turbulence models since variations of turbulence length and time scales and flow interactions in different regions are generally inadequately predicted. The data indicate that the flow in the free shear layer region behaves like the plane mixing layer up to about 2/3 of the mean reattachment length when the flow interaction with the wall commences the flow recovery to that of an ordinary turbulent boundary layer structure. These changes of the flow do not occur abruptly with the change of boundary conditions. A reattachment region represents a transitional region where the flow undergoes the most dramatic adjustments to the new boundary conditions. Large eddies, created in the upstream free-shear layer region, are being torn, recirculated, reentrained back into the main stream interacting with the incoming flow structure. It is foreseeable that it is quite difficult to describe the physics of this region in a rational and quantitative manner other than statistical. Downstream of the reattachment point the flow recovers at different rates near the wall, in the newly developing internal boundary layer, and in the outer part of the flow. It appears that Reynolds stresses do not fully recover up to the longest recovery length of 114 h.

Characterization of pulsed flow attenuation on a regulated montane river

NASA Astrophysics Data System (ADS)

Fong, C. S.; Yarnell, S. M.; Fleenor, W. E.; Viers, J. H.

2013-12-01

A major benefit of hydropower is its ability to respond quickly to fluctuating electrical loads. However, the sharp changes in discharge caused by this practice have detrimental environmental effects downstream. This study investigated the effects of hydrograph shape on attenuation of regulated pulsed flow events by first categorizing, then modeling the downstream movement of representative pulses on the upper Tuolumne River below Holm Powerhouse in the Sierra Nevada mountains of California. This system was managed by a public utility and produced flow pulses primarily for hydroelectricity generation and/or whitewater recreation. Operations were highly influenced by a system-wide "Water First" policy, which prioritized drinking water supply and quality over other beneficial uses. Pulses were therefore associated with a spectrum of time scales, from predetermined schedules decided far in advance to hydropeaking operations responding to real-time demands. We extracted underlying hydrograph shape patterns using principal component analysis on individual pulsed flow events released from 1988-2012 (n=4439). From principal component loadings, six shape categories were determined: rectangular, front-step, back-step, goalpost, centered tower, and other. The rectangular and stepped shapes were the most frequent, composing 62% and 24% of total events, respectively. The rectangular shape was often produced by 'standard' hydropeaking or recreational releases, while the stepped shapes were often used for water conservation or were recreational flows bordered by periods of electricity generation. The stepped shape increased in occurrence after the "Water First" policy took effect in 1993 and dominated two drier years (2007 and 2009). After categorization by shape, magnitude and durational indices were used to fabricate representative pulsed flow events. Attenuation of these representative pulses was then modeled using a 1D hydraulic model of 42 river km prepared in HEC-RAS. As no operational measures or physical structures existed within the system to counter the adverse effects of pulsed flow events, natural attenuation was the only potential major mitigation agent. However, model results demonstrated a clear durational threshold for representative pulses (~ 3-5 hrs) over which the degree of attenuation of ramping rates and peak discharge approached a limit. These thresholds were unique to the study reach and were dependent upon river morphology, bed characteristics, and flow rates. Increasing baseflows did not necessarily increase attenuation of pulses, most likely due to minimal increases in bed friction forces in this fairly steep and confined channel. Simulations of front and back-step representative pulses showed trade-offs between attenuation of peak magnitudes and steepness of ramping rates. Finally, a range of rising ramping rates were shown to steepen downstream above initial rates due to the study reach's channel morphology. Reshaping pulses to be more ecologically benign at all points downstream was infeasible if the system was required to maintain current electricity production and recreational service levels.

Masters change, slaves remain.

PubMed

Graham, Patricia; Penn, Jill K M; Schedl, Paul

2003-01-01

Sex determination offers an opportunity to address many classic questions of developmental biology. In addition, because sex determination evolves rapidly, it offers an opportunity to investigate the evolution of genetic hierarchies. Sex determination in Drosophila melanogaster is controlled by the master regulatory gene, Sex lethal (Sxl). DmSxl controls the alternative splicing of a downstream gene, transformer (tra), which acts with tra2 to control alternative splicing of doublesex (dsx). DmSxl also controls its own splicing, creating an autoregulatory feedback loop that ensures expression of Sxl in females, but not males. A recent paper has shown that in the dipteran Ceratitis capitata later (downstream) steps in the regulatory hierarchy are conserved, while earlier (upstream) steps are not. Cctra is regulated by alternative splicing and apparently controls the alternative splicing of Ccdsx. However, Cctra is not regulated by CcSxl. Instead it appears to autoregulate in a manner similar to the autoregulation seen with DmSxl. Copyright 2002 Wiley Periodicals, Inc.

Microwave-accelerated method for ultra-rapid extraction of Neisseria gonorrhoeae DNA for downstream detection.

PubMed

Melendez, Johan H; Santaus, Tonya M; Brinsley, Gregory; Kiang, Daniel; Mali, Buddha; Hardick, Justin; Gaydos, Charlotte A; Geddes, Chris D

2016-10-01

Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by detection of the genomic target often involving polymerase chain reaction (PCR)-based approaches. In an effort to improve on current detection approaches, we have developed a unique two-step microwave-accelerated approach for rapid extraction and detection of Neisseria gonorrhoeae (gonorrhea, GC) DNA. Our approach is based on the use of highly focused microwave radiation to rapidly lyse bacterial cells, release, and subsequently fragment microbial DNA. The DNA target is then detected by a process known as microwave-accelerated metal-enhanced fluorescence (MAMEF), an ultra-sensitive direct DNA detection analytical technique. In the current study, we show that highly focused microwaves at 2.45 GHz, using 12.3-mm gold film equilateral triangles, are able to rapidly lyse both bacteria cells and fragment DNA in a time- and microwave power-dependent manner. Detection of the extracted DNA can be performed by MAMEF, without the need for DNA amplification, in less than 10 min total time or by other PCR-based approaches. Collectively, the use of a microwave-accelerated method for the release and detection of DNA represents a significant step forward toward the development of a point-of-care (POC) platform for detection of gonorrhea infections. Copyright © 2016 Elsevier Inc. All rights reserved.

Robotic platform for parallelized cultivation and monitoring of microbial growth parameters in microwell plates.

PubMed

Knepper, Andreas; Heiser, Michael; Glauche, Florian; Neubauer, Peter

2014-12-01

The enormous variation possibilities of bioprocesses challenge process development to fix a commercial process with respect to costs and time. Although some cultivation systems and some devices for unit operations combine the latest technology on miniaturization, parallelization, and sensing, the degree of automation in upstream and downstream bioprocess development is still limited to single steps. We aim to face this challenge by an interdisciplinary approach to significantly shorten development times and costs. As a first step, we scaled down analytical assays to the microliter scale and created automated procedures for starting the cultivation and monitoring the optical density (OD), pH, concentrations of glucose and acetate in the culture medium, and product formation in fed-batch cultures in the 96-well format. Then, the separate measurements of pH, OD, and concentrations of acetate and glucose were combined to one method. This method enables automated process monitoring at dedicated intervals (e.g., also during the night). By this approach, we managed to increase the information content of cultivations in 96-microwell plates, thus turning them into a suitable tool for high-throughput bioprocess development. Here, we present the flowcharts as well as cultivation data of our automation approach. © 2014 Society for Laboratory Automation and Screening.

Separation of porcine parvovirus from bovine serum albumin using PEG-salt aqueous two-phase system.

PubMed

Vijayaragavan, K Saagar; Zahid, Amna; Young, Jonathan W; Heldt, Caryn L

2014-09-15

Vaccine production faces a challenge in adopting conventional downstream processing steps that can efficiently purify large viral particles. Some major issues that plague vaccine purification are purity, potency, and quality. The industry currently considers 30% as an acceptable virus recovery for a vaccine purification process, including all downstream processes, whereas antibody recovery from CHO cell culture is generally around 80-85%. A platform technology with an improved virus recovery would revolutionize vaccine production. In a quest to fulfill this goal, we have been exploring aqueous two-phase systems (ATPSs) as an optional mechanism to purify virus. ATPS has been unable to gain wide implementation mainly due to loss of virus infectivity, co-purification of proteins, and difficulty of polymer recycling. Non-enveloped viruses are chemically resistant enough to withstand the high polymer and salt concentrations that are required for effective ATPS separations. We used infectious porcine parvovirus (PPV), a non-enveloped, DNA virus as a model virus to test and develop an ATPS separation method. We successfully tackled two of the three main disadvantages of ATPS previously stated; we achieved a high infectious yield of 64% in a PEG-citrate ATPS process while separating out the main contaminate protein, bovine serum albumin (BSA). The most dominant forces in the separation were biomolecule charge, virus surface hydrophobicity, and the ATPS surface tension. Highly hydrophobic viruses are likely to benefit from the discovered ATPS for high-purity vaccine production and ease of implementation. Copyright © 2014 Elsevier B.V. All rights reserved.

Modeling and simulation of anion-exchange membrane chromatography for purification of Sf9 insect cell-derived virus-like particles.

PubMed

Ladd Effio, Christopher; Hahn, Tobias; Seiler, Julia; Oelmeier, Stefan A; Asen, Iris; Silberer, Christine; Villain, Louis; Hubbuch, Jürgen

2016-01-15

Recombinant protein-based virus-like particles (VLPs) are steadily gaining in importance as innovative vaccines against cancer and infectious diseases. Multiple VLPs are currently evaluated in clinical phases requiring a straightforward and rational process design. To date, there is no generic platform process available for the purification of VLPs. In order to accelerate and simplify VLP downstream processing, there is a demand for novel development approaches, technologies, and purification tools. Membrane adsorbers have been identified as promising stationary phases for the processing of bionanoparticles due to their large pore sizes. In this work, we present the potential of two strategies for designing VLP processes following the basic tenet of 'quality by design': High-throughput experimentation and process modeling of an anion-exchange membrane capture step. Automated membrane screenings allowed the identification of optimal VLP binding conditions yielding a dynamic binding capacity of 5.7 mg/mL for human B19 parvovirus-like particles derived from Spodoptera frugiperda Sf9 insect cells. A mechanistic approach was implemented for radial ion-exchange membrane chromatography using the lumped-rate model and stoichiometric displacement model for the in silico optimization of a VLP capture step. For the first time, process modeling enabled the in silico design of a selective, robust and scalable process with minimal experimental effort for a complex VLP feedstock. The optimized anion-exchange membrane chromatography process resulted in a protein purity of 81.5%, a DNA clearance of 99.2%, and a VLP recovery of 59%. Copyright © 2015 Elsevier B.V. All rights reserved.

Practicing safe cell culture: applied process designs for minimizing virus contamination risk.

PubMed

Kiss, Robert D

2011-01-01

CONFERENCE PROCEEDING Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop in Bethesda, MD, USA; December 1-3, 2010 Guest Editors: Arifa Khan (Bethesda, MD), Patricia Hughes (Bethesda, MD) and Michael Wiebe (San Francisco, CA) Genentech responded to a virus contamination in its biologics manufacturing facility by developing and implementing a series of barriers specifically designed to prevent recurrence of this significant and impactful event. The barriers included steps to inactivate or remove potential virus particles from the many raw materials used in cell culture processing. Additionally, analytical testing barriers provided protection of the downstream processing areas should a culture contamination occur, and robust virus clearance capability provided further assurance of virus safety should a low level contamination go undetected. This conference proceeding will review Genentech's approach, and lessons learned, in minimizing virus contamination risk in cell culture processes through multiple layers of targeted barriers designed to deliver biologics products with high success rates.

Acoustic investigation of wall jet over a backward-facing step using a microphone phased array

NASA Astrophysics Data System (ADS)

Perschke, Raimund F.; Ramachandran, Rakesh C.; Raman, Ganesh

2015-02-01

The acoustic properties of a wall jet over a hard-walled backward-facing step of aspect ratios 6, 3, 2, and 1.5 are studied using a 24-channel microphone phased array at Mach numbers up to M=0.6. The Reynolds number based on inflow velocity and step height assumes values from Reh = 3.0 ×104 to 7.2 ×105. Flow without and with side walls is considered. The experimental setup is open in the wall-normal direction and the expansion ratio is effectively 1. In case of flow through a duct, symmetry of the flow in the spanwise direction is lost downstream of separation at all but the largest aspect ratio as revealed by oil paint flow visualization. Hydrodynamic scattering of turbulence from the trailing edge of the step contributes significantly to the radiated sound. Reflection of acoustic waves from the bottom plate results in a modulation of power spectral densities. Acoustic source localization has been conducted using a 24-channel microphone phased array. Convective mean-flow effects on the apparent source origin have been assessed by placing a loudspeaker underneath a perforated flat plate and evaluating the displacement of the beamforming peak with inflow Mach number. Two source mechanisms are found near the step. One is due to interaction of the turbulent wall jet with the convex edge of the step. Free-stream turbulence sound is found to be peaked downstream of the step. Presence of the side walls increases free-stream sound. Results of the flow visualization are correlated with acoustic source maps. Trailing-edge sound and free-stream turbulence sound can be discriminated using source localization.

Compound immobilization and drug-affinity chromatography.

PubMed

Rix, Uwe; Gridling, Manuela; Superti-Furga, Giulio

2012-01-01

Bioactive small molecules act through modulating a yet unpredictable number of targets. It is therefore of critical importance to define the cellular target proteins of a compound as an entry point to understanding its mechanism of action. Often, this can be achieved in a direct fashion by chemical proteomics. As with any affinity chromatography, immobilization of the bait to a solid support is one of the earliest and most crucial steps in the process. Interfering with structural features that are important for identification of a target protein will be detrimental to binding affinity. Also, many molecules are sensitive to heat or to certain chemicals, such as acid or base, and might be destroyed during the process of immobilization, which therefore needs to be not only efficient, but also mild. The subsequent affinity chromatography step needs to preserve molecular and conformational integrity of both bait compound and proteins in order to result in the desired specific enrichment while ensuring a high level of compatibility with downstream analysis by mass spectrometry. Thus, the right choice of detergent, buffer, and protease inhibitors is also essential. This chapter describes a widely applicable procedure for the immobilization of small molecule drugs and for drug-affinity chromatography with subsequent protein identification by mass spectrometry.

Acid-base characterization of 5-hydroxypyrazine-2-carboxylic acid and the role of ionic equilibria in the optimization of some process conditions for its biocatalytic production.

PubMed

Sak-Bosnar, M; Kovar, K

2005-10-01

This paper describes the use of potentiometric titration to determine the relevant acid-base properties of 5-hydroxypyrazine-2-carboxylic acid (5OH-PYCA), an important intermediate in the production of tuberculostatics. The data obtained were used for calculation of the dissociation constants of 5OH-PYCA. It was found that 5OH-PYCA dissociates in two steps, with the corresponding dissociation constants pK (a1)=3.42 and pK (a2)=7.96, designating 5OH-PYCA as a medium weak acid (1st step). The distribution diagram of dissociated species and the buffer-strength diagram of 5OH-PYCA provide useful information about its behaviour at different pH. The ionic equilibria data obtained can be used for selection of the optimum pH for biotransformation of pyrazine-2-carboxylic acid (PYCA) and for prediction of pH changes during the biotransformation. These data can also be used for selection of the optimum pH for precipitating 5OH-PYCA in downstream processing. All computations have been optimized by mathematical modelling using Solver.

Use of mep HyperCel for polishing of human serum albumin.

PubMed

McCann, Karl B; Vucica, Yvonne; Wu, John; Bertolini, Joseph

2014-10-15

The manufacture of human serum albumin by chromatographic procedures involves gel filtration chromatography as a final polishing step. Despite this step being essential to remove high molecular weight impurity proteins and thus ensure a stable and safe final product, it is relatively inefficient. This paper explores the use of hydrophobic charge induction chromatographic media, MEP HyperCel as an alternative to Sephacryl S200HR gel filtration for the polishing of human serum albumin derived by ion exchange chromatographic purification of Cohn Supernatant I. The use of MEP HyperCel results in a product with a higher purity than achieved with gel filtration and in a less time consuming manner and with potential resource savings. MEP HyperCel appears to have great potential for incorporation into downstream processes in the plasma fractionation industry as an efficient means of achieving polishing of intermediates or capture of proteins of interest. Copyright © 2014 Elsevier B.V. All rights reserved.

Triggers of key calcium signals during erythrocyte invasion by Plasmodium falciparum

PubMed Central

Gao, Xiaohong; Gunalan, Karthigayan; Yap, Sally Shu Lin; Preiser, Peter R.

2013-01-01

Invasion of erythrocytes by Plasmodium falciparum merozoites is a complex multi-step process mediated by specific interactions between host receptors and parasite ligands. Reticulocyte-binding protein homologues (RHs) and erythrocyte-binding-like (EBL) proteins are discharged from specialized organelles and used in early steps of invasion. Here we show that monoclonal antibodies against PfRH1 (an RH) block merozoite invasion by specifically inhibiting calcium signalling in the parasite, whereas invasion-inhibiting monoclonal antibodies targeting EBA175 (an EBL protein) have no effect on signalling. We further show that inhibition of this calcium signalling prevents EBA175 discharge and thereby formation of the junction between parasite and host cell. Our results indicate that PfRH1 has an initial sensing as well as signal transduction role that leads to the subsequent release of EBA175. They also provide new insights on how RH–host cell interactions lead to essential downstream signalling events in the parasite, suggesting new targets for malaria intervention. PMID:24280897

Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells.

PubMed

Nordlund, Henri R; Laitinen, Olli H; Uotila, Sanna T H; Kulmala, Minna; Kalkkinen, Nisse; Kulomaa, Markku S

2005-10-14

The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches.

2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation.

PubMed

Choi, Junhong; Indrisiunaite, Gabriele; DeMirci, Hasan; Ieong, Ka-Weng; Wang, Jinfan; Petrov, Alexey; Prabhakar, Arjun; Rechavi, Gideon; Dominissini, Dan; He, Chuan; Ehrenberg, Måns; Puglisi, Joseph D

2018-03-01

Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2'-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2'-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2'-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

Numerical modelling study of gully recharge and debris flows in Haida Gwaii, British Columbia

NASA Astrophysics Data System (ADS)

Martin, Yvonne; Johnson, Edward; Chaikina, Olga

2015-04-01

In high mountains, debris flows are a major process responsible for transferring sediment to more downstream fluvial reaches. This sediment transfer begins on mountain hillslopes where various mass wasting processes move sediment from hillslopes to uppermost reaches of the channel system (these reaches are herein referred to as gullies and only experience water flow during high intensity precipitation events). Sediment recharge into gullies, which has received minimal attention in the scientific literature, refers to the transfer of sediment and other debris from surrounding hillslopes into gullies (Jakob and Oden, 2005). Debris flow occurrence and debris flow volumes depend on some precipitation threshold as well as volumes of material contained in the particular gully. For example, if one debris flow has removed all of the accumulated material from the gully, then any subsequent debris flow will be smaller if enough time has not yet passed for notable sediment recharge. Herein, we utilize the numerical model of landscape development, LandMod (Martin, 1998; Dadson and Church, 2005; Martin, 2007), to explore connections between hillslope processes, gully recharge rates, and transfer of sediment to downstream channel reaches in the Haida Gwaii, British Columbia. Hillslope processes in the model include shallow landsliding, bedrock failures and weathering. The updated debris flow algorithm is based on extensive field data available for debris flows in Haida Gwaii (e.g., Rood, 1984; Oden, 1994; Jakob and Oden, 2005), as well as theoretical considerations based on debris flow studies. The most significant model extension is the calculation of gully recharge rates; for each gully, the total accumulated sediment in gullies at each time step is determined using a power-law relation for area-normalized recharge rate versus elapsed time since the last debris flow. Thus, when the stochastic driver for debris flow occurrence triggers an event, the amount of stored material is known and can be transferred and deposited along the channel system. Results show that the size distribution of debris flows and sediment transfers from gullies to downstream reaches are modified by the inclusion of a module that accounts for sediment recharge when compared to model runs that do not consider gully recharge.

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

Frederix, Marijke; Mingardon, Florence; Hu, Matthew

Biological production of chemicals and fuels using microbial transformation of sustainable carbon sources, such as pretreated and saccharified plant biomass, is a multi-step process. Typically, each segment of the workflow is optimized separately, often generating conditions that may not be suitable for integration or consolidation with the upstream or downstream steps. While significant effort has gone into developing solutions to incompatibilities at discrete steps, very few studies report the consolidation of the multi-step workflow into a single pot reactor system. Here we demonstrate a one-pot biofuel production process that uses the ionic liquid 1-ethyl-3-methylimidazolium acetate (C 2C 1Im][OAc] ) formore » pretreatment of switchgrass biomass. [C 2C 1Im][OAc] is highly effective in deconstructing lignocellulose, but nonetheless leaves behind residual reagents that are toxic to standard saccharification enzymes and the microbial production host. We report the discovery of an [C 2C 1Im]-tolerant E. coli strain, where [C 2C 1Im] tolerance is bestowed by a P7Q mutation in the transcriptional regulator encoded by rcdA. We establish that the causal impact of this mutation is the derepression of a hitherto uncharacterized major facilitator family transporter, YbjJ. To develop the strain for a one-pot process we engineered this [C 2C 1Im]-tolerant strain to express a recently reported d-limonene production pathway. We also screened previously reported [C 2C 1Im]-tolerant cellulases to select one that would function with the range of E. coli cultivation conditions and expressed it in the [C 2C 1 Im]-tolerant E. coli strain so as to secrete this [C 2C 1Im]-tolerant cellulase. The final strain digests pretreated biomass, and uses the liberated sugars to produce the bio-jet fuel candidate precursor d-limonene in a one-pot process.« less

Development of an E. coli strain for one-pot biofuel production from ionic liquid pretreated cellulose and switchgrass

DOE PAGES

Frederix, Marijke; Mingardon, Florence; Hu, Matthew; ...

2016-04-11

Biological production of chemicals and fuels using microbial transformation of sustainable carbon sources, such as pretreated and saccharified plant biomass, is a multi-step process. Typically, each segment of the workflow is optimized separately, often generating conditions that may not be suitable for integration or consolidation with the upstream or downstream steps. While significant effort has gone into developing solutions to incompatibilities at discrete steps, very few studies report the consolidation of the multi-step workflow into a single pot reactor system. Here we demonstrate a one-pot biofuel production process that uses the ionic liquid 1-ethyl-3-methylimidazolium acetate (C 2C 1Im][OAc] ) formore » pretreatment of switchgrass biomass. [C 2C 1Im][OAc] is highly effective in deconstructing lignocellulose, but nonetheless leaves behind residual reagents that are toxic to standard saccharification enzymes and the microbial production host. We report the discovery of an [C 2C 1Im]-tolerant E. coli strain, where [C 2C 1Im] tolerance is bestowed by a P7Q mutation in the transcriptional regulator encoded by rcdA. We establish that the causal impact of this mutation is the derepression of a hitherto uncharacterized major facilitator family transporter, YbjJ. To develop the strain for a one-pot process we engineered this [C 2C 1Im]-tolerant strain to express a recently reported d-limonene production pathway. We also screened previously reported [C 2C 1Im]-tolerant cellulases to select one that would function with the range of E. coli cultivation conditions and expressed it in the [C 2C 1 Im]-tolerant E. coli strain so as to secrete this [C 2C 1Im]-tolerant cellulase. The final strain digests pretreated biomass, and uses the liberated sugars to produce the bio-jet fuel candidate precursor d-limonene in a one-pot process.« less

Identification of a sequence element on the 3' side of AAUAAA which is necessary for simian virus 40 late mRNA 3'-end processing.

PubMed Central

Sadofsky, M; Connelly, S; Manley, J L; Alwine, J C

1985-01-01

Our previous studies of the 3'-end processing of simian virus 40 late mRNAs indicated the existence of an essential element (or elements) downstream of the AAUAAA signal. We report here the use of transient expression analysis to study a functional element which we located within the sequence AGGUUUUUU, beginning 59 nucleotides downstream of the recognized signal AAUAAA. Deletion of this element resulted in (i) at least a 75% drop in 3'-end processing at the normal site and (ii) appearance of readthrough transcripts with alternate 3' ends. Some flexibility in the downstream position of this element relative to the AAUAAA was noted by deletion analysis. Using computer sequence comparison, we located homologous regions within downstream sequences of other genes, suggesting a generalized sequence element. In addition, specific complementarity is noted between the downstream element and U4 RNA. The possibility that this complementarity could participate in 3'-end site selection is discussed. Images PMID:3016512

Knock-out of the magnesium protoporphyrin IX methyltransferase gene in Arabidopsis. Effects on chloroplast development and on chloroplast-to-nucleus signaling

PubMed Central

Pontier, Dominique; Albrieux, Catherine; Joyard, Jacques; Lagrange, Thierry; Block, Maryse

2007-01-01

Protoporphyrin IX is the last common intermediate between the haem and chlorophyll biosynthesis pathways. The addition of Mg directs this molecule toward chlorophyll biosynthesis. The first step downstream from the branchpoint is catalyzed by the Mg chelatase and is a highly regulated process. The corresponding product, Mg protoporphyrin IX, has been proposed to play an important role as a signaling molecule implicated in plastid-to-nucleus communication. In order to get more information on the chlorophyll biosynthesis pathway and on Mg protoporphyrin IX derivative functions, we have identified an Mg protoporphyrin IX methyltransferase (CHLM) knock-out mutant in Arabidopsis in which the mutation induces a blockage downstream from Mg protoporphyrin IX and an accumulation of this chlorophyll biosynthesis intermediate. Our results demonstrate that the CHLM gene is essential for the formation of chlorophyll and subsequently for the formation of photosystems I and II and cyt b6f complexes. Analysis of gene expression in the chlm mutant provides an independent indication that Mg protoporphyrin IX is a negative effector of nuclear photosynthetic gene expression, as previously reported. Moreover, it suggests the possible implication of Mg protoporphyrin IX methylester, the product of CHLM, in chloroplast-to-nucleus signaling. Finally, post-transcriptional up-regulation of the level of the CHLH subunit of the Mg chelatase has been detected in the chlm mutant and most likely corresponds to specific accumulation of this protein inside plastids. This result suggests that the CHLH subunit might play an important regulatory role when the chlorophyll biosynthetic pathway is disrupted at this particular step. PMID:17135235

On-line casein micelle disruption for downstream purification of recombinant human myelin basic protein produced in the milk of transgenic cows.

PubMed

Al-Ghobashy, Medhat A; Williams, Martin A K; Brophy, Brigid; Laible, Götz; Harding, David R K

2009-06-01

Downstream purification of a model recombinant protein (human myelin basic protein) from milk of transgenic cows is described. The recombinant protein was expressed as a His tagged fusion protein in the milk of transgenic cows and was found associated with the casein micellar phase. While difficulties in obtaining good recoveries were found when employing conventional micelle disruption procedures, direct capture using the cation exchanger SP Sepharose Big Beads was found successful in the extraction of the recombinant protein. Early breakthrough suggested a slow release of the recombinant protein from the micelles and dictated micelle disruption in order to obtain good yields. A new approach for deconstruction of the calcium core of the casein micelles, employing the interaction between the micellar calcium and the active sites of the cation exchanger resin was developed. Milk samples were loaded to the column in aliquots with a column washing step after each aliquot. This sequential loading approach successfully liberated the recombinant protein from the micelles and was found superior to the conventional sample loading approach. It increased the recovery by more than 25%, reduced fouling due to milk components and improved the column hydrodynamic properties as compared to the conventional sample loading approach. Hardware and software modifications to the chromatography system were necessary in order to keep the whole process automated. A second purification step using a Ni2+ affinity column was used to isolate the recombinant protein at purity more than 90% and a recovery percentage of 78%.

Particle Image Velocimetry Measurements of a Two/Three-dimensional Separating/Reattaching Boundary Layer Downstream of an Axisymmetric Backward-facing Step

NASA Technical Reports Server (NTRS)

Hudy, Laura M.; Naguib, Ahmed M.; Humphreys, William M.; Bartram, Scott M.

2005-01-01

Planar Particle Image Velocimetry measurements were obtained in the separating/reattaching flow region downstream of an axisymmetric backward-facing step. Data were acquired for a two-dimensional (2D) separating boundary layer at five different Reynolds numbers based on step height (Re(sub h)), spanning 5900-33000, and for a three-dimensional (3D) separating boundary layer at Re(sub h) = 5980 and 8081. Reynolds number effects were investigated in the 2D cases using mean-velocity field, streamwise and wall-normal turbulent velocity, and Reynolds stress statistics. Results show that both the reattachment length (x(sub r)) and the secondary separation point are Reynolds number dependent. The reattachment length increased with rising Re(sub h) while the secondary recirculation region decreased in size. These and other Re(sub h) effects were interpreted in terms of changes in the separating boundary layer thickness and wall-shear stress. On the other hand, in the 3D case, it was found that the imposed cross-flow component was relatively weak in comparison to the streamwise component. As a result, the primary influences of three dimensionality only affected the near-separation region rather than the entire separation bubble.

SWATH2stats: An R/Bioconductor Package to Process and Convert Quantitative SWATH-MS Proteomics Data for Downstream Analysis Tools.

PubMed

Blattmann, Peter; Heusel, Moritz; Aebersold, Ruedi

2016-01-01

SWATH-MS is an acquisition and analysis technique of targeted proteomics that enables measuring several thousand proteins with high reproducibility and accuracy across many samples. OpenSWATH is popular open-source software for peptide identification and quantification from SWATH-MS data. For downstream statistical and quantitative analysis there exist different tools such as MSstats, mapDIA and aLFQ. However, the transfer of data from OpenSWATH to the downstream statistical tools is currently technically challenging. Here we introduce the R/Bioconductor package SWATH2stats, which allows convenient processing of the data into a format directly readable by the downstream analysis tools. In addition, SWATH2stats allows annotation, analyzing the variation and the reproducibility of the measurements, FDR estimation, and advanced filtering before submitting the processed data to downstream tools. These functionalities are important to quickly analyze the quality of the SWATH-MS data. Hence, SWATH2stats is a new open-source tool that summarizes several practical functionalities for analyzing, processing, and converting SWATH-MS data and thus facilitates the efficient analysis of large-scale SWATH/DIA datasets.

Urate Oxidase Purification by Salting-in Crystallization: Towards an Alternative to Chromatography

PubMed Central

Giffard, Marion; Ferté, Natalie; Ragot, François; El Hajji, Mohamed; Castro, Bertrand; Bonneté, Françoise

2011-01-01

Background Rasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step. It compares the efficacy of two crystallization techniques that have proven successful on pure urate oxidase, testing them on impure urate oxidase solutions. Methodology/Principal Findings Here we investigate the possibility of purifying urate oxidase directly by crystallization from the fermentation broth. Based on attractive interaction potentials which are known to drive urate oxidase crystallization, two crystallization routes are compared: a) by increased polymer concentration, which induces a depletion attraction and b) by decreased salt concentration, which induces attractive interactions via a salting-in effect. We observe that adding polymer, a very efficient way to crystallize pure urate oxidase through the depletion effect, is not an efficient way to grow crystals from impure solution. On the other hand, we show that dialysis, which decreases salt concentration through its strong salting-in effect, makes purification of urate oxidase from the fermentation broth possible. Conclusions The aim of this study is to compare purification efficacy of two crystallization methods. Our findings show that crystallization of urate oxidase from the fermentation broth provides purity comparable to what can be achieved with one chromatography step. This suggests that, in the case of urate oxidase, crystallization could be implemented not only for polishing or concentration during the last steps of purification, but also as an initial capture step, with minimal changes to the current process. PMID:21589929

Stem cell-dependent formation of a functional anterior regeneration pole in planarians requires Zic and Forkhead transcription factors.

PubMed

Vogg, Matthias C; Owlarn, Suthira; Pérez Rico, Yuvia A; Xie, Jianlei; Suzuki, Yoko; Gentile, Luca; Wu, Wei; Bartscherer, Kerstin

2014-06-15

Planarians can regenerate their head within days. This process depends on the direction of adult stem cells to wound sites and the orchestration of their progenitors to commit to appropriate lineages and to arrange into patterned tissues. We identified a zinc finger transcription factor, Smed-ZicA, as a downstream target of Smed-FoxD, a Forkhead transcription factor required for head regeneration. Smed-zicA and Smed-FoxD are co-expressed with the Wnt inhibitor notum and the Activin inhibitor follistatin in a cluster of cells at the anterior-most tip of the regenerating head - the anterior regeneration pole - and in surrounding stem cell progeny. Depletion of Smed-zicA and Smed-FoxD by RNAi abolishes notum and follistatin expression at the pole and inhibits head formation downstream of initial polarity decisions. We suggest a model in which ZicA and FoxD transcription factors synergize to control the formation of Notum- and Follistatin-producing anterior pole cells. Pole formation might constitute an early step in regeneration, resulting in a signaling center that orchestrates cellular events in the growing tissue. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

An essential role of a FoxD gene in notochord induction in Ciona embryos.

PubMed

Imai, Kaoru S; Satoh, Nori; Satou, Yutaka

2002-07-01

A key issue for understanding the early development of the chordate body plan is how the endoderm induces notochord formation. In the ascidian Ciona, nuclear accumulation of beta-catenin is the first step in the process of endoderm specification. We show that nuclear accumulation of beta-catenin directly activates the gene (Cs-FoxD) for a winged helix/forkhead transcription factor and that this gene is expressed transiently at the 16- and 32-cell stages in endodermal cells. The function of Cs-FoxD, however, is not associated with differentiation of the endoderm itself but is essential for notochord differentiation or induction. In addition, it is likely that the inductive signal that appears to act downstream of Cs-FoxD does not act over a long range. It has been suggested that FGF or Notch signal transduction pathway mediates ascidian notochord induction. Our previous study suggests that Cs-FGF4/6/9 is partially involved in the notochord induction. The present experimental results suggest that the expression and function of Cs-FGF4/6/9 and Cs-FoxD are not interdependent, and that the Notch pathway is involved in B-line notochord induction downstream of Cs-FoxD.

Downstream processing from melt granulation towards tablets: In-depth analysis of a continuous twin-screw melt granulation process using polymeric binders.

PubMed

Grymonpré, W; Verstraete, G; Vanhoorne, V; Remon, J P; De Beer, T; Vervaet, C

2018-03-01

The concept of twin-screw melt granulation (TSMG) has steadily (re)-gained interest in pharmaceutical formulation development as an intermediate step during tablet manufacturing. However, to be considered as a viable processing option for solid oral dosage forms there is a need to understand all critical sources of variability which could affect this granulation technique. The purpose of this study was to provide an in-depth analysis of the continuous TSMG process in order to expose the critical process parameters (CPP) and elucidate the impact of process and formulation parameters on the critical quality attributes (CQA) of granules and tablets during continuous TSMG. A first part of the study dealt with the screening of various amorphous polymers as binder for producing high-dosed melt granules of two model drug (i.e. acetaminophen and hydrochlorothiazide). The second part of this study described a quality-by-design (QbD) approach for melt granulation of hydrochlorothiazide in order to thoroughly evaluate TSMG, milling and tableting stage of the continuous TSMG line. Using amorphous polymeric binders resulted in melt granules with high milling efficiency due to their brittle behaviour without producing excessive amounts of fines, providing high granule yields with low friability. Therefore, it makes them extremely suitable for further downstream processing. One of the most important CPP during TSMG with polymeric binders was the granulation-torque, which - in case of polymers with high T g - increased during longer granulation runs to critical levels endangering the continuous process flow. However, by optimizing both screw speed and throughput or changing to polymeric binders with lower T g it was possible to significantly reduce this risk. This research paper highlighted that TSMG must be considered as a viable option during formulation development of solid oral dosage forms based on the robustness of the CQA of both melt granules and tablets. Copyright © 2017 Elsevier B.V. All rights reserved.

Geophysical bed sediment characterization of the Androscoggin River from the former Chlor-Alkali Facility Superfund Site, Berlin, New Hampshire, to the state border with Maine, August 2009

USGS Publications Warehouse

Degnan, James R.; Teeple, Andrew; Johnston, Craig M.; Marvin-DiPasquale, Mark C.; Luce, Darryl

2011-01-01

The former Chlor-Alkali Facility in Berlin, New Hampshire, was listed on the U.S. Environmental Protection Agency National Priorities List in 2005 as a Superfund site. The Chlor-Alkali Facility lies on the east bank of the Androscoggin River. Elemental mercury currently discharges from that bank into the Androscoggin River. The nature, extent, and the speciation of mercury and the production of methyl mercury contamination in the adjacent Androscoggin River is the subject of continuing investigations. The U.S. Geological Survey, in cooperation with Region I of the U.S. Environmental Protection Agency, used geophysical methods to determine the distribution, thickness, and physical properties of sediments in the Androscoggin River channel at a small area of an upstream reference reach and downstream from the site to the New Hampshire–Maine State border. Separate reaches of the Androscoggin River in the study area were surveyed with surface geophysical methods including ground-penetrating radar and step-frequency electromagnetics. Results were processed to assess sediment characteristics including grain size, electrical conductivity, and pore-water specific conductance. Specific conductance measured during surface- and pore-water sampling was used to help interpret the results of the geophysical surveys. The electrical resistivity of sediment samples was measured in the laboratory with intact pore water for comparison with survey results. In some instances, anthropogenic features and land uses, such as roads and power lines affected the detection of riverbed properties using geophysical methods; when this occurred, the data were removed. Through combining results, detailed riverbed sediment characterizations were made. Results from ground-penetrating radar surveys were used to image and measure the depth to the riverbed, depth to buried riverbeds, riverbed thickness and to interpret material-type variations in terms of relative grain size. Fifty two percent of the riverbed in the study area was covered with gravel and finer sediments. The electrically resistive river water and sediment in this study area were conducive to the penetration of the ground-penetrating radar and step-frequency electromagnetic signals and allowed for effective sediment characterization by geophysical methods. The reach between the former Chlor-Alkali Facility and the Riverside Dam, had small areas of fine sediment (estimated 11 percent of riverbed area), found on the upstream left bank and the downstream right bank, with an electromagnetic conductivity (31.4 millisiemens per meter (mS/m) maximum) that was higher than the upstream reference reach. The greatest electromagnetic conductivity (195 mS/m), pore-water specific conductance (324 mS/m) and lab measured sediment conductivity of (76.8 mS/m, measured with a direct-current resistivity test box) in the study were measured approximately 1 mile (mi) downstream of the site from a sandbar on the left bank. Reaches adjacent to and within 2 mi downstream from the site had elevated electromagnetic conductivity despite having lower estimated percentages of riverbed area covered in sediment (11, 25, and 61 percent, respectively) than the reference reach (97). Typically finer grained sediment with similar mineralogy will be more conductive. The Shelburne Reservoir is approximately 8 mi downstream from the site had the second greatest pore-water specific conductance measured, 45.8 mS/m. Many of the locations with the largest step-frequency electromagnetic values have not been sampled for pore water and sediment.

A novel microalgal lipid extraction method using biodiesel (fatty acid methyl esters) as an extractant.

PubMed

Huang, Wen-Can; Park, Chan Woo; Kim, Jong-Duk

2017-02-01

Although microalgae are considered promising renewable sources of biodiesel, the high cost of the downstream process is a significant obstacle in large-scale biodiesel production. In this study, a novel approach for microalgal biodiesel production was developed by using the biodiesel as an extractant. First, wet microalgae with 70% water content were incubated with a mixture of biodiesel/methanol and penetration of the mixture through the cell membrane and swelling of the lipids contained in microalgae was confirmed. Significant increases of lipid droplets were observed by confocal microscopy. Second, the swelled lipid droplets in microalgae were squeezed out using mechanical stress across the cell membrane and washed with methanol. The lipid extraction efficiency reached 68%. This process does not require drying of microalgae or solvent recovery, which the most energy-intensive step in solvent-based biodiesel production. Copyright © 2016 Elsevier Ltd. All rights reserved.

Flow chemistry vs. flow analysis.

PubMed

Trojanowicz, Marek

2016-01-01

The flow mode of conducting chemical syntheses facilitates chemical processes through the use of on-line analytical monitoring of occurring reactions, the application of solid-supported reagents to minimize downstream processing and computerized control systems to perform multi-step sequences. They are exactly the same attributes as those of flow analysis, which has solid place in modern analytical chemistry in several last decades. The following review paper, based on 131 references to original papers as well as pre-selected reviews, presents basic aspects, selected instrumental achievements and developmental directions of a rapidly growing field of continuous flow chemical synthesis. Interestingly, many of them might be potentially employed in the development of new methods in flow analysis too. In this paper, examples of application of flow analytical measurements for on-line monitoring of flow syntheses have been indicated and perspectives for a wider application of real-time analytical measurements have been discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Computation of turbulent flows over backward and forward-facing steps using a near-wall Reynolds stress model

NASA Technical Reports Server (NTRS)

Ko, Sung HO

1993-01-01

Separation and reattachment of turbulent shear layers is observed in many important engineering applications, yet it is poorly understood. This has motivated many studies on understanding and predicting the processes of separation and reattachment of turbulent shear layers. Both of the situations in which separation is induced by adverse pressure gradient, or by discontinuities of geometry, have attracted attention of turbulence model developers. Formulation of turbulence closure models to describe the essential features of separated turbulent flows accurately is still a formidable task. Computations of separated flows associated with sharp-edged bluff bodies are described. For the past two decades, the backward-facing step flow, the simplest separated flow, has been a popular test case for turbulence models. Detailed studies on the performance of many turbulence models, including two equation turbulence models and Reynolds stress models, for flows over steps can be found in the papers by Thangam & Speziale and Lasher & Taulbee). These studies indicate that almost all the existing turbulence models fail to accurately predict many important features of back step flow such as reattachment length, recovery rate of the redeveloping boundary layers downstream of the reattachment point, streamlines near the reattachment point, and the skin friction coefficient. The main objectives are to calculate flows over backward and forward-facing steps using the NRSM and to make use of the newest DNS data for detailed comparison. This will give insights for possible improvements of the turbulence model.

Single step production of Cas9 mRNA for zygote injection.

PubMed

Redel, Bethany K; Beaton, Benjamin P; Spate, Lee D; Benne, Joshua A; Murphy, Stephanie L; O'Gorman, Chad W; Spate, Anna M; Prather, Randall S; Wells, Kevin D

2018-03-01

Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

Direct measurements and analysis of skin friction and cooling downstream of multiple flush-slot injection into a turbulent Mach 6 boundary layer

NASA Technical Reports Server (NTRS)

Howard, F. G.; Strokowski, A. J.

1978-01-01

Experiments were conducted to determine the reduction in surface skin friction and the effectiveness of surface cooling downstream of one to four successive flush slots injecting cold air at an angle of 10 deg into a turbulent Mach 6 boundary layer. Data were obtained by direct measurement of surface shear and equilibrium temperatures, respectively. Increasing the number of slots decreased the skin friction, but the incremental improvement in skin-friction reduction decreased as the number of slots was increased. Cooling effectiveness was found to improve, for a given total mass injection, as the number of slots was increased from one to four. Comparison with previously reported step-slot data, however, indicated that step slots with tangential injection are more effective for both reducing skin friction and cooling than the present flush-slot configuration. Finite-difference predictions are in reasonable agreement with skin-friction data and with boundary-layer profile data.

Characterization of Non-Infectious Virus-Like Particle Surrogates for Viral Clearance Applications.

PubMed

Johnson, Sarah; Brorson, Kurt A; Frey, Douglas D; Dhar, Arun K; Cetlin, David A

2017-09-01

Viral clearance is a critical aspect of biopharmaceutical manufacturing process validation. To determine the viral clearance efficacy of downstream chromatography and filtration steps, live viral "spiking" studies are conducted with model mammalian viruses such as minute virus of mice (MVM). However, due to biosafety considerations, spiking studies are costly and typically conducted in specialized facilities. In this work, we introduce the concept of utilizing a non-infectious MVM virus-like particle (MVM-VLP) as an economical surrogate for live MVM during process development and characterization. Through transmission electron microscopy, size exclusion chromatography with multi-angle light scattering, chromatofocusing, and a novel solute surface hydrophobicity assay, we examined and compared the size, surface charge, and hydrophobic properties of MVM and MVM-VLP. The results revealed that MVM and MVM-VLP exhibited nearly identical physicochemical properties, indicating the potential utility of MVM-VLP as an accurate and economical surrogate to live MVM during chromatography and filtration process development and characterization studies.

Glacial Hazards in Chile: Processes, Assessment, Mitigation and Risk Management Strategies

NASA Astrophysics Data System (ADS)

Glasser, N. F.; Wilson, R.; Casassa, G., Sr.; Reynolds, J.; Harrison, S.; Shannon, S. R.; Schaefer, M.; Iribarran, P.

2017-12-01

Glacial Lake Outburst Floods (GLOFs) are capable of travelling considerable distances from their source and they represent one of the most important glacial hazards. In line with observations in other parts of the world, the frequency of GLOF events in Chile has increased in recent decades highlighting the need to quantify the flood risk posed to downstream areas. This poster presents the work of the `Glacial Hazards in Chile' project which aims to (1) better understand the processes that govern the development of GLOFs in Chile, (2) estimate the socio-economic effects of GLOFs in Chile, and (3) provide a GLOF risk assessment framework that can be applied to Chile and other lower income countries globally. As an initial step towards the completion of these aims, we have recently compiled the first glacial lake inventory for the central and Patagonian Andes, which details the temporal development of glacial lakes in this region over the past three decades. This analysis was used to identify two lakes of interest that were visited during a fieldwork expedition in February 2017. The first of these, Lago Chileno in Patagonia, has recently produced a large GLOF causing significant damage to the downstream floodplain, whilst the second was identified as one of the fastest growing lakes in the central Andes. Both these lakes were surveyed using aerial imagery acquired with a drone and a custom-built bathymetry boat, data from which will help to improve our understanding of the physical processes associated with glacial lake development and failure within the Chilean Andes.

Aptamer-based downstream processing of his-tagged proteins utilizing magnetic beads.

PubMed

Kökpinar, Öznur; Walter, Johanna-Gabriela; Shoham, Yuval; Stahl, Frank; Scheper, Thomas

2011-10-01

Aptamers are synthetic nucleic acid-based high affinity ligands that are able to capture their corresponding target via molecular recognition. Here, aptamer-based affinity purification for His-tagged proteins was developed. Two different aptamers directed against the His-tag were immobilized on magnetic beads covalently. The resulting aptamer-modified magnetic beads were characterized and successfully applied for purification of different His-tagged proteins from complex E. coli cell lysates. Purification effects comparable to conventional immobilized metal affinity chromatography were achieved in one single purification step. Moreover, we have investigated the possibility to regenerate and reuse the aptamer-modified magnetic beads and have shown their long-term stability over a period of 6 months. Copyright © 2011 Wiley Periodicals, Inc.

Rac-WAVE-mediated actin reorganization is required for organization and maintenance of cell-cell adhesion.

PubMed

Yamazaki, Daisuke; Oikawa, Tsukasa; Takenawa, Tadaomi

2007-01-01

During cadherin-dependent cell-cell adhesion, the actin cytoskeleton undergoes dynamic reorganization in epithelial cells. Rho-family small GTPases, which regulate actin dynamics, play pivotal roles in cadherin-dependent cell-cell adhesion; however, the precise molecular mechanisms that underlie cell-cell adhesion formation remain unclear. Here we show that Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE)-mediated reorganization of actin, downstream of Rac plays an important role in normal development of cadherin-dependent cell-cell adhesions in MDCK cells. Rac-induced development of cadherin-dependent adhesions required WAVE2-dependent actin reorganization. The process of cell-cell adhesion is divided into three steps: formation of new cell-cell contacts, stabilization of these new contacts and junction maturation. WAVE1 and WAVE2 were expressed in MDCK cells. The functions of WAVE1 and WAVE2 were redundant in this system but WAVE2 appeared to play a more significant role. During the first step, WAVE2-dependent lamellipodial protrusions facilitated formation of cell-cell contacts. During the second step, WAVE2 recruited actin filaments to new cell-cell contacts and stabilized newly formed cadherin clusters. During the third step, WAVE2-dependent actin reorganization was required for organization and maintenance of mature cell-cell adhesions. Thus, Rac-WAVE-dependent actin reorganization is not only involved in formation of cell-cell adhesions but is also required for their maintenance.

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts.

PubMed

Buyel, Johannes F; Hubbuch, Jürgen; Fischer, Rainer

2016-08-08

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 °C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Next steps in determining the overall sustainability of perennial bioenergy crops

USDA-ARS?s Scientific Manuscript database

Perennial bioenergy crops are being developed and evaluated in the United States to partially offset petroleum transport fuels. Accurate accounting of upstream and downstream greenhouse gas (GHG) emissions is necessary to measure the overall carbon intensity of new biofuel feedstocks. For example, c...

Headwater Stream Management Dichotomies: Local Amphibian Habitat vs. Downstream Fish Habitat

NASA Astrophysics Data System (ADS)

Jackson, C. R.

2002-12-01

Small headwater streams in mountainous areas of the Pacific Northwest often do not harbor fish populations because of low water depth and high gradients. Rather, these streams provide habitat for dense assemblages of stream-dwelling amphibians. A variety of management goals have been suggested for such streams such as encouraging large woody debris recruitment to assist in sediment trapping and valley floor formation, encouraging large woody debris recruitment to provide downstream wood when debris flows occur, providing continuous linear stream buffers within forest harvest areas to provide shade and bank stability, etc. A basic problem with analying the geomorphic or biotic benefits of any of these strategies is the lack of explicit management goals for such streams. Should managers strive to optimize downstream fish habitat, local amphibian habitat, or both? Through observational data and theoretical considerations, it will be shown that these biotic goals will lead to very different geomorphic management recommendations. For instance, woody debris greater than 60 cm diameter may assist in valley floor development, but it is likely to create subsurface channel flow of unknown value to amphibians. Trapping and retention of fine sediments within headwater streams may improve downstream spawning gravels, but degrades stream-dwelling amphibian habitat. In response to the need for descriptive information on habitat and channel morphology specific to small, non-fish-bearing streams in the Pacific Northwest, morphologies and wood frequencies in forty-two first- and second-order forested streams less than four meters wide were surveyed. Frequencies and size distributions of woody debris were compared between small streams and larger fish-bearing streams as well as between second-growth and virgin timber streams. Statistical models were developed to explore dominant factors affecting channel morphology and habitat. Findings suggest geomorphological relationships, specifically the role of woody debris in habitat formation, documented for larger streams do not apply to headwater streams. Relatively small wood (diameters between 10 and 40 cm), inorganic material, and organic debris (diameters less than 10 cm) were major step-forming agents while big woody debris pieces (> 40 cm dia.) created less than 10% of steps. Streams in virgin and managed stands did not differ in relative importance of very large woody debris. Due to low fluvial power, pool habitat was rare. These streams featured mostly step-riffle morphology, not step-pool, indicating insufficient flow for pool-scour. Stream power and unit stream power were dominant channel shaping factors.

A Hierarchical Multivariate Bayesian Approach to Ensemble Model output Statistics in Atmospheric Prediction

DTIC Science & Technology

2017-09-01

efficacy of statistical post-processing methods downstream of these dynamical model components with a hierarchical multivariate Bayesian approach to...Bayesian hierarchical modeling, Markov chain Monte Carlo methods , Metropolis algorithm, machine learning, atmospheric prediction 15. NUMBER OF PAGES...scale processes. However, this dissertation explores the efficacy of statistical post-processing methods downstream of these dynamical model components

Laminar supersonic flow over a backstep - A numerical solution at higher Reynolds numbers

NASA Technical Reports Server (NTRS)

Kronzon, Y.; Rom, J.; Seginer, A.

1976-01-01

The Allen-Cheng solution of the flow over a backward facing step is extended to Reynolds numbers up to 16,000 and to inflow boundary-layer height ratios as low as 0.1 by moving the downstream boundary into the recompression region and by smoothing the resulting errors. The boundary conditions in the supersonic outer flow and the downstream boundary conditions in the wake are determined by an extrapolation procedure. Computational results are compared with relevant experimental data. Fair agreement is found between the calculated base pressures and the experimental values, whereas agreement between heat transfer rates appears to be qualitative only.

RNA from the 5' end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site.

PubMed

Christensen, Shawn M; Ye, Junqiang; Eickbush, Thomas H

2006-11-21

Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5' end of the R2 element. In the absence of this 5' RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3' untranslated region of the R2 transcript is present. In the presence of the 320-nt 5' RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5' RNA is removed from this subunit. We postulate that the removal of the 5' RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction.

CE-UV/VIS and CE-MS for monitoring organic impurities during the downstream processing of fermentative-produced lactic acid from second-generation renewable feedstocks.

PubMed

Laube, Hendrik; Matysik, Frank-Michael; Schmidberger, Andreas; Mehlmann, Kerstin; Toursel, Andreas; Boden, Jana

2016-01-01

During the downstream process of bio-based bulk chemicals, organic impurities, mostly residues from the fermentation process, must be separated to obtain a pure and ready-to-market chemical. In this study, capillary electrophoresis was investigated for the non-targeting downstream process monitoring of organic impurities and simultaneous quantitative detection of lactic acid during the purification process of fermentatively produced lactic acid. The downstream process incorporated 11 separation units, ranging from filtration, adsorption and ion exchange to electrodialysis and distillation, and 15 different second-generation renewable feedstocks were processed into lactic acid. The identification of organic impurities was established through spiking and the utilization of an advanced capillary electrophoresis mass spectrometry system. A total of 53 % of the organic impurities were efficiently removed via bipolar electrodialysis; however, one impurity, pyroglutamic acid, was recalcitrant to separation. It was demonstrated that the presence of pyroglutamic acid disrupts the polymerization of lactic acid into poly lactic acid. Pyroglutamic acid was present in all lactic acid solutions, independent of the type of renewable resource or the bacterium applied. Pyroglutamic acid, also known as 5-oxoproline, is a metabolite in the glutathione cycle, which is present in all living microorganisms. pyroglutamic acid is found in many proteins, and during intracellular protein metabolism, N-terminal glutamic acid and glutamine residues can spontaneously cyclize to become pyroglutamic acid. Hence, the concentration of pyroglutamic acid in the lactic acid solution can only be limited to a certain amount. The present study proved the capillary electrophoresis system to be an important tool for downstream process monitoring. The high product concentration encountered in biological production processes did not hinder the capillary electrophoresis from separating and detecting organic impurities, even at minor concentrations. The coupling of the capillary electrophoresis with a mass spectrometry system allowed for the straightforward identification of the remaining critical impurity, pyroglutamic acid. Although 11 separation units were applied during the downstream process, the pyroglutamic acid concentration remained at 12,900 ppm, which was comparatively high. All organic impurities found were tracked by the capillary electrophoresis, allowing for further separation optimization.

Downstream Processing of Synechocystis for Biofuel Production

NASA Astrophysics Data System (ADS)

Sheng, Jie

Lipids and free fatty acids (FFA) from cyanobacterium Synechocystis can be used for biofuel (e.g. biodiesel or renewable diesel) production. In order to utilize and scale up this technique, downstream processes including culturing and harvest, cell disruption, and extraction were studied. Several solvents/solvent systems were screened for lipid extraction from Synechocystis. Chloroform + methanol-based Folch and Bligh & Dyer methods were proved to be "gold standard" for small-scale analysis due to their highest lipid recoveries that were confirmed by their penetration of the cell membranes, higher polarity, and stronger interaction with hydrogen bonds. Less toxic solvents, such as methanol and MTBE, or direct transesterification of biomass (without preextraction step) gave only slightly lower lipid-extraction yields and can be considered for large-scale application. Sustained exposure to high and low temperature extremes severely lowered the biomass and lipid productivity. Temperature stress also triggered changes of lipid quality such as the degree of unsaturation; thus, it affected the productivities and quality of Synechocystis-derived biofuel. Pulsed electric field (PEF) was evaluated for cell disruption prior to lipid extraction. A treatment intensity > 35 kWh/m3 caused significant damage to the plasma membrane, cell wall, and thylakoid membrane, and it even led to complete disruption of some cells into fragments. Treatment by PEF enhanced the potential for the low-toxicity solvent isopropanol to access lipid molecules during subsequent solvent extraction, leading to lower usage of isopropanol for the same extraction efficiency. Other cell-disruption methods also were tested. Distinct disruption effects to the cell envelope, plasma membrane, and thylakoid membranes were observed that were related to extraction efficiency. Microwave and ultrasound had significant enhancement of lipid extraction. Autoclaving, ultrasound, and French press caused significant release of lipid into the medium, which may increase solvent usage and make medium recycling difficult. Production of excreted FFA by mutant Synechocystis has the potential of reducing the complexity of downstream processing. Major problems, such as FFA precipitation and biodegradation by scavengers, account for FFA loss in operation. Even a low concentration of FFA scavengers could consume FFA at a high rate that outpaced FFA production rate. Potential strategies to overcome FFA loss include high pH, adsorptive resin, and sterilization techniques.

Achieving continuous manufacturing for final dosage formation: challenges and how to meet them. May 20-21, 2014 Continuous Manufacturing Symposium.

PubMed

Byrn, Stephen; Futran, Maricio; Thomas, Hayden; Jayjock, Eric; Maron, Nicola; Meyer, Robert F; Myerson, Allan S; Thien, Michael P; Trout, Bernhardt L

2015-03-01

We describe the key issues and possibilities for continuous final dosage formation, otherwise known as downstream processing or drug product manufacturing. A distinction is made between heterogeneous processing and homogeneous processing, the latter of which is expected to add more value to continuous manufacturing. We also give the key motivations for moving to continuous manufacturing, some of the exciting new technologies, and the barriers to implementation of continuous manufacturing. Continuous processing of heterogeneous blends is the natural first step in converting existing batch processes to continuous. In heterogeneous processing, there are discrete particles that can segregate, versus in homogeneous processing, components are blended and homogenized such that they do not segregate. Heterogeneous processing can incorporate technologies that are closer to existing technologies, where homogeneous processing necessitates the development and incorporation of new technologies. Homogeneous processing has the greatest potential for reaping the full rewards of continuous manufacturing, but it takes long-term vision and a more significant change in process development than heterogeneous processing. Heterogeneous processing has the detriment that, as the technologies are adopted rather than developed, there is a strong tendency to incorporate correction steps, what we call below "The Rube Goldberg Problem." Thus, although heterogeneous processing will likely play a major role in the near-term transformation of heterogeneous to continuous processing, it is expected that homogeneous processing is the next step that will follow. Specific action items for industry leaders are: Form precompetitive partnerships, including industry (pharmaceutical companies and equipment manufacturers), government, and universities. These precompetitive partnerships would develop case studies of continuous manufacturing and ideally perform joint-technology development, including development of small-scale equipment and processes. Develop ways to invest internally in continuous manufacturing. How best to do this will depend on the specifics of a given organization, in particular the current development projects. Upper managers will need to energize their process developers to incorporate continuous manufacturing in at least part of their processes to gain experience and demonstrate directly the benefits. Training of continuous manufacturing technologies, organizational approaches, and regulatory approaches is a key area that industrial leaders should pursue together. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Verification and Validation (V&V) Methodologies for Multiphase Turbulent and Explosive Flows. V&V Case Studies of Computer Simulations from Los Alamos National Laboratory GMFIX codes

NASA Astrophysics Data System (ADS)

Dartevelle, S.

2006-12-01

Large-scale volcanic eruptions are inherently hazardous events, hence cannot be described by detailed and accurate in situ measurements; hence, volcanic explosive phenomenology is inadequately constrained in terms of initial and inflow conditions. Consequently, little to no real-time data exist to Verify and Validate computer codes developed to model these geophysical events as a whole. However, code Verification and Validation remains a necessary step, particularly when volcanologists use numerical data for mitigation of volcanic hazards as more often performed nowadays. The Verification and Validation (V&V) process formally assesses the level of 'credibility' of numerical results produced within a range of specific applications. The first step, Verification, is 'the process of determining that a model implementation accurately represents the conceptual description of the model', which requires either exact analytical solutions or highly accurate simplified experimental data. The second step, Validation, is 'the process of determining the degree to which a model is an accurate representation of the real world', which requires complex experimental data of the 'real world' physics. The Verification step is rather simple to formally achieve, while, in the 'real world' explosive volcanism context, the second step, Validation, is about impossible. Hence, instead of validating computer code against the whole large-scale unconstrained volcanic phenomenology, we rather suggest to focus on the key physics which control these volcanic clouds, viz., momentum-driven supersonic jets and multiphase turbulence. We propose to compare numerical results against a set of simple but well-constrained analog experiments, which uniquely and unambiguously represent these two key-phenomenology separately. Herewith, we use GMFIX (Geophysical Multiphase Flow with Interphase eXchange, v1.62), a set of multiphase- CFD FORTRAN codes, which have been recently redeveloped to meet the strict Quality Assurance, verification, and validation requirements from the Office of Civilian Radioactive Waste Management of the US Dept of Energy. GMFIX solves Navier-Stokes and energy partial differential equations for each phase with appropriate turbulence and interfacial coupling between phases. For momentum-driven single- to multi-phase underexpanded jets, the position of the first Mach disk is known empirically as a function of both the pressure ratio, K, and the particle mass fraction, Phi at the nozzle. Namely, the higher K, the further downstream the Mach disk and the higher Phi, the further upstream the first Mach disk. We show that GMFIX captures these two essential features. In addition, GMFIX displays all the properties found in these jets, such as expansion fans, incident and reflected shocks, and subsequent downstream mach discs, which make this code ideal for further investigations of equivalent volcanological phenomena. One of the other most challenging aspects of volcanic phenomenology is the multiphase nature of turbulence. We also validated GMFIX in comparing the velocity profiles and turbulence quantities against well constrained analog experiments. The velocity profiles agree with the analog ones as well as these of production of turbulent quantities. Overall, the Verification and the Validation experiments although inherently challenging suggest GMFIX captures the most essential dynamical properties of multiphase and supersonic flows and jets.

Sample preservation, transport and processing strategies for honeybee RNA extraction: Influence on RNA yield, quality, target quantification and data normalization.

PubMed

Forsgren, Eva; Locke, Barbara; Semberg, Emilia; Laugen, Ane T; Miranda, Joachim R de

2017-08-01

Viral infections in managed honey bees are numerous, and most of them are caused by viruses with an RNA genome. Since RNA degrades rapidly, appropriate sample management and RNA extraction methods are imperative to get high quality RNA for downstream assays. This study evaluated the effect of various sampling-transport scenarios (combinations of temperature, RNA stabilizers, and duration) of transport on six RNA quality parameters; yield, purity, integrity, cDNA synthesis efficiency, target detection and quantification. The use of water and extraction buffer were also compared for a primary bee tissue homogenate prior to RNA extraction. The strategy least affected by time was preservation of samples at -80°C. All other regimens turned out to be poor alternatives unless the samples were frozen or processed within 24h. Chemical stabilizers have the greatest impact on RNA quality and adding an extra homogenization step (a QIAshredder™ homogenizer) to the extraction protocol significantly improves the RNA yield and chemical purity. This study confirms that RIN values (RNA Integrity Number), should be used cautiously with bee RNA. Using water for the primary homogenate has no negative effect on RNA quality as long as this step is no longer than 15min. Copyright © 2017 Elsevier B.V. All rights reserved.

Energy dissipation on flat-sloped stepped spillways: Part 2. Downstream of the inception point

USDA-ARS?s Scientific Manuscript database

Many small earthen embankments are faced with inadequate spillway capacity due to filled sediment pools filled with sediment and sediment now filling flood pools. Additionally, hydrologic conditions have changed as a result of urbanization; thereby causing changes in the hazard classification of th...

Effects of Forward- and Backward-Facing Steps on the Crossflow Receptivity and Stability in Supersonic Boundary Layers

NASA Technical Reports Server (NTRS)

Balakumar, P.; King, Rudolph A.; Eppink, Jenna L.

2014-01-01

The effects of forward- and backward-facing steps on the receptivity and stability of three-dimensional supersonic boundary layers over a swept wing with a blunt leading edge are numerically investigated for a freestream Mach number of 3 and a sweep angle of 30 degrees. The flow fields are obtained by solving the full Navier-Stokes equations. The evolution of instability waves generated by surface roughness is simulated with and without the forward- and backward-facing steps. The separation bubble lengths are about 5-10 step heights for the forward-facing step and are about 10 for the backward-facing step. The linear stability calculations show very strong instability in the separated region with a large frequency domain. The simulation results show that the presence of backward-facing steps decreases the amplitude of the stationary crossflow vortices with longer spanwise wavelengths by about fifty percent and the presence of forward-facing steps does not modify the amplitudes noticeably across the steps. The waves with the shorter wavelengths grow substantially downstream of the step in agreement with the linear stability prediction.

Purification of adenoviral vectors by combined anion exchange and gel filtration chromatography.

PubMed

Eglon, Marc N; Duffy, Aoife M; O'Brien, Timothy; Strappe, Padraig M

2009-11-01

Adenoviral vectors are used extensively in human gene therapy trials and in vaccine development. Large-scale GMP production requires a downstream purification process, and liquid chromatography is emerging as the most powerful mode of purification, enabling the production of vectors at a clinically relevant scale and quality. The present study describes the development of a two-step high-performance liquid chromatography (HPLC) process combining anion exchange (AIEX) and gel filtration (GF) in comparison with the caesium chloride density gradient method. HEK-293 cells were cultured in ten-layer CellStacks() and infected with 10 pfu/cell of adenoviral vector expressing green fluorescent protein (Ad5-GFP). Cell-bound virus was harvested and benzonase added to digest DNA, crude lysate was clarified by centrifugation and filtration prior to HPLC. Chromatography fractions were added to HEK-293 cells and GFP expression measured using a fluorescent plate reader. Using AIEX then GF resulted in an adenoviral vector with purity comparable to Ad5-GFP purified by CsCl, whereas the reverse process (GF-AIEX) showed a reduced purity by electrophoresis and required further buffer exchange of the product. The optimal process (AIEX-GF) resulted in a vector yield of 2.3 x 10(7) pfu/cm(2) of cell culture harvested compared to 3.3 x 10(7) pfu/cm(2) for CsCl. The process recovery for the HPLC process was 36% compared to 27.5% for CsCl and total virion to infectious particle ratios of 18 and 11, respectively, were measured. We present a simple two-step chromatography process that is capable of producing high-quality adenovirus at a titre suitable for scale-up and clinical translation.

Production of alpha-amylase from Aspergillus oryzae for several industrial applications in a single step.

PubMed

Porfirif, María C; Milatich, Esteban J; Farruggia, Beatriz M; Romanini, Diana

2016-06-01

A one-step method as a strategy of alpha-amylase concentration and purification was developed in this work. This methodology requires the use of a very low concentration of biodegradable polyelectrolyte (Eudragit(®) E-PO) and represents a low cost, fast, easy to scale up and non-polluting technology. Besides, this methodology allows recycling the polymer after precipitation. The formation of reversible soluble/insoluble complexes between alpha-amylase and the polymer Eudragit(®) E-PO was studied, and their precipitation in selected conditions was applied with bioseparation purposes. Turbidimetric assays allowed to determine the pH range where the complexes are insoluble (4.50-7.00); pH 5.50 yielded the highest turbidity of the system. The presence of NaCl (0.05M) in the medium totally dissociates the protein-polymer complexes. When the adequate concentration of polymer was added under these conditions to a liquid culture of Aspergillus oryzae, purification factors of alpha-amylase up to 7.43 and recoveries of 88% were obtained in a simple step without previous clarification. These results demonstrate that this methodology is suitable for the concentration and production of alpha-amylase from this source and could be applied at the beginning of downstream processing. Copyright © 2016 Elsevier B.V. All rights reserved.

Diffusive Shock Acceleration and Reconnection Acceleration Processes

NASA Astrophysics Data System (ADS)

Zank, G. P.; Hunana, P.; Mostafavi, P.; Le Roux, J. A.; Li, Gang; Webb, G. M.; Khabarova, O.; Cummings, A.; Stone, E.; Decker, R.

2015-12-01

Shock waves, as shown by simulations and observations, can generate high levels of downstream vortical turbulence, including magnetic islands. We consider a combination of diffusive shock acceleration (DSA) and downstream magnetic-island-reconnection-related processes as an energization mechanism for charged particles. Observations of electron and ion distributions downstream of interplanetary shocks and the heliospheric termination shock (HTS) are frequently inconsistent with the predictions of classical DSA. We utilize a recently developed transport theory for charged particles propagating diffusively in a turbulent region filled with contracting and reconnecting plasmoids and small-scale current sheets. Particle energization associated with the anti-reconnection electric field, a consequence of magnetic island merging, and magnetic island contraction, are considered. For the former only, we find that (i) the spectrum is a hard power law in particle speed, and (ii) the downstream solution is constant. For downstream plasmoid contraction only, (i) the accelerated spectrum is a hard power law in particle speed; (ii) the particle intensity for a given energy peaks downstream of the shock, and the distance to the peak location increases with increasing particle energy, and (iii) the particle intensity amplification for a particular particle energy, f(x,c/{c}0)/f(0,c/{c}0), is not 1, as predicted by DSA, but increases with increasing particle energy. The general solution combines both the reconnection-induced electric field and plasmoid contraction. The observed energetic particle intensity profile observed by Voyager 2 downstream of the HTS appears to support a particle acceleration mechanism that combines both DSA and magnetic-island-reconnection-related processes.

C, N, P export regimes from headwater catchments to downstream reaches

NASA Astrophysics Data System (ADS)

Dupas, R.; Musolff, A.; Jawitz, J. W.; Rao, P. S.; Jaeger, C. G.; Fleckenstein, J. H.; Rode, M.; Borchardt, D.

2017-12-01

Excessive amounts of nutrients and dissolved organic matter in freshwater bodies affect aquatic ecosystems. In this study, the spatial and temporal variability in nitrate (NO3), dissolved organic carbon (DOC) and soluble reactive phosphorus (SRP) was analyzed in the Selke river continuum from headwaters draining 1 - 3 km² catchments to downstream reaches representing spatially integrated signals from 184 - 456 km² catchments (part of TERENO - Terrestrial Environmental Observatories, in Germany). Three headwater catchments were selected as archetypes of the main landscape units (land use x lithology) present in the Selke catchment. Export regimes in headwater catchments were interpreted in terms of NO3, DOC and SRP land-to-stream transfer processes. Headwater signals were subtracted from downstream signals, with the differences interpreted in terms of in-stream processes and contribution of point-source emissions. The seasonal dynamics for NO3 were opposite those of DOC and SRP in all three headwater catchments, and spatial differences also showed NO3 contrasting with DOC and SRP. These dynamics were interpreted as the result of the interplay of hydrological and biogeochemical processes, for which riparian zones were hypothesized to play a determining role. In the two downstream reaches, NO3 was transported almost conservatively, whereas DOC was consumed and produced in the upper and lower river sections, respectively. The natural export regime of SRP in the three headwater catchments mimicked a point-source signal, which may lead to overestimation of domestic contributions in the downstream reaches. Monitoring the river continuum from headwaters to downstream reaches proved effective to investigate jointly land-to-stream and in-stream transport and transformation processes.

N-terminal domains of fibrillin 1 and fibrillin 2 direct the formation of homodimers: a possible first step in microfibril assembly.

PubMed Central

Trask, T M; Ritty, T M; Broekelmann, T; Tisdale, C; Mecham, R P

1999-01-01

Aggregation of fibrillin molecules via disulphide bonds is postulated to be an early step in microfibril assembly. By expressing fragments of fibrillin 1 and fibrillin 2 in a mammalian expression system, we found that the N-terminal region of each protein directs the formation of homodimers and that disulphide bonds stabilize this interaction. A large fragment of fibrillin 1 containing much of the region downstream from the N-terminus remained as a monomer when expressed in the same cell system, indicating that this region of the protein lacks dimerization domains. This finding also confirms that the overexpression of fibrillin fragments does not in itself lead to spurious dimer formation. Pulse-chase analysis demonstrated that dimer formation occurred intracellularly, suggesting that the process of fibrillin aggregation is initiated early after biosynthesis of the molecules. These findings also implicate the N-terminal region of fibrillin 1 and fibrillin 2 in directing the formation of a dimer intermediate that aggregates to form the functional microfibril. PMID:10359653

Production of human insulin in an E. coli system with Met-Lys-human proinsulin as the expressed precursor

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

Jin-Qiu Chen; Hong-Tao Zhang; Mei-Hao Hu

1995-10-01

The construction of a gene encoding Lys-human proinsulin, its direct expression in E.coli, and the simple purification procedure are described here. The temperature inducible promotor was employed for induction in a very short time. The expression level could reach 20-30%. After a simple downstream processing and only one step of Sephadex G50 purification, 150 mg recombinant Lys-human proinsulin with a purity of up to 90% could be obtained easily from 1 L of high density fermentation medium. The obtained product is in the form of Met-Lys-human proinsulin because of the failure of the bacterial host to remove the initiator methioninemore » residue. The Lys-human proinsulin could be changed into human insulin by tryspin and carboxypeptidase B treatment in later steps. After separation with DEAE Sephadex A25, human insulin with expected amino acid composition and full native biological activity could be obtained with a yield of 50 mg/L fermentation medium. 20 refs., 5 figs., 4 tabs.« less

A coupled modelling effort to study the fate of contaminated sediments downstream of the Coles Hill deposit, Virginia, USA

NASA Astrophysics Data System (ADS)

Castro-Bolinaga, C. F.; Zavaleta, E. R.; Diplas, P.

2015-03-01

This paper presents the preliminary results of a coupled modelling effort to study the fate of tailings (radioactive waste-by product) downstream of the Coles Hill uranium deposit located in Virginia, USA. The implementation of the overall modelling process includes a one-dimensional hydraulic model to qualitatively characterize the sediment transport process under severe flooding conditions downstream of the potential mining site, a two-dimensional ANSYS Fluent model to simulate the release of tailings from a containment cell located partially above the local ground surface into the nearby streams, and a one-dimensional finite-volume sediment transport model to examine the propagation of a tailings sediment pulse in the river network located downstream. The findings of this investigation aim to assist in estimating the potential impacts that tailings would have if they were transported into rivers and reservoirs located downstream of the Coles Hill deposit that serve as municipal drinking water supplies.

Source-to-sink sediment transfers, environmental engineering and hazard mitigation in the steep Var River catchment, French Riviera, southeastern France

NASA Astrophysics Data System (ADS)

Anthony, Edward J.; Julian, Maurice

1999-12-01

Steep coastal margins are potentially subject to mass wasting processes involving notable landslide activity and sediment evacuation downstream by steep-gradient streams. Sediment transfer from short source-to-sink segments, coupled with mountain hydrological regimes, regulate patterns of river channel aggradation and coastal sediment supply in such geomorphic settings. On the steep French Riviera margin, sediment transfers from existing landslides or from various minor mass wasting processes to stream channels may result following bursts of heavy, concentrated rainfall. High-magnitude flooding and massive sediment transport downstream are generally related to unpredictable extreme rainfalls. Both mass movements and channel sediment storage pose serious hazards to downvalley settlements and infrastructure. A consideration of channel sediment storage patterns in the Var River catchment, the most important catchment in this area, highlights two important shortcomings relative to environmental engineering and hazard mitigation practices. In the first place, the appreciation of geomorphic processes is rather poor. This is illustrated by the undersized nature of engineering works constructed to mitigate hazards in the upstream bedload-dominated channels, and by the unforeseen effects that ten rock dams, constructed in the early 1970s, have had on downstream and coastal sediment storage and on sediment dispersal patterns and, consequently, valley flooding. Secondly, planners and environmental engineers have lacked foresight in valley and coastal management issues on this steep setting, notably as regards the reclaimed areas of the lower Var channel and delta liable to flooding. Urbanization and transport and environmental engineering works have progressively affected patterns of storage and transport of fine-grained sediments in the lower Var channel and delta. Meanwhile the problems raised by these changes have not been adequately addressed in terms of scientific research. A necessary future step in bettering the engineering solutions implemented to contain natural hazards or to harness water and sediment resources is that of fine-scale analysis of source-to-sink sediment transfer processes, of sediment budgets, of time-scales of storage in stream channels, and, finally, of high-magnitude hydrometeorological forcing events in this area. The way all these aspects have been modulated by engineering practices and socioeconomic development should also be an important part of such an analysis.

Simulation-based comprehensive benchmarking of RNA-seq aligners

PubMed Central

Baruzzo, Giacomo; Hayer, Katharina E; Kim, Eun Ji; Di Camillo, Barbara; FitzGerald, Garret A; Grant, Gregory R

2018-01-01

Alignment is the first step in most RNA-seq analysis pipelines, and the accuracy of downstream analyses depends heavily on it. Unlike most steps in the pipeline, alignment is particularly amenable to benchmarking with simulated data. We performed a comprehensive benchmarking of 14 common splice-aware aligners for base, read, and exon junction-level accuracy and compared default with optimized parameters. We found that performance varied by genome complexity, and accuracy and popularity were poorly correlated. The most widely cited tool underperforms for most metrics, particularly when using default settings. PMID:27941783

Instream wood in a steep headwater channel: geomorphic significance of large and small wood

NASA Astrophysics Data System (ADS)

Galia, Tomáš; Šilhán, Karel; Ruiz-Villanueva, Virginia; Tichavský, Radek

2016-04-01

Besides the well-known significance of large wood (LW), also small woody pieces (SW; here defined as pieces with dimensions at least 0.5 m length and 0.05 m diameter), can play an important role in steep narrow headwaters. We inventoried instream wood in the 0.4 km long Mazák headwater channel, Moravskoslezské Beskydy Mts, Czech Republic (2

Modification of suburban carbon and nitrogen fluxes by a coupled channel/floodplain system assessed using in situ sensors

NASA Astrophysics Data System (ADS)

Wollheim, W. M.; Pellerin, B. A.; Saraceno, J.; Hopkinson, C.; Hope, A.; Morse, N.

2010-12-01

Biogeochemical fluxes in human dominated streams and rivers are highly impacted, but effects can be attenuated downstream through natural ecosystem processes. We deployed in situ nitrate, fdom, and chlorophyll sensors to characterize biogeochemical fluxes draining a suburban catchment, and modifications by a channel-floodplain system located immediately downstream. The upstream site reflects the suburban signal; the downstream site reflects the influence of the channel/floodplain on the suburban signal. FDOM showed a diurnal signal at both sites, but was stronger downstream, likely indicating new DOC production within the channel-floodplain system, which contained a small pond. In situ chlorophyll concentrations were also highly correlated with FDOM. FDOM showed a stronger storm response upstream than downstream, indicating terrestrial sources are mobilized by storms and subsequent dampening of the pulse by the floodplain. Nitrate concentrations consistently dropped from 0.6 to 0.7 mg/l upstream to less than 0.4 mg/l downstream, indicating likely nitrogen retention or removal over a relatively short distance (~500m). Use of in situ sensors is likely to greatly advance our understanding of biogeochemical processes in aquatic systems.

Optimization of miRNA-seq data preprocessing.

PubMed

Tam, Shirley; Tsao, Ming-Sound; McPherson, John D

2015-11-01

The past two decades of microRNA (miRNA) research has solidified the role of these small non-coding RNAs as key regulators of many biological processes and promising biomarkers for disease. The concurrent development in high-throughput profiling technology has further advanced our understanding of the impact of their dysregulation on a global scale. Currently, next-generation sequencing is the platform of choice for the discovery and quantification of miRNAs. Despite this, there is no clear consensus on how the data should be preprocessed before conducting downstream analyses. Often overlooked, data preprocessing is an essential step in data analysis: the presence of unreliable features and noise can affect the conclusions drawn from downstream analyses. Using a spike-in dilution study, we evaluated the effects of several general-purpose aligners (BWA, Bowtie, Bowtie 2 and Novoalign), and normalization methods (counts-per-million, total count scaling, upper quartile scaling, Trimmed Mean of M, DESeq, linear regression, cyclic loess and quantile) with respect to the final miRNA count data distribution, variance, bias and accuracy of differential expression analysis. We make practical recommendations on the optimal preprocessing methods for the extraction and interpretation of miRNA count data from small RNA-sequencing experiments. © The Author 2015. Published by Oxford University Press.

Effects of secondary structure on pre-mRNA splicing: hairpins sequestering the 5' but not the 3' splice site inhibit intron processing in Nicotiana plumbaginifolia.

PubMed

Liu, H X; Goodall, G J; Kole, R; Filipowicz, W

1995-01-16

We have performed a systematic study of the effect of artificial hairpins on pre-mRNA splicing in protoplasts of a dicot plant, Nicotiana plumbaginifolia. Hairpins with a potential to form 18 or 24 bp stems strongly inhibit splicing when they sequester the 5' splice site or are placed in the middle of short introns. However, similar 24 bp hairpins sequestering the 3' splice site do not prevent this site from being used as an acceptor. Utilization of the stem-located 3' site requires that the base of the stem is separated from the upstream 5' splice site by a minimum of approximately 45 nucleotides and that another 'helper' 3' splice site is present downstream of the stem. The results indicate that the spliceosome or factors associated with it may have a potential to unfold secondary structure present in the downstream portion of the intron, prior to or at the step of the 3' splice site selection. The finding that the helper 3' site is required for utilization of the stem-located acceptor confirms and extends previous observations, obtained with HeLa cell in vitro splicing systems, indicating that the 3' splice site may be recognized at least twice during spliceosome assembly.

Efficacy of hybrid adsorption/membrane pretreatment for low pressure membrane.

PubMed

Malczewska, B; Benjamin, M M

2016-08-01

Fouling by natural organic matter (NOM) is a major obstacle when water from natural sources is treated using low-pressure membranes. Prior research by our group has demonstrated that passing natural water through a thin, pre-deposited layer of heated aluminum oxide particles (HAOPs) can remove substantial amounts of NOM from the feed and thereby reduce the fouling rate of downstream membranes. The work reported here explored the technical efficacy of such a pretreatment process under more challenging (and therefore realistic) conditions than reported earlier. Several analytical techniques were applied to the feed and permeate in an attempt to identify the key fouling components. The results demonstrate that a HAOPs layer can be pre-deposited on a stainless steel mesh and then be readily washed off at the end of a filtration cycle with very little irreversible fouling due to residual NOM or HAOPs left on the mesh. In addition, the pretreatment step removes enough foulant to allow a downstream UF membrane to operate at significantly higher fluxes than when conventional pretreatment is applied. HAOPs pretreatment also reduced the formation of chlorinated and brominated trihalomethanes (THM4) by more than 67% and of haloacetic acids (HAA9) by 64%-88% in simulated distribution system (SDS) tests. Copyright © 2016 Elsevier Ltd. All rights reserved.

One-Step Partially Purified Lipases (ScLipA and ScLipB) from Schizophyllum commune UTARA1 Obtained via Solid State Fermentation and Their Applications.

PubMed

Kam, Yew Chee; Woo, Kwan Kit; Ong, Lisa Gaik Ai

2017-12-08

Lipases with unique characteristics are of value in industrial applications, especially those targeting cost-effectiveness and less downstream processes. The aims of this research were to: (i) optimize the fermentation parameters via solid state fermentation (SSF); and (ii) study the performance in hydrolysis and esterification processes of the one-step partially purified Schizophyllum commune UTARA1 lipases. Lipase was produced by cultivating S. commune UTARA1 on sugarcane bagasse (SB) with used cooking oil (UCO) via SSF and its production was optimized using Design-Expert ® 7.0.0. Fractions 30% ( Sc LipA) and 70% ( Sc LipB) which contained high lipase activity were obtained by stepwise (NH₄)₂SO₄ precipitation. Crude fish oil, coconut oil and butter were used to investigate the lipase hydrolysis capabilities by a free glycerol assay. Results showed that Sc LipA has affinities for long, medium and short chain triglycerides, as all the oils investigated were degraded, whereas Sc LipB has affinities for long chain triglycerides as it only degrades crude fish oil. During esterification, Sc LipA was able to synthesize trilaurin and triacetin. Conversely, Sc LipB was specific towards the formation of 2-mono-olein and triacetin. From the results obtained, it was determined that Sc LipA and Sc LipB are sn -2 regioselective lipases. Hence, the one-step partial purification strategy proved to be feasible for partial purification of S. commune UTARA1 lipases that has potential use in industrial applications.

Ecological linkages between headwaters and downstream ecosystems: Transport of organic matter, invertebrates, and wood down headwater channels

USGS Publications Warehouse

Wipfli, M.S.; Richardson, J.S.; Naiman, R.J.

2007-01-01

Headwater streams make up a large proportion of the total length and watershed area of fluvial networks, and are partially characterized by the large volume of organic matter (large wood, detritus, and dissolved organic matter) and invertebrate inputs from the riparian forest, relative to stream size. Much of those inputs are exported to downstream reaches through time where they potentially subsidize river communities. The relative rates, timing, and conversion processes that carry inputs from small streams to downstream reaches are reasonably well quantified. For example, larger particles are converted to smaller particles, which are more easily exported. Also, dissolved organic matter and surface biofilms are converted to larger particles which can be more easily intercepted by consumers. However, the quality of these materials as it affects biological activity downstream is not well known, nor is the extent to which timing permits biological use of those particles. These ecological unknowns need to be resolved. Further, land uses may disrupt and diminish material transport to downstream reaches by removing sources (e.g., forest harvest), by affecting transport and decomposition processes (e.g., flow regulation, irrigation, changes in biotic communities), and by altering mechanisms of storage within headwaters (e.g., channelization). We present conceptual models of energy and nutrient fluxes that outline small stream processes and pathways important to downstream communities, and we identify informational gaps that, if filled, could significantly advance the understanding of linkages between headwater streams and larger rivers. The models, based on empirical evidence and best professional judgment, suggest that navigable waters are significantly influenced by headwater streams through hydrological and ecological connectivities, and land use can dramatically influence these natural connectivities, impacting downstream riverine ecosystems. ?? 2007 American Water Resources Association.

Push-Pull and Feedback Mechanisms Can Align Signaling System Outputs with Inputs.

PubMed

Andrews, Steven S; Peria, William J; Yu, Richard C; Colman-Lerner, Alejandro; Brent, Roger

2016-11-23

Many cell signaling systems, including the yeast pheromone response system, exhibit "dose-response alignment" (DoRA), in which output of one or more downstream steps closely matches the fraction of occupied receptors. DoRA can improve the fidelity of transmitted dose information. Here, we searched systematically for biochemical network topologies that produced DoRA. Most networks, including many containing feedback and feedforward loops, could not produce DoRA. However, networks including "push-pull" mechanisms, in which the active form of a signaling species stimulates downstream activity and the nominally inactive form reduces downstream activity, enabled perfect DoRA. Networks containing feedbacks enabled DoRA, but only if they also compared feedback to input and adjusted output to match. Our results establish push-pull as a non-feedback mechanism to align output with variable input and maximize information transfer in signaling systems. They also suggest genetic approaches to determine whether particular signaling systems use feedback or push-pull control. Copyright © 2016 Elsevier Inc. All rights reserved.

Integration and Visualization of Translational Medicine Data for Better Understanding of Human Diseases

PubMed Central

Satagopam, Venkata; Gu, Wei; Eifes, Serge; Gawron, Piotr; Ostaszewski, Marek; Gebel, Stephan; Barbosa-Silva, Adriano; Balling, Rudi; Schneider, Reinhard

2016-01-01

Abstract Translational medicine is a domain turning results of basic life science research into new tools and methods in a clinical environment, for example, as new diagnostics or therapies. Nowadays, the process of translation is supported by large amounts of heterogeneous data ranging from medical data to a whole range of -omics data. It is not only a great opportunity but also a great challenge, as translational medicine big data is difficult to integrate and analyze, and requires the involvement of biomedical experts for the data processing. We show here that visualization and interoperable workflows, combining multiple complex steps, can address at least parts of the challenge. In this article, we present an integrated workflow for exploring, analysis, and interpretation of translational medicine data in the context of human health. Three Web services—tranSMART, a Galaxy Server, and a MINERVA platform—are combined into one big data pipeline. Native visualization capabilities enable the biomedical experts to get a comprehensive overview and control over separate steps of the workflow. The capabilities of tranSMART enable a flexible filtering of multidimensional integrated data sets to create subsets suitable for downstream processing. A Galaxy Server offers visually aided construction of analytical pipelines, with the use of existing or custom components. A MINERVA platform supports the exploration of health and disease-related mechanisms in a contextualized analytical visualization system. We demonstrate the utility of our workflow by illustrating its subsequent steps using an existing data set, for which we propose a filtering scheme, an analytical pipeline, and a corresponding visualization of analytical results. The workflow is available as a sandbox environment, where readers can work with the described setup themselves. Overall, our work shows how visualization and interfacing of big data processing services facilitate exploration, analysis, and interpretation of translational medicine data. PMID:27441714

Performance optimization in mass production of volume holographic optical elements (vHOEs) using Bayfol HX photopolymer film

NASA Astrophysics Data System (ADS)

Bruder, Friedrich-Karl; Fäcke, Thomas; Grote, Fabian; Hagen, Rainer; Hönel, Dennis; Koch, Eberhard; Rewitz, Christian; Walze, Günther; Wewer, Brita

2017-05-01

Volume Holographic Optical Elements (vHOEs) gained wide attention as optical combiners for the use in smart glasses and augmented reality (SG and AR, respectively) consumer electronics and automotive head-up display applications. The unique characteristics of these diffractive grating structures - being lightweight, thin and flat - make them perfectly suitable for use in integrated optical components like spectacle lenses and car windshields. While being transparent in Off-Bragg condition, they provide full color capability and adjustable diffraction efficiency. The instant developing photopolymer Bayfol® HX film provides an ideal technology platform to optimize the performance of vHOEs in a wide range of applications. Important for any commercialization are simple and robust mass production schemes. In this paper, we present an efficient and easy to control one-beam recording scheme to copy a so-called master vHOE in a step-and-repeat process. In this contact-copy scheme, Bayfol® HX film is laminated to a master stack before being exposed by a scanning laser line. Subsequently, the film is delaminated in a controlled fashion and bleached. We explain working principles of the one-beam copy concept, discuss the opto-mechanical construction and outline the downstream process of the installed vHOE replication line. Moreover, we focus on aspects like performance optimization of the copy vHOE, the bleaching process and the suitable choice of protective cover film in the re-lamination step, preparing the integration of the vHOE into the final device.

A Systems Biology Approach Reveals Converging Molecular Mechanisms that Link Different POPs to Common Metabolic Diseases.

PubMed

Ruiz, Patricia; Perlina, Ally; Mumtaz, Moiz; Fowler, Bruce A

2016-07-01

A number of epidemiological studies have identified statistical associations between persistent organic pollutants (POPs) and metabolic diseases, but testable hypotheses regarding underlying molecular mechanisms to explain these linkages have not been published. We assessed the underlying mechanisms of POPs that have been associated with metabolic diseases; three well-known POPs [2,3,7,8-tetrachlorodibenzodioxin (TCDD), 2,2´,4,4´,5,5´-hexachlorobiphenyl (PCB 153), and 4,4´-dichlorodiphenyldichloroethylene (p,p´-DDE)] were studied. We used advanced database search tools to delineate testable hypotheses and to guide laboratory-based research studies into underlying mechanisms by which this POP mixture could produce or exacerbate metabolic diseases. For our searches, we used proprietary systems biology software (MetaCore™/MetaDrug™) to conduct advanced search queries for the underlying interactions database, followed by directional network construction to identify common mechanisms for these POPs within two or fewer interaction steps downstream of their primary targets. These common downstream pathways belong to various cytokine and chemokine families with experimentally well-documented causal associations with type 2 diabetes. Our systems biology approach allowed identification of converging pathways leading to activation of common downstream targets. To our knowledge, this is the first study to propose an integrated global set of step-by-step molecular mechanisms for a combination of three common POPs using a systems biology approach, which may link POP exposure to diseases. Experimental evaluation of the proposed pathways may lead to development of predictive biomarkers of the effects of POPs, which could translate into disease prevention and effective clinical treatment strategies. Ruiz P, Perlina A, Mumtaz M, Fowler BA. 2016. A systems biology approach reveals converging molecular mechanisms that link different POPs to common metabolic diseases. Environ Health Perspect 124:1034-1041; http://dx.doi.org/10.1289/ehp.1510308.

Effect of Temperature and Fluid Flow on Dendrite Growth During Solidification of Al-3 Wt Pct Cu Alloy by the Two-Dimensional Cellular Automaton Method

NASA Astrophysics Data System (ADS)

Gu, Cheng; Wei, Yanhong; Liu, Renpei; Yu, Fengyi

2017-12-01

A two-dimensional cellular automaton-finite volume model was developed to simulate dendrite growth of Al-3 wt pct Cu alloy during solidification to investigate the effect of temperature and fluid flow on dendrite morphology, solute concentration distribution, and dendrite growth velocity. Different calculation conditions that may influence the results of the simulation, including temperature and flow, were considered. The model was also employed to study the effect of different undercoolings, applied temperature fields, and forced flow velocities on solute segregation and dendrite growth. The initial temperature and fluid flow have a significant impact on the dendrite morphologies and solute profiles during solidification. The release of energy is operated with solidification and results in the increase of temperature. A larger undercooling leads to larger solute concentration near the solid/liquid interface and solute concentration gradient at the same time-step. Solute concentration in the solid region tends to increase with the increase of undercooling. Four vortexes appear under the condition when natural flow exists: the two on the right of the dendrite rotate clockwise, and those on the left of the dendrite rotate counterclockwise. With the increase of forced flow velocity, the rejected solute in the upstream region becomes easier to be washed away and enriched in the downstream region, resulting in acceleration of the growth of the dendrite in the upstream and inhibiting the downstream dendrite growth. The dendrite perpendicular to fluid flow shows a coarser morphology in the upstream region than that of the downstream. Almost no secondary dendrite appears during the calculation process.

Passive control of coherent structures in a modified backwards-facing step flow

NASA Astrophysics Data System (ADS)

Ormonde, Pedro C.; Cavalieri, André V. G.; Silva, Roberto G. A. da; Avelar, Ana C.

2018-05-01

We study a modified backwards-facing step flow, with the addition of two different plates; one is a baseline, impermeable plate and the second a perforated one. An experimental investigation is carried out for a turbulent reattaching shear layer downstream of the two plates. The proposed setup is a model configuration to study how the plate characteristics affect the separated shear layer and how turbulent kinetic energies and large-scale coherent structures are modified. Measurements show that the perforated plate changes the mean flow field, mostly by reducing the intensity of reverse flow close to the bottom wall. Disturbance amplitudes are significantly reduced up to five step heights downstream of the trailing edge of the plate, more specifically in the recirculation region. A loudspeaker is then used to introduce phase-locked, low-amplitude perturbations upstream of the plates, and phase-averaged measurements allow a quantitative study of large-scale structures in the shear-layer. The evolution of such coherent structures is evaluated in light of linear stability theory, comparing the eigenfunction of the Kelvin-Helmholtz mode to the experimental results. We observe a close match of linear-stability eigenfunctions with phase-averaged amplitudes for the two tested Strouhal numbers. The perforated plate is found to reduce the amplitude of the Kelvin-Helmholtz coherent structures in comparison to the baseline, impermeable plate, a behavior consistent with the predicted amplification trends from linear stability.

Progress and Challenges in Microalgal Biodiesel Production.

PubMed

Mallick, Nirupama; Bagchi, Sourav K; Koley, Shankha; Singh, Akhilesh K

2016-01-01

The last decade has witnessed a tremendous impetus on biofuel research due to the irreversible diminution of fossil fuel reserves for enormous demands of transportation vis-a-vis escalating emissions of green house gasses (GHGs) into the atmosphere. With an imperative need of CO2 reduction and considering the declining status of crude oil, governments in various countries have not only diverted substantial funds for biofuel projects but also have introduced incentives to vendors that produce biofuels. Currently, biodiesel production from microalgal biomass has drawn an immense importance with the potential to exclude high-quality agricultural land use and food safe-keeping issues. Moreover, microalgae can grow in seawater or wastewater and microalgal oil can exceed 50-60% (dry cell weight) as compared with some best agricultural oil crops of only 5-10% oil content. Globally, microalgae are the highest biomass producers and neutral lipid accumulators contending any other terrestrial oil crops. However, there remain many hurdles in each and every step, starting from strain selection and lipid accumulation/yield, algae mass cultivation followed by the downstream processes such as harvesting, drying, oil extraction, and biodiesel conversion (transesterification), and overall, the cost of production. Isolation and screening of oleaginous microalgae is one pivotal important upstream factor which should be addressed according to the need of freshwater or marine algae with a consideration that wild-type indigenous isolate can be the best suited for the laboratory to large scale exploitation. Nowadays, a large number of literature on microalgal biodiesel production are available, but none of those illustrate a detailed step-wise description with the pros and cons of the upstream and downstream processes of biodiesel production from microalgae. Specifically, harvesting and drying constitute more than 50% of the total production costs; however, there are quite a less number of detailed study reports available. In this review, a pragmatic and critical analysis was tried to put forward with the on-going researches on isolation and screening of oleaginous microalgae, microalgal large scale cultivation, biomass harvesting, drying, lipid extraction and finally biodiesel production.

Progress and Challenges in Microalgal Biodiesel Production

PubMed Central

Mallick, Nirupama; Bagchi, Sourav K.; Koley, Shankha; Singh, Akhilesh K.

2016-01-01

The last decade has witnessed a tremendous impetus on biofuel research due to the irreversible diminution of fossil fuel reserves for enormous demands of transportation vis-a-vis escalating emissions of green house gasses (GHGs) into the atmosphere. With an imperative need of CO2 reduction and considering the declining status of crude oil, governments in various countries have not only diverted substantial funds for biofuel projects but also have introduced incentives to vendors that produce biofuels. Currently, biodiesel production from microalgal biomass has drawn an immense importance with the potential to exclude high-quality agricultural land use and food safe-keeping issues. Moreover, microalgae can grow in seawater or wastewater and microalgal oil can exceed 50–60% (dry cell weight) as compared with some best agricultural oil crops of only 5–10% oil content. Globally, microalgae are the highest biomass producers and neutral lipid accumulators contending any other terrestrial oil crops. However, there remain many hurdles in each and every step, starting from strain selection and lipid accumulation/yield, algae mass cultivation followed by the downstream processes such as harvesting, drying, oil extraction, and biodiesel conversion (transesterification), and overall, the cost of production. Isolation and screening of oleaginous microalgae is one pivotal important upstream factor which should be addressed according to the need of freshwater or marine algae with a consideration that wild-type indigenous isolate can be the best suited for the laboratory to large scale exploitation. Nowadays, a large number of literature on microalgal biodiesel production are available, but none of those illustrate a detailed step-wise description with the pros and cons of the upstream and downstream processes of biodiesel production from microalgae. Specifically, harvesting and drying constitute more than 50% of the total production costs; however, there are quite a less number of detailed study reports available. In this review, a pragmatic and critical analysis was tried to put forward with the on-going researches on isolation and screening of oleaginous microalgae, microalgal large scale cultivation, biomass harvesting, drying, lipid extraction and finally biodiesel production. PMID:27446055

Advances in primary recovery: centrifugation and membrane technology.

PubMed

Roush, David J; Lu, Yuefeng

2008-01-01

Significant and continual improvements in upstream processing for biologics have resulted in challenges for downstream processing, both primary recovery and purification. Given the high cell densities achievable in both microbial and mammalian cell culture processes, primary recovery can be a significant bottleneck in both clinical and commercial manufacturing. The combination of increased product titer and low viability leads to significant relative increases in the levels of process impurities such as lipids, intracellular proteins and nucleic acid versus the product. In addition, cell culture media components such as soy and yeast hydrolysates have been widely applied to achieve the cell culture densities needed for higher titers. Many of the process impurities can be negatively charged at harvest pH and can form colloids during the cell culture and harvest processes. The wide size distribution of these particles and the potential for additional particles to be generated by shear forces within a centrifuge may result in insufficient clarification to prevent fouling of subsequent filters. The other residual process impurities can lead to precipitation and increased turbidity during processing and even interference with the performance of the capturing chromatographic step. Primary recovery also poses significant challenges owing to the necessity to execute in an expedient manner to minimize both product degradation and bioburden concerns. Both microfiltration and centrifugation coupled with depth filtration have been employed successfully as primary recovery processing steps. Advances in the design and application of membrane technology for microfiltration and dead-end filtration have contributed to significant improvements in process performance and integration, in some cases allowing for a combination of multiple unit operations in a given step. Although these advances have increased productivity and reliability, the net result is that optimization of primary recovery processes has become substantially more complicated. Ironically, the application of classical chemical engineering approaches to overcome issues in primary recovery and purification (e.g., turbidity and trace impurity removal) are just recently gaining attention. Some of these techniques (e.g., membrane cascades, pretreatment, precipitation, and the use of affinity tags) are now seen almost as disruptive technologies. This paper will review the current and potential future state of research on primary recovery, including relevant papers presented at the 234th American Chemical Society (ACS) National Meeting in Boston.

Harvesting of Chlorella sorokiniana by co-culture with the filamentous fungus Isaria fumosorosea: A potential sustainable feedstock for hydrothermal gasification.

PubMed

Mackay, Stephen; Gomes, Eduardo; Holliger, Christof; Bauer, Rolene; Schwitzguébel, Jean-Paul

2015-06-01

Despite recent advances in down-stream processing, production of microalgae remains substantially limited because of economical reasons. Harvesting and dewatering are the most energy-intensive processing steps in their production and contribute 20-30% of total operational cost. Bio-flocculation of microalgae by co-cultivation with filamentous fungi relies on the development of large structures that facilitate cost effective harvesting. A yet unknown filamentous fungus was isolated as a contaminant from a microalgal culture and identified as Isaria fumosorosea. Blastospores production was optimized in minimal medium and the development of pellets, possibly lichens, was followed when co-cultured with Chlorella sorokiniana under strict autotrophic conditions. Stable pellets (1-2mm) formed rapidly at pH 7-8, clearing the medium of free algal cells. Biomass was harvested with large inexpensive filters, generating wet slurry suitable for hydrothermal gasification. Nutrient rich brine from the aqueous phase of hydrothermal gasification supported growth of the fungus and may increase the process sustainability. Copyright © 2015 Elsevier Ltd. All rights reserved.

Promoting clinical and laboratory interaction by harmonization.

PubMed

Plebani, Mario; Panteghini, Mauro

2014-05-15

The lack of interchangeable results in current practice among clinical laboratories has underpinned greater attention to standardization and harmonization projects. Although the focus was mainly on the standardization and harmonization of measurement procedures and their results, the scope of harmonization goes beyond method and analytical results: it includes all other aspects of laboratory testing, including terminology and units, report formats, reference limits and decision thresholds, as well as test profiles and criteria for the interpretation of results. In particular, as evidence collected in last decades demonstrates that pre-pre- and post-post-analytical steps are more vulnerable to errors, harmonization initiatives should be performed to improve procedures and processes at the laboratory-clinical interface. Managing upstream demand, down-stream interpretation of laboratory results, and subsequent appropriate action through close relationships between laboratorians and clinicians remains a crucial issue of the laboratory testing process. Therefore, initiatives to improve test demand management from one hand and to harmonize procedures to improve physicians' acknowledgment of laboratory data and their interpretation from the other hand are needed in order to assure quality and safety in the total testing process. © 2013.

A comparative study between spiral-filter press and belt press implemented in a cloudy apple juice production process.

PubMed

De Paepe, Domien; Coudijzer, Katleen; Noten, Bart; Valkenborg, Dirk; Servaes, Kelly; De Loose, Marc; Diels, Ludo; Voorspoels, Stefan; Van Droogenbroeck, Bart

2015-04-15

In this study, advantages and disadvantages of the innovative, low-oxygen spiral-filter press system were studied in comparison with the belt press, commonly applied in small and medium size enterprises for the production of cloudy apple juice. On the basis of equivalent throughput, a higher juice yield could be achieved with spiral-filter press. Also a more turbid juice with a higher content of suspended solids could be produced. The avoidance of enzymatic browning during juice extraction led to an attractive yellowish juice with an elevated phenolic content. Moreover, it was found that juice produced with spiral-filter press demonstrates a higher retention of phenolic compounds during the downstream processing steps and storage. The results demonstrates the advantage of the use of a spiral-filter press in comparison with belt press in the production of a high quality cloudy apple juice rich in phenolic compounds, without the use of oxidation inhibiting additives. Copyright © 2014 Elsevier Ltd. All rights reserved.

Rapid, chemical-free breaking of microfluidic emulsions with a hand-held antistatic gun

PubMed Central

Shahi, Payam; Abate, Adam R.

2017-01-01

Droplet microfluidics can form and process millions of picoliter droplets with speed and ease, allowing the execution of huge numbers of biological reactions for high-throughput studies. However, at the conclusion of most experiments, the emulsions must be broken to recover and analyze their contents. This is usually achieved with demulsifiers, like perfluorooctanol and chloroform, which can interfere with downstream reactions and harm cells. Here, we describe a simple approach to rapidly and efficiently break microfluidic emulsions, which requires no chemicals. Our method allows one-pot multi-step reactions, making it useful for large scale automated processing of reactions requiring demulsification. Using a hand-held antistatic gun, we pulse emulsions with the electric field, coalescing ∼100 μl of droplets in ∼10 s. We show that while emulsions broken with chemical demulsifiers exhibit potent PCR inhibition, the antistatic-broken emulsions amplify efficiently. The ability to break emulsions quickly without chemicals should make our approach valuable for most demulsification needs in microfluidics. PMID:28794817

Global Analysis of Yeast Endosomal Transport Identifies the Vps55/68 Sorting Complex

PubMed Central

Schluter, Cayetana; Lam, Karen K.Y.; Brumm, Jochen; Wu, Bella W.; Saunders, Matthew; Stevens, Tom H.

2008-01-01

Endosomal transport is critical for cellular processes ranging from receptor down-regulation and retroviral budding to the immune response. A full understanding of endosome sorting requires a comprehensive picture of the multiprotein complexes that orchestrate vesicle formation and fusion. Here, we use unsupervised, large-scale phenotypic analysis and a novel computational approach for the global identification of endosomal transport factors. This technique effectively identifies components of known and novel protein assemblies. We report the characterization of a previously undescribed endosome sorting complex that contains two well-conserved proteins with four predicted membrane-spanning domains. Vps55p and Vps68p form a complex that acts with or downstream of ESCRT function to regulate endosomal trafficking. Loss of Vps68p disrupts recycling to the TGN as well as onward trafficking to the vacuole without preventing the formation of lumenal vesicles within the MVB. Our results suggest the Vps55/68 complex mediates a novel, conserved step in the endosomal maturation process. PMID:18216282

Continuous Manufacturing of Recombinant Therapeutic Proteins: Upstream and Downstream Technologies.

PubMed

Patil, Rohan; Walther, Jason

2017-03-07

Continuous biomanufacturing of recombinant therapeutic proteins offers several potential advantages over conventional batch processing, including reduced cost of goods, more flexible and responsive manufacturing facilities, and improved and consistent product quality. Although continuous approaches to various upstream and downstream unit operations have been considered and studied for decades, in recent years interest and application have accelerated. Researchers have achieved increasingly higher levels of process intensification, and have also begun to integrate different continuous unit operations into larger, holistically continuous processes. This review first discusses approaches for continuous cell culture, with a focus on perfusion-enabling cell separation technologies including gravitational, centrifugal, and acoustic settling, as well as filtration-based techniques. We follow with a review of various continuous downstream unit operations, covering categories such as clarification, chromatography, formulation, and viral inactivation and filtration. The review ends by summarizing case studies of integrated and continuous processing as reported in the literature.

At-line process analytical technology (PAT) for more efficient scale up of biopharmaceutical microfiltration unit operations.

PubMed

Watson, Douglas S; Kerchner, Kristi R; Gant, Sean S; Pedersen, Joseph W; Hamburger, James B; Ortigosa, Allison D; Potgieter, Thomas I

2016-01-01

Tangential flow microfiltration (MF) is a cost-effective and robust bioprocess separation technique, but successful full scale implementation is hindered by the empirical, trial-and-error nature of scale-up. We present an integrated approach leveraging at-line process analytical technology (PAT) and mass balance based modeling to de-risk MF scale-up. Chromatography-based PAT was employed to improve the consistency of an MF step that had been a bottleneck in the process used to manufacture a therapeutic protein. A 10-min reverse phase ultra high performance liquid chromatography (RP-UPLC) assay was developed to provide at-line monitoring of protein concentration. The method was successfully validated and method performance was comparable to previously validated methods. The PAT tool revealed areas of divergence from a mass balance-based model, highlighting specific opportunities for process improvement. Adjustment of appropriate process controls led to improved operability and significantly increased yield, providing a successful example of PAT deployment in the downstream purification of a therapeutic protein. The general approach presented here should be broadly applicable to reduce risk during scale-up of filtration processes and should be suitable for feed-forward and feed-back process control. © 2015 American Institute of Chemical Engineers.

Contamination pathways of spore-forming bacteria in a vegetable cannery.

PubMed

Durand, Loïc; Planchon, Stella; Guinebretiere, Marie-Hélène; André, Stéphane; Carlin, Frédéric; Remize, Fabienne

2015-06-02

Spoilage of low-acid canned food during prolonged storage at high temperatures is caused by heat resistant thermophilic spores of strict or facultative bacteria. Here, we performed a bacterial survey over two consecutive years on the processing line of a French company manufacturing canned mixed green peas and carrots. In total, 341 samples were collected, including raw vegetables, green peas and carrots at different steps of processing, cover brine, and process environment samples. Thermophilic and highly-heat-resistant thermophilic spores growing anaerobically were counted. During vegetable preparation, anaerobic spore counts were significantly decreased, and tended to remain unchanged further downstream in the process. Large variation of spore levels in products immediately before the sterilization process could be explained by occasionally high spore levels on surfaces and in debris of vegetable combined with long residence times in conditions suitable for growth and sporulation. Vegetable processing was also associated with an increase in the prevalence of highly-heat-resistant species, probably due to cross-contamination of peas via blanching water. Geobacillus stearothermophilus M13-PCR genotypic profiling on 112 isolates determined 23 profile-types and confirmed process-driven cross-contamination. Taken together, these findings clarify the scheme of contamination pathway by thermophilic spore-forming bacteria in a vegetable cannery. Copyright © 2015 Elsevier B.V. All rights reserved.

Examining the freezing process of an intermediate bulk containing an industrially relevant protein

PubMed Central

Reinsch, Holger; Spadiut, Oliver; Heidingsfelder, Johannes; Herwig, Christoph

2015-01-01

Numerous biopharmaceuticals are produced in recombinant microorganisms in the controlled environment of a bioreactor, a process known as Upstream Process. To minimize product loss due to physico-chemical and enzymatic degradation, the Upstream Process should be directly followed by product purification, known as Downstream Process. However, the Downstream Process can be technologically complex and time-consuming which is why Upstream and Downstream Process usually have to be decoupled temporally and spatially. Consequently, the product obtained after the Upstream Process, known as intermediate bulk, has to be stored. In those circumstances, a freezing procedure is often performed to prevent product loss. However, the freezing process itself is inseparably linked to physico-chemical changes of the intermediate bulk which may in turn damage the product. The present study analysed the behaviour of a Tris-buffered intermediate bulk containing a biopharmaceutically relevant protein during a bottle freezing process. Major damaging mechanisms, like the spatiotemporal redistribution of ion concentrations and pH, and their influence on product stability were investigated. Summarizing, we show the complex events which happen in an intermediate bulk during freezing and explain the different causes for product loss. PMID:25765305

In situ affinity purification of his-tagged protein A from Bacillus megaterium cultivation using recyclable superparamagnetic iron oxide nanoparticles.

PubMed

Gädke, Johannes; Kleinfeldt, Lennart; Schubert, Chris; Rohde, Manfred; Biedendieck, Rebekka; Garnweitner, Georg; Krull, Rainer

2017-01-20

This paper discusses the use of recyclable functionalized nanoparticles for an improved downstream processing of recombinant products. The Gram-positive bacterium Bacillus megaterium was used to secrete recombinant protein A fused to a histidine tag into the culture supernatant in shaker flasks. Superparamagnetic iron oxide nanoparticles functionalized with 3-glycidoxypropyl-trimethoxysilane-coupled-nitrilotriacetic-acid groups (GNTA-SPION) were synthesized and added directly to the growing culture. After 10min incubation time, >85% of the product was adsorbed onto the particles. The particles were magnetically separated using handheld neodymium magnets and the product was eluted. The GNTA-SPION were successfully regenerated and reused in five consecutive cycles. In the one-step purification, the purity of the product reached >99.9% regarding protein A. A very low particle concentration of 0.5g/L was sufficient for effective product separation. Bacterial growth was not influenced negatively by this concentration. Particle analysis showed similar properties between freshly synthesized and regenerated GNTA-SPION. The overall process efficiency was however influenced by partial disintegration of particle agglomerates and thus loss of particles. The demonstration of very fast in situ product removal from growing bacterial culture combined with a very high product purity within one step shows possibilities for automated large scale purification combined with recycling of biomass. Copyright © 2016 Elsevier B.V. All rights reserved.

The Evolutionary Fate of the Genes Encoding the Purine Catabolic Enzymes in Hominoids, Birds, and Reptiles

PubMed Central

Keebaugh, Alaine C.; Thomas, James W.

2010-01-01

Gene loss has been proposed to play a major role in adaptive evolution, and recent studies are beginning to reveal its importance in human evolution. However, the potential consequence of a single gene-loss event upon the fates of functionally interrelated genes is poorly understood. Here, we use the purine metabolic pathway as a model system in which to explore this important question. The loss of urate oxidase (UOX) activity, a necessary step in this pathway, has occurred independently in the hominoid and bird/reptile lineages. Because the loss of UOX would have removed the functional constraint upon downstream genes in this pathway, these downstream genes are generally assumed to have subsequently deteriorated. In this study, we used a comparative genomics approach to empirically determine the fate of UOX itself and the downstream genes in five hominoids, two birds, and a reptile. Although we found that the loss of UOX likely triggered the genetic deterioration of the immediate downstream genes in the hominoids, surprisingly in the birds and reptiles, the UOX locus itself and some of the downstream genes were present in the genome and predicted to encode proteins. To account for the variable pattern of gene retention and loss after the inactivation of UOX, we hypothesize that although gene loss is a common fate for genes that have been rendered obsolete due to the upstream loss of an enzyme a metabolic pathway, it is also possible that same lack of constraint will foster the evolution of new functions or allow the optimization of preexisting alternative functions in the downstream genes, thereby resulting in gene retention. Thus, adaptive single-gene losses have the potential to influence the long-term evolutionary fate of functionally interrelated genes. PMID:20106906

The evolutionary fate of the genes encoding the purine catabolic enzymes in hominoids, birds, and reptiles.

PubMed

Keebaugh, Alaine C; Thomas, James W

2010-06-01

Gene loss has been proposed to play a major role in adaptive evolution, and recent studies are beginning to reveal its importance in human evolution. However, the potential consequence of a single gene-loss event upon the fates of functionally interrelated genes is poorly understood. Here, we use the purine metabolic pathway as a model system in which to explore this important question. The loss of urate oxidase (UOX) activity, a necessary step in this pathway, has occurred independently in the hominoid and bird/reptile lineages. Because the loss of UOX would have removed the functional constraint upon downstream genes in this pathway, these downstream genes are generally assumed to have subsequently deteriorated. In this study, we used a comparative genomics approach to empirically determine the fate of UOX itself and the downstream genes in five hominoids, two birds, and a reptile. Although we found that the loss of UOX likely triggered the genetic deterioration of the immediate downstream genes in the hominoids, surprisingly in the birds and reptiles, the UOX locus itself and some of the downstream genes were present in the genome and predicted to encode proteins. To account for the variable pattern of gene retention and loss after the inactivation of UOX, we hypothesize that although gene loss is a common fate for genes that have been rendered obsolete due to the upstream loss of an enzyme a metabolic pathway, it is also possible that same lack of constraint will foster the evolution of new functions or allow the optimization of preexisting alternative functions in the downstream genes, thereby resulting in gene retention. Thus, adaptive single-gene losses have the potential to influence the long-term evolutionary fate of functionally interrelated genes.

The evolution of a colluvial hollow to a fluvial channel with periodic steps following two transformational disturbances: A wildfire and a historic flood

NASA Astrophysics Data System (ADS)

Rengers, F. K.; McGuire, L. A.; Ebel, B. A.; Tucker, G. E.

2018-05-01

The transition of a colluvial hollow to a fluvial channel with discrete steps was observed after two landscape-scale disturbances. The first disturbance, a high-severity wildfire, changed the catchment hydrology to favor overland flow, which incised a colluvial hollow, creating a channel in the same location. This incised channel became armored with cobbles and boulders following repeated post-wildfire overland flow events. Three years after the fire, a record rainstorm produced regional flooding and generated sufficient fluvial erosion and sorting to produce a fluvial channel with periodically spaced steps. An analysis of the step spacing shows that after the flood, newly formed steps retained a similar spacing to the topographic roughness spacing in the original colluvial hollow (prior to channelization). This suggests that despite a distinct change in channel form roughness and bedform morphology, the endogenous roughness periodicity was conserved. Variations in sediment erodibility helped to create the emergent steps as the largest particles (>D84) remained immobile, becoming step features, and downstream soil was easily winnowed away.

The evolution of a colluvial hollow to a fluvial channel with periodic steps following two transformational disturbances: A wildfire and a historic flood

USGS Publications Warehouse

Rengers, Francis K.; McGuire, Luke; Ebel, Brian A.; Tucker, G. E.

2018-01-01

The transition of a colluvial hollow to a fluvial channel with discrete steps was observed after two landscape-scale disturbances. The first disturbance, a high-severity wildfire, changed the catchment hydrology to favor overland flow, which incised a colluvial hollow, creating a channel in the same location. This incised channel became armored with cobbles and boulders following repeated post-wildfire overland flow events. Three years after the fire, a record rainstorm produced regional flooding and generated sufficient fluvial erosion and sorting to produce a fluvial channel with periodically spaced steps. An analysis of the step spacing shows that after the flood, newly formed steps retained a similar spacing to the topographic roughness spacing in the original colluvial hollow (prior to channelization). This suggests that despite a distinct change in channel form roughness and bedform morphology, the endogenous roughness periodicity was conserved. Variations in sediment erodibility helped to create the emergent steps as the largest particles ( >D84) remained immobile, becoming step features, and downstream soil was easily winnowed away.

Physical modeling of vortical cross-step flow in the American paddlefish, Polyodon spathula

PubMed Central

Brooks, Hannah; Haines, Grant E.; Lin, M. Carly

2018-01-01

Vortical cross-step filtration in suspension-feeding fish has been reported recently as a novel mechanism, distinct from other biological and industrial filtration processes. Although crossflow passing over backward-facing steps generates vortices that can suspend, concentrate, and transport particles, the morphological factors affecting this vortical flow have not been identified previously. In our 3D-printed models of the oral cavity for ram suspension-feeding fish, the angle of the backward-facing step with respect to the model’s dorsal midline affected vortex parameters significantly, including rotational, tangential, and axial speed. These vortices were comparable to those quantified downstream of the backward-facing steps that were formed by the branchial arches of preserved American paddlefish in a recirculating flow tank. Our data indicate that vortices in cross-step filtration have the characteristics of forced vortices, as the flow of water inside the oral cavity provides the external torque required to sustain forced vortices. Additionally, we quantified a new variable for ram suspension feeding termed the fluid exit ratio. This is defined as the ratio of the total open pore area for water leaving the oral cavity via spaces between branchial arches that are not blocked by gill rakers, divided by the total area for water entering through the gape during ram suspension feeding. Our experiments demonstrated that the fluid exit ratio in preserved paddlefish was a significant predictor of the flow speeds that were quantified anterior of the rostrum, at the gape, directly dorsal of the first ceratobranchial, and in the forced vortex generated by the first ceratobranchial. Physical modeling of vortical cross-step filtration offers future opportunities to explore the complex interactions between structural features of the oral cavity, vortex parameters, motile particle behavior, and particle morphology that determine the suspension, concentration, and transport of particles within the oral cavity of ram suspension-feeding fish. PMID:29561890

Defining process design space for monoclonal antibody cell culture.

PubMed

Abu-Absi, Susan Fugett; Yang, LiYing; Thompson, Patrick; Jiang, Canping; Kandula, Sunitha; Schilling, Bernhard; Shukla, Abhinav A

2010-08-15

The concept of design space has been taking root as a foundation of in-process control strategies for biopharmaceutical manufacturing processes. During mapping of the process design space, the multidimensional combination of operational variables is studied to quantify the impact on process performance in terms of productivity and product quality. An efficient methodology to map the design space for a monoclonal antibody cell culture process is described. A failure modes and effects analysis (FMEA) was used as the basis for the process characterization exercise. This was followed by an integrated study of the inoculum stage of the process which includes progressive shake flask and seed bioreactor steps. The operating conditions for the seed bioreactor were studied in an integrated fashion with the production bioreactor using a two stage design of experiments (DOE) methodology to enable optimization of operating conditions. A two level Resolution IV design was followed by a central composite design (CCD). These experiments enabled identification of the edge of failure and classification of the operational parameters as non-key, key or critical. In addition, the models generated from the data provide further insight into balancing productivity of the cell culture process with product quality considerations. Finally, process and product-related impurity clearance was evaluated by studies linking the upstream process with downstream purification. Production bioreactor parameters that directly influence antibody charge variants and glycosylation in CHO systems were identified.

Automatic Phase Calibration for RF Cavities using Beam-Loading Signals

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

Edelen, J. P.; Chase, B. E.

Precise calibration of the cavity phase signals is necessary for the operation of any particle accelerator. For many systems this requires human in the loop adjustments based on measurements of the beam parameters downstream. Some recent work has developed a scheme for the calibration of the cavity phase using beam measurements and beam-loading however this scheme is still a multi-step process that requires heavy automation or human in the loop. In this paper we analyze a new scheme that uses only RF signals reacting to beam-loading to calculate the phase of the beam relative to the cavity. This technique couldmore » be used in slow control loops to provide real-time adjustment of the cavity phase calibration without human intervention thereby increasing the stability and reliability of the accelerator.« less

Increased sediment loads cause non-linear decreases in seagrass suitable habitat extent

PubMed Central

Atkinson, Scott; Klein, Carissa Joy; Weber, Tony; Possingham, Hugh P.

2017-01-01

Land-based activities, including deforestation, agriculture, and urbanisation, cause increased erosion, reduced inland and coastal water quality, and subsequent loss or degradation of downstream coastal marine ecosystems. Quantitative approaches to link sediment loads from catchments to metrics of downstream marine ecosystem state are required to calculate the cost effectiveness of taking conservation actions on land to benefits accrued in the ocean. Here we quantify the relationship between sediment loads derived from landscapes to habitat suitability of seagrass meadows in Moreton Bay, Queensland, Australia. We use the following approach: (1) a catchment hydrological model generates sediment loads; (2) a statistical model links sediment loads to water clarity at monthly time-steps; (3) a species distribution model (SDM) factors in water clarity, bathymetry, wave height, and substrate suitability to predict seagrass habitat suitability at monthly time-steps; and (4) a statistical model quantifies the effect of sediment loads on area of seagrass suitable habitat in a given year. The relationship between sediment loads and seagrass suitable habitat is non-linear: large increases in sediment have a disproportionately large negative impact on availability of seagrass suitable habitat. Varying the temporal scale of analysis (monthly vs. yearly), or varying the threshold value used to delineate predicted seagrass presence vs. absence, both affect the magnitude, but not the overall shape, of the relationship between sediment loads and seagrass suitable habitat area. Quantifying the link between sediment produced from catchments and extent of downstream marine ecosystems allows assessment of the relative costs and benefits of taking conservation actions on land or in the ocean, respectively, to marine ecosystems. PMID:29125843

Pilot scale production of highly efficacious and stable enterovirus 71 vaccine candidates.

PubMed

Chou, Ai-Hsiang; Liu, Chia-Chyi; Chang, Cheng-Peng; Guo, Meng-Shin; Hsieh, Shih-Yang; Yang, Wen-Hsueh; Chao, Hsin-Ju; Wu, Chien-Long; Huang, Ju-Lan; Lee, Min-Shi; Hu, Alan Yung-Chi; Lin, Sue-Chen; Huang, Yu-Yun; Hu, Mei-Hua; Chow, Yen-Hung; Chiang, Jen-Ron; Chang, Jui-Yuan; Chong, Pele

2012-01-01

Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial. In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7-10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30-43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37 °C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4 °C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates. These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials.

Energy Secretary Dedicates ESIF at NREL | News | NREL

Science.gov Websites

-like screen how wind turbine blades can alter the wind flow as it approaches other turbines downstream flows downstream when the blades hit it. A man with his back to the camera explains the process, while

Cortical Specializations Underlying Fast Computations

PubMed Central

Volgushev, Maxim

2016-01-01

The time course of behaviorally relevant environmental events sets temporal constraints on neuronal processing. How does the mammalian brain make use of the increasingly complex networks of the neocortex, while making decisions and executing behavioral reactions within a reasonable time? The key parameter determining the speed of computations in neuronal networks is a time interval that neuronal ensembles need to process changes at their input and communicate results of this processing to downstream neurons. Theoretical analysis identified basic requirements for fast processing: use of neuronal populations for encoding, background activity, and fast onset dynamics of action potentials in neurons. Experimental evidence shows that populations of neocortical neurons fulfil these requirements. Indeed, they can change firing rate in response to input perturbations very quickly, within 1 to 3 ms, and encode high-frequency components of the input by phase-locking their spiking to frequencies up to 300 to 1000 Hz. This implies that time unit of computations by cortical ensembles is only few, 1 to 3 ms, which is considerably faster than the membrane time constant of individual neurons. The ability of cortical neuronal ensembles to communicate on a millisecond time scale allows for complex, multiple-step processing and precise coordination of neuronal activity in parallel processing streams, while keeping the speed of behavioral reactions within environmentally set temporal constraints. PMID:25689988

Transport of sediment through a channel network during a post-fire debris flow

NASA Astrophysics Data System (ADS)

Nyman, P.; Box, W. A. C.; Langhans, C.; Stout, J. C.; Keesstra, S.; Sheridan, G. J.

2017-12-01

Transport processes linking sediment in steep headwaters with rivers during high magnitude events are rarely examined in detail, particularly in forested settings where major erosion events are rare and opportunities for collecting data are limited. Yet high magnitude events in headwaters are known to drive landscape change. This study examines how a debris flow after wildfire impacts on sediment transport from small headwaters (0.02 km2) through a step pool stream system within a larger 14 km2 catchment, which drains into the East Ovens River in SE Australia. Sediment delivery from debris flows was modelled and downstream deposition of sediment was measured using a combination of aerial imagery and field surveys. Particle size distributions were measured for all major deposits. These data were summarised to map sediment flux as a continuous variable over the drainage network. Total deposition throughout the stream network was 39 x 103 m3. Catchment efflux was 61 x 103 m3 (specific sediment yield of 78 ton ha-1), which equates to 400-800 years of background erosion, based on measurements in nearby catchments. Despite the low gradient (ca. 0.1 m m-1) of the main channel there was no systematic downstream sorting in sediment deposits in the catchment. This is due to debris flow processes operating throughout the stream network, with lateral inputs sustaining the process in low gradient channels, except in the most downstream reaches where the flow transitioned towards hyper-concentrated flow. Overall, a large proportion ( 88%) of the eroded fine fraction (<63 micron) exited the catchment, when compared to the overall ratio (55%) of erosion to deposition. The geomorphic legacy of this post-wildfire event depends on scale. In the lower channels (steam order 4-5), where erosion was nearly equal to deposition, the event had no real impact on total sediment volumes stored. In upper channels (stream orders < 3) erosion was widespread but deposition rates were low. So debris flows are really effective at removing sediment from headwaters, but at some scale (between 3th and 4th order channels) they are equally effective at depositing sediment. In these lower reaches the geomorphic legacy of the post-wildfire debris flow is about how channel sediment is distributed rather than how much volume is stored.

Cyclic steps due to the surge-type turbidity currents in flume experiments: effect of surge duration on the topography of steps

NASA Astrophysics Data System (ADS)

Yokokawa, Miwa; Yamano, Junpei; Miyai, Masatomo; Hughes Clarke, John; Izumi, Norihiro

2017-04-01

Field observations of turbidity currents and seabed topography on the Squamish delta in British Columbia, Canada revealed that cyclic steps formed by the surge-type turbidity currents (e.g., Hughes Clarke et al., 2014). The high-density portion of the flow, which affects the sea floor morphology, lasted only 30-60 seconds. We are doing flume experiments aiming to investigate the relationship between the condition of surges and topography of resultant steps. In this presentation, we are going to discuss about the effect of surge duration on the topography of steps. The experiments have been performed at Osaka Institute of Technology. A flume, which is 7.0 m long, 0.3 m deep and 2 cm wide, was suspended in a larger tank, which is 7.6 m long, 1.2 m deep and 0.3 m wide, filled with water. The inner flume tilted at 7 degrees. As a source of turbidity currents, mixture of salt water (1.17 g/cm^3) and plastic particles (1.3 g/cm^3, 0.1-0.18 mm in diameter) was prepared. The concentration of the sediments was 6.1 weight % (5.5 volume %) in the head tank. This mixture of salt water and plastic particles poured into the upstream end of the inner flume from head tank for 3 seconds or 7 seconds. 140 surges were made respectively. Discharge of the currents were fluctuated but range from 306 to 870 mL for 3s-surge, and from 1134 to 2030 mL for 7s-surge. As a result, five or six steps were formed respectively. At the case of 3s-surge, steps located at upstream portion of the flume moved vigorously toward upstream direction, whereas steps at downstream portion of the flume moved toward upstream direction at the case of 7s-surge. The wavelengths and wave heights of the steps by 3s-surge are larger than those of 7s-surge at the upstream portion of the flume, but the size of steps of 3s-surge are smaller than those of 7s-surge at the downstream portion of the flume. In this condition of slope and concentration, the longer surge duration, i.e. larger discharge of the current transports the sediment further and makes the steps larger and active at the further location from the source of the currents.

Investigation of chromatography and polymer/salt aqueous two-phase processes for downstream processing development of recombinant phenylalanine dehydrogenase.

PubMed

Omidinia, Eskandar; Shahbaz Mohamadi, Hamid; Dinarvand, Rassoul; Taherkhani, Heshmat-Allah

2010-03-01

This work presents a comprehensive study between the polymer/salt aqueous two-phase systems (ATPS) and chromatography process for downstream processing of recombinant Bacillus badius phenylalanine dehydrogenase (PheDH). First, the partitioning behavior of recombinant PheDH in polyethylene glycol (PEG)/K2HPO4 ATPS was examined. For comparative purpose, a classical chromatographic protocol was performed as well. Investigation of chromatography and ATPS procedures revealed that the ATPS comprising of 9% (w/w) PEG-6000, 16% (w/w) K2HPO4 and 16% (w/w) KCl with pH of 8.0, volume ratio (V ( R )) of 0.25, temperature of 25 degrees C and 40% (w/w) cell lysate ensured the most favorable approach for PheDH downstream process. A specific activity of 4,231.4 U/mg, a yield of 96.7% and a recovery of 162.0% were obtained. Furthermore, the shorter process time (4 vs. 48 h) and the lower total cost (4 vs. 20 euro) were additionally features that confirmed the suitability of proposed technique.

Morphodynamics of Travertine Dam/Waterfall Growth due to the Interaction of Biological Activity, Water Flow and Limestone Emplacement

NASA Astrophysics Data System (ADS)

Izumi, N.; Parker, G.

2012-12-01

Plitvice Lakes in Croatia are characterized by a step-like train of lakes and waterfalls. The waterfalls are located at the crests of naturally-emplaced dams. The top of each dam grows upward at the rate of a few millimeters per year. It is thought that the upward growth of these dams is caused by the interaction of water flow and biological activity, resulting in the precipitation of dissolved limestone. Dam evolution is initiated by the growth of mosses that favor swift, shallow water. Bacteria that inhabit the roots of the moss excrete solid limestone (travertine) from the water. The limestone fossilizes the moss, and then more moss grows on top of the travertine deposit. In this way, the natural dam can grow over to 10 m high, impounding the water behind it to form a lake. We propose a simple model to explain the formation of natural limestone dams by the interaction between water flow and biologically-mediated travertine deposition. We assume for simplicity that light is the only factor determining the growth of moss, which is then colonized by travertine-emplacing bacteria. We also assume that the water is saturated with dissolved limestone, so that the process is not limited by limestone availability. Photosynthesis, and thus the growth rate of moss are crudely approximated as decreasing linearly with depth. We employ the shallow water equations to describe water flow over the dam. In order to obtain a profile of permanent form for a dam migrating upward and downstream at constant speed, we solve the problem in a moving coordinate system. When water flows over the dam, it is accelerated in the streamwise direction, and the water surface forms a backwater curve. The flow regime changes from Froude-subcritical to Froude-supercritical at a point slightly downstream of the crest of the dam. Farther downstream, the flow attains a threshold velocity beyond which moss is detached. This threshold point defines the downstream end of the active part of the dam. The analysis provides a first-order morphodynamic model of natural dam/waterfall evolution.

The role of headwater streams in downstream water quality

USGS Publications Warehouse

Alexander, R.B.; Boyer, E.W.; Smith, R.A.; Schwarz, G.E.; Moore, R.B.

2007-01-01

Knowledge of headwater influences on the water-quality and flow conditions of downstream waters is essential to water-resource management at all governmental levels; this includes recent court decisions on the jurisdiction of the Federal Clean Water Act (CWA) over upland areas that contribute to larger downstream water bodies. We review current watershed research and use a water-quality model to investigate headwater influences on downstream receiving waters. Our evaluations demonstrate the intrinsic connections of headwaters to landscape processes and downstream waters through their influence on the supply, transport, and fate of water and solutes in watersheds. Hydrological processes in headwater catchments control the recharge of subsurface water stores, flow paths, and residence times of water throughout landscapes. The dynamic coupling of hydrological and biogeochemical processes in upland streams further controls the chemical form, timing, and longitudinal distances of solute transport to downstream waters. We apply the spatially explicit, mass-balance watershed model SPARROW to consider transport and transformations of water and nutrients throughout stream networks in the northeastern United States. We simulate fluxes of nitrogen, a primary nutrient that is a water-quality concern for acidification of streams and lakes and eutrophication of coastal waters, and refine the model structure to include literature observations of nitrogen removal in streams and lakes. We quantify nitrogen transport from headwaters to downstream navigable waters, where headwaters are defined within the model as first-order, perennial streams that include flow and nitrogen contributions from smaller, intermittent and ephemeral streams. We find that first-order headwaters contribute approximately 70% of the mean-annual water volume and 65% of the nitrogen flux in second-order streams. Their contributions to mean water volume and nitrogen flux decline only marginally to about 55% and 40% in fourth- and higher-order rivers that include navigable waters and their tributaries. These results underscore the profound influence that headwater areas have on shaping downstream water quantity and water quality. The results have relevance to water-resource management and regulatory decisions and potentially broaden understanding of the spatial extent of Federal CWA jurisdiction in U.S. waters. ?? 2007 American Water Resources Association.

The Role of Headwater Streams in Downstream Water Quality1

PubMed Central

Alexander, Richard B; Boyer, Elizabeth W; Smith, Richard A; Schwarz, Gregory E; Moore, Richard B

2007-01-01

Knowledge of headwater influences on the water-quality and flow conditions of downstream waters is essential to water-resource management at all governmental levels; this includes recent court decisions on the jurisdiction of the Federal Clean Water Act (CWA) over upland areas that contribute to larger downstream water bodies. We review current watershed research and use a water-quality model to investigate headwater influences on downstream receiving waters. Our evaluations demonstrate the intrinsic connections of headwaters to landscape processes and downstream waters through their influence on the supply, transport, and fate of water and solutes in watersheds. Hydrological processes in headwater catchments control the recharge of subsurface water stores, flow paths, and residence times of water throughout landscapes. The dynamic coupling of hydrological and biogeochemical processes in upland streams further controls the chemical form, timing, and longitudinal distances of solute transport to downstream waters. We apply the spatially explicit, mass-balance watershed model SPARROW to consider transport and transformations of water and nutrients throughout stream networks in the northeastern United States. We simulate fluxes of nitrogen, a primary nutrient that is a water-quality concern for acidification of streams and lakes and eutrophication of coastal waters, and refine the model structure to include literature observations of nitrogen removal in streams and lakes. We quantify nitrogen transport from headwaters to downstream navigable waters, where headwaters are defined within the model as first-order, perennial streams that include flow and nitrogen contributions from smaller, intermittent and ephemeral streams. We find that first-order headwaters contribute approximately 70% of the mean-annual water volume and 65% of the nitrogen flux in second-order streams. Their contributions to mean water volume and nitrogen flux decline only marginally to about 55% and 40% in fourth- and higher-order rivers that include navigable waters and their tributaries. These results underscore the profound influence that headwater areas have on shaping downstream water quantity and water quality. The results have relevance to water-resource management and regulatory decisions and potentially broaden understanding of the spatial extent of Federal CWA jurisdiction in U.S. waters. PMID:22457565

Metabolic pathways as possible therapeutic targets for progressive multiple sclerosis.

PubMed

Heidker, Rebecca M; Emerson, Mitchell R; LeVine, Steven M

2017-08-01

Unlike relapsing remitting multiple sclerosis, there are very few therapeutic options for patients with progressive forms of multiple sclerosis. While immune mechanisms are key participants in the pathogenesis of relapsing remitting multiple sclerosis, the mechanisms underlying the development of progressive multiple sclerosis are less well understood. Putative mechanisms behind progressive multiple sclerosis have been put forth: insufficient energy production via mitochondrial dysfunction, activated microglia, iron accumulation, oxidative stress, activated astrocytes, Wallerian degeneration, apoptosis, etc . Furthermore, repair processes such as remyelination are incomplete. Experimental therapies that strive to improve metabolism within neurons and glia, e.g. , oligodendrocytes, could act to counter inadequate energy supplies and/or support remyelination. Most experimental approaches have been examined as standalone interventions; however, it is apparent that the biochemical steps being targeted are part of larger pathways, which are further intertwined with other metabolic pathways. Thus, the potential benefits of a tested intervention, or of an established therapy, e.g. , ocrelizumab, could be undermined by constraints on upstream and/or downstream steps. If correct, then this argues for a more comprehensive, multifaceted approach to therapy. Here we review experimental approaches to support neuronal and glial metabolism, and/or promote remyelination, which may have potential to lessen or delay progressive multiple sclerosis.

Tissue-aware RNA-Seq processing and normalization for heterogeneous and sparse data.

PubMed

Paulson, Joseph N; Chen, Cho-Yi; Lopes-Ramos, Camila M; Kuijjer, Marieke L; Platig, John; Sonawane, Abhijeet R; Fagny, Maud; Glass, Kimberly; Quackenbush, John

2017-10-03

Although ultrahigh-throughput RNA-Sequencing has become the dominant technology for genome-wide transcriptional profiling, the vast majority of RNA-Seq studies typically profile only tens of samples, and most analytical pipelines are optimized for these smaller studies. However, projects are generating ever-larger data sets comprising RNA-Seq data from hundreds or thousands of samples, often collected at multiple centers and from diverse tissues. These complex data sets present significant analytical challenges due to batch and tissue effects, but provide the opportunity to revisit the assumptions and methods that we use to preprocess, normalize, and filter RNA-Seq data - critical first steps for any subsequent analysis. We find that analysis of large RNA-Seq data sets requires both careful quality control and the need to account for sparsity due to the heterogeneity intrinsic in multi-group studies. We developed Yet Another RNA Normalization software pipeline (YARN), that includes quality control and preprocessing, gene filtering, and normalization steps designed to facilitate downstream analysis of large, heterogeneous RNA-Seq data sets and we demonstrate its use with data from the Genotype-Tissue Expression (GTEx) project. An R package instantiating YARN is available at http://bioconductor.org/packages/yarn .

M13 bacteriophage purification using poly(ionic liquids) as alternative separation matrices.

PubMed

Jacinto, Maria João; Patinha, David J S; Marrucho, Isabel M; Gonçalves, João; Willson, Richard C; Azevedo, Ana M; Aires-Barros, M Raquel

2018-01-12

M13 is a filamentous, non-lytic bacteriophage that infects Escherichia coli via the F pilus. Currently, phage M13 is widely used in phage display technology and bio-nanotechnology, and is considered a possible antibacterial therapeutic agent, among other applications. Conventional phage purification involves 5-7 operational steps, with high operational costs and significant product loss (approximately 60%). In this work, we propose a scalable purification process for M13 bacteriophage using a novel stationary phase based on a polymeric ionic liquid (PIL) with a positively charged backbone structure. Poly (1-vinyl-3-ethyl imidazolium bis(trifluoromethylsulfonyl) imide) - poly(VEIM-TFSI) predominantly acted as an anion exchanger under binding-elution mode. This revealed to be a rapid and simple method for the recovery of phage M13 with an overall separation yield of over 70% after a single downstream step. To the best of our knowledge, PILs have never been used as separation matrices for biological products and the results obtained, together with the large number of cations and anions available to prepare PILs, illustrate well the large potential of the proposed methodology. Copyright © 2017 Elsevier B.V. All rights reserved.

G-protein-coupled receptors participate in cytokinesis

PubMed Central

Zhang, Xin; Bedigian, Anne V.; Wang, Wenchao; Eggert, Ulrike S.

2014-01-01

Cytokinesis, the last step during cell division, is a highly coordinated process that involves the relay of signals from both the outside and inside of the cell. We have a basic understanding of how cells regulate internal events, but how cells respond to extracellular cues is less explored. In a systematic RNAi screen of G-protein-coupled receptors (GPCRs) and their effectors, we found that some GPCRs are involved in cytokinesis. RNAi knockdown of these GPCRs caused increased binucleated cell formation, and live cell imaging showed that most formed midbodies but failed at the abscission stage. OR2A4 localized to cytokinetic structures in cells and its knockdown caused cytokinesis failure at an earlier stage, likely due to effects on the actin cytoskeleton. Identifying the downstream components that transmit GPCR signals during cytokinesis will be the next step and we show that GIPC1, an adaptor protein for GPCRs, may play a part. RNAi knockdown of GIPC1 significantly increased binucleated cell formation. Understanding the molecular details of GPCRs and their interaction proteins in cytokinesis regulation will give us important clues about GPCRs signaling as well as how cells communicate with their environment during division. PMID:22888021

A Model for Direction Sensing in Dictyostelium discoideum: Ras Activity and Symmetry Breaking Driven by a Gβγ-Mediated, Gα2-Ric8 -- Dependent Signal Transduction Network

PubMed Central

Cheng, Yougan; Othmer, Hans

2016-01-01

Chemotaxis is a dynamic cellular process, comprised of direction sensing, polarization and locomotion, that leads to the directed movement of eukaryotic cells along extracellular gradients. As a primary step in the response of an individual cell to a spatial stimulus, direction sensing has attracted numerous theoretical treatments aimed at explaining experimental observations in a variety of cell types. Here we propose a new model of direction sensing based on experiments using Dictyostelium discoideum (Dicty). The model is built around a reaction-diffusion-translocation system that involves three main component processes: a signal detection step based on G-protein-coupled receptors (GPCR) for cyclic AMP (cAMP), a transduction step based on a heterotrimetic G protein Gα2βγ, and an activation step of a monomeric G-protein Ras. The model can predict the experimentally-observed response of cells treated with latrunculin A, which removes feedback from downstream processes, under a variety of stimulus protocols. We show that Gα2βγ cycling modulated by Ric8, a nonreceptor guanine exchange factor for Gα2 in Dicty, drives multiple phases of Ras activation and leads to direction sensing and signal amplification in cAMP gradients. The model predicts that both Gα2 and Gβγ are essential for direction sensing, in that membrane-localized Gα2*, the activated GTP-bearing form of Gα2, leads to asymmetrical recruitment of RasGEF and Ric8, while globally-diffusing Gβγ mediates their activation. We show that the predicted response at the level of Ras activation encodes sufficient ‘memory’ to eliminate the ‘back-of-the wave’ problem, and the effects of diffusion and cell shape on direction sensing are also investigated. In contrast with existing LEGI models of chemotaxis, the results do not require a disparity between the diffusion coefficients of the Ras activator GEF and the Ras inhibitor GAP. Since the signal pathways we study are highly conserved between Dicty and mammalian leukocytes, the model can serve as a generic one for direction sensing. PMID:27152956

Edge detection and localization with edge pattern analysis and inflection characterization

NASA Astrophysics Data System (ADS)

Jiang, Bo

2012-05-01

In general edges are considered to be abrupt changes or discontinuities in two dimensional image signal intensity distributions. The accuracy of front-end edge detection methods in image processing impacts the eventual success of higher level pattern analysis downstream. To generalize edge detectors designed from a simple ideal step function model to real distortions in natural images, research on one dimensional edge pattern analysis to improve the accuracy of edge detection and localization proposes an edge detection algorithm, which is composed by three basic edge patterns, such as ramp, impulse, and step. After mathematical analysis, general rules for edge representation based upon the classification of edge types into three categories-ramp, impulse, and step (RIS) are developed to reduce detection and localization errors, especially reducing "double edge" effect that is one important drawback to the derivative method. But, when applying one dimensional edge pattern in two dimensional image processing, a new issue is naturally raised that the edge detector should correct marking inflections or junctions of edges. Research on human visual perception of objects and information theory pointed out that a pattern lexicon of "inflection micro-patterns" has larger information than a straight line. Also, research on scene perception gave an idea that contours have larger information are more important factor to determine the success of scene categorization. Therefore, inflections or junctions are extremely useful features, whose accurate description and reconstruction are significant in solving correspondence problems in computer vision. Therefore, aside from adoption of edge pattern analysis, inflection or junction characterization is also utilized to extend traditional derivative edge detection algorithm. Experiments were conducted to test my propositions about edge detection and localization accuracy improvements. The results support the idea that these edge detection method improvements are effective in enhancing the accuracy of edge detection and localization.

The lower San Pedro River: hydrology and flow restoration for biodiversity conservation

Treesearch

Jeanmarie Haney

2005-01-01

The lower San Pedro River, downstream from Benson, is a nearly unfragmented habitat containing perennial flow reaches that support riparian vegetation that serve as “stepping stones” for migratory species. The Nature Conservancy has purchased farm properties and retired agricultural pumping along the lower river, based largely on results from hydrologic analyses...

Omega-3 Fatty acids and inflammation: novel interactions reveal a new step in neutrophil recruitment.

PubMed

Tull, Samantha P; Yates, Clara M; Maskrey, Benjamin H; O'Donnell, Valerie B; Madden, Jackie; Grimble, Robert F; Calder, Philip C; Nash, Gerard B; Rainger, G Ed

2009-08-01

Inflammation is a physiological response to tissue trauma or infection, but leukocytes, which are the effector cells of the inflammatory process, have powerful tissue remodelling capabilities. Thus, to ensure their precise localisation, passage of leukocytes from the blood into inflamed tissue is tightly regulated. Recruitment of blood borne neutrophils to the tissue stroma occurs during early inflammation. In this process, peptide agonists of the chemokine family are assumed to provide a chemotactic stimulus capable of supporting the migration of neutrophils across vascular endothelial cells, through the basement membrane of the vessel wall, and out into the tissue stroma. Here, we show that, although an initial chemokine stimulus is essential for the recruitment of flowing neutrophils by endothelial cells stimulated with the inflammatory cytokine tumour necrosis factor-alpha, transit of the endothelial monolayer is regulated by an additional and downstream stimulus. This signal is supplied by the metabolism of the omega-6-polyunsaturated fatty acid (n-6-PUFA), arachidonic acid, into the eicosanoid prostaglandin-D(2) (PGD(2)) by cyclooxygenase (COX) enzymes. This new step in the neutrophil recruitment process was revealed when the dietary n-3-PUFA, eicosapentaenoic acid (EPA), was utilised as an alternative substrate for COX enzymes, leading to the generation of PGD(3). This alternative series eicosanoid inhibited the migration of neutrophils across endothelial cells by antagonising the PGD(2) receptor. Here, we describe a new step in the neutrophil recruitment process that relies upon a lipid-mediated signal to regulate the migration of neutrophils across endothelial cells. PGD(2) signalling is subordinate to the chemokine-mediated activation of neutrophils, but without the sequential delivery of this signal, neutrophils fail to penetrate the endothelial cell monolayer. Importantly, the ability of the dietary n-3-PUFA, EPA, to inhibit this process not only revealed an unsuspected level of regulation in the migration of inflammatory leukocytes, it also contributes to our understanding of the interactions of this bioactive lipid with the inflammatory system. Moreover, it indicates the potential for novel therapeutics that target the inflammatory system with greater affinity and/or specificity than supplementing the diet with n-3-PUFAs.

The river as a chemostat: fresh perspectives on dissolved organic matter flowing down the river continuum

USGS Publications Warehouse

Creed, Irena F.; McKnight, Diane M.; Pellerin, Brian; Green, Mark B.; Bergamaschi, Brian; Aiken, George R.; Burns, Douglas A.; Findlay, Stuart E G; Shanley, James B.; Striegl, Robert G.; Aulenbach, Brent T.; Clow, David W.; Laudon, Hjalmar; McGlynn, Brian L.; McGuire, Kevin J.; Smith, Richard A.; Stackpoole, Sarah M.

2015-01-01

A better understanding is needed of how hydrological and biogeochemical processes control dissolved organic carbon (DOC) concentrations and dissolved organic matter (DOM) composition from headwaters downstream to large rivers. We examined a large DOM dataset from the National Water Information System of the US Geological Survey, which represents approximately 100 000 measurements of DOC concentration and DOM composition at many sites along rivers across the United States. Application of quantile regression revealed a tendency towards downstream spatial and temporal homogenization of DOC concentrations and a shift from dominance of aromatic DOM in headwaters to more aliphatic DOM downstream. The DOC concentration–discharge (C-Q) relationships at each site revealed a downstream tendency towards a slope of zero. We propose that despite complexities in river networks that have driven many revisions to the River Continuum Concept, rivers show a tendency towards chemostasis (C-Q slope of zero) because of a downstream shift from a dominance of hydrologic drivers that connect terrestrial DOM sources to streams in the headwaters towards a dominance of instream and near-stream biogeochemical processes that result in preferential losses of aromatic DOM and preferential gains of aliphatic DOM.

Analysis of Water Recovery Rate from the Heat Melt Compactor

NASA Technical Reports Server (NTRS)

Balasubramaniam, R.; Hegde, U.; Gokoglu, S.

2013-01-01

Human space missions generate trash with a substantial amount of plastic (20% or greater by mass). The trash also contains water trapped in food residue and paper products and other trash items. The Heat Melt Compactor (HMC) under development by NASA Ames Research Center (ARC) compresses the waste, dries it to recover water and melts the plastic to encapsulate the compressed trash. The resulting waste disk or puck represents an approximately ten-fold reduction in the volume of the initial trash loaded into the HMC. In the current design concept being pursued, the trash is compressed by a piston after it is loaded into the trash chamber. The piston face, the side walls of the waste processing chamber and the end surface in contact with the waste can be heated to evaporate the water and to melt the plastic. Water is recovered by the HMC in two phases. The first is a pre-process compaction without heat or with the heaters initially turned on but before the waste heats up. Tests have shown that during this step some liquid water may be expelled from the chamber. This water is believed to be free water (i.e., not bound with or absorbed in other waste constituents) that is present in the trash. This phase is herein termed Phase A of the water recovery process. During HMC operations, it is desired that liquid water recovery in Phase A be eliminated or minimized so that water-vapor processing equipment (e.g., condensers) downstream of the HMC are not fouled by liquid water and its constituents (i.e., suspended or dissolved matter) exiting the HMC. The primary water recovery process takes place next where the trash is further compacted while the heated surfaces reach their set temperatures for this step. This step will be referred to herein as Phase B of the water recovery process. During this step the waste chamber may be exposed to different selected pressures such as ambient, low pressure (e.g., 0.2 atm), or vacuum. The objective for this step is to remove both bound and any remaining free water in the trash by evaporation. The temperature settings of the heated surfaces are usually kept above the saturation temperature of water but below the melting temperature of the plastic in the waste during this step to avoid any encapsulation of wet trash which would reduce the amount of recovered water by blocking the vapor escape. In this paper, we analyze the water recovery rate during Phase B where the trash is heated and water leaves the waste chamber as vapor, for operation of the HMC in reduced gravity. We pursue a quasi-one-dimensional model with and without sidewall heating to determine the water recovery rate and the trash drying time. The influences of the trash thermal properties, the amount of water loading, and the distribution of the water in the trash on the water recovery rates are determined.

Wildfire disturbance, erosion and sedimentation risks following the Waldo Canyon Fire in Colorado

NASA Astrophysics Data System (ADS)

Flint, K.; Kinoshita, A. M.; Chin, A.; Florsheim, J. L.; Nourbakhshbeidokhti, S.

2016-12-01

Wildfire is a landscape-scale disturbance that causes abrupt changes to hydrological responses and sediment flux during subsequent storms. Burning hillslope vegetation during wildfires induces changes to sediment supply and stream flow magnitude. Altered post-fire processes such as channel erosion and sedimentation or flooding enhance downstream hazards that may threaten human populations and physical aquatic habitat over various time scales. Using data from a small drainage basin (Williams Canyon, 4.7 km2) in the Colorado front range burned by the 2012 Waldo Fire as a case study, we investigate post-fire recovery and assess changes in fire-related risks to downstream areas. Our local ground-based precipitation, field measurements, terrestrial Light Detection and Ranging (LiDAR) scanning together with satellite-based remote sensing data (i.e. Landsat) provide a basis for time series analyses of reach-scale erosion and sedimentation response to rainfall patterns as vegetation patterns change following the wildfire. As a first step in quantifying the likelihood and consequences of specific risk scenarios, we examine changes in the combined probability of storm flows and post-fire erosion and sedimentation as vegetation recovers within the study watershed. We explore possible feedbacks and thresholds related to vegetation-hydrology-sediment interactions following wildfire under changing climate regimes. This information is needed to assist in post-fire management to promote sustainability of wildland fluvial systems.

Chemical loading into surface water along a hydrological, biogeochemical, and land use gradient: A holistic watershed approach

USGS Publications Warehouse

Barber, L.B.; Murphy, S.F.; Verplanck, P.L.; Sandstrom, M.W.; Taylor, Howard E.; Furlong, E.T.

2006-01-01

Identifying the sources and impacts of organic and inorganic contaminants at the watershed scale is a complex challenge because of the multitude of processes occurring in time and space. Investigation of geochemical transformations requires a systematic evaluation of hydrologic, landscape, and anthropogenic factors. The 1160 km2 Boulder Creek Watershed in the Colorado Front Range encompasses a gradient of geology, ecotypes, climate, and urbanization. Streamflow originates primarily as snowmelt and shows substantial annual variation. Water samples were collected along a 70-km transect during spring-runoff and base-flow conditions, and analyzed for major elements, trace elements, bulk organics, organic wastewater contaminants (OWCs), and pesticides. Major-element and trace-element concentrations were low in the headwaters, increased through the urban corridor, and had a step increase downstream from the first major wastewater treatment plant (WWTP). Boron, gadolinium, and lithium were useful inorganic tracers of anthropogenic inputs. Effluent from the WWTP accounted for as much as 75% of the flow in Boulder Creek and was the largest chemical input. Under both hydrological conditions, OWCs and pesticides were detected in Boulder Creek downstream from the WWTP outfall as well as in the headwater region, and loads of anthropogenic-derived contaminants increased as basin population density increased. This report documents a suite of potential endocrine-disrupting chemicals in a reach of stream with native fish populations showing indication of endocrine disruption.

Natural Origin Lycopene and Its "Green" Downstream Processing.

PubMed

Papaioannou, Emmanouil H; Liakopoulou-Kyriakides, Maria; Karabelas, Anastasios J

2016-01-01

Lycopene is an abundant natural carotenoid pigment with several biological functions (well-known for its antioxidant properties) which is under intensive investigation in recent years. Lycopene chemistry, its natural distribution, bioavailability, biological significance, and toxicological effects are briefly outlined in the first part of this review. The second, major part, deals with various modern downstream processing techniques, which are assessed in order to identify promising approaches for the recovery of lycopene and of similar lipophilic compounds. Natural lycopene is synthesized in plants and by microorganisms, with main representatives of these two categories (for industrial production) tomato and its by-products and the fungus Blakeslea trispora, respectively. Currently, there is a great deal of effort to develop efficient downstream processing for large scale production of natural-origin lycopene, with trends strongly indicating the necessity for "green" and mild extraction conditions. In this review, emphasis is placed on final product safety and ecofriendly processing, which are expected to totally dominate in the field of natural-origin lycopene extraction and purification.

Morphology analysis in middle-downstream area of Progo River due to the debris flow

NASA Astrophysics Data System (ADS)

Fitriadin, Ahmad Azmi; Ikhsan, Jaza'ul; Harsanto, Puji

2017-06-01

One of the problems that occur in Progo River is the formation of sediment in the downstream section. The sediment material in the upstream becomes the source of sediment at the downstream area. Excess sediment supply from the upstream causes morphological changes in a relatively short time. The morphological changes in riverbed will affect hydraulics conditions. Hydraulic has an important role in the process of aggradation and degradation in the riverbed. Furthermore, the process of erosion and sedimentation will affect the stability of the construction in the water. In Progo River, there are some buildings of infrastructure such as revetment, bridge, irrigation intake, groundsill, and weir. Based on the results of a numerical model of the hydraulic analysis system, there was approximately 87,000,000 m3 of sediment on Progo River in 2015. In fact, aggradation and degradation occurred very intensively in the middle-downstream area of Progo River. Sediment movement simulation also showed that the sediment supply of lava could prevent excessive bed degradation. Nevertheless, the absence of sediment supply will lead to bed degradation process. It indicates that the management of the sediment supply in the upstream area must be managed properly.

Association of ice and river channel morphology determined using ground-penetrationg radar in the Kuparuk River, Alaska

USGS Publications Warehouse

Best, Heather; McNamara, J.P.; Liberty, Lee M.

2005-01-01

We collected ground-penetrating radar data at 10 sites along the Kuparuk River and its main tributary, the Toolik River, to detect unfrozen water beneath river ice. We used 250 MHz and 500 MHz antennas to image both the ice-water interface and the river channel in late April 2001, when daily high temperatures were consistently freezing and river ice had attained its maximum seasonal thickness. The presence of water below the river ice appears as a strong, horizontal reflection observed in the radar data and is confirmed by drill hole data. A downstream transition occurs from ice that is frozen to the bed, called bedfast ice, to ice that is floating on unfrozen water, called floating ice. This transition in ice type corresponds to a downstream change in channel size that was detected in previously conducted hydraulic geometry surveys of the Kuparuk River. We propose a conceptual model wherein the downstream transition from bedfast ice to floating ice is responsible for an observed step change in channel size due to enhanced bank erosion in large channels by floating ice.

Pharmacologic inhibition of squalene synthase and other downstream enzymes of the cholesterol synthesis pathway: a new therapeutic approach to treatment of hypercholesterolemia.

PubMed

Seiki, Stephanie; Frishman, William H

2009-01-01

Hypercholesterolemia is a major risk factor for the development of atherosclerotic vascular diseases. The most popular agents for cholesterol reduction are the statin drugs, which are competitive inhibitors of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, the primary rate-limiting enzyme in the hepatic biosynthesis of cholesterol. Although relatively safe and effective, the available statins can cause elevations in liver enzymes and myopathy. Squalene synthase is another enzyme that is downstream to HMG-CoA reductase in the cholesterol synthesis pathway and modulates the first committed step of hepatic cholesterol biosynthesis at the final branch point of the cholesterol biosynthetic pathway. Squalene epoxidase and oxidosqualene cyclase are other enzymes that act distally to squalene synthase. Pharmacologic inhibitors of these downstream enzymes have been developed, which may reduce low-density lipoprotein cholesterol and reduce the myopathy side effect seen with upstream inhibition of HMG-CoA. At this juncture, one squalene synthase inhibitor, lapaquistat (TAK-475) is in active clinical trials as a monotherapy, but there have been suggestions of increased hepatotoxicity with the drug.

Bioprocess integration for human mesenchymal stem cells: From up to downstream processing scale-up to cell proteome characterization.

PubMed

Cunha, Bárbara; Aguiar, Tiago; Carvalho, Sofia B; Silva, Marta M; Gomes, Ricardo A; Carrondo, Manuel J T; Gomes-Alves, Patrícia; Peixoto, Cristina; Serra, Margarida; Alves, Paula M

2017-04-20

To deliver the required cell numbers and doses to therapy, scaling-up production and purification processes (at least to the liter-scale) while maintaining cells' characteristics is compulsory. Therefore, the aim of this work was to prove scalability of an integrated streamlined bioprocess compatible with current good manufacturing practices (cGMP) comprised by cell expansion, harvesting and volume reduction unit operations using human mesenchymal stem cells (hMSC) isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC). BM-MSC and AT-MSC expansion and harvesting steps were scaled-up from spinner flasks to 2L scale stirred tank single-use bioreactor using synthetic microcarriers and xeno-free medium, ensuring high cellular volumetric productivities (50×10 6 cellL -1 day -1 ), expansion factors (14-16 fold) and cell recovery yields (80%). For the concentration step, flat sheet cassettes (FSC) and hollow fiber cartridges (HF) were compared showing a fairly linear scale-up, with a need to slightly decrease the permeate flux (30-50 LMH, respectively) to maximize cell recovery yield. Nonetheless, FSC allowed to recover 18% more cells after a volume reduction factor of 50. Overall, at the end of the entire bioprocess more than 65% of viable (>95%) hMSC could be recovered without compromising cell's critical quality attributes (CQA) of viability, identity and differentiation potential. Alongside the standard quality assays, a proteomics workflow based on mass spectrometry tools was established to characterize the impact of processing on hMSC's CQA; These analytical tools constitute a powerful tool to be used in process design and development. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of antibody disulfide bond reduction on purification process performance and final drug substance stability

PubMed Central

Russell, Brian; Yang, Yanhong; Handlogten, Michael; Hudak, Suzanne; Cao, Mingyan; Wang, Jihong; Robbins, David; Ahuja, Sanjeev; Zhu, Min

2017-01-01

ABSTRACT Antibody disulfide bond reduction during monoclonal antibody (mAb) production is a phenomenon that has been attributed to the reducing enzymes from CHO cells acting on the mAb during the harvest process. However, the impact of antibody reduction on the downstream purification process has not been studied. During the production of an IgG2 mAb, antibody reduction was observed in the harvested cell culture fluid (HCCF), resulting in high fragment levels. In addition, aggregate levels increased during the low pH treatment step in the purification process. A correlation between the level of free thiol in the HCCF (as a result of antibody reduction) and aggregation during the low pH step was established, wherein higher levels of free thiol in the starting sample resulted in increased levels of aggregates during low pH treatment. The elevated levels of free thiol were not reduced over the course of purification, resulting in carry‐over of high free thiol content into the formulated drug substance. When the drug substance with high free thiols was monitored for product degradation at room temperature and 2–8°C, faster rates of aggregation were observed compared to the drug substance generated from HCCF that was purified immediately after harvest. Further, when antibody reduction mitigations (e.g., chilling, aeration, and addition of cystine) were applied, HCCF could be held for an extended period of time while providing the same product quality/stability as material that had been purified immediately after harvest. Biotechnol. Bioeng. 2017;114: 1264–1274. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc. PMID:28186329

An experimental study of turbine vane heat transfer with leading edge and downstream film cooling

NASA Astrophysics Data System (ADS)

Nirmalan, V.; Hylton, L. D.

1989-06-01

This paper presents the effects of downstream film cooling, with and without leading edge showerhead film cooling, on turbine-vane external heat transfer. Steady-state experimental measurements were made in a three-vane linear two-dimensional cascade. The principal independent parameters were maintained over ranges consistent with actual engine conditions. The test matrix was structured to provide an assessment of the independent influence of parameters of interest, namely, exit Mach number, exit Reynolds number, coolant-to-gas temperature ratio, and coolant-to-gas pressure ratio. The data obtained indicate that considerable cooling benefits can be achieved by utilizing downstream film cooling. The downstream film cooling process was shown to be a complex interaction of two competing mechanisms. The thermal dilution effect, associated with the injection of relatively cold fluid, results in a decrease in the heat transfer to the airfoil. Conversely, the turbulence augmentation, produced by the injection process, results in increased heat transfer to the airfoil.

An experimental study of turbine vane heat transfer with leading edge and downstream film cooling

NASA Technical Reports Server (NTRS)

Nirmalan, V.; Hylton, L. D.

1989-01-01

This paper presents the effects of downstream film cooling, with and without leading edge showerhead film cooling, on turbine-vane external heat transfer. Steady-state experimental measurements were made in a three-vane linear two-dimensional cascade. The principal independent parameters were maintained over ranges consistent with actual engine conditions. The test matrix was structured to provide an assessment of the independent influence of parameters of interest, namely, exit Mach number, exit Reynolds number, coolant-to-gas temperature ratio, and coolant-to-gas pressure ratio. The data obtained indicate that considerable cooling benefits can be achieved by utilizing downstream film cooling. The downstream film cooling process was shown to be a complex interaction of two competing mechanisms. The thermal dilution effect, associated with the injection of relatively cold fluid, results in a decrease in the heat transfer to the airfoil. Conversely, the turbulence augmentation, produced by the injection process, results in increased heat transfer to the airfoil.

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

PubMed Central

Rothrock, Michael J.; Hiett, Kelli L.; Gamble, John; Caudill, Andrew C.; Cicconi-Hogan, Kellie M.; Caporaso, J. Gregory

2014-01-01

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the “gold standard” enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples. PMID:25548939

hUTP24 is essential for processing of the human rRNA precursor at site A1, but not at site A0

PubMed Central

Tomecki, Rafal; Labno, Anna; Drazkowska, Karolina; Cysewski, Dominik; Dziembowski, Andrzej

2015-01-01

Production of ribosomes relies on more than 200 accessory factors to ensure the proper sequence of steps and faultless assembly of ribonucleoprotein machinery. Among trans-acting factors are numerous enzymes, including ribonucleases responsible for processing the large rRNA precursor synthesized by RNA polymerase I that encompasses sequences corresponding to mature 18S, 5.8S, and 25/28S rRNA. In humans, the identity of most enzymes responsible for individual processing steps, including endoribonucleases that cleave pre-rRNA at specific sites within regions flanking and separating mature rRNA, remains largely unknown. Here, we investigated the role of hUTP24 in rRNA maturation in human cells. hUTP24 is a human homolog of the Saccharomyces cerevisiae putative PIN domain-containing endoribonuclease Utp24 (yUtp24), which was suggested to participate in the U3 snoRNA-dependent processing of yeast pre-rRNA at sites A0, A1, and A2. We demonstrate that hUTP24 interacts to some extent with proteins homologous to the components of the yeast small subunit (SSU) processome. Moreover, mutation in the putative catalytic site of hUTP24 results in slowed growth of cells and reduced metabolic activity. These effects are associated with a defect in biogenesis of the 40S ribosomal subunit, which results from decreased amounts of 18S rRNA as a consequence of inaccurate pre-rRNA processing at the 5′-end of the 18S rRNA segment (site A1). Interestingly, and in contrast to yeast, site A0 located upstream of A1 is efficiently processed upon UTP24 dysfunction. Finally, hUTP24 inactivation leads to aberrant processing of 18S rRNA 2 nucleotides downstream of the normal A1 cleavage site. PMID:26237581

Hot Film Velocity Measurements Downstream of a Swept Backward-Facing Step

DTIC Science & Technology

1990-08-01

WR ~4’ nol w of tdl. O64" $l"uwe. suite 424 i lV~lZdO . 00 to effice at UAqeww0fl" S4 aud ’mpeom t, 0edUOM ’l~c 004 S~ a lleq~k Dc 2010). I. AGENCY USE ...Velocity measurements were made using a single component hot film anemometer. Results from this study indicate the following: the flow in the recirculation...velocity component parallel to the step face retains a finite magnitude. Coles’ law of the wall and wake was used to corralate the data from this study. The

Non-Gaussian PDF Modeling of Turbulent Boundary Layer Fluctuating Pressure Excitation

NASA Technical Reports Server (NTRS)

Steinwolf, Alexander; Rizzi, Stephen A.

2003-01-01

The purpose of the study is to investigate properties of the probability density function (PDF) of turbulent boundary layer fluctuating pressures measured on the exterior of a supersonic transport aircraft. It is shown that fluctuating pressure PDFs differ from the Gaussian distribution even for surface conditions having no significant discontinuities. The PDF tails are wider and longer than those of the Gaussian model. For pressure fluctuations upstream of forward-facing step discontinuities and downstream of aft-facing step discontinuities, deviations from the Gaussian model are more significant and the PDFs become asymmetrical. Various analytical PDF distributions are used and further developed to model this behavior.

High-throughput micro-scale cultivations and chromatography modeling: Powerful tools for integrated process development.

PubMed

Baumann, Pascal; Hahn, Tobias; Hubbuch, Jürgen

2015-10-01

Upstream processes are rather complex to design and the productivity of cells under suitable cultivation conditions is hard to predict. The method of choice for examining the design space is to execute high-throughput cultivation screenings in micro-scale format. Various predictive in silico models have been developed for many downstream processes, leading to a reduction of time and material costs. This paper presents a combined optimization approach based on high-throughput micro-scale cultivation experiments and chromatography modeling. The overall optimized system must not necessarily be the one with highest product titers, but the one resulting in an overall superior process performance in up- and downstream. The methodology is presented in a case study for the Cherry-tagged enzyme Glutathione-S-Transferase from Escherichia coli SE1. The Cherry-Tag™ (Delphi Genetics, Belgium) which can be fused to any target protein allows for direct product analytics by simple VIS absorption measurements. High-throughput cultivations were carried out in a 48-well format in a BioLector micro-scale cultivation system (m2p-Labs, Germany). The downstream process optimization for a set of randomly picked upstream conditions producing high yields was performed in silico using a chromatography modeling software developed in-house (ChromX). The suggested in silico-optimized operational modes for product capturing were validated subsequently. The overall best system was chosen based on a combination of excellent up- and downstream performance. © 2015 Wiley Periodicals, Inc.

Economic feasibility of biochemical processes for the upgrading of crudes and the removal of sulfur, nitrogen, and trace metals from crude oil -- Benchmark cost establishment of biochemical processes on the basis of conventional downstream technologies. Final report FY95

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

Premuzic, E.T.

1996-08-01

During the past several years, a considerable amount of work has been carried out showing that microbially enhanced oil recovery (MEOR) is promising and the resulting biotechnology may be deliverable. At Brookhaven National Laboratory (BNL), systematic studies have been conducted which dealt with the effects of thermophilic and thermoadapted bacteria on the chemical and physical properties of selected types of crude oils at elevated temperatures and pressures. Current studies indicate that during the biotreatment several chemical and physical properties of crude oils are affected. The oils are (1) emulsified; (2) acidified; (3) there is a qualitative and quantitative change inmore » light and heavy fractions of the crudes; (4) there are chemical changes in fractions containing sulfur compounds; (5) there is an apparent reduction in the concentration of trace metals; and (6) the qualitative and quantitative changes appear to be microbial species dependent; and (7) there is a distinction between biodegraded and biotreated oils. The downstream biotechnological crude oil processing research performed thus far is of laboratory scale and has focused on demonstrating the technical feasibility of downstream processing with different types of biocatalysts under a variety of processing conditions. Quantitative economic analysis is the topic of the present project which investigates the economic feasibility of the various biochemical downstream processes which hold promise in upgrading of heavy crudes, such as those found in California, e.g., Monterey-type, Midway Sunset, Honda crudes, and others.« less

Coordination of the recruitment of the FANCD2 and PALB2 Fanconi anemia proteins by an ubiquitin signaling network.

PubMed

Bick, Gregory; Zhang, Fan; Meetei, A Ruhikanta; Andreassen, Paul R

2017-06-01

Fanconi anemia (FA) is a chromosome instability syndrome and the 20 identified FA proteins are organized into two main arms which are thought to function at distinct steps in the repair of DNA interstrand crosslinks (ICLs). These two arms include the upstream FA pathway, which culminates in the monoubiquitination of FANCD2 and FANCI, and downstream breast cancer (BRCA)-associated proteins that interact in protein complexes. How, and whether, these two groups of FA proteins are integrated is unclear. Here, we show that FANCD2 and PALB2, as indicators of the upstream and downstream arms, respectively, colocalize independently of each other in response to DNA damage induced by mitomycin C (MMC). We also show that ubiquitin chains are induced by MMC and colocalize with both FANCD2 and PALB2. Our finding that the RNF8 E3 ligase has a role in recruiting FANCD2 and PALB2 also provides support for the hypothesis that the two branches of the FA-BRCA pathway are coordinated by ubiquitin signaling. Interestingly, we find that the RNF8 partner, MDC1, as well as the ubiquitin-binding protein, RAP80, specifically recruit PALB2, while a different ubiquitin-binding protein, FAAP20, functions only in the recruitment of FANCD2. Thus, FANCD2 and PALB2 are not recruited in a single linear pathway, rather we define how their localization is coordinated and integrated by a network of ubiquitin-related proteins. We propose that such regulation may enable upstream and downstream FA proteins to act at distinct steps in the repair of ICLs.

Optimization of the extraction and purification of the compatible solute ectoine from Halomonas elongate in the laboratory experiment of a commercial production project.

PubMed

Chen, Ruifeng; Zhu, Lijun; Lv, Lihuo; Yao, Su; Li, Bin; Qian, Junqing

2017-06-01

Optimization of compatible solutes (ectoine) extraction and purification from Halomonas elongata cell fermentation had been investigated in the laboratory tests of a large scale commercial production project. After culturing H. elongata cells in developed medium at 28 °C for 23-30 h, we obtained an average yield and biomass of ectoine for 15.9 g/L and 92.9 (OD 600 ), respectively. Cell lysis was performed with acid treatment at moderate high temperature (60-70 °C). The downstream processing operations were designed to be as follows: filtration, desalination, cation exchange, extraction of crude product and three times of refining. Among which the cation exchange and extraction of crude product acquired a high average recovery rate of 95 and 96%; whereas a great loss rate of 19 and 15% was observed during the filtration and desalination, respectively. Combined with the recovering of ectoine from the mother liquor of the three times refining, the average of overall yield (referring to the amount of ectoine synthesized in cells) and purity of final product obtained were 43% and over 98%, respectively. However, key factors that affected the production efficiency were not yields but the time used in the extraction of crude product, involving the crystallization step from water, which spended 24-72 h according to the production scale. Although regarding to the productivity and simplicity on laboratory scale, the method described here can not compete with other investigations, in this study we acquired higher purity of ectoine and provided downstream processes that are capable of operating on industrial scale.

Evaluation of different pulverisation methods for RNA extraction in squash fruit: lyophilisation, cryogenic mill and mortar grinding.

PubMed

Román, Belén; González-Verdejo, Clara I; Peña, Francisco; Nadal, Salvador; Gómez, Pedro

2012-01-01

Quality and integrity of RNA are critical for transcription studies in plant molecular biology. In squash fruit and other high water content crops, the grinding of tissue with mortar and pestle in liquid nitrogen fails to produce a homogeneous and fine powered sample desirable to ensure a good penetration of the extraction reagent. To develop an improved pulverisation method to facilitate the homogenisation process of squash fruit tissue prior to RNA extraction without reducing quality and yield of the extracted RNA. Three methods of pulverisation, each followed by the same extraction protocol, were compared. The first approach consisted of the lyophilisation of the sample in order to remove the excess of water before grinding, the second one used a cryogenic mill and the control one a mortar grinding of frozen tissue. The quality of the isolated RNA was tested by carrying out a quantitative real time downstream amplification. In the three situations considered, mean values for A(260) /A(280) indicated minimal interference by proteins and RNA quality indicator (RQI) values were considered appropriate for quantitative real-time polymerase chain reaction (qRT-PCR) amplification. Successful qRT-PCR amplifications were obtained with cDNA isolated with the three protocols. Both apparatus can improve and facilitate the grinding step in the RNA extraction process in zucchini, resulting in isolated RNA of high quality and integrity as revealed by qRT-PCR downstream application. This is apparently the first time that a cryogenic mill has been used to prepare fruit samples for RNA extraction, thereby improving the sampling strategy because the fine powder obtained represents a homogeneous mix of the organ tissue. Copyright © 2012 John Wiley & Sons, Ltd.

Method for determining molten metal pool level in twin-belt continuous casting machines

DOEpatents

Kaiser, Timothy D.; Daniel, Sabah S.; Dykes, Charles D.

1989-03-21

A method for determining level of molten metal in the input of a continuous metal casting machine having at least one endless, flexible, revolving casting belt with a surface which engages the molten metal to be cast and a reverse, cooled surface along which is directed high velocity liquid coolant includes the steps of predetermining the desired range of positions of the molten metal pool and positioning at least seven heat-sensing transducers in bearing contact with the moving reverse belt surface and spaced in upstream-downstream relationship relative to belt travel spanning the desired pool levels. A predetermined temperature threshold is set, somewhat above coolant temperature and the output signals of the transducer sensors are scanned regarding their output signals indicative of temperatures of the moving reverse belt surface. Position of the molten pool is determined using temperature interpolation between any successive pair of upstream-downstream spaced sensors, which follows confirmation that two succeeding downstream sensors are at temperature levels exceeding threshold temperature. The method accordingly provides high resolution for determining pool position, and verifies the determined position by utilizing full-strength signals from two succeeding downstream sensors. In addition, dual sensors are used at each position spanning the desired range of molten metal pool levels to provide redundancy, wherein only the higher temperature of each pair of sensors at a station is utilized.

esATAC: An Easy-to-use Systematic pipeline for ATAC-seq data analysis.

PubMed

Wei, Zheng; Zhang, Wei; Fang, Huan; Li, Yanda; Wang, Xiaowo

2018-03-07

ATAC-seq is rapidly emerging as one of the major experimental approaches to probe chromatin accessibility genome-wide. Here, we present "esATAC", a highly integrated easy-to-use R/Bioconductor package, for systematic ATAC-seq data analysis. It covers essential steps for full analyzing procedure, including raw data processing, quality control and downstream statistical analysis such as peak calling, enrichment analysis and transcription factor footprinting. esATAC supports one command line execution for preset pipelines, and provides flexible interfaces for building customized pipelines. esATAC package is open source under the GPL-3.0 license. It is implemented in R and C ++. Source code and binaries for Linux, MAC OS X and Windows are available through Bioconductor https://www.bioconductor.org/packages/release/bioc/html/esATAC.html). xwwang@tsinghua.edu.cn. Supplementary data are available at Bioinformatics online.

A revised velocity-reversal and sediment-sorting model for a high-gradient, pool-riffle stream

USGS Publications Warehouse

Thompson, D.M.; Wohl, E.E.; Jarrett, R.D.

1996-01-01

Sediment-sorting processes related to varying channel-bed morphology were investigated from April to November 1993 along a 1-km pool-riffle and step-pool reach of North Saint Vrain Creek, a small mountain stream in the Rocky Mountains of northern Colorado. Measured cross-sectional areas of flow were used to suggest higher velocities in pools than in riffles at high flow. Three hundred and sixteen tracer particles, ranging in size from 16 mm to 256 mm, were placed in two separate pool-riffle-pool sequences and used to assess sediment-sorting patterns and sediment-transport competence variations. Tracer-particle depositional evidence indicated higher sediment-transport competence in pools than in riffles at high flow. Pool-riffle sediment sorting may be created by velocity reversals, and more localized sorting results from gravitational forces along the upstream sloping portion of the channel bed located at the downstream end of pools.

Cell disruption for microalgae biorefineries.

PubMed

Günerken, E; D'Hondt, E; Eppink, M H M; Garcia-Gonzalez, L; Elst, K; Wijffels, R H

2015-01-01

Microalgae are a potential source for various valuable chemicals for commercial applications ranging from nutraceuticals to fuels. Objective in a biorefinery is to utilize biomass ingredients efficiently similarly to petroleum refineries in which oil is fractionated in fuels and a variety of products with higher value. Downstream processes in microalgae biorefineries consist of different steps whereof cell disruption is the most crucial part. To maintain the functionality of algae biochemicals during cell disruption while obtaining high disruption yields is an important challenge. Despite this need, studies on mild disruption of microalgae cells are limited. This review article focuses on the evaluation of conventional and emerging cell disruption technologies, and a comparison thereof with respect to their potential for the future microalgae biorefineries. The discussed techniques are bead milling, high pressure homogenization, high speed homogenization, ultrasonication, microwave treatment, pulsed electric field treatment, non-mechanical cell disruption and some emerging technologies. Copyright © 2015 Elsevier Inc. All rights reserved.

Extraction of oil from microalgae for biodiesel production: A review.

PubMed

Halim, Ronald; Danquah, Michael K; Webley, Paul A

2012-01-01

The rapid increase of CO(2) concentration in the atmosphere combined with depleted supplies of fossil fuels has led to an increased commercial interest in renewable fuels. Due to their high biomass productivity, rapid lipid accumulation, and ability to survive in saline water, microalgae have been identified as promising feedstocks for industrial-scale production of carbon-neutral biodiesel. This study examines the principles involved in lipid extraction from microalgal cells, a crucial downstream processing step in the production of microalgal biodiesel. We analyze the different technological options currently available for laboratory-scale microalgal lipid extraction, with a primary focus on the prospect of organic solvent and supercritical fluid extraction. The study also provides an assessment of recent breakthroughs in this rapidly developing field and reports on the suitability of microalgal lipid compositions for biodiesel conversion. Copyright © 2012 Elsevier Inc. All rights reserved.

Structure of a reattaching supersonic shear flow

NASA Technical Reports Server (NTRS)

Samimy, M.; Abu-Hijleh, B. A. K.

1988-01-01

A Mach 1.83 fully developed turbulent boundary layer with boundary layer thickness, free stream velocity, and Reynolds number of 7.5 mm, 476 m/s, and 6.2 x 10 to the 7th/m, respectively, was separated at a 25.4-mm backward step and formed a shear layer. Fast-response pressure transducers, schlieren photography, and LDV were used to study the structure of this reattaching shear flow. The preliminary results show that large-scale relatively organized structures with limited spanwise extent form in the free shear layer. Some of these structures appear to survive the recompression and reattachment processes, while others break down into smaller scales and the flow becomes increasingly three-dimensional. The survived large-scale structures lose their organization through recompression/reattachment, but regain it after reattachment. The structures after reattachment form a 40-45-degree angle relative to the free stream and deteriorate gradually as they move downstream.

Combining metabolic engineering and electrocatalysis: Application to the production of polyamides from sugar

DOE PAGES

Suastegui, Miguel; Matthiesen, John E.; Carraher, Jack M.; ...

2016-01-14

Here, biorefineries aim to convert biomass into a spectrum of products ranging from biofuels to specialty chemicals. To achieve economically sustainable conversion, it is crucial to streamline the catalytic and downstream processing steps. In this work, a route that combines bio- and electrocatalysis to convert glucose into bio-based unsaturated nylon-6,6 is reported. An engineered strain of Saccharomyces cerevisiae was used as the initial biocatalyst for the conversion of glucose into muconic acid, with the highest reported muconic acid titer of 559.5 mg L –1 in yeast. Without any separation, muconic acid was further electrocatalytically hydrogenated to 3-hexenedioic acid in 94more » % yield despite the presence of biogenic impurities. Bio-based unsaturated nylon-6,6 (unsaturated polyamide-6,6) was finally obtained by polymerization of 3-hexenedioic acid with hexamethylenediamine.« less

Coordinated DNA dynamics during the human telomerase catalytic cycle

NASA Astrophysics Data System (ADS)

Parks, Joseph W.; Stone, Michael D.

2014-06-01

The human telomerase reverse transcriptase (hTERT) utilizes a template within the integral RNA subunit (hTR) to direct extension of telomeres. Telomerase exhibits repeat addition processivity (RAP) and must therefore translocate the nascent DNA product into a new RNA:DNA hybrid register to prime each round of telomere repeat synthesis. Here, we use single-molecule FRET and nuclease protection assays to monitor telomere DNA structure and dynamics during the telomerase catalytic cycle. DNA translocation during RAP proceeds through a previously uncharacterized kinetic substep during which the 3‧-end of the DNA substrate base pairs downstream within the hTR template. The rate constant for DNA primer realignment reveals this step is not rate limiting for RAP, suggesting a second slow conformational change repositions the RNA:DNA hybrid into the telomerase active site and drives the extrusion of the 5‧-end of the DNA primer out of the enzyme complex.

Programming Many-Core Systems with GRAMPS

DTIC Science & Technology

2010-08-01

12 3.2 Hypothetical GRAMPS cookie dough application . . . . . . . . . . . 13 3.3 Using a queue set...programming model. As shown, dough preparation is broken into individual stages corresponding to logical steps in 3.2. A GRAMPS EXAMPLE 13 Scoop Cookies Put on...chocolate chip cookie dough . the recipe [7]. Each stage sends its output downstream and takes as input the in- gredients and/or batter from its prior

Advances in downstream processing of biologics - Spectroscopy: An emerging process analytical technology.

PubMed

Rüdt, Matthias; Briskot, Till; Hubbuch, Jürgen

2017-03-24

Process analytical technologies (PAT) for the manufacturing of biologics have drawn increased interest in the last decade. Besides being encouraged by the Food and Drug Administration's (FDA's) PAT initiative, PAT promises to improve process understanding, reduce overall production costs and help to implement continuous manufacturing. This article focuses on spectroscopic tools for PAT in downstream processing (DSP). Recent advances and future perspectives will be reviewed. In order to exploit the full potential of gathered data, chemometric tools are widely used for the evaluation of complex spectroscopic information. Thus, an introduction into the field will be given. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Evaluating the aggregate effect of geographical isolated wetlands and associated spatial and size distributions on downstream hydrologic flows

EPA Science Inventory

Geographically isolated wetlands (GIW), defined as depressional wetlands completely surrounded by uplands, support an array of ecological processes. A solid scientific understanding of the hydrologic effects of GIWs upon downstream waterways is important for legal and policy-mak...

From Process Development to Manufacturing: Lab-Intensive Courses in Downstream Bioprocessing

ERIC Educational Resources Information Center

Gilleskie, Gary L.; Reeves, Baley A.

2014-01-01

Most chemical engineering graduates work in industry, a fact that underscores the need for courses to provide experiences that prepare them for industry. The Biomanufacturing Training and Education Center (BTEC) at North Carolina State University has addressed this need by developing and delivering a comprehensive downstream bioprocessing program…

Rational design and optimization of downstream processes of virus particles for biopharmaceutical applications: current advances.

PubMed

Vicente, Tiago; Mota, José P B; Peixoto, Cristina; Alves, Paula M; Carrondo, Manuel J T

2011-01-01

The advent of advanced therapies in the pharmaceutical industry has moved the spotlight into virus-like particles and viral vectors produced in cell culture holding great promise in a myriad of clinical targets, including cancer prophylaxis and treatment. Even though a couple of cases have reached the clinic, these products have yet to overcome a number of biological and technological challenges before broad utilization. Concerning the manufacturing processes, there is significant research focusing on the optimization of current cell culture systems and, more recently, on developing scalable downstream processes to generate material for pre-clinical and clinical trials. We review the current options for downstream processing of these complex biopharmaceuticals and underline current advances on knowledge-based toolboxes proposed for rational optimization of their processing. Rational tools developed to increase the yet scarce knowledge on the purification processes of complex biologicals are discussed as alternative to empirical, "black-boxed" based strategies classically used for process development. Innovative methodologies based on surface plasmon resonance, dynamic light scattering, scale-down high-throughput screening and mathematical modeling for supporting ion-exchange chromatography show great potential for a more efficient and cost-effective process design, optimization and equipment prototyping. Copyright © 2011 Elsevier Inc. All rights reserved.

BER-Myriant Succinic Acid Biorefinery

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

Shmorhun, Mark

Myriant Corporation (Myriant) has successfully produced the bioproduct succinic acid by the fermentation of glucose at a commercial scale operation in Lake Providence, Louisiana. The MySAB facility (Myriant Succinic Acid Biorefinery) came on stream in May 2013 and has been producing product since then. The MySAB facility is a demonstration-scale plant, capable of utilizing sorghum grits and commercially available dextrose, to ferment glucose into succinic acid. A downstream processing train has demonstrated the ability to produce an industrial, a standard and a polymer grade product. It consists of cell separation, membrane filtration, continuous chromatography, polishing to remove ionic and colormore » bodies impurities, and final evaporation and crystallization. A by-product of the process is ammonium sulfate which is sold as a liquid fertilizer product. Since 2007 when development work began in the Woburn, Massachusetts R&D laboratories, the succinic acid bio-process has evolved through: Process development (microbiology, fermentation, and downstream) – R&D development laboratories; Piloting efforts at Fermic S.A. de C.V., Mexico City, Mexico – upstream and downstream processes; Design, construction, commissioning, and commercial production operations at the MySAB facility Additionally, Myriant became a wholly-owned subsidiary of the PTT Global Chemical Plc., Thailand, in late 2015, their investment into and support of Myriant goes back to 2011. The support of PTT Global Chemical Plc. helped to improve the upstream and downstream processes, and produce significant metric ton quantities of high quality bio-based succinic acid. The product has gone into a number of commercial markets worldwide for customer applications, development and production. The experience base gained via operations at the MySAB facility since May 2013, along with continued R&D development efforts involving Microbiology, Fermentation, and Downstream processes, at both the Woburn, Massachusetts and PTT Global Chemical Plc. Thailand laboratories, positions the company well for future production at the plant and commercialization of new bio-based products. This will be especially important and valuable as the green chemistry business climate continues to take root and flourish.« less

Streamflow gain-loss characteristics of Elkhead Creek downstream from Elkhead Reservoir near Craig, Colorado, 2009

USGS Publications Warehouse

Ruddy, Barbara C.

2010-01-01

The U.S. Geological Survey (USGS), in cooperation with the Colorado Water Conservation Board, the Upper Colorado River Endangered Fish Recovery Program (UCREFRP), Colorado Division of Water Resources, and City of Craig studied the gain-loss characteristics of Elkhead Creek downstream from Elkhead Reservoir to the confluence with the Yampa River during August through October 2009. Earlier qualitative interpretation of streamflow data downstream from the reservoir indicated that there could be a transit loss of nearly 10 percent. This potential loss could be a significant portion of the releases from Elkhead Reservoir requested by UCREFRP during late summer and early fall for improving critical habitat for endangered fish downstream in the Yampa River. Information on the gain-loss characteristics was needed for the effective management of the reservoir releases. In order to determine streamflow gain-loss characteristics for Elkhead Creek, eight measurement sets were made at four strategic instream sites and at one diversion from August to early October 2009. An additional measurement set was made after the study period during low-flow conditions in November 2009. Streamflow measurements were made using an Acoustic Doppler Velocimeter to provide high accuracy and consistency, especially at low flows. During this study, streamflow ranged from about 5 cubic feet per second up to more than 90 cubic feet per second with step increments in between. Measurements were made at least 24 hours after a change in reservoir release (streamflow) during steady-state conditions. The instantaneous streamflow measurements and the streamflow volume comparisons show the reach of Elkhead Creek immediately downstream from Elkhead Reservoir to the streamflow-gaging station 09246500, Elkhead Creek near Craig, CO, is neither a gaining nor losing reach. The instantaneous measurements immediately downstream from the dam and the combined measurements of Norvell ditch plus streamflow-gaging station 09246500 are mostly within the plus or minus 5-percent measurement error of each other. The variability of data is such that sometimes the streamflow is greater upstream than downstream and sometimes the streamflow is greater downstream than upstream. Streamflow volumes were calculated for multiple time periods as determined by a change in release from the reservoir. Streamflow volumes were greater downstream than upstream for all but one time period. The predominance of greater streamflows downstream is due to the difference between the USGS instantaneous measurements and record computation with the Supervisory Control and Data Acquisition (SCADA) record at the dam. Immediately following an increase in streamflow from the reservoir, the downstream volume was smaller than the upstream volume, but this was an artifact of the traveltime between the two sites and possibly small amounts of water entering the streambank. Traveltimes were shorter at higher streamflows and when streamflow was increasing.

A self-regulating biomolecular comparator for processing oscillatory signals

PubMed Central

Agrawal, Deepak K.; Franco, Elisa; Schulman, Rebecca

2015-01-01

While many cellular processes are driven by biomolecular oscillators, precise control of a downstream on/off process by a biochemical oscillator signal can be difficult: over an oscillator's period, its output signal varies continuously between its amplitude limits and spends a significant fraction of the time at intermediate values between these limits. Further, the oscillator's output is often noisy, with particularly large variations in the amplitude. In electronic systems, an oscillating signal is generally processed by a downstream device such as a comparator that converts a potentially noisy oscillatory input into a square wave output that is predominantly in one of two well-defined on and off states. The comparator's output then controls downstream processes. We describe a method for constructing a synthetic biochemical device that likewise produces a square-wave-type biomolecular output for a variety of oscillatory inputs. The method relies on a separation of time scales between the slow rate of production of an oscillatory signal molecule and the fast rates of intermolecular binding and conformational changes. We show how to control the characteristics of the output by varying the concentrations of the species and the reaction rates. We then use this control to show how our approach could be applied to process different in vitro and in vivo biomolecular oscillators, including the p53-Mdm2 transcriptional oscillator and two types of in vitro transcriptional oscillators. These results demonstrate how modular biomolecular circuits could, in principle, be combined to build complex dynamical systems. The simplicity of our approach also suggests that natural molecular circuits may process some biomolecular oscillator outputs before they are applied downstream. PMID:26378119

Ionic liquid-tolerant microorganisms and microbial communities for lignocellulose conversion to bioproducts

DOE PAGES

Yu, Chaowei; Simmons, Blake A.; Singer, Steven W.; ...

2016-11-12

Chemical and physical pretreatment of biomass is a critical step in the conversion of lignocellulose to biofuels and bioproducts. Ionic liquid (IL) pretreatment has attracted significant attention due to the unique ability of certain ILs to solubilize some or all components of the plant cell wall. However, these ILs inhibit not only the enzyme activities but also the growth and productivity of microorganisms used in downstream hydrolysis and fermentation processes. While pretreated biomass can be washed to remove residual IL and reduce inhibition, extensive washing is costly and not feasible in large-scale processes. IL-tolerant microorganisms and microbial communities have beenmore » discovered from environmental samples and studies begun to elucidate mechanisms of IL tolerance. The discovery of IL tolerance in environmental microbial communities and individual microbes has lead to the proposal of molecular mechanisms of resistance. Here, we review recent progress on discovering IL-tolerant microorganisms, identifying metabolic pathways and mechanisms of tolerance, and engineering microorganisms for IL tolerance. Research in these areas will yield new approaches to overcome inhibition in lignocellulosic biomass bioconversion processes and increase opportunities for the use of ILs in biomass pretreatment.« less

Ionic liquid-tolerant microorganisms and microbial communities for lignocellulose conversion to bioproducts.

PubMed

Yu, Chaowei; Simmons, Blake A; Singer, Steven W; Thelen, Michael P; VanderGheynst, Jean S

2016-12-01

Chemical and physical pretreatment of biomass is a critical step in the conversion of lignocellulose to biofuels and bioproducts. Ionic liquid (IL) pretreatment has attracted significant attention due to the unique ability of certain ILs to solubilize some or all components of the plant cell wall. However, these ILs inhibit not only the enzyme activities but also the growth and productivity of microorganisms used in downstream hydrolysis and fermentation processes. While pretreated biomass can be washed to remove residual IL and reduce inhibition, extensive washing is costly and not feasible in large-scale processes. IL-tolerant microorganisms and microbial communities have been discovered from environmental samples and studies begun to elucidate mechanisms of IL tolerance. The discovery of IL tolerance in environmental microbial communities and individual microbes has lead to the proposal of molecular mechanisms of resistance. In this article, we review recent progress on discovering IL-tolerant microorganisms, identifying metabolic pathways and mechanisms of tolerance, and engineering microorganisms for IL tolerance. Research in these areas will yield new approaches to overcome inhibition in lignocellulosic biomass bioconversion processes and increase opportunities for the use of ILs in biomass pretreatment.

Ionic liquid-tolerant microorganisms and microbial communities for lignocellulose conversion to bioproducts

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

Yu, Chaowei; Simmons, Blake A.; Singer, Steven W.

Chemical and physical pretreatment of biomass is a critical step in the conversion of lignocellulose to biofuels and bioproducts. Ionic liquid (IL) pretreatment has attracted significant attention due to the unique ability of certain ILs to solubilize some or all components of the plant cell wall. However, these ILs inhibit not only the enzyme activities but also the growth and productivity of microorganisms used in downstream hydrolysis and fermentation processes. While pretreated biomass can be washed to remove residual IL and reduce inhibition, extensive washing is costly and not feasible in large-scale processes. IL-tolerant microorganisms and microbial communities have beenmore » discovered from environmental samples and studies begun to elucidate mechanisms of IL tolerance. The discovery of IL tolerance in environmental microbial communities and individual microbes has lead to the proposal of molecular mechanisms of resistance. Here, we review recent progress on discovering IL-tolerant microorganisms, identifying metabolic pathways and mechanisms of tolerance, and engineering microorganisms for IL tolerance. Research in these areas will yield new approaches to overcome inhibition in lignocellulosic biomass bioconversion processes and increase opportunities for the use of ILs in biomass pretreatment.« less

How females of chirping and trilling field crickets integrate the 'what' and 'where' of male acoustic signals during decision making.

PubMed

Gabel, Eileen; Gray, David A; Matthias Hennig, R

2016-11-01

In crickets acoustic communication serves mate selection. Female crickets have to perceive and integrate male cues relevant for mate choice while confronted with several different signals in an acoustically diverse background. Overall female decisions are based on the attractiveness of the temporal pattern (informative about the 'what') and on signal intensity (informative about the 'where') of male calling songs. Here, we investigated how the relevant cues for mate choice are integrated during the decision process by females of five different species of chirping and trilling field crickets. Using a behavioral design, female preferences in no-choice and choice situations for male calling songs differing in pulse rate, modulation depth, intensities, chirp/trill arrangements and temporal shifts were examined. Sensory processing underlying decisions in female field crickets is rather similar as combined evidence suggested that incoming song patterns were analyzed separately by bilaterally paired networks for pattern attractiveness and pattern intensity. A downstream gain control mechanism leads to a weighting of the intensity cue by pattern attractiveness. While remarkable differences between species were observed with respect to specific processing steps, closely related species exhibited more similar preferences than did more distantly related species.

QuickRNASeq lifts large-scale RNA-seq data analyses to the next level of automation and interactive visualization.

PubMed

Zhao, Shanrong; Xi, Li; Quan, Jie; Xi, Hualin; Zhang, Ying; von Schack, David; Vincent, Michael; Zhang, Baohong

2016-01-08

RNA sequencing (RNA-seq), a next-generation sequencing technique for transcriptome profiling, is being increasingly used, in part driven by the decreasing cost of sequencing. Nevertheless, the analysis of the massive amounts of data generated by large-scale RNA-seq remains a challenge. Multiple algorithms pertinent to basic analyses have been developed, and there is an increasing need to automate the use of these tools so as to obtain results in an efficient and user friendly manner. Increased automation and improved visualization of the results will help make the results and findings of the analyses readily available to experimental scientists. By combing the best open source tools developed for RNA-seq data analyses and the most advanced web 2.0 technologies, we have implemented QuickRNASeq, a pipeline for large-scale RNA-seq data analyses and visualization. The QuickRNASeq workflow consists of three main steps. In Step #1, each individual sample is processed, including mapping RNA-seq reads to a reference genome, counting the numbers of mapped reads, quality control of the aligned reads, and SNP (single nucleotide polymorphism) calling. Step #1 is computationally intensive, and can be processed in parallel. In Step #2, the results from individual samples are merged, and an integrated and interactive project report is generated. All analyses results in the report are accessible via a single HTML entry webpage. Step #3 is the data interpretation and presentation step. The rich visualization features implemented here allow end users to interactively explore the results of RNA-seq data analyses, and to gain more insights into RNA-seq datasets. In addition, we used a real world dataset to demonstrate the simplicity and efficiency of QuickRNASeq in RNA-seq data analyses and interactive visualizations. The seamless integration of automated capabilites with interactive visualizations in QuickRNASeq is not available in other published RNA-seq pipelines. The high degree of automation and interactivity in QuickRNASeq leads to a substantial reduction in the time and effort required prior to further downstream analyses and interpretation of the analyses findings. QuickRNASeq advances primary RNA-seq data analyses to the next level of automation, and is mature for public release and adoption.

Conversion of cellulosic materials into glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma spp. under SHF and SSF processes.

PubMed

Faria, Nuno Torres; Santos, Marisa; Ferreira, Carla; Marques, Susana; Ferreira, Frederico Castelo; Fonseca, César

2014-11-04

Mannosylerythritol lipids (MEL) are glycolipids with unique biosurfactant properties and are produced by Pseudozyma spp. from different substrates, preferably vegetable oils, but also sugars, glycerol or hydrocarbons. However, solvent intensive downstream processing and the relatively high prices of raw materials currently used for MEL production are drawbacks in its sustainable commercial deployment. The present work aims to demonstrate MEL production from cellulosic materials and investigate the requirements and consequences of combining commercial cellulolytic enzymes and Pseudozyma spp. under separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes. MEL was produced from cellulosic substrates, Avicel® as reference (>99% cellulose) and hydrothermally pretreated wheat straw, using commercial cellulolytic enzymes (Celluclast 1.5 L® and Novozyme 188®) and Pseudozyma antarctica PYCC 5048(T) or Pseudozyma aphidis PYCC 5535(T). The strategies included SHF, SSF and fed-batch SSF with pre-hydrolysis. While SSF was isothermal at 28°C, in SHF and fed-batch SSF, yeast fermentation was preceded by an enzymatic (pre-)hydrolysis step at 50°C for 48 h. Pseudozyma antarctica showed the highest MEL yields from both cellulosic substrates, reaching titres of 4.0 and 1.4 g/l by SHF of Avicel® and wheat straw (40 g/l glucan), respectively, using enzymes at low dosage (3.6 and 8.5 FPU/gglucan at 28°C and 50°C, respectively) with prior dialysis. Higher MEL titres were obtained by fed-batch SSF with pre-hydrolysis, reaching 4.5 and 2.5 g/l from Avicel® and wheat straw (80 g/l glucan), respectively. This work reports for the first time MEL production from cellulosic materials. The process was successfully performed through SHF, SSF or Fed-batch SSF, requiring, for maximal performance, dialysed commercial cellulolytic enzymes. The use of inexpensive lignocellulosic substrates associated to straightforward downstream processing from sugary broths is expected to have a great impact in the economy of MEL production for the biosurfactant market, inasmuch as low enzyme dosage is sufficient for good systems performance.

Towards a Mobile Ecogenomic sensor: the Third Generation Environmental Sample Processor (3G-ESP).

NASA Astrophysics Data System (ADS)

Birch, J. M.; Pargett, D.; Jensen, S.; Roman, B.; Preston, C. M.; Ussler, W.; Yamahara, K.; Marin, R., III; Hobson, B.; Zhang, Y.; Ryan, J. P.; Scholin, C. A.

2016-02-01

Researchers are increasingly using one or more autonomous platforms to characterize ocean processes that change in both space and time. Conceptually, studying processes that change quickly both spatially and temporally seems relatively straightforward. One needs to sample in many locations synoptically over time, or follow a coherent water mass and sample it repeatedly. However, implementing either approach presents many challenges. For example, acquiring samples over days to weeks far from shore, without human intervention, requires multiple systems to work together seamlessly, and the level of autonomy, navigation and communications needed to conduct the work exposes the complexity of these requirements. We are addressing these challenges by developing a new generation of robotic systems that are primarily aimed at studies of microbial-mediated processes. As a step towards realizing this new capability, we have taken lessons learned from our second-generation Environmental Sample Processor (2G-ESP), a robotic microbiology "lab-in-a-can" and have re-engineered the system for use on a Tethys-class Long Range AUV (LRAUV). The new instrument is called the third-generation ESP (3G-ESP), and its integration with the LRAUV provides mobility and a persistent presence not seen before in microbial oceanography. The 3G-ESP autonomously filters a water sample and then either preserves that material for eventual return to a laboratory, or processes the sample in real-time for further downstream molecular analytical analyses. The 3G ESP modularizes hardware needed for the collection and preparation of a sample from subsequent molecular analyses by the use of self-contained "cartridges". Cartridges currently come in two forms: one for the preservation of a sample, and the other for onboard homogenization and handoff for downstream processing via one or more analytical devices. The 3G-ESP is designed as a stand-alone instrument, and thus could be deployed on a variety of platforms. This presentation will focus on results from early deployments of the prototype 3G-ESP/LRAUV, the challenges encountered in cartridge design, ESP/LRAUV integration, and operational capabilities that show the potential of mobile, ecogenomic sensors in the ocean sciences.

Balancing hydropower production and river bed incision in operating a run-of-river hydropower scheme along the River Po

NASA Astrophysics Data System (ADS)

Denaro, Simona; Dinh, Quang; Bizzi, Simone; Bernardi, Dario; Pavan, Sara; Castelletti, Andrea; Schippa, Leonardo; Soncini-Sessa, Rodolfo

2013-04-01

Water management through dams and reservoirs is worldwide necessary to support key human-related activities ranging from hydropower production to water allocation, and flood risk mitigation. Reservoir operations are commonly planned in order to maximize these objectives. However reservoirs strongly influence river geomorphic processes causing sediment deficit downstream, altering the flow regime, leading, often, to process of river bed incision: for instance the variations of river cross sections over few years can notably affect hydropower production, flood mitigation, water supply strategies and eco-hydrological processes of the freshwater ecosystem. The river Po (a major Italian river) has experienced severe bed incision in the last decades. For this reason infrastructure stability has been negatively affected, and capacity to derive water decreased, navigation, fishing and tourism are suffering economic damages, not to mention the impact on the environment. Our case study analyzes the management of Isola Serafini hydropower plant located on the main Po river course. The plant has a major impact to the geomorphic river processes downstream, affecting sediment supply, connectivity (stopping sediment upstream the dam) and transport capacity (altering the flow regime). Current operation policy aims at maximizing hydropower production neglecting the effects in term of geomorphic processes. A new improved policy should also consider controlling downstream river bed incision. The aim of this research is to find suitable modeling framework to identify an operating policy for Isola Serafini reservoir able to provide an optimal trade-off between these two conflicting objectives: hydropower production and river bed incision downstream. A multi-objective simulation-based optimization framework is adopted. The operating policy is parameterized as a piecewise linear function and the parameters optimized using an interactive response surface approach. Global and local response surface are comparatively assessed. Preliminary results show that a range of potentially interesting trade-off policies exist able to better control river bed incision downstream without significantly decreasing hydropower production.

Spatio-temporal changes in river bank mass failures in the Lockyer Valley, Queensland, Australia

NASA Astrophysics Data System (ADS)

Thompson, Chris; Croke, Jacky; Grove, James; Khanal, Giri

2013-06-01

Wet-flow river bank failure processes are poorly understood relative to the more commonly studied processes of fluvial entrainment and gravity-induced mass failures. Using high resolution topographic data (LiDAR) and near coincident aerial photography, this study documents the downstream distribution of river bank mass failures which occurred as a result of a catastrophic flood in the Lockyer Valley in January 2011. In addition, this distribution is compared with wet flow mass failure features from previous large floods. The downstream analysis of these two temporal data sets indicated that they occur across a range of river lengths, catchment areas, bank heights and angles and do not appear to be scale-dependent or spatially restricted to certain downstream zones. The downstream trends of each bank failure distribution show limited spatial overlap with only 17% of wet flows common to both distributions. The modification of these features during the catastrophic flood of January 2011 also indicated that such features tend to form at some 'optimum' shape and show limited evidence of subsequent enlargement even when flow and energy conditions within the banks and channel were high. Elevation changes indicate that such features show evidence for infilling during subsequent floods. The preservation of these features in the landscape for a period of at least 150 years suggests that the seepage processes dominant in their initial formation appear to have limited role in their continuing enlargement over time. No evidence of gully extension or headwall retreat is evident. It is estimated that at least 12 inundation events would be required to fill these failures based on the average net elevation change recorded for the 2011 event. Existing conceptual models of downstream bank erosion process zones may need to consider a wider array of mass failure processes to accommodate for wet flow failures.

Destination bioeconomy - The path towards a smarter, more sustainable future.

PubMed

Dupont-Inglis, Joanna; Borg, Agnes

2018-01-25

Five years following the publication of the EU Bioeconomy Strategy, this article discusses the state of play of the bioeconomy in Europe. Placing specific focus on Industrial Biotech, it outlines ten pragmatic recommendations from BIO-TIC [BIO-TIC, A roadmap to a thriving industrial biotechnology sector in Europe, 2015], an EU FP7 funded project completed in 2015 and coordinated by EuropaBio, comprehensively examining the hurdles to the development of a bioeconomy in Europe, enabled by industrial biotech. These include improving opportunities for feedstock producers within the bioeconomy; investigating the scope for using novel biomass; developing a workforce which can maintain Europe's competitiveness in industrial biotechnology; introducing a long-term, stable and transparent policy and incentive framework to promote the bioeconomy; improving public perception and awareness of industrial biotechnology and bio-based products; identifying, leveraging and building upon EU capabilities for pilot and demonstration facilities; promoting the use of co-products from processing; improving the bioconversion and downstream processing steps; improving access to financing for large-scale biorefinery projects; developing stronger relationships between conventional and non-conventional players. Copyright © 2017. Published by Elsevier B.V.

Phospho-H1 Decorates the Inter-chromatid Axis and Is Evicted along with Shugoshin by SET during Mitosis.

PubMed

Krishnan, Swathi; Smits, Arne H; Vermeulen, Michiel; Reinberg, Danny

2017-08-17

Precise control of sister chromatid separation during mitosis is pivotal to maintaining genomic integrity. Yet, the regulatory mechanisms involved are not well understood. Remarkably, we discovered that linker histone H1 phosphorylated at S/T18 decorated the inter-chromatid axial DNA on mitotic chromosomes. Sister chromatid resolution during mitosis required the eviction of such H1S/T18ph by the chaperone SET, with this process being independent of and most likely downstream of arm-cohesin dissociation. SET also directed the disassembly of Shugoshins in a polo-like kinase 1-augmented manner, aiding centromere resolution. SET ablation compromised mitotic fidelity as evidenced by unresolved sister chromatids with marked accumulation of H1S/T18ph and centromeric Shugoshin. Thus, chaperone-assisted eviction of linker histones and Shugoshins is a fundamental step in mammalian mitotic progression. Our findings also elucidate the functional implications of the decades-old observation of mitotic linker histone phosphorylation, serving as a paradigm to explore the role of linker histones in bio-signaling processes. Copyright © 2017 Elsevier Inc. All rights reserved.

Genomic Insights into Geothermal Spring Community Members using a 16S Agnostic Single-Cell Approach

NASA Astrophysics Data System (ADS)

Bowers, R. M.

2016-12-01

INSTUTIONS (ALL): DOE Joint Genome Institute, Walnut Creek, CA USA. Bigelow Laboratory for Ocean Sciences, East Boothbay, ME USA. Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada. ABSTRACT BODY: With recent advances in DNA sequencing, rapid and affordable screening of single-cell genomes has become a reality. Single-cell sequencing is a multi-step process that takes advantage of any number of single-cell sorting techniques, whole genome amplification (WGA), and 16S rRNA gene based PCR screening to identify the microbes of interest prior to shotgun sequencing. However, the 16S PCR based screening step is costly and may lead to unanticipated losses of microbial diversity, as cells that do not produce a clean 16S amplicon are typically omitted from downstream shotgun sequencing. While many of the sorted cells that fail the 16S PCR step likely originate from poor quality amplified DNA, some of the cells with good WGA kinetics may instead represent bacteria or archaea with 16S genes that fail to amplify due to primer mis-matches or the presence of intervening sequences. Using cell material from Dewar Creek, a hot spring in British Columbia, we sequenced all sorted cells with good WGA kinetics irrespective of their 16S amplification success. We show that this high-throughput approach to single-cell sequencing (i) can reduce the overall cost of single-cell genome production, and (ii). may lead to the discovery of previously unknown branches on the microbial tree of life.

Definition and dynamic control of a continuous chromatography process independent of cell culture titer and impurities.

PubMed

Chmielowski, Rebecca A; Mathiasson, Linda; Blom, Hans; Go, Daniel; Ehring, Hanno; Khan, Heera; Li, Hong; Cutler, Collette; Lacki, Karol; Tugcu, Nihal; Roush, David

2017-12-01

Advances in cell culture technology have enabled the production of antibody titers upwards of 30g/L. These highly productive cell culture systems can potentially lead to productivity bottlenecks in downstream purification due to lower column loadings, especially in the primary capture chromatography step. Alternative chromatography solutions to help remedy this bottleneck include the utilization of continuous processing systems such as periodic counter-current chromatography (PCC). Recent studies have provided methods to optimize and improve the design of PCC for cell culture titers up to about 3g/L. This paper defines a continuous loading strategy for PCC that is independent of cell culture background and encompasses cell culture titers up to about 31g/L. Initial experimentation showed a challenge with determining a difference in change in UV280nm signal (ie. ΔUV) between cell culture feed and monoclonal antibody (mAb) concentration. Further investigation revealed UV280nm absorbance of the cell culture feedstock without antibody was outside of the linear range of detection for a given cell pathlength. Additional experimentation showed the difference in ΔUV for various cell culture feeds can be either theoretically predicted by Beer's Law given a known absorbance of the media background and impurities or experimentally determined using various UV280nm cell pathlengths. Based on these results, a 0.35mm pathlength at UV280nm was chosen for dynamic control to overcome the background signal. The pore diffusion model showed good agreement with the experimental frontal analysis data, which resulted in definition of a ΔUV setpoint range between 20 and 70% for 3C-PCC experiments. Product quality of the elution pools was acceptable between various cell culture feeds and titers up to about 41g/L. Results indicated the following ΔUV setpoints to achieve robust dynamic control and maintain 3C-PCC yield: ∼20-45% for titers greater than 10g/L depending on UV absorbance of the HCCF and ∼45-70% for titers of up to 10g/L independent of UV absorbance of the HCCF. The strategy and results presented in this paper show column loading in a continuous chromatography step can be dynamically controlled independent of the cell culture feedstock and titer, and allow for enhanced process control built into the downstream continuous operations. Copyright © 2017 Elsevier B.V. All rights reserved.

Downstream processing of biopharmaceutical proteins produced in plants: the pros and cons of flocculants.

PubMed

Buyel, Johannes Felix; Fischer, Rainer

2014-01-01

All biological platforms for the manufacture of biopharmaceutical proteins produce an initially turbid extract that must be clarified to avoid fouling sensitive media such as chromatography resins. Clarification is more challenging if the feed stream contains large amounts of dispersed particles, because these rapidly clog the filter media typically used to remove suspended solids. Charged polymers (flocculants) can increase the apparent size of the dispersed particles by aggregation, facilitating the separation of solids and liquids, and thus reducing process costs. However, many different factors can affect the behavior of flocculants, including the pH and conductivity of the medium, the size and charge distribution of the particulates, and the charge density and molecular mass of the polymer. Importantly, these properties can also affect the recovery of the target protein and the overall safety profile of the process. We therefore used a design of experiments approach to establish reliable predictive models that characterize the impact of flocculants during the downstream processing of biopharmaceutical proteins. We highlight strategies for the selection of flocculants during process optimization. These strategies will contribute to the quality by design aspects of process development and facilitate the development of safe and efficient downstream processes for plant-derived pharmaceutical proteins.

Seamless editing of the chloroplast genome in plants.

PubMed

Martin Avila, Elena; Gisby, Martin F; Day, Anil

2016-07-29

Gene editing technologies enable the precise insertion of favourable mutations and performance enhancing trait genes into chromosomes whilst excluding all excess DNA from modified genomes. The technology gives rise to a new class of biotech crops which is likely to have widespread applications in agriculture. Despite progress in the nucleus, the seamless insertions of point mutations and non-selectable foreign genes into the organelle genomes of crops have not been described. The chloroplast genome is an attractive target to improve photosynthesis and crop performance. Current chloroplast genome engineering technologies for introducing point mutations into native chloroplast genes leave DNA scars, such as the target sites for recombination enzymes. Seamless editing methods to modify chloroplast genes need to address reversal of site-directed point mutations by template mediated repair with the vast excess of wild type chloroplast genomes that are present early in the transformation process. Using tobacco, we developed an efficient two-step method to edit a chloroplast gene by replacing the wild type sequence with a transient intermediate. This was resolved to the final edited gene by recombination between imperfect direct repeats. Six out of 11 transplastomic plants isolated contained the desired intermediate and at the second step this was resolved to the edited chloroplast gene in five of six plants tested. Maintenance of a single base deletion mutation in an imperfect direct repeat of the native chloroplast rbcL gene showed the limited influence of biased repair back to the wild type sequence. The deletion caused a frameshift, which replaced the five C-terminal amino acids of the Rubisco large subunit with 16 alternative residues resulting in a ~30-fold reduction in its accumulation. We monitored the process in vivo by engineering an overlapping gusA gene downstream of the edited rbcL gene. Translational coupling between the overlapping rbcL and gusA genes resulted in relatively high GUS accumulation (~0.5 % of leaf protein). Editing chloroplast genomes using transient imperfect direct repeats provides an efficient method for introducing point mutations into chloroplast genes. Moreover, we describe the first synthetic operon allowing expression of a downstream overlapping gene by translational coupling in chloroplasts. Overlapping genes provide a new mechanism for co-ordinating the translation of foreign proteins in chloroplasts.

Subsequent to suppression: Downstream comprehension consequences of noun/verb ambiguity in natural reading

PubMed Central

Stites, Mallory C.; Federmeier, Kara D.

2015-01-01

We used eye-tracking to investigate the downstream processing consequences of encountering noun/verb (NV) homographs (i.e., park) in semantically neutral but syntactically constraining contexts. Target words were followed by a prepositional phrase containing a noun that was plausible for only one meaning of the homograph. Replicating previous work, we found increased first fixation durations on NV homographs compared to unambiguous words, which persisted into the next sentence region. At the downstream noun, we found plausibility effects following ambiguous words that were correlated with the size of a reader's first fixation effect, suggesting that this effect reflects the recruitment of processing resources necessary to suppress the homograph's context-inappropriate meaning. Using these same stimuli, Lee and Federmeier (2012) found a sustained frontal negativity to the NV homographs, and, on the downstream noun, found a plausibility effect that was also positively correlated with the size of a reader's ambiguity effect. Together, these findings suggest that when only syntactic constraints are available, meaning selection recruits inhibitory mechanisms that can be measured in both first fixation slowdown and ERP ambiguity effects. PMID:25961358

Quantitative proton imaging from multiple physics processes: a proof of concept

NASA Astrophysics Data System (ADS)

Bopp, C.; Rescigno, R.; Rousseau, M.; Brasse, D.

2015-07-01

Proton imaging is developed in order to improve the accuracy of charged particle therapy treatment planning. It makes it possible to directly map the relative stopping powers of the materials using the information on the energy loss of the protons. In order to reach a satisfactory spatial resolution in the reconstructed images, the position and direction of each particle is recorded upstream and downstream from the patient. As a consequence of individual proton detection, information on the transmission rate and scattering of the protons is available. Image reconstruction processes are proposed to make use of this information. A proton tomographic acquisition of an anthropomorphic head phantom was simulated. The transmission rate of the particles was used to reconstruct a map of the macroscopic cross section for nuclear interactions of the materials. A two-step iterative reconstruction process was implemented to reconstruct a map of the inverse scattering length of the materials using the scattering of the protons. Results indicate that, while the reconstruction processes should be optimized, it is possible to extract quantitative information from the transmission rate and scattering of the protons. This suggests that proton imaging could provide additional knowledge on the materials that may be of use to further improve treatment planning.

Quantitative proton imaging from multiple physics processes: a proof of concept.

PubMed

Bopp, C; Rescigno, R; Rousseau, M; Brasse, D

2015-07-07

Proton imaging is developed in order to improve the accuracy of charged particle therapy treatment planning. It makes it possible to directly map the relative stopping powers of the materials using the information on the energy loss of the protons. In order to reach a satisfactory spatial resolution in the reconstructed images, the position and direction of each particle is recorded upstream and downstream from the patient. As a consequence of individual proton detection, information on the transmission rate and scattering of the protons is available. Image reconstruction processes are proposed to make use of this information. A proton tomographic acquisition of an anthropomorphic head phantom was simulated. The transmission rate of the particles was used to reconstruct a map of the macroscopic cross section for nuclear interactions of the materials. A two-step iterative reconstruction process was implemented to reconstruct a map of the inverse scattering length of the materials using the scattering of the protons. Results indicate that, while the reconstruction processes should be optimized, it is possible to extract quantitative information from the transmission rate and scattering of the protons. This suggests that proton imaging could provide additional knowledge on the materials that may be of use to further improve treatment planning.

Iteratively improving Hi-C experiments one step at a time.

PubMed

Golloshi, Rosela; Sanders, Jacob T; McCord, Rachel Patton

2018-06-01

The 3D organization of eukaryotic chromosomes affects key processes such as gene expression, DNA replication, cell division, and response to DNA damage. The genome-wide chromosome conformation capture (Hi-C) approach can characterize the landscape of 3D genome organization by measuring interaction frequencies between all genomic regions. Hi-C protocol improvements and rapid advances in DNA sequencing power have made Hi-C useful to study diverse biological systems, not only to elucidate the role of 3D genome structure in proper cellular function, but also to characterize genomic rearrangements, assemble new genomes, and consider chromatin interactions as potential biomarkers for diseases. Yet, the Hi-C protocol is still complex and subject to variations at numerous steps that can affect the resulting data. Thus, there is still a need for better understanding and control of factors that contribute to Hi-C experiment success and data quality. Here, we evaluate recently proposed Hi-C protocol modifications as well as often overlooked variables in sample preparation and examine their effects on Hi-C data quality. We examine artifacts that can occur during Hi-C library preparation, including microhomology-based artificial template copying and chimera formation that can add noise to the downstream data. Exploring the mechanisms underlying Hi-C artifacts pinpoints steps that should be further optimized in the future. To improve the utility of Hi-C in characterizing the 3D genome of specialized populations of cells or small samples of primary tissue, we identify steps prone to DNA loss which should be considered to adapt Hi-C to lower cell numbers. Copyright © 2018 Elsevier Inc. All rights reserved.

Has dyke development in the Vietnamese Mekong Delta shifted flood hazard downstream?

NASA Astrophysics Data System (ADS)

Van Khanh Triet, Nguyen; Viet Dung, Nguyen; Fujii, Hideto; Kummu, Matti; Merz, Bruno; Apel, Heiko

2017-08-01

In the Vietnamese part of the Mekong Delta (VMD) the areas with three rice crops per year have been expanded rapidly during the last 15 years. Paddy-rice cultivation during the flood season has been made possible by implementing high-dyke flood defenses and flood control structures. However, there are widespread claims that the high-dyke system has increased water levels in downstream areas. Our study aims at resolving this issue by attributing observed changes in flood characteristics to high-dyke construction and other possible causes. Maximum water levels and duration above the flood alarm level are analysed for gradual trends and step changes at different discharge gauges. Strong and robust increasing trends of peak water levels and duration downstream of the high-dyke areas are found with a step change in 2000/2001, i.e. immediately after the disastrous flood which initiated the high-dyke development. These changes are in contrast to the negative trends detected at stations upstream of the high-dyke areas. This spatially different behaviour of changes in flood characteristics seems to support the public claims. To separate the impact of the high-dyke development from the impact of the other drivers - i.e. changes in the flood hydrograph entering the Mekong Delta, and changes in the tidal dynamics - hydraulic model simulations of the two recent large flood events in 2000 and 2011 are performed. The hydraulic model is run for a set of scenarios whereas the different drivers are interchanged. The simulations reveal that for the central VMD an increase of 9-13 cm in flood peak and 15 days in duration can be attributed to high-dyke development. However, for this area the tidal dynamics have an even larger effect in the range of 19-32 cm. However, the relative contributions of the three drivers of change vary in space across the delta. In summary, our study confirms the claims that the high-dyke development has raised the flood hazard downstream. However, it is not the only and not the most important driver of the observed changes. It has to be noted that changes in tidal levels caused by sea level rise in combination with the widely observed land subsidence and the temporal coincidence of high water levels and spring tides have even larger impacts. It is recommended to develop flood risk management strategies using the high-dyke areas as retention zones to mitigate the flood hazard downstream.

Two-step membrane binding by the bacterial SRP receptor enable efficient and accurate Co-translational protein targeting.

PubMed

Hwang Fu, Yu-Hsien; Huang, William Y C; Shen, Kuang; Groves, Jay T; Miller, Thomas; Shan, Shu-Ou

2017-07-28

The signal recognition particle (SRP) delivers ~30% of the proteome to the eukaryotic endoplasmic reticulum, or the bacterial plasma membrane. The precise mechanism by which the bacterial SRP receptor, FtsY, interacts with and is regulated at the target membrane remain unclear. Here, quantitative analysis of FtsY-lipid interactions at single-molecule resolution revealed a two-step mechanism in which FtsY initially contacts membrane via a Dynamic mode, followed by an SRP-induced conformational transition to a Stable mode that activates FtsY for downstream steps. Importantly, mutational analyses revealed extensive auto-inhibitory mechanisms that prevent free FtsY from engaging membrane in the Stable mode; an engineered FtsY pre-organized into the Stable mode led to indiscriminate targeting in vitro and disrupted FtsY function in vivo. Our results show that the two-step lipid-binding mechanism uncouples the membrane association of FtsY from its conformational activation, thus optimizing the balance between the efficiency and fidelity of co-translational protein targeting.

Engineering metabolic pathways in plants by multigene transformation.

PubMed

Zorrilla-López, Uxue; Masip, Gemma; Arjó, Gemma; Bai, Chao; Banakar, Raviraj; Bassie, Ludovic; Berman, Judit; Farré, Gemma; Miralpeix, Bruna; Pérez-Massot, Eduard; Sabalza, Maite; Sanahuja, Georgina; Vamvaka, Evangelia; Twyman, Richard M; Christou, Paul; Zhu, Changfu; Capell, Teresa

2013-01-01

Metabolic engineering in plants can be used to increase the abundance of specific valuable metabolites, but single-point interventions generally do not improve the yields of target metabolites unless that product is immediately downstream of the intervention point and there is a plentiful supply of precursors. In many cases, an intervention is necessary at an early bottleneck, sometimes the first committed step in the pathway, but is often only successful in shifting the bottleneck downstream, sometimes also causing the accumulation of an undesirable metabolic intermediate. Occasionally it has been possible to induce multiple genes in a pathway by controlling the expression of a key regulator, such as a transcription factor, but this strategy is only possible if such master regulators exist and can be identified. A more robust approach is the simultaneous expression of multiple genes in the pathway, preferably representing every critical enzymatic step, therefore removing all bottlenecks and ensuring completely unrestricted metabolic flux. This approach requires the transfer of multiple enzyme-encoding genes to the recipient plant, which is achieved most efficiently if all genes are transferred at the same time. Here we review the state of the art in multigene transformation as applied to metabolic engineering in plants, highlighting some of the most significant recent advances in the field.

Cyclic Steps and Antidunes : Relating Their Features to a Suspension Index

NASA Astrophysics Data System (ADS)

Yokokawa, M.; Kishima, Y.; Parker, G.

2010-12-01

Cyclic Steps and Antidunes : Relating Their Features to a Suspension Index Miwa Yokokawa (1), Yasushi Kishima (1), Gary Parker (2, 3) 1: Osaka Institute of Technology, Hirakata, Osaka, Japan 2: Dept. of Civil & Environmental Engineering, University of Illinois, Urbana, Illinois, U.S.A. 3: Dept. of Geology, University of Illinois, Urbana, Illinois, U.S.A. There are very few comparative studies of the differences in hydraulic conditions and morphologic features of bed- and water-surface-waves associated with cyclic steps and antidunes. In this study, the features of both the bed and the water surface, as well as hydraulic conditions are examined over the spectrum from antidune to cyclic steps. Experiments were performed using a flume at the Osaka Institute of Technology. The resultant features of the bedforms are as follows. In the case of antidunes, bed waves and water surface waves are in phase except when they collapse. Antidunes show several kinds of behavior; migrating downstream, standing, or migrating upstream. Upstream-migrating antidunes are divided into non-breaking, and breaking-types. Breaking antidunes appear alternatively with the plane bed state. Cyclic steps migrate upstream regularly associated with trains of hydraulic jumps, which divide each step. There is a significant change in water depth at the hydraulic jump, so that the phasing between the bed waves and water surface waves break at the each hydraulic jump. There is a kind of compromise between cyclic steps and antidunes, which we designate as “intermediate steps”. They move upstream and are associated with regular trains of hydraulic jumps. The jumps, however, occasionally collapse toward upstream. When this happens, bed waves move rapidly upstream; low-amplitude water surface waves and bed waves become in phase all over the bed shortly after the collapse. Then after some time, water surface waves become sufficiently prominent to yield regular hydraulic jumps. This cycle is then repeated.The hydraulic conditions for these bedfoms were examined using three non-dimensional parameters, i.e. the Froude Number, the Suspension Index, and the dimensionless particle size. The suspension index is a newly introduced parameter which is the ratio of the shear velocity divided by the settling velocity of the sediment (u*/Vs). Data from previous experimental studies are examined together with the present data in studying the characteristic regimes of bedform formation. In a diagram of Froude Number v.s. Suspension Index, antidunes, intermediate steps and cyclic steps can be divided along the axis of the Suspension Index. In the lowest range of the suspension index, downstream-migrating antidunes and upstream-migrating antidunes that do not break are found. The intermediate steps discussed above are located in the middle range. The highest range corresponds to cyclic steps and breaking antidunes. As described above, the Suspension Index can serve as a scale to quantify the spectrum between antidunes and cyclic steps. The use of the parameter also helps verify that suspension plays an important role in the formation and maintenance of cyclic steps.

Downstream process options for the ABE fermentation.

PubMed

Friedl, Anton

2016-05-01

Butanol is a very interesting substance both for the chemical industry and as a biofuel. The classical distillation process for the removal of butanol is far too energy demanding, at a factor of 220% of the energy content of butanol. Alternative separation processes studied are hybrid processes of gas-stripping, liquid-liquid extraction and pervaporation with distillation and a novel adsorption/drying/desorption hybrid process. Compared with the energy content of butanol, the resulting energy demand for butanol separation and concentration of optimized hybrid processes is 11%-22% for pervaporation/distillation and 11%-17% for liquid-liquid extraction/distillation. For a novel adsorption/drying/desorption process, the energy demand is 9.4%. But all downstream process options need further proof of industrial applicability. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Allogeneic cell therapy bioprocess economics and optimization: downstream processing decisions.

PubMed

Hassan, Sally; Simaria, Ana S; Varadaraju, Hemanthram; Gupta, Siddharth; Warren, Kim; Farid, Suzanne S

2015-01-01

To develop a decisional tool to identify the most cost effective process flowsheets for allogeneic cell therapies across a range of production scales. A bioprocess economics and optimization tool was built to assess competing cell expansion and downstream processing (DSP) technologies. Tangential flow filtration was generally more cost-effective for the lower cells/lot achieved in planar technologies and fluidized bed centrifugation became the only feasible option for handling large bioreactor outputs. DSP bottlenecks were observed at large commercial lot sizes requiring multiple large bioreactors. The DSP contribution to the cost of goods/dose ranged between 20-55%, and 50-80% for planar and bioreactor flowsheets, respectively. This analysis can facilitate early decision-making during process development.

Purification of Proteins Fused to Maltose-Binding Protein.

PubMed

Lebendiker, Mario; Danieli, Tsafi

2017-01-01

Maltose-Binding Protein (MBP) is one of the most popular fusion partners being used for producing recombinant proteins in bacterial cells. MBP allows the use of a simple capture affinity step on Amylose-Agarose or Dextrin-Sepharose columns, resulting in a protein that is often 70-90 % pure in a single step. In addition to protein isolation applications, MBP provides a high degree of translation, and facilitates the proper folding and solubility of the target protein. This paper describes efficient procedures for isolating highly purified MBP target proteins. Special attention is given to considerations for downstream applications such as structural determination studies, protein activity assays, and assessing the chemical characteristics of the target protein.

Large-eddy simulation of a backward facing step flow using a least-squares spectral element method

NASA Technical Reports Server (NTRS)

Chan, Daniel C.; Mittal, Rajat

1996-01-01

We report preliminary results obtained from the large eddy simulation of a backward facing step at a Reynolds number of 5100. The numerical platform is based on a high order Legendre spectral element spatial discretization and a least squares time integration scheme. A non-reflective outflow boundary condition is in place to minimize the effect of downstream influence. Smagorinsky model with Van Driest near wall damping is used for sub-grid scale modeling. Comparisons of mean velocity profiles and wall pressure show good agreement with benchmark data. More studies are needed to evaluate the sensitivity of this method on numerical parameters before it is applied to complex engineering problems.

Physiological and endocrine changes in Atlantic salmon smolts during hatchery rearing, downstream migration and ocean entry

USGS Publications Warehouse

McCormick, Stephen D.; Sheehan, Timothy F.; Björnsson, Björn Thrandur; Lipsky, Christine; Kocik, John F.; Regish, Amy M.; O'Dea, Michael F.

2013-01-01

Billions of hatchery salmon smolts are released annually in an attempt to mitigate anthropogenic impacts on freshwater habitats, often with limited success. Mortality of wild and hatchery fish is high during downstream and early ocean migration. To understand changes that occur during migration, we examined physiological and endocrine changes in Atlantic salmon (Salmo salar) smolts during hatchery rearing, downstream migration, and early ocean entry in two successive years. Gill Na+/K+-ATPase activity increased in the hatchery during spring, increased further after river release, and was slightly lower after recapture in the ocean. Plasma growth hormone levels increased in the hatchery, were higher in the river, and increased further in the ocean. Plasma IGF-I remained relatively constant in the hatchery, increased in the river, then decreased in the ocean. Plasma thyroid hormones were variable in the hatchery, but increased in both river- and ocean-captured smolts. Naturally reared fish had lower condition factor, gill NKA activity, and plasma thyroxine than hatchery fish in the river but were similar in the ocean. This novel data set provides a vital first step in understanding the role and norms of endocrine function in smolts and the metrics of successful marine entry.

Comprehensive analysis of line-edge and line-width roughness for EUV lithography

NASA Astrophysics Data System (ADS)

Bonam, Ravi; Liu, Chi-Chun; Breton, Mary; Sieg, Stuart; Seshadri, Indira; Saulnier, Nicole; Shearer, Jeffrey; Muthinti, Raja; Patlolla, Raghuveer; Huang, Huai

2017-03-01

Pattern transfer fidelity is always a major challenge for any lithography process and needs continuous improvement. Lithographic processes in semiconductor industry are primarily driven by optical imaging on photosensitive polymeric material (resists). Quality of pattern transfer can be assessed by quantifying multiple parameters such as, feature size uniformity (CD), placement, roughness, sidewall angles etc. Roughness in features primarily corresponds to variation of line edge or line width and has gained considerable significance, particularly due to shrinking feature sizes and variations of features in the same order. This has caused downstream processes (Etch (RIE), Chemical Mechanical Polish (CMP) etc.) to reconsider respective tolerance levels. A very important aspect of this work is relevance of roughness metrology from pattern formation at resist to subsequent processes, particularly electrical validity. A major drawback of current LER/LWR metric (sigma) is its lack of relevance across multiple downstream processes which effects material selection at various unit processes. In this work we present a comprehensive assessment of Line Edge and Line Width Roughness at multiple lithographic transfer processes. To simulate effect of roughness a pattern was designed with periodic jogs on the edges of lines with varying amplitudes and frequencies. There are numerous methodologies proposed to analyze roughness and in this work we apply them to programmed roughness structures to assess each technique's sensitivity. This work also aims to identify a relevant methodology to quantify roughness with relevance across downstream processes.

A data-model integration approach toward improved understanding on wetland functions and hydrological benefits at the catchment scale

NASA Astrophysics Data System (ADS)

Yeo, I. Y.; Lang, M.; Lee, S.; Huang, C.; Jin, H.; McCarty, G.; Sadeghi, A.

2017-12-01

The wetland ecosystem plays crucial roles in improving hydrological function and ecological integrity for the downstream water and the surrounding landscape. However, changing behaviours and functioning of wetland ecosystems are poorly understood and extremely difficult to characterize. Improved understanding on hydrological behaviours of wetlands, considering their interaction with surrounding landscapes and impacts on downstream waters, is an essential first step toward closing the knowledge gap. We present an integrated wetland-catchment modelling study that capitalizes on recently developed inundation maps and other geospatial data. The aim of the data-model integration is to improve spatial prediction of wetland inundation and evaluate cumulative hydrological benefits at the catchment scale. In this paper, we highlight problems arising from data preparation, parameterization, and process representation in simulating wetlands within a distributed catchment model, and report the recent progress on mapping of wetland dynamics (i.e., inundation) using multiple remotely sensed data. We demonstrate the value of spatially explicit inundation information to develop site-specific wetland parameters and to evaluate model prediction at multi-spatial and temporal scales. This spatial data-model integrated framework is tested using Soil and Water Assessment Tool (SWAT) with improved wetland extension, and applied for an agricultural watershed in the Mid-Atlantic Coastal Plain, USA. This study illustrates necessity of spatially distributed information and a data integrated modelling approach to predict inundation of wetlands and hydrologic function at the local landscape scale, where monitoring and conservation decision making take place.

Simultaneous automatic scoring and co-registration of hormone receptors in tumor areas in whole slide images of breast cancer tissue slides.

PubMed

Trahearn, Nicholas; Tsang, Yee Wah; Cree, Ian A; Snead, David; Epstein, David; Rajpoot, Nasir

2017-06-01

Automation of downstream analysis may offer many potential benefits to routine histopathology. One area of interest for automation is in the scoring of multiple immunohistochemical markers to predict the patient's response to targeted therapies. Automated serial slide analysis of this kind requires robust registration to identify common tissue regions across sections. We present an automated method for co-localized scoring of Estrogen Receptor and Progesterone Receptor (ER/PR) in breast cancer core biopsies using whole slide images. Regions of tumor in a series of fifty consecutive breast core biopsies were identified by annotation on H&E whole slide images. Sequentially cut immunohistochemical stained sections were scored manually, before being digitally scanned and then exported into JPEG 2000 format. A two-stage registration process was performed to identify the annotated regions of interest in the immunohistochemistry sections, which were then scored using the Allred system. Overall correlation between manual and automated scoring for ER and PR was 0.944 and 0.883, respectively, with 90% of ER and 80% of PR scores within in one point or less of agreement. This proof of principle study indicates slide registration can be used as a basis for automation of the downstream analysis for clinically relevant biomarkers in the majority of cases. The approach is likely to be improved by implantation of safeguarding analysis steps post registration. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Inhibition of STAT-3 Results in Radiosensitization of Human Squamous Cell Carcinoma

PubMed Central

Bonner, James A.; Trummell, Hoa Q.; Willey, Christopher D.; Plants, Brian A.; Raisch, Kevin P.

2009-01-01

Background Signal Transducer and Activator of Transcription – 3 (STAT-3) is a downstream component of the Epidermal Growth Factor Receptor (EGFr) signaling process that may facilitate the resistance of tumor cells to conventional cancer treatments. Studies were performed to determine if inhibition of this downstream protein may produce radiosensitization. Methods/Results A431 cells (human squamous cell carcinoma cells with EGFr overexpression) were found to be sensitized to radiation after treatment with STAT-3 small interfering RNA (siRNA). Therefore, a short hairpin RNA (shRNA) against STAT-3 was designed and cloned into a pBABE vector system modified for shRNA expression. Following transfection, clone 2.1 was selected for further study as it showed a dramatic reduction of STAT-3 protein (and mRNA) when compared to A431 parental cells or a negative control shRNA cell line (transfected with STAT-3 shRNA with 2 base pairs mutated). A431 2.1 showed doubling times of 25-31 h as compared to 18-24 h for the parental cell line. The A431 shRNA knockdown STAT-3 cells A431 were more sensitive to radiation than A431 parental or negative STAT-3 control cells. Conclusion A431 cells stably transfected with shRNA against STAT-3 resulted in enhanced radiosensitivity. Further work will be necessary to determine whether inhibition of STAT-3 phosphorylation is a necessary step for the radiosensitization that is induced by inhibition of EGFr. PMID:19616333

Cultivation of the Marine Macroalgae Chaetomorpha linum in Municipal Wastewater for Nutrient Recovery and Biomass Production.

PubMed

Ge, Shijian; Champagne, Pascale

2017-03-21

Compared to microalgae, macroalgae are larger in size, thereby imposing lower separation and drying costs. This study demonstrates the feasibility of cultivating macroalgae Chaetomorpha linum in different types of municipal wastewaters, their ability to remove nutrient and their biomass composition for downstream biofuel production. Screening experiments indicated that C. linum grew well on primary (PW) and secondary wastewaters (SW), as well as centrate wastewater (CW) diluted to less than 20%. In a subsequent experiment, a step feeding approach was found to significantly increase biomass productivity to 10.7 ± 0.2 g AFDW·m -2 ·d -1 (p < 0.001), a 26.5% improvement in comparison to the control with single feeding, when grown on 10-CW; meanwhile, nitrogen and phosphorus removal efficiencies rose to 86.8 ± 1.1% (p < 0.001) and 92.6 ± 0.2% (p < 0.001), respectively. The CO 2 -supplemented SW cultures (10.1 ± 0.4 g AFDW·m -2 ·d -1 ) were 1.20 times more productive than the corresponding controls without CO 2 supplementation (p < 0.001); however, similar improvements were not observed in PW (p = 0.07) and 10-CW cultures (p = 0.07). Moreover, wastewater type and nutrient concentration influenced biomass composition (protein, carbohydrate and lipid). These findings indicate that the application of the macroalgae C. linum could represent an effective wastewater treatment alternative that could also provide a feedstock for downstream processing to biofuels.

Influence of macromolecular precipitants on phase behavior of monoclonal antibodies.

PubMed

Rakel, Natalie; Galm, Lara; Bauer, Katharina Christin; Hubbuch, Juergen

2015-01-01

For the successful application of protein crystallization as a downstream step, a profound knowledge of protein phase behavior in solutions is needed. Therefore, a systematic screening was conducted to analyze the influence of macromolecular precipitants in the form of polyethylene glycol (PEG). First, the influence of molecular weight and concentration of PEG at different pH-values were investigated and analyzed in three-dimensional (3-D) phase diagrams to find appropriate conditions in terms of a fast kinetic and crystal size for downstream processing. In comparison to the use of salts as precipitant, PEG was more suitable to obtain compact 3-D crystals over a broad range of conditions, whereby the molecular weight of PEG is, besides the pH-value, the most important parameter. Second, osmotic second virial coefficients as parameters for protein interactions are experimentally determined with static light scattering to gain a deep insight view in the phase behavior on a molecular basis. The PEG-protein solutions were analyzed as a pseudo-one-compartment system. As the precipitant is also a macromolecule, the new approach of analyzing cross-interactions between the protein and the macromolecule PEG in form of the osmotic second cross-virial coefficient (B23 ) was applied. Both parameters help to understand the protein phase behavior. However, a predictive description of protein phase behavior for systems consisting of monoclonal antibodies and PEG as precipitant is not possible, as kinetic phenomena and concentration dependencies were not taken into account. © 2014 American Institute of Chemical Engineers.

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

PubMed

Bova, G Steven; Eltoum, Isam A; Kiernan, John A; Siegal, Gene P; Frost, Andra R; Best, Carolyn J M; Gillespie, John W; Su, Gloria H; Emmert-Buck, Michael R

2005-02-01

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This article reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies, and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing, and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high quality, appropriately anatomically tagged scientific results. In optimized protocols is a source of inefficiency in current life science research. Improvement in this area will significantly increase life science quality and productivity. The article is divided into introduction, materials, protocols, and notes sections. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this article, readers are advised to read through the entire article first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

PubMed

Rodriguez-Canales, Jaime; Hanson, Jeffrey C; Hipp, Jason D; Balis, Ulysses J; Tangrea, Michael A; Emmert-Buck, Michael R; Bova, G Steven

2013-01-01

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This updated chapter reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high-quality, appropriately anatomically tagged scientific results. Improvement in this area will significantly increase life science quality and productivity. The chapter is divided into introduction, materials, protocols, and notes subheadings. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this chapter, readers are advised to read through the entire chapter first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.

Physical mechanisms in shock-induced turbulent separated flow

NASA Astrophysics Data System (ADS)

Dolling, D. S.

1987-12-01

It has been demonstrated that the flow downstream of the moving shock is separated and that the foot of the shock is effectively the instantaneous separation point. The shock induced turbulent separation is an intermittant process and the separation line indicated by surface tracer methods, such as kerosene-lampblack, is a downstream boundary of a region of intermittent separation.

Reduction of product-related species during the fermentation and purification of a recombinant IL-1 receptor antagonist at the laboratory and pilot scale.

PubMed

Schirmer, Emily B; Golden, Kathryn; Xu, Jin; Milling, Jesse; Murillo, Alec; Lowden, Patricia; Mulagapati, Srihariraju; Hou, Jinzhao; Kovalchin, Joseph T; Masci, Allyson; Collins, Kathryn; Zarbis-Papastoitsis, Gregory

2013-08-01

Through a parallel approach of tracking product quality through fermentation and purification development, a robust process was designed to reduce the levels of product-related species. Three biochemically similar product-related species were identified as byproducts of host-cell enzymatic activity. To modulate intracellular proteolytic activity, key fermentation parameters (temperature, pH, trace metals, EDTA levels, and carbon source) were evaluated through bioreactor optimization, while balancing negative effects on growth, productivity, and oxygen demand. The purification process was based on three non-affinity steps and resolved product-related species by exploiting small charge differences. Using statistical design of experiments for elution conditions, a high-resolution cation exchange capture column was optimized for resolution and recovery. Further reduction of product-related species was achieved by evaluating a matrix of conditions for a ceramic hydroxyapatite column. The optimized fermentation process was transferred from the 2-L laboratory scale to the 100-L pilot scale and the purification process was scaled accordingly to process the fermentation harvest. The laboratory- and pilot-scale processes resulted in similar process recoveries of 60 and 65%, respectively, and in a product that was of equal quality and purity to that of small-scale development preparations. The parallel approach for up- and downstream development was paramount in achieving a robust and scalable clinical process. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detection of ASC Speck Formation by Flow Cytometry and Chemical Cross-linking.

PubMed

Hoss, Florian; Rolfes, Verena; Davanso, Mariana R; Braga, Tarcio T; Franklin, Bernardo S

2018-01-01

Assembly of a relatively large protein aggregate or "speck" formed by the adaptor protein ASC is a common downstream step in the activation of most inflammasomes. This unique feature of ASC allows its visualization by several imaging techniques and constitutes a reliable and feasible readout for inflammasome activation in cells and tissues. We have previously described step-by-step protocols to generate immortalized cell lines stably expressing ASC fused to a fluorescent protein for measuring inflammasome activation by confocal microscopy, and immunofluorescence of endogenous ASC in primary cells. Here, we present two more methods to detect ASC speck formation: (1) Assessment of ASC speck formation by flow cytometry; and (2) Chemical cross-linking of ASC followed by immunoblotting. These methods allow for the discrimination of inflammasome-activated versus non-activated cells, the identification of lineage-specific inflammasome activation in complex cell mixtures, and sorting of inflammasome-activated cells for further analysis.

The influence of processing on the microbial risk associated with Rooibos (Aspalathus linearis) tea.

PubMed

Gouws, Pieter; Hartel, Toni; van Wyk, Rudean

2014-12-01

This review discusses the influence of processing on the microbial risk associated with Salmonella in Rooibos tea, the identification of Salmonella and preventative and control measures to control microbial contamination. Rooibos tea, like other plant products, naturally contains a high microbial load. Downstream processing steps of these products usually help in reducing any contaminants present. Due to the delicate flavour properties and nature of Rooibos, gentle processing techniques are necessary for the production of good quality tea. However, this has a major influence on the microbiological status of the product. The presence of Salmonella in Rooibos is poorly understood. The ubiquitous distribution of Salmonella in the natural environment and its prevalence in the global food chain, the physiological adaptability, virulence of the bacterial pathogen and its serious economic impact on the food industry, emphasises the need for continued awareness and stringent controls at all levels of food production. With the advances of technology and information at hand, the processing of Rooibos needs to be re-evaluated. Since the delicate nature of Rooibos prohibits the use of harsh methods to control Salmonella, alternative methods for the steam pasteurisation of Rooibos show great potential to control Salmonella in a fast, efficient and cost-effective manner. These alternative methods will significantly improve the microbiological quality of Rooibos and provide a product that is safe to consumers. © 2014 Society of Chemical Industry.

Interactions of a co-rotating vortex pair at multiple offsets

NASA Astrophysics Data System (ADS)

Forster, Kyle J.; Barber, Tracie J.; Diasinos, Sammy; Doig, Graham

2017-05-01

Two NACA0012 vanes at various lateral offsets were investigated by wind tunnel testing to observe the interactions between the streamwise vortices. The vanes were separated by nine chord lengths in the streamwise direction to allow the upstream vortex to impact on the downstream geometry. These vanes were evaluated at an angle of incidence of 8° and a Reynolds number of 7 ×104 using particle image velocimetry. A helical motion of the vortices was observed, with rotational rate increasing as the offset was reduced to the point of vortex merging. Downstream meandering of the weaker vortex was found to increase in magnitude near the point of vortex merging. The merging process occurred more rapidly when the upstream vortex was passed on the pressure side of the vane, with the downstream vortex being produced with less circulation and consequently merging into the upstream vortex. The merging distance was found to be statistical rather than deterministic quantity, indicating that the meandering of the vortices affected their separations and energies. This resulted in a fluctuation of the merging location. A loss of circulation associated with the merging process was identified, with the process of achieving vortex circularity causing vorticity diffusion, however all merged cases maintained higher circulation than a single vortex condition. The presence of the upstream vortex was found to reduce the strength of the downstream vortex in all offsets evaluated.

The effects of liming an Adirondack lake watershed on downstream water chemistry: Effects of liming on stream chemistry

USGS Publications Warehouse

Burns, Douglas A.

1996-01-01

Calcite treatment of chronically acidic lakes has improved fish habitat, but the effects on downstream water quality have not previously been examined. In this study, the spatial and temporal effects of watershed CaCO3 treatment on the chemistry of a lake outlet stream in the Adirondack Mountains of New York were examined. Before CaCO3 treatment, the stream was chronically acidic. During spring snowmelt before treatment, pH and acid-neutralizing capacity (ANC) in the outlet stream declined, and NO3- and inorganic monomeric aluminum (AlIM) concentrations increased sharply. During that summer, SO42- and NO3- concentrations decreased downstream, and dissolved organic carbon (DOC) concentrations and ANC increased, in association with the seasonal increase in decomposition of organic matter and the attendant SO42--reduction process. A charge-balance ANC calculation closely matched measured downstream changes in ANC in the summer and indicated that SO42- reduction was the major process contributing to summer increases in ANC. Increases in Ca2+ concentration and ANC began immediately after CaCO3 application, and within 3 months, exceeded their pretreatment values by more than 130 ??eq/L. Within 2 months after treatment, downstream decreases in Ca2+ concentration, ANC, and pH, were noted. Stream mass balances between the lake and the sampling site 1.5 km downstream revealed that the transport of all chemical constituents was dominated by conservative mixing with tributaries and ground water; however, non-conservative processes resulted in significant Ca2+ losses during the 13-month period after CaCO3 treatment. Comparison of substrate samples from the buffered outlet stream with those from its untreated tributaries showed that the percentage of cation-exchange sites occupied by Ca2+, as well as non-exchangeable Ca, were higher in the outlet-stream substrate than in tributary-stream substrate. Mass-balance data for Ca2+, H+, AlIM, and DOC revealed net downstream losses of these constituents and indicated that a reasonable set of hypothesized reactions involving AlIM, HCO3-, Ca2+, SO42-, NO3-7, and DOC could have caused the measured changes in stream acid/base chemistry. In the summer, the sharp decrease in ANC continued despite significant downstream decreases in SO42- concentrations. After CaCO3 treatment, reduction of SO42- was only a minor contributor to ANC changes relative to those caused by Ca2+ dilution from acidic tributaries and acidic ground water, and Ca2+ interactions with stream substrate. ?? 1996 Kluwer Academic Publishers.

Integrating DNA strand displacement circuitry to the nonlinear hybridization chain reaction.

PubMed

Zhang, Zhuo; Fan, Tsz Wing; Hsing, I-Ming

2017-02-23

Programmable and modular attributes of DNA molecules allow one to develop versatile sensing platforms that can be operated isothermally and enzyme-free. In this work, we present an approach to integrate upstream DNA strand displacement circuits that can be turned on by a sequence-specific microRNA analyte with a downstream nonlinear hybridization chain reaction for a cascading hyperbranched nucleic acid assembly. This system provides a two-step amplification strategy for highly sensitive detection of the miRNA analyte, conducive for multiplexed detection. Multiple miRNA analytes were tested with our integrated circuitry using the same downstream signal amplification setting, showing the decoupling of nonlinear self-assembly with the analyte sequence. Compared with the reported methods, our signal amplification approach provides an additional control module for higher-order DNA self-assembly and could be developed into a promising platform for the detection of critical nucleic-acid based biomarkers.

Genetic variation in insulin-induced kinase signaling

PubMed Central

Wang, Isabel Xiaorong; Ramrattan, Girish; Cheung, Vivian G

2015-01-01

Individual differences in sensitivity to insulin contribute to disease susceptibility including diabetes and metabolic syndrome. Cellular responses to insulin are well studied. However, which steps in these response pathways differ across individuals remains largely unknown. Such knowledge is needed to guide more precise therapeutic interventions. Here, we studied insulin response and found extensive individual variation in the activation of key signaling factors, including ERK whose induction differs by more than 20-fold among our subjects. This variation in kinase activity is propagated to differences in downstream gene expression response to insulin. By genetic analysis, we identified cis-acting DNA variants that influence signaling response, which in turn affects downstream changes in gene expression and cellular phenotypes, such as protein translation and cell proliferation. These findings show that polymorphic differences in signal transduction contribute to individual variation in insulin response, and suggest kinase modulators as promising therapeutics for diseases characterized by insulin resistance. PMID:26202599

Downscaling catchment scale flood risk to contributing sub-catchments to determine the optimum location for flood management.

NASA Astrophysics Data System (ADS)

Pattison, Ian; Lane, Stuart; Hardy, Richard; Reaney, Sim

2010-05-01

The recent increase in flood frequency and magnitude has been hypothesised to have been caused by either climate change or land management. Field scale studies have found that changing land management practices does affect local runoff and streamflow, but upscaling these effects to the catchment scale continues to be problematic, both conceptually and more importantly methodologically. The impact on downstream flood risk is highly dependent upon where the changes are in the catchment, indicating that some areas of the catchment are more important in determining downstream flood risk than others. This is a major flaw in the traditional approach to studying the effect of land use on downstream flood risk: catchment scale hydrological models, which treat every cell in the model equally. We are proposing an alternative ideological approach for doing flood management research, which is underpinned by downscaling the downstream effect (problem i.e. flooding) to the upstream causes (contributing sub-catchments). It is hoped that this approach could have several benefits over the traditional upscaling approach. Firstly, it provides an efficient method to prioritise areas for land use management changes to be implemented to reduce downstream flood risk. Secondly, targets for sub-catchment hydrograph change can be determined which will deliver the required downstream effect. Thirdly, it may be possible to detect the effect of land use changes in upstream areas on downstream flood risk, by weighting the areas of most importance in hydrological models. Two methods for doing this downscaling are proposed; 1) data-based statistical analysis; and 2) hydraulic modelling-based downscaling. These will be outlined using the case study of the River Eden, Cumbria, NW England. The data-based methodology uses the timing and magnitude of floods for each sub-catchment. Principal components analysis (PCA) is used to simplify sub-catchment interactions and optimising stepwise regression is used to predict downstream flood magnitude from the significant principal components. Two particular sub-catchments, the Eamont and the Upper Eden were highlighted as explaining the highest proportion of downstream flood risk, with 21.0% and 19.6% respectively. This approach uses the concept of data mining, whereby commonly available discharge data is used in an innovative way to learn about catchment behaviour. An alternative downscaling approach is hydraulic modelling whereby the input hydrographs from each tributary are changed in turn, both in terms of the magnitudes and the timing of the flows. This basic scenario testing approach can be used to assess the sensitivity of downstream flood risk to upstream contributing tributaries. This approach also highlighted the Upper Eden and Eamont as the most sensitive sub-catchments. A 25% reduction in the flows from these sub-catchments resulted in a 33.1cm and 21.9cm stage reduction downstream respectively, while an 8 hour delay of the peak flow caused a 32.3cm and 27.4cm decrease in downstream stage respectively. This alternative flood management approach is not a replacement to traditional hydrological modelling (upscaling), but a pre-step which allows for more focussed and informed investigation of land management scenarios, in the area where they are most likely to have beneficial impacts on downstream flooding.

Molecular mechanism of ERK dephosphorylation by striatal-enriched protein tyrosine phosphatase (STEP)

PubMed Central

Li, Hui; Li, Kang-shuai; Su, Jing; Chen, Lai-Zhong; Xu, Yun-Fei; Wang, Hong-Mei; Gong, Zheng; Cui, Guo-Ying; Yu, Xiao; Wang, Kai; Yao, Wei; Xin, Tao; Li, Min-Yong; Xiao, Kun-Hong; An, Xiao-fei; Huo, Yuqing; Xu, Zhi-gang; Sun, Jin-Peng; Pang, Qi

2013-01-01

Striatal-enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal-regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho-ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho-ERK by STEP is not known. Therefore, we examined STEP activity toward pNPP, phospho-tyrosine-containing peptides, and the full-length phospho-ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N-terminal regulatory region and key residues in its active site. Specifically, both KIM and KIS of STEP were required for ERK interaction. In addition to the N-terminal KIS region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho-ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho-ERK peptide sequence through its active site, and the contact of STEP F311 with phospho-ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP-ERK recognition, which could serve as a potential therapy for neurological disorders. PMID:24117863

STRIATAL-ENRICHED PROTEIN TYROSINE PHOSPHATASE (STEP) KNOCKOUT MICE HAVE ENHANCED HIPPOCAMPAL MEMORY

PubMed Central

Venkitaramani, Deepa V.; Moura, Paula J.; Picciotto, Marina R.; Lombroso, Paul J.

2011-01-01

STEP is a brain-specific phosphatase that opposes synaptic strengthening by the regulation of key synaptic signaling proteins. Previous studies suggest a possible role for STriatal-Enriched protein tyrosine Phosphatase (STEP) in learning and memory. To demonstrate the functional importance of STEP in learning and memory, we generated STEP knockout (KO) mice and examined the effect of deletion of STEP on behavioral performance, as well as the phosphorylation and expression of its substrates. Here we report that loss of STEP leads to significantly enhanced performance in hippocampal-dependent learning and memory tasks. In addition, STEP KO mice displayed greater dominance behavior, although they were normal in their motivation, motor coordination, visual acuity and social interactions. STEP KO mice displayed enhanced tyrosine phosphorylation of extracellular-signal regulated kinase 1/2 (ERK1/2), the NR2B subunit of the N-methyl-D-aspartate receptor (NMDAR), Proline-rich tyrosine kinase (Pyk2), as well as an increased phosphorylation of ERK1/2 substrates. Concomitant to the increased phosphorylation of NR2B, synaptosomal expression of NR1/NR2B NMDARs was increased in STEP KO mice, as was the GluR1/GluR2 containing α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPAR), providing a potential molecular mechanism for the improved cognitive performance. The data support a role for STEP in the regulation of synaptic strengthening. The absence of STEP improves cognitive performance, and may do so by the regulation of downstream effectors necessary for synaptic transmission. PMID:21501258

Waste Minimization Study on Pyrochemical Reprocessing Processes

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

Boussier, H.; Conocar, O.; Lacquement, J.

2006-07-01

Ideally a new pyro-process should not generate more waste, and should be at least as safe and cost effective as the hydrometallurgical processes currently implemented at industrial scale. This paper describes the thought process, the methodology and some results obtained by process integration studies to devise potential pyro-processes and to assess their capability of achieving this challenging objective. As example the assessment of a process based on salt/metal reductive extraction, designed for the reprocessing of Generation IV carbide spent fuels, is developed. Salt/metal reductive extraction uses the capability of some metals, aluminum in this case, to selectively reduce actinide fluoridesmore » previously dissolved in a fluoride salt bath. The reduced actinides enter the metal phase from which they are subsequently recovered; the fission products remain in the salt phase. In fact, the process is not so simple, as it requires upstream and downstream subsidiary steps. All these process steps generate secondary waste flows representing sources of actinide leakage and/or FP discharge. In aqueous processes the main solvent (nitric acid solution) has a low boiling point and evaporate easily or can be removed by distillation, thereby leaving limited flow containing the dissolved substance behind to be incorporated in a confinement matrix. From the point of view of waste generation, one main handicap of molten salt processes, is that the saline phase (fluoride in our case) used as solvent is of same nature than the solutes (radionuclides fluorides) and has a quite high boiling point. So it is not so easy, than it is with aqueous solutions, to separate solvent and solutes in order to confine only radioactive material and limit the final waste flows. Starting from the initial block diagram devised two years ago, the paper shows how process integration studies were able to propose process fittings which lead to a reduction of the waste variety and flows leading at an 'ideal' new block diagram allowing internal solvent recycling, and self eliminating reactants. This new flowsheet minimizes the quantity of inactive inlet flows that would have inevitably to be incorporated in a final waste form. The study identifies all knowledge gaps to be filled and suggest some possible R and D issues to confirm or infirm the feasibility of the proposed process fittings. (authors)« less

The Splicing ATPase Prp43p Is a Component of Multiple Preribosomal Particles

PubMed Central

Lebaron, Simon; Froment, Carine; Fromont-Racine, Micheline; Rain, Jean-Christophe; Monsarrat, Bernard; Caizergues-Ferrer, Michèle; Henry, Yves

2005-01-01

Prp43p is a putative helicase of the DEAH family which is required for the release of the lariat intron from the spliceosome. Prp43p could also play a role in ribosome synthesis, since it accumulates in the nucleolus. Consistent with this hypothesis, we find that depletion of Prp43p leads to accumulation of 35S pre-rRNA and strongly reduces levels of all downstream pre-rRNA processing intermediates. As a result, the steady-state levels of mature rRNAs are greatly diminished following Prp43p depletion. We present data arguing that such effects are unlikely to be solely due to splicing defects. Moreover, we demonstrate by a combination of a comprehensive two-hybrid screen, tandem-affinity purification followed by mass spectrometry, and Northern analyses that Prp43p is associated with 90S, pre-60S, and pre-40S ribosomal particles. Prp43p seems preferentially associated with Pfa1p, a novel specific component of pre-40S ribosomal particles. In addition, Prp43p interacts with components of the RNA polymerase I (Pol I) transcription machinery and with mature 18S and 25S rRNAs. Hence, Prp43p might be delivered to nascent 90S ribosomal particles during pre-rRNA transcription and remain associated with preribosomal particles until their final maturation steps in the cytoplasm. Our data also suggest that the ATPase activity of Prp43p is required for early steps of pre-rRNA processing and normal accumulation of mature rRNAs. PMID:16227579

Application of aqueous two-phase micellar system to improve extraction of adenoviral particles from cell lysate.

PubMed

Molino, João Vitor Dutra; Lopes, André Moreni; Viana Marques, Daniela de Araújo; Mazzola, Priscila Gava; da Silva, Joas Lucas; Hirata, Mario Hiroyuki; Hirata, Rosário Dominguez Crespo; Gatti, Maria Silvia Viccari; Pessoa, Adalberto

2017-12-04

Viral vectors are important in medical approaches, such as disease prevention and gene therapy, and their production depends on efficient prepurification steps. In the present study, an aqueous two-phase micellar system (ATPMS) was evaluated to extract human adenovirus type 5 particles from a cell lysate. Adenovirus was cultured in human embryonic kidney 293 (HEK-293) cells to a concentration of 1.4 × 10 10 particles/mL. Cells were lysed, and the system formed by direct addition of Triton X-114 in a 2 3 full factorial design with center points. The systems were formed with Triton X-114 at a final concentration of 1.0, 6.0, and 11.0% (w/w), cell lysate pH of 6.0, 6.5, and 7.0, and incubation temperatures at 33, 35, and 37 °C. Adenovirus particles recovered from partition phases were measured by qPCR. The best system condition was with 11.0% (w/w) of Triton X-114, a cell lysate pH of 7.0, and an incubation temperature at 33 °C, yielding 3.51 × 10 10 adenovirus particles/mL, which increased the initial adenovirus particles concentration by 2.3-fold, purifying it by 2.2-fold from the cell lysate, and removing cell debris. In conclusion, these results demonstrated that the use of an aqueous two-phase micellar system in the early steps of downstream processing could improve viral particle extraction from cultured cells while integrating clarification, concentration, and prepurification steps. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Stability of spanwise-modulated flows behind backward-facing steps

NASA Astrophysics Data System (ADS)

Boiko, A. V.; Dovgal, A. V.; Sorokin, A. M.

2017-10-01

An overview and synthesis of researches on development of local vortical disturbances in laminar separated flows downstream of backward-facing steps, in which the velocity field depends essentially on two variables are given. Peculiarities of transition to turbulence in such spatially inhomogeneous separated zones are discussed. The experimental data are supplemented by the linear stability characteristics of model velocity profiles of the separated flow computed using both the classical local formulation and the nonlocal approach based on the Floquet theory for partial differential equations with periodic coefficients. The results clarify the response of the local separated flows to their modulation with stationary geometrical and temperature inhomogeneities. The results can be useful for the development of new methods of laminar separation control.

Improving efficiency and safety in external beam radiation therapy treatment delivery using a Kaizen approach.

PubMed

Kapur, Ajay; Adair, Nilda; O'Brien, Mildred; Naparstek, Nikoleta; Cangelosi, Thomas; Zuvic, Petrina; Joseph, Sherin; Meier, Jason; Bloom, Beatrice; Potters, Louis

Modern external beam radiation therapy treatment delivery processes potentially increase the number of tasks to be performed by therapists and thus opportunities for errors, yet the need to treat a large number of patients daily requires a balanced allocation of time per treatment slot. The goal of this work was to streamline the underlying workflow in such time-interval constrained processes to enhance both execution efficiency and active safety surveillance using a Kaizen approach. A Kaizen project was initiated by mapping the workflow within each treatment slot for 3 Varian TrueBeam linear accelerators. More than 90 steps were identified, and average execution times for each were measured. The time-consuming steps were stratified into a 2 × 2 matrix arranged by potential workflow improvement versus the level of corrective effort required. A work plan was created to launch initiatives with high potential for workflow improvement but modest effort to implement. Time spent on safety surveillance and average durations of treatment slots were used to assess corresponding workflow improvements. Three initiatives were implemented to mitigate unnecessary therapist motion, overprocessing of data, and wait time for data transfer defects, respectively. A fourth initiative was implemented to make the division of labor by treating therapists as well as peer review more explicit. The average duration of treatment slots reduced by 6.7% in the 9 months following implementation of the initiatives (P = .001). A reduction of 21% in duration of treatment slots was observed on 1 of the machines (P < .001). Time spent on safety reviews remained the same (20% of the allocated interval), but the peer review component increased. The Kaizen approach has the potential to improve operational efficiency and safety with quick turnaround in radiation therapy practice by addressing non-value-adding steps characteristic of individual department workflows. Higher effort opportunities are identified to guide continual downstream quality improvements. Copyright © 2017 American Society for Radiation Oncology. Published by Elsevier Inc. All rights reserved.

Research on the resurrection evolution mechanism of Gendakan ancient landslide in the upstream on Lancang River, China

NASA Astrophysics Data System (ADS)

H, D.

2017-12-01

The Gendakan ancient landslide is located on the West bank of the upstream on Lancang River and about 4 km downstream from the Gushui hydropower station dam site. The ancient landslide is 850 m long and 700 m wide, the drill cores show that the maximum thickness of the landslide body is 107 m, with a mean thickness of approximately 80 m. Thus, the overall volume is about 3000×104m3. At present, the landslide has obvious deformation and failure signs, the leading edge is collapsing step by step. Once the landslide is unstable, it will affect the construction and operation of the Gushui hydropower station. In this paper, the development characteristics of the landslide accumulation body and the characteristics of the resurrection deformation are summarized in detail from the regional geological environment of the Gandakan landslide accumulation body. The three-dimensional geological model is established to analyze the stress and strain, displacement change and deformation failure characteristics and further evaluate its resurrection evolution trend , Combined with the developmental characteristics of the typical rock mass in the nearshore slope of the engineering area, analyzes the process of the resurrection and evolution of the rooted landslide accumulation. The FLAC-3D finite difference software was used to analyze the shear strain increment, displacement and plastic zone of the landslide accumulation body under natural conditions and rainfall conditions. The results show: the Gendakan landslide is stable in the natural state, and its deformation and failure are mainly caused by the tensile and shearing of the surface, under the rainfall condition, the local deformation and failure of the landslide accumulation body is obvious and the resurrection deformation Intensified. The resurrection evolutionary process of Gendakan ancient landslide includes three steps below. 1) The landslide body trailing edge creep cracking, leading edge shear deformation. 2) Sliding surface, accelerate the decline. 3)Disintegration of collision and impact into the dam.

Downstream mixing of sediment and tracers in agricultural catchments: Evidence of changing sediment sources and fluvial processes?

NASA Astrophysics Data System (ADS)

Ralph, Timothy; Wethered, Adam; Smith, Hugh; Heijnis, Henk

2014-05-01

Land clearance, soil tillage and grazing in agricultural catchments have liberated sediment and altered hydrological connectivity between hillslopes and channels, leading to increased sediment availability, mobilisation and delivery to rivers. The type and amount of sediment supplied to rivers is critical for fluvial geomorphology and aquatic ecosystem health. Contemporary sediment dynamics are routinely investigated using environmental radionuclides such as caesium-137 (Cs-137) and excess lead-210 (Pb-210ex), which can provide information regarding sediment source types and fluvial processes if sediment sources can be distinguished from one another and mixing models applied to representative samples. However, downstream transport, mixing and dilution of radionuclide-labelled sediment (especially from sources with low initial concentrations) can obliterate the tracer signal; sometimes before anything of geomorphological importance happens in the catchment. Can these findings be used as evidence of sediment source variations and fluvial processes when the limits of detection (of Cs-137 in particular) are being exceeded so rapidly downstream? Sediment sources and downstream sediment dynamics were investigated in Coolbaggie Creek, a major supplier of sediment to the Macquarie River in an agricultural catchment with temperate to semi-arid climate in Australia. Radionuclides were used to discriminate between the <63 micron fraction of sediment sources including forested topsoils (Cs-137 11.28 +/- 0.75 Bq/kg; Pb-210ex 181.87 +/- 20.00 Bq/kg), agricultural topsoils (Cs-137 3.21 +/- 0.26 Bq/kg; Pb-210ex 29.59 +/- 10.94 Bq/kg) and sub-soils from channel banks and gullies (Cs-137 1.45 +/- 0.47 Bq/kg; Pb-210ex 4.67 +/- 1.93 Bq/kg). Within the trunk stream, suspended sediment, organic matter and Cs-137 and Pb-210ex concentrations declined downstream. Results from a mixing model suggest that agricultural topsoils account for 95% of fine sediment entering the channel in the upper reach (<10 km long), while sub-soils account for 90 to 100% of sediment entering and being transported in the remaining ~50 km of the system. This shift in dominant sediment source material coincided with a large increase in channel cross sectional area (~20 to >200 m2) downstream, with channel expansion and gullies contributing fine sediment to the system. A lack of topsoil being supplied to the channel suggests minimal lateral connectivity between the catchment and the trunk stream in all areas apart from the upper catchment. The enlargement and entrenchment of the channel downstream has also resulted in lateral disconnection between the channel and floodplain. In this case, a rapid reduction in radionuclide concentrations downstream does coincide with hydrogeomorphic changes, supporting their use for studying short-term sediment dynamics. These findings highlight the importance of understanding hydrogeomorphic processes and connectivity when interpreting sediment source and tracer data.

Laboratory Modelling of the Effect of Bend Orientation on the Morphological Development of Alluvial Channels

NASA Astrophysics Data System (ADS)

Good, R. G. R.; Sullivan, C.; Binns, A. D.

2017-12-01

Bend orientation, or skewness, in natural streams is often caused by riparian vegetation or underlying geology that lead to a meandering stream following a non-sinuous path. The bend orientation affects how the fluid momentum interacts with the bed and banks, which can alter the location and shape of bedforms as well as the channel planform geometry. An experimental study in a laboratory sand flume with movable bed and banks (5.6 m long, 1.9 m wide; D50 = 0.7 mm; B = 0.2m; 3 wavelengths) was carried out to quantify the effect of bend orientation on bedform development and planform changes. While previous research in the literature has found that channels with an upstream bend orientation had a less developed secondary flow than a downstream orientation, few studies on the morphological development of streams having varying bend orientation have been conducted. In total, three runs were carried out using channels with upstream, downstream, and no skewness. The runs progressed in a series of time-steps to monitor the morphological evolution of the streams with time. Sediment transport rates were quantified at the outlet, flow was measured using an ultrasonic flow meter at the inlet, flow depths were measured at the apex of the bends, and channel morphology was measured at each time step using Structure-from-Motion photogrammetry with Agisoft Photoscan. Bend orientation was found to influence the position of the point bar development as well as the locations of maximum and minimum channel migration. Relative to the bend apex, point bars tended to be positioned in the same direction as the channel skewness. Channel width showed the greatest variation with the upstream orientation, with the channel narrowing before the apex where the channel flows in the up-valley direction, and widening downstream of the apex. These results show that the channel orientation influences the morphological development of the channel bed and banks. The effect of velocity structure and turbulence regime on the morphological development in the three bend orientations was analysed by comparing morphological and flow data at each time step. Results from this research will benefit the design of future engineered channels, as certain channel orientations may be preferable for managing erosion and sediment transport within a watershed.

Strategic funding priorities in the pharmaceutical sciences allied to Quality by Design (QbD) and Process Analytical Technology (PAT).

PubMed

Aksu, Buket; De Beer, Thomas; Folestad, Staffan; Ketolainen, Jarkko; Lindén, Hans; Lopes, Joao Almeida; de Matas, Marcel; Oostra, Wim; Rantanen, Jukka; Weimer, Marco

2012-09-29

Substantial changes in Pharmaceutical R&D strategy are required to address existing issues of low productivity, imminent patent expirations and pressures on pricing. Moves towards personalized healthcare and increasing diversity in the nature of portfolios including the rise of biopharmaceuticals however have the potential to provide considerable challenges to the establishment of cost effective and robust supply chains. To guarantee product quality and surety of supply for essential medicines it is necessary that manufacturing science keeps pace with advances in pharmaceutical R&D. In this position paper, the EUFEPS QbD and PAT Sciences network make recommendations that European industry, academia and health agencies focus attention on delivering step changes in science and technology in a number of key themes. These subject areas, all underpinned by the sciences allied to QbD and PAT, include product design and development for personalized healthcare, continuous-processing in pharmaceutical product manufacture, quantitative quality risk assessment for pharmaceutical development including life cycle management and the downstream processing of biopharmaceutical products. Plans are being established to gain commitment for inclusion of these themes into future funding priorities for the Innovative Medicines Initiative (IMI). Copyright © 2012 Elsevier B.V. All rights reserved.

Adapting Wave-front Algorithms to Efficiently Utilize Systems with Deep Communication Hierarchies

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

Kerbyson, Darren J.; Lang, Michael; Pakin, Scott

2011-09-30

Large-scale systems increasingly exhibit a differential between intra-chip and inter-chip communication performance especially in hybrid systems using accelerators. Processorcores on the same socket are able to communicate at lower latencies, and with higher bandwidths, than cores on different sockets either within the same node or between nodes. A key challenge is to efficiently use this communication hierarchy and hence optimize performance. We consider here the class of applications that contains wavefront processing. In these applications data can only be processed after their upstream neighbors have been processed. Similar dependencies result between processors in which communication is required to pass boundarymore » data downstream and whose cost is typically impacted by the slowest communication channel in use. In this work we develop a novel hierarchical wave-front approach that reduces the use of slower communications in the hierarchy but at the cost of additional steps in the parallel computation and higher use of on-chip communications. This tradeoff is explored using a performance model. An implementation using the Reverse-acceleration programming model on the petascale Roadrunner system demonstrates a 27% performance improvement at full system-scale on a kernel application. The approach is generally applicable to large-scale multi-core and accelerated systems where a differential in system communication performance exists.« less

Qualis-SIS: automated standard curve generation and quality assessment for multiplexed targeted quantitative proteomic experiments with labeled standards.

PubMed

Mohammed, Yassene; Percy, Andrew J; Chambers, Andrew G; Borchers, Christoph H

2015-02-06

Multiplexed targeted quantitative proteomics typically utilizes multiple reaction monitoring and allows the optimized quantification of a large number of proteins. One challenge, however, is the large amount of data that needs to be reviewed, analyzed, and interpreted. Different vendors provide software for their instruments, which determine the recorded responses of the heavy and endogenous peptides and perform the response-curve integration. Bringing multiplexed data together and generating standard curves is often an off-line step accomplished, for example, with spreadsheet software. This can be laborious, as it requires determining the concentration levels that meet the required accuracy and precision criteria in an iterative process. We present here a computer program, Qualis-SIS, that generates standard curves from multiplexed MRM experiments and determines analyte concentrations in biological samples. Multiple level-removal algorithms and acceptance criteria for concentration levels are implemented. When used to apply the standard curve to new samples, the software flags each measurement according to its quality. From the user's perspective, the data processing is instantaneous due to the reactivity paradigm used, and the user can download the results of the stepwise calculations for further processing, if necessary. This allows for more consistent data analysis and can dramatically accelerate the downstream data analysis.

Next generation calmodulin affinity purification: Clickable calmodulin facilitates improved protein purification

PubMed Central

Kinzer-Ursem, Tamara L.

2018-01-01

As the proteomics field continues to expand, scientists are looking to integrate cross-disciplinary tools for studying protein structure, function, and interactions. Protein purification remains a key tool for many characterization studies. Calmodulin (CaM) is a calcium-binding messenger protein with over a hundred downstream binding partners, and is involved in a host of physiological processes, from learning and memory to immune and cardiac function. To facilitate biophysical studies of calmodulin, researchers have designed a site-specific labeling process for use in bioconjugation applications while maintaining high levels of protein activity. Here, we present a platform for selective conjugation of calmodulin directly from clarified cell lysates under bioorthogonal reaction conditions. Using a chemoenzymatically modified calmodulin, we employ popular click chemistry reactions for the conjugation of calmodulin to Sepharose resin, thereby streamlining a previously multi-step purification and conjugation process. We show that this “next-generation” calmodulin-Sepharose resin is not only easy to produce, but is also able to purify more calmodulin-binding proteins per volume of resin than traditional calmodulin-Sepharose resins. We expect these methods to be translatable to other proteins of interest and to other conjugation applications such as surface-based assays for the characterization of protein-protein interaction dynamics. PMID:29864125

Standardization and quality management in next-generation sequencing.

PubMed

Endrullat, Christoph; Glökler, Jörn; Franke, Philipp; Frohme, Marcus

2016-09-01

DNA sequencing continues to evolve quickly even after > 30 years. Many new platforms suddenly appeared and former established systems have vanished in almost the same manner. Since establishment of next-generation sequencing devices, this progress gains momentum due to the continually growing demand for higher throughput, lower costs and better quality of data. In consequence of this rapid development, standardized procedures and data formats as well as comprehensive quality management considerations are still scarce. Here, we listed and summarized current standardization efforts and quality management initiatives from companies, organizations and societies in form of published studies and ongoing projects. These comprise on the one hand quality documentation issues like technical notes, accreditation checklists and guidelines for validation of sequencing workflows. On the other hand, general standard proposals and quality metrics are developed and applied to the sequencing workflow steps with the main focus on upstream processes. Finally, certain standard developments for downstream pipeline data handling, processing and storage are discussed in brief. These standardization approaches represent a first basis for continuing work in order to prospectively implement next-generation sequencing in important areas such as clinical diagnostics, where reliable results and fast processing is crucial. Additionally, these efforts will exert a decisive influence on traceability and reproducibility of sequence data.

The role of anaerobic digestion in the emerging energy economy.

PubMed

Batstone, Damien John; Virdis, Bernardino

2014-06-01

Anaerobic digestion is the default process for biological conversion of residue organics to renewable energy and biofuel in the form of methane. However, its scope of application is expanding, due to availability of new technologies, and the emerging drivers of energy and nutrient conservation and recovery. Here, we outline two of these new application areas, namely wastewater nutrient and energy recovery, and generation of value added chemicals through mixed culture biotechnology. There exist two options for nutrient and energy recovery from domestic wastewater: low energy mainline and partition-release-recovery. Both are heavily dependent on anaerobic digestion as an energy generating and nutrient release step, and have been enabled by new technologies such as low emission anaerobic membrane processes. The area of mixed culture biotechnology has been previously identified as a key industrial opportunity, but is now moving closer to application due application of existing and new technologies. As well as acting as a core technology option in bioproduction, anaerobic digestion has a key role in residual waste valorization and generation of energy for downstream processing. These new application areas and technologies are emerging simultaneously with substantial advances in knowledge of underlying mechanisms such as electron transfer, understanding of which is critical to development of the new application areas. Copyright © 2014 Elsevier Ltd. All rights reserved.

Microfluidic Device for Studying Tumor Cell Extravasation in Cancer Metastasis

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

Lin, Henry K; Thundat, Thomas George; Evans III, Boyd Mccutchen

Metastasis is the process by which cancer spreads to form secondary tumors at downstream locations throughout the body. This uncontrolled spreading is the leading cause of death in patients with epithelial cancers and is the main reason that suppressing and targeting cancer has proven to be so challenging. Tumor cell extravasation is one of the key steps in cancer s progression towards a metastatic state. This occurs when circulating tumor cells found within the blood stream are able to transmigrate through the endothelium lining and basement membrane of the vasculature to form metastatic tumors at secondary sites within the body.more » Predicting the likelihood of this occurrence in patients, or being able to determine specific markers involved in this process could lead to preventative measures targeting these types of cancer; moreover, this may lead to the discovery of novel anti-metastatic drugs. We have developed a microfluidic device that has shown the extravasation of fluorescently labeled tumor cells across an endothelial cell lined membrane coated with matrigel followed by the formation of colonies. This device provides the advantages of combining a controlled environment, mimicking that found within the body, with real-time monitoring capabilities allowing for the study of these biomarkers and cellular interactions along with other potential mechanisms involved in the process of extravasation.« less

On-chip integrated labelling, transport and detection of tumour cells.

PubMed

Woods, Jane; Docker, Peter T; Dyer, Charlotte E; Haswell, Stephen J; Greenman, John

2011-11-01

Microflow cytometry represents a promising tool for the investigation of diagnostic and prognostic cellular cancer markers, particularly if integrated within a device that allows primary cells to be freshly isolated from the solid tumour biopsies that more accurately reflect patient-specific in vivo tissue microenvironments at the time of staining. However, current tissue processing techniques involve several sequential stages with concomitant cell losses, and as such are inappropriate for use with small biopsies. Accordingly, we present a simple method for combined antibody-labelling and dissociation of heterogeneous cells from a tumour mass, which reduces the number of processing steps. Perfusion of ex vivo tissue at 4°C with antibodies and enzymes slows cellular activity while allowing sufficient time for the diffusion of minimally active enzymes. In situ antibody-labelled cells are then dissociated at 37°C from the tumour mass, whereupon hydrogel-filled channels allow the release of relatively low cell numbers (<1000) into a biomimetic microenvironment. This novel approach to sample processing is then further integrated with hydrogel-based electrokinetic transport of the freshly liberated fluorescent cells for downstream detection. It is anticipated that this integrated microfluidic methodology will have wide-ranging biomedical and clinical applications. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantifying sediment dynamics on alluvial fans, Iglesia basin, south Central Argentine Andes

NASA Astrophysics Data System (ADS)

Harries, Rebekah; Kirstein, Linda; Whittaker, Alex; Attal, Mikael; Peralta, Silvio

2017-04-01

Qualitative interpretations of environmental change drawn from alluvial fan stratigraphy typically tie the deposition of greater volumes of coarser sediment to wetter climatic periods. For example, step changes in sediment flux and discharge associated with glacial-interglacial cycles are often linked to the progradation and back stepping of a fan's toe (Harvey et al, 2002). Indeed, more recent quantitative stratigraphic models demonstrate changes in the volume and calibre of sediment fluxed from an uplifted catchment can produce predictable shifts in the rate at which fluvial deposits fine downstream (Duller et al. 2010, Armitage et al. 2011). These interpretations, however, make three important assumptions: 1) the volume and calibre of the sediment transferred from an eroding mountain belt to a depositional basin is directly related to climate through some value of time-averaged discharge or catchment wetness; 2) lateral sources of sediment, such as tributaries, do not significantly influence the pattern of deposition in a basin and, similarly, 3) the reworking of older fan surfaces is minimal and does not impact the depositional pattern of younger deposits. Here we demonstrate each of these assumptions underestimates the importance of variance in transportable grain sizes in influencing the local and basin-wide deposited grain size trends. Using the Iglesia basin in the Argentine south Central Andes as a natural laboratory, we compare three large, adjacent, alluvial fan systems whose catchments experience the same background tectonic and climatic forcing. We find regional climate forcing is not expressed uniformly in the downstream grain size fining rates of their modern systems. Furthermore, we observe the variance in transportable grain sizes supplied from each primary catchment and the variance of material introduced by tributaries and fan surfaces downstream can act as first order controls on the rate of downstream fining. We also raise the importance of considering factors such as climate storminess and degree of glacial cover in having a dominant control on the variance of sediment released. These findings have significant implications for our ability to invert the fluvial stratigraphy for climatically driven changes in discharge and highlight a need to quantify the impact of sediment dynamics on modern systems so that we may better understand the limitations in applying quantitative models to ancient stratigraphy.

Status of downstream fish passage at hydroelectric projects in the northeast, USA

USGS Publications Warehouse

Odeh, Mufeed; Orvis, Curtis

1997-01-01

In the northeastern United States several guidance, protection, and conveyance methods have been employed to assist downstream migrating fish. Overlay racks, standard bar racks with close spacing, louvers, curtain walls, guide walls, netting, and other means have been used to guide and protect fish from entrainment. The design process of these facilities comprises consideration of various factors, including flow approach, attraction flow, guidance and protection devices, bypass location, conveyance mechanism, and plunge pool conditions. This paper presents the status of the design criteria for downstream fish passage facilities at hydroelectric sites in the northeast part of the United States. Examples of existing facilities are given.

The Relative Frequency of Rossby Wave Train Triggering Mechanisms Associated with Downstream Development Over Europe

DTIC Science & Technology

2014-06-01

subsequent to the beginning of the transformation stage. Means ± standard deviation of SLP are listed for each step of transformation, and for the...labeled every 6 dam, and solid contours are SLP every 4 hPa (unpublished figure provided by H. Archambault...53.  A 1-day SLP anomaly (color shading, hPa) from the NCEP operational dataset for 31 Oct 2010. (Image provided by the NOAA/ESRL Physical Sciences

Influence of reservoirs on solute transport: A regional-scale approach

USGS Publications Warehouse

Kelly, V.J.

2001-01-01

Regional transport of water and dissolved constituents through heavily regulated river systems is influenced by the presence of reservoirs. Analysis of seasonal patterns in solute fluxes for salinity and nutrients indicates that in-reservoir processes within large storage reservoirs in the Rio Grande and Colorado basins (southwestern USA) are superimposed over the underlying watershed processes that predominate in relatively unregulated stream reaches. Connectivity of the aquatic system with the landscape is apparently disrupted by processes within the reservoir systems; these processes result in large changes in characteristics for solute transport that persist downstream in the absence of significant inputs. Additionally, reservoir processes may be linked for upstream/downstream reservoirs that are located relatively close in a series. In contrast, the regional effect of in-reservoir processes is negligible for solute transport through run-of-river reservoirs in the lower Columbia River (northwestern USA).

Wood and Sediment Dynamics in River Corridors

NASA Astrophysics Data System (ADS)

Wohl, E.; Scott, D.

2015-12-01

Large wood along rivers influences entrainment, transport, and storage of mineral sediment and particulate organic matter. We review how wood alters sediment dynamics and explore patterns among volumes of instream wood, sediment storage, and residual pools for dispersed pieces of wood, logjams, and beaver dams. We hypothesized that: volume of sediment per unit area of channel stored in association with wood is inversely proportional to drainage area; the form of sediment storage changes downstream; sediment storage correlates most strongly with wood load; and volume of sediment stored behind beaver dams correlates with pond area. Lack of data from larger drainage areas limits tests of these hypotheses, but analyses suggest a negative correlation between sediment volume and drainage area and a positive correlation between wood and sediment volume. The form of sediment storage in relation to wood changes downstream, with wedges of sediment upstream from jammed steps most prevalent in small, steep channels and more dispersed sediment storage in lower gradient channels. Use of a published relation between sediment volume, channel width, and gradient predicted about half of the variation in sediment stored upstream from jammed steps. Sediment volume correlates well with beaver pond area. Historically more abundant instream wood and beaver populations likely equated to greater sediment storage within river corridors. This review of the existing literature on wood and sediment dynamics highlights the lack of studies on larger rivers.

Long-range effect of cyanide on mercury methylation in a gold mining area in southern Ecuador.

PubMed

Guimaraes, Jean Remy Davée; Betancourt, Oscar; Miranda, Marcio Rodrigues; Barriga, Ramiro; Cueva, Edwin; Betancourt, Sebastián

2011-11-01

Small-scale gold mining in Portovelo-Zaruma, Southern Equador, performed by mercury amalgamation and cyanidation, yields 9-10 t of gold/annum, resulting in annual releases of around 0.65 t of inorganic mercury and 6000 t of sodium cyanide in the local river system. The release of sediments, cyanide, mercury, and other metals present in the ore such as lead, manganese and arsenic significantly reduces biodiversity downstream the processing plants and enriches metals in bottom sediments and biota. However, methylmercury concentrations in sediments downstream the mining area were recently found to be one order of magnitude lower than upstream or in small tributaries. In this study we investigated cyanide, bacterial activity in water and sediment and mercury methylation potentials in sediments along the Puyango river watershed, measured respectively by in-situ spectrophotometry and incubation with (3)H-leucine and (203)Hg(2+). Free cyanide was undetectable (<1 μg·L(-1)) upstream mining activities, reached 280 μg·L(-1) a few km downstream the processing plants area and was still detectable about 100 km downstream. At stations with detectable free cyanide in unfiltered water, 50% of it was dissolved and 50% associated to suspended particles. Bacterial activity and mercury methylation in sediment showed a similar spatial pattern, inverse to the one found for free cyanide in water, i.e. with significant values in pristine upstream sampling points (respectively 6.4 to 22 μgC·mg wet weight(-1)·h(-1) and 1.2 to 19% of total (203) Hg·gdry weight(-1)·day(-1)) and undetectable downstream the processing plants, returning to upstream values only in the most distant downstream stations. The data suggest that free cyanide oxidation was slower than would be expected from the high water turbulence, resulting in a long-range inhibition of bacterial activity and hence mercury methylation. The important mercury fluxes resultant from mining activities raise concerns about its biomethylation in coastal areas where many mangrove areas have been converted to shrimp farming. Copyright © 2011. Published by Elsevier B.V.

A cell culture-derived whole virus influenza A vaccine based on magnetic sulfated cellulose particles confers protection in mice against lethal influenza A virus infection.

PubMed

Pieler, Michael M; Frentzel, Sarah; Bruder, Dunja; Wolff, Michael W; Reichl, Udo

2016-12-07

Downstream processing and formulation of viral vaccines employs a large number of different unit operations to achieve the desired product qualities. The complexity of individual process steps involved, the need for time consuming studies towards the optimization of virus yields, and very high requirements regarding potency and safety of vaccines results typically in long lead times for the establishment of new processes. To overcome such obstacles, to enable fast screening of potential vaccine candidates, and to explore options for production of low cost veterinary vaccines a new platform for whole virus particle purification and formulation based on magnetic particles has been established. Proof of concept was carried out with influenza A virus particles produced in suspension Madin Darby canine kidney (MDCK) cells. The clarified, inactivated, concentrated, and diafiltered virus particles were bound to magnetic sulfated cellulose particles (MSCP), and directly injected into mice for immunization including positive and negative controls. We show here, that in contrast to the mock-immunized group, vaccination of mice with antigen-loaded MSCP (aMSCP) resulted in high anti-influenza A antibody responses and full protection against a lethal challenge with replication competent influenza A virus. Antiviral protection correlated with a 400-fold reduced number of influenza nucleoprotein gene copies in the lungs of aMSCP immunized mice compared to mock-treated animals, indicating the efficient induction of antiviral immunity by this novel approach. Thus, our data proved the use of MSCP for purification and formulation of the influenza vaccine to be fast and efficient, and to confer protection of mice against influenza A virus infection. Furthermore, the method proposed has the potential for fast purification of virus particles directly from bioreactor harvests with a minimum number of process steps towards formulation of low-cost veterinary vaccines, and for screening studies requiring fast purification protocols. Copyright © 2016 Elsevier Ltd. All rights reserved.

LITERATURE REVIEW FOR OXALATE OXIDATION PROCESSES AND PLUTONIUM OXALATE SOLUBILITY

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

Nash, C.

2012-02-03

A literature review of oxalate oxidation processes finds that manganese(II)-catalyzed nitric acid oxidation of oxalate in precipitate filtrate is a viable and well-documented process. The process has been operated on the large scale at Savannah River in the past, including oxidation of 20 tons of oxalic acid in F-Canyon. Research data under a variety of conditions show the process to be robust. This process is recommended for oxalate destruction in H-Canyon in the upcoming program to produce feed for the MOX facility. Prevention of plutonium oxalate precipitation in filtrate can be achieved by concentrated nitric acid/ferric nitrate sequestration of oxalate.more » Organic complexants do not appear practical to sequester plutonium. Testing is proposed to confirm the literature and calculation findings of this review at projected operating conditions for the upcoming campaign. H Canyon plans to commence conversion of plutonium metal to low-fired plutonium oxide in 2012 for eventual use in the Mixed Oxide Fuel (MOX) Facility. The flowsheet includes sequential operations of metal dissolution, ion exchange, elution, oxalate precipitation, filtration, and calcination. All processes beyond dissolution will occur in HB-Line. The filtration step produces an aqueous filtrate that may have as much as 4 M nitric acid and 0.15 M oxalate. The oxalate needs to be removed from the stream to prevent possible downstream precipitation of residual plutonium when the solution is processed in H Canyon. In addition, sending the oxalate to the waste tank farm is undesirable. This report addresses the processing options for destroying the oxalate in existing H Canyon equipment.« less

Combustion efficiency determined from wall pressure and temperature measurement in a Mach 2 combustor

NASA Technical Reports Server (NTRS)

Segal, Corin; Mcdaniel, James C.; Whitehurst, Robert B.; Krauss, Roland H.

1991-01-01

A study of transverse hydrogen injection behind a rearward facing step in a Mach 2 airflow was conducted to determine the combustion efficiency and the combustor/inlet interactions at the low temperature lean-mixture operational end of a scramjet combustor model. The fuel was injected at sonic conditions into the electrically heated airstream, which was maintained at 850 K or below. The static pressure delivered at the entrance of the combustor ranged between 0.25 to 0.5 atm. Injector configurations included single and staged injectors placed at 3 or 3-and-7 step-heights downstream of the step, respectively, with injector diameters of 1, 1.5, and 2 mm. Ignition was achieved by initially unstarting the test section. The constant area combustor and the low initial temperatures caused thermal choking and upstream interaction to occur at very low equivalence ratios. Typically, most of the fuel was burned in the recirculation region behind the step and around the jets. The effects of initial conditions (temperature and pressure), fuel-to-air dynamic pressure ratio, and boundaries (thermal vs adiabatic) are presented.

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

Tan, Eric; Talmadge, M.; Dutta, Abhijit

The U.S. Department of Energy (DOE) promotes research for enabling cost-competitive liquid fuels production from lignocellulosic biomass feedstocks. The research is geared to advance the state of technology (SOT) of biomass feedstock supply and logistics, conversion, and overall system sustainability. As part of their involvement in this program, the National Renewable Energy Laboratory (NREL) and the Pacific Northwest National Laboratory (PNNL) investigate the economics of conversion pathways through the development of conceptual biorefinery process models. This report describes in detail one potential conversion process for the production of high octane gasoline blendstock via indirect liquefaction (IDL). The steps involve themore » conversion of biomass to syngas via indirect gasification followed by gas cleanup and catalytic syngas conversion to a methanol intermediate; methanol is then further catalytically converted to high octane hydrocarbons. The conversion process model leverages technologies previously advanced by research funded by the Bioenergy Technologies Office (BETO) and demonstrated in 2012 with the production of mixed alcohols from biomass. Biomass-derived syngas cleanup via tar and hydrocarbons reforming was one of the key technology advancements as part of that research. The process described in this report evaluates a new technology area with downstream utilization of clean biomass-syngas for the production of high octane hydrocarbon products through a methanol intermediate, i.e., dehydration of methanol to dimethyl ether (DME) which subsequently undergoes homologation to high octane hydrocarbon products.« less

A multiphysics and multiscale model for low frequency electromagnetic direct-chill casting

NASA Astrophysics Data System (ADS)

Košnik, N.; Guštin, A. Z.; Mavrič, B.; Šarler, B.

2016-03-01

Simulation and control of macrosegregation, deformation and grain size in low frequency electromagnetic (EM) direct-chill casting (LFEMC) is important for downstream processing. Respectively, a multiphysics and multiscale model is developed for solution of Lorentz force, temperature, velocity, concentration, deformation and grain structure of LFEMC processed aluminum alloys, with focus on axisymmetric billets. The mixture equations with lever rule, linearized phase diagram, and stationary thermoelastic solid phase are assumed, together with EM induction equation for the field imposed by the coil. Explicit diffuse approximate meshless solution procedure [1] is used for solving the EM field, and the explicit local radial basis function collocation method [2] is used for solving the coupled transport phenomena and thermomechanics fields. Pressure-velocity coupling is performed by the fractional step method [3]. The point automata method with modified KGT model is used to estimate the grain structure [4] in a post-processing mode. Thermal, mechanical, EM and grain structure outcomes of the model are demonstrated. A systematic study of the complicated influences of the process parameters can be investigated by the model, including intensity and frequency of the electromagnetic field. The meshless solution framework, with the implemented simplest physical models, will be further extended by including more sophisticated microsegregation and grain structure models, as well as a more realistic solid and solid-liquid phase rheology.

Economic feasibility and environmental impact of synthetic spider silk production from escherichia coli.

PubMed

Edlund, Alan M; Jones, Justin; Lewis, Randolph; Quinn, Jason C

2018-05-25

Major ampullate spider silk represents a promising protein-based biomaterial with diverse commercial potential ranging from textiles to medical devices due to its excellent physical and thermal properties. Recent advancements in synthetic biology have facilitated the development of recombinant spider silk proteins from Escherichia coli (E. coli). This study specifically investigates the economic feasibility and environmental impact of synthetic spider silk manufacturing. Pilot scale data was used to validate an engineering process model that includes all of the required sub-processing steps for synthetic fiber manufacture: production, harvesting, purification, drying, and spinning. Modeling was constructed modularly to support assessment of alternative downstream processing technologies. The techno-economic analysis indicates a minimum sale price from pioneer and optimized E. coli plants of $761 kg -1 and $23 kg -1 with greenhouse gas emissions of 572 kg CO 2-eq. kg -1 and 55 kg CO 2-eq. kg -1 , respectively. Elevated costs and emissions from the pioneer plant can be directly tied to the high material consumption and low protein yield. Decreased production costs associated with the optimized plant includes improved protein yield, process optimization, and an N th plant assumption. Discussion focuses on the commercial potential of spider silk, the production performance requirements for commercialization, and the impact of alternative technologies on the system. Copyright © 2018 Elsevier B.V. All rights reserved.

Jet formation of SF6 bubble induced by incident and reflected shock waves

NASA Astrophysics Data System (ADS)

Zhu, Yuejin; Yu, Lei; Pan, Jianfeng; Pan, Zhenhua; Zhang, Penggang

2017-12-01

The computational results of two different cases on the evolution of the shock-SF6 heavy bubble interaction are presented. The shock focusing processes and jet formation mechanisms are analyzed by using the high resolution of computation schemes, and the influence of reflected shock waves is also investigated. It is concluded that there are two steps in the shock focusing process behind the incident shock wave, and the density and pressure values increase distinctly when the shock focusing process is completed. The local high pressure and vorticities in the vicinity of the downstream pole can propel the formation of the jet behind the incident shock wave. In addition, the gas is with the rightward velocity before the reflected shock wave impinges on the bubble; therefore, the evolutions of the waves and the bubble are more complicated when the reflected shock wave impinges on the SF6 bubble. Furthermore, the different end wall distances would affect the deformation degree of the bubble before the interaction of the reflected shock wave; therefore, the different left jet formation processes are found after the impingement of reflected shock waves when L = 27 mm. The local high pressure zones in the vicinity of the left bubble interface and the impingement of different shock waves can induce the local gas to shift the rightward velocity to the leftward velocity, which can further promote the formation of jets.

Imaging immune response of skin mast cells in vivo with two-photon microscopy

NASA Astrophysics Data System (ADS)

Li, Chunqiang; Pastila, Riikka K.; Lin, Charles P.

2012-02-01

Intravital multiphoton microscopy has provided insightful information of the dynamic process of immune cells in vivo. However, the use of exogenous labeling agents limits its applications. There is no method to perform functional imaging of mast cells, a population of innate tissue-resident immune cells. Mast cells are widely recognized as the effector cells in allergy. Recently their roles as immunoregulatory cells in certain innate and adaptive immune responses are being actively investigated. Here we report in vivo mouse skin mast cells imaging with two-photon microscopy using endogenous tryptophan as the fluorophore. We studied the following processes. 1) Mast cells degranulation, the first step in the mast cell activation process in which the granules are released into peripheral tissue to trigger downstream reactions. 2) Mast cell reconstitution, a procedure commonly used to study mast cells functioning by comparing the data from wild type mice, mast cell-deficient mice, and mast-cell deficient mice reconstituted with bone marrow-derived mast cells (BMMCs). Imaging the BMMCs engraftment in tissue reveals the mast cells development and the efficiency of BMMCs reconstitution. We observed the reconstitution process for 6 weeks in the ear skin of mast cell-deficient Kit wsh/ w-sh mice by two-photon imaging. Our finding is the first instance of imaging mast cells in vivo with endogenous contrast.

Derivation of Lake Areas and Elevations for the Mackenzie Basin Using Satellite Remote Sensing

NASA Technical Reports Server (NTRS)

Birkett, Charon; Kite, Geoff

1997-01-01

Modelling hydrological processes in large watersheds flowing to the Arctic ocean is one step towards larger-scale modelling of the global water and energy cycles. Models of the Mackenzie River Basin (Northern Canada) are currently available but omit explicit routing of river flows through the three main lakes - Athabasca, Great Slave Lake and Great Bear Lake (Kite et al, 1994). These lakes occupy an area of 65,000 sq km but little gauge information is available. The levels of the lakes are only measured at a few points on the circumferences and river flows are only measured downstream. The hydraulic relationships between level/discharge and level/area/volume are uncertain. It has been previously shown that satellite remote sensing can be utilised in providing measurements of both lake surface area using imaging techniques and lake level using radar altimetry (Birkett, 1994). Here, we explore the application of these techniques to derive the lake levels and areas for the Mackenzie Basin lakes.

Molecular basis of the activity of the phytopathogen pectin methylesterase

PubMed Central

Fries, Markus; Ihrig, Jessica; Brocklehurst, Keith; Shevchik, Vladimir E; Pickersgill, Richard W

2007-01-01

We provide a mechanism for the activity of pectin methylesterase (PME), the enzyme that catalyses the essential first step in bacterial invasion of plant tissues. The complexes formed in the crystal using specifically methylated pectins, together with kinetic measurements of directed mutants, provide clear insights at atomic resolution into the specificity and the processive action of the Erwinia chrysanthemi enzyme. Product complexes provide additional snapshots along the reaction coordinate. We previously revealed that PME is a novel aspartic-esterase possessing parallel β-helix architecture and now show that the two conserved aspartates are the nucleophile and general acid-base in the mechanism, respectively. Other conserved residues at the catalytic centre are shown to be essential for substrate binding or transition state stabilisation. The preferential binding of methylated sugar residues upstream of the catalytic site, and demethylated residues downstream, drives the enzyme along the pectin molecule and accounts for the sequential pattern of demethylation produced by both bacterial and plant PMEs. PMID:17717531

A Practical Approach to Governance and Optimization of Structured Data Elements.

PubMed

Collins, Sarah A; Gesner, Emily; Morgan, Steven; Mar, Perry; Maviglia, Saverio; Colburn, Doreen; Tierney, Diana; Rocha, Roberto

2015-01-01

Definition and configuration of clinical content in an enterprise-wide electronic health record (EHR) implementation is highly complex. Sharing of data definitions across applications within an EHR implementation project may be constrained by practical limitations, including time, tools, and expertise. However, maintaining rigor in an approach to data governance is important for sustainability and consistency. With this understanding, we have defined a practical approach for governance of structured data elements to optimize data definitions given limited resources. This approach includes a 10 step process: 1) identification of clinical topics, 2) creation of draft reference models for clinical topics, 3) scoring of downstream data needs for clinical topics, 4) prioritization of clinical topics, 5) validation of reference models for clinical topics, and 6) calculation of gap analyses of EHR compared against reference model, 7) communication of validated reference models across project members, 8) requested revisions to EHR based on gap analysis, 9) evaluation of usage of reference models across project, and 10) Monitoring for new evidence requiring revisions to reference model.

RhoC and ROCKs regulate cancer cell interactions with endothelial cells.

PubMed

Reymond, Nicolas; Im, Jae Hong; Garg, Ritu; Cox, Susan; Soyer, Magali; Riou, Philippe; Colomba, Audrey; Muschel, Ruth J; Ridley, Anne J

2015-06-01

RhoC is a member of the Rho GTPase family that is implicated in cancer progression by stimulating cancer cell invasiveness. Here we report that RhoC regulates the interaction of cancer cells with vascular endothelial cells (ECs), a crucial step in the metastatic process. RhoC depletion by RNAi reduces PC3 prostate cancer cell adhesion to ECs, intercalation between ECs as well as transendothelial migration in vitro. Depletion of the kinases ROCK1 and ROCK2, two known RhoC downstream effectors, similarly decreases cancer interaction with ECs. RhoC also regulates the extension of protrusions made by cancer cells on vascular ECs in vivo. Transient RhoC depletion is sufficient to reduce both early PC3 cell retention in the lungs and experimental metastasis formation in vivo. Our results indicate RhoC plays a central role in cancer cell interaction with vascular ECs, which is a critical event for cancer progression. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

The Drosophila homologue of SRF acts as a boosting mechanism to sustain FGF-induced terminal branching in the tracheal system.

PubMed

Gervais, Louis; Casanova, Jordi

2011-04-01

Recent data have demonstrated a crucial role for the transcription factor SRF (serum response factor) downstream of VEGF and FGF signalling during branching morphogenesis. This is the case for sprouting angiogenesis in vertebrates, axonal branching in mammals and terminal branching of the Drosophila tracheal system. However, the specific functions of SRF in these processes remain unclear. Here, we establish the relative contributions of the Drosophila homologues of FGF [Branchless (BNL)] and SRF [Blistered (BS)] in terminal tracheal branching. Conversely to an extended view, we show that BNL triggers terminal branching initiation in a DSRF-independent mechanism and that DSRF transcription induced by BNL signalling is required to maintain terminal branch elongation. Moreover, we report that increased and continuous FGF signalling can trigger tracheal cells to develop full-length terminal branches in the absence of DSRF transcription. Our results indicate that DSRF acts as an amplifying step to sustain the progression of terminal branch elongation even in the wild-type conditions of FGF signalling.

Cryo-EM structures of the eukaryotic replicative helicase bound to a translocation substrate

NASA Astrophysics Data System (ADS)

Abid Ali, Ferdos; Renault, Ludovic; Gannon, Julian; Gahlon, Hailey L.; Kotecha, Abhay; Zhou, Jin Chuan; Rueda, David; Costa, Alessandro

2016-02-01

The Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function. Whether CMG translocation occurs on single- or double-stranded DNA and how ATP hydrolysis drives DNA unwinding remain open questions. Here we use cryo-electron microscopy to describe two subnanometre resolution structures of the CMG helicase trapped on a DNA fork. In the predominant state, the ring-shaped C-terminal ATPase of MCM is compact and contacts single-stranded DNA, via a set of pre-sensor 1 hairpins that spiral around the translocation substrate. In the second state, the ATPase module is relaxed and apparently substrate free, while DNA intimately contacts the downstream amino-terminal tier of the MCM motor ring. These results, supported by single-molecule FRET measurements, lead us to suggest a replication fork unwinding mechanism whereby the N-terminal and AAA+ tiers of the MCM work in concert to translocate on single-stranded DNA.

The Cryptococcus neoformans Capsule: a Sword and a Shield

PubMed Central

O'Meara, Teresa R.

2012-01-01

Summary: The human fungal pathogen Cryptococcus neoformans is characterized by its ability to induce a distinct polysaccharide capsule in response to a number of host-specific environmental stimuli. The induction of capsule is a complex biological process encompassing regulation at multiple steps, including the biosynthesis, transport, and maintenance of the polysaccharide at the cell surface. By precisely regulating the composition of its cell surface and secreted polysaccharides, C. neoformans has developed intricate ways to establish chronic infection and dormancy in the human host. The plasticity of the capsule structure in response to various host conditions also underscores the complex relationship between host and parasite. Much of this precise regulation of capsule is achieved through the transcriptional responses of multiple conserved signaling pathways that have been coopted to regulate this C. neoformans-specific virulence-associated phenotype. This review focuses on specific host stimuli that trigger the activation of the signal transduction cascades and on the downstream transcriptional responses that are required for robust encapsulation around the cell. PMID:22763631

Automated Web-Based Request Mechanism for Workflow Enhancement in an Academic Customer-Focused Biorepository.

PubMed

McDonald, Sandra A; Ryan, Benjamin J; Brink, Amy; Holtschlag, Victoria L

2012-02-01

Informatics systems, particularly those that provide capabilities for data storage, specimen tracking, retrieval, and order fulfillment, are critical to the success of biorepositories and other laboratories engaged in translational medical research. A crucial item-one easily overlooked-is an efficient way to receive and process investigator-initiated requests. A successful electronic ordering system should allow request processing in a maximally efficient manner, while also allowing streamlined tracking and mining of request data such as turnaround times and numerical categorizations (user groups, funding sources, protocols, and so on). Ideally, an electronic ordering system also facilitates the initial contact between the laboratory and customers, while still allowing for downstream communications and other steps toward scientific partnerships. We describe here the recently established Web-based ordering system for the biorepository at Washington University Medical Center, along with its benefits for workflow, tracking, and customer service. Because of the system's numerous value-added impacts, we think our experience can serve as a good model for other customer-focused biorepositories, especially those currently using manual or non-Web-based request systems. Our lessons learned also apply to the informatics developers who serve such biobanks.

Automated Web-Based Request Mechanism for Workflow Enhancement in an Academic Customer-Focused Biorepository

PubMed Central

Ryan, Benjamin J.; Brink, Amy; Holtschlag, Victoria L.

2012-01-01

Informatics systems, particularly those that provide capabilities for data storage, specimen tracking, retrieval, and order fulfillment, are critical to the success of biorepositories and other laboratories engaged in translational medical research. A crucial item—one easily overlooked—is an efficient way to receive and process investigator-initiated requests. A successful electronic ordering system should allow request processing in a maximally efficient manner, while also allowing streamlined tracking and mining of request data such as turnaround times and numerical categorizations (user groups, funding sources, protocols, and so on). Ideally, an electronic ordering system also facilitates the initial contact between the laboratory and customers, while still allowing for downstream communications and other steps toward scientific partnerships. We describe here the recently established Web-based ordering system for the biorepository at Washington University Medical Center, along with its benefits for workflow, tracking, and customer service. Because of the system's numerous value-added impacts, we think our experience can serve as a good model for other customer-focused biorepositories, especially those currently using manual or non-Web–based request systems. Our lessons learned also apply to the informatics developers who serve such biobanks. PMID:23386921

Engineering a Synthetic Dual-Organism System for Hydrogen Production▿

PubMed Central

Waks, Zeev; Silver, Pamela A.

2009-01-01

Molecular hydrogen produced biologically from renewable biomass is an attractive replacement for fossil fuels. One potential route for biological hydrogen production is the conversion of biomass into formate, which can subsequently be processed into hydrogen by Escherichia coli. Formate is also a widely used commodity chemical, making its bioproduction even more attractive. Here we demonstrate the implementation of a formate-overproducing pathway in Saccharomyces cerevisiae, a well-established industrial organism. By expressing the anaerobic enzyme pyruvate formate lyase from E. coli, we engineered a strain of yeast that overproduced formate relative to undetectable levels in the wild type. The addition of a downstream enzyme, AdhE of E. coli, resulted in an additional 4.5-fold formate production increase as well as an increase in growth rate and biomass yield. Overall, an 18-fold formate increase was achieved in a strain background whose formate degradation pathway had been deleted. Finally, as a proof of concept, we were able to produce hydrogen from this formate-containing medium by using E. coli as a catalyst in a two-step process. With further optimizations, it may be feasible to use S. cerevisiae on a larger scale as the foundation for yeast-based biohydrogen. PMID:19201964

Evaluation of Novel Large Cut-Off Ultrafiltration Membranes for Adenovirus Serotype 5 (Ad5) Concentration

PubMed Central

Peixoto, Cristina; Roederstein, Susanne; Schleuss, Tobias; Alves, Paula M.; Mota, José P. B.; Carrondo, Manuel J. T.

2014-01-01

The purification of virus particles and viral vectors for vaccine and gene therapy applications is gaining increasing importance in order to deliver a fast, efficient, and reliable production process. Ultrafiltration (UF) is a widely employed unit operation in bioprocessing and its use is present in several steps of the downstream purification train of biopharmaceuticals. However, to date few studies have thoroughly investigated the performance of several membrane materials and cut-offs for virus concentration/diafiltration. The present study aimed at developing a novel class of UF cassettes for virus concentration/diafiltration. A detailed study was conducted to evaluate the effects of (i) membrane materials, namely polyethersulfone (PES), regenerated cellulose (RC), and highly cross-linked RC (xRC), (ii) nominal cut-off, and (iii) UF device geometry at different production scales. The results indicate that the xRC cassettes with a cut-off of approximately 500 kDa are able to achieve a 10-fold concentration factor with 100% recovery of particles with a process time twice as fast as that of a commercially available hollow fiber. DNA and host cell protein clearances, as well as hydraulic permeability and fouling behavior, were also assessed. PMID:25546428

2,3-Butanediol recovery from fermentation broth by alcohol precipitation and vacuum distillation.

PubMed

Jeon, Sangjun; Kim, Duk-Ki; Song, Hyohak; Lee, Hee Jong; Park, Sunghoon; Seung, Doyoung; Chang, Yong Keun

2014-04-01

This study presents a new and effective downstream process to recover 2,3-butanediol (2,3-BD) from fermentation broth which is produced by a recombinant Klebsiella pneumoniae strain. The ldhA-deficient K. pneumoniae strain yielded about 90 g/L of 2,3-BD, along with a number of by-products, such as organic acids and alcohols, in a 65 h fed-batch fermentation. The pH-adjusted cell-free fermentation broth was firstly concentrated until 2,3-BD reached around 500 g/L by vacuum evaporation at 50°C and 50 mbar vacuum pressure. The concentrated solution was further treated using light alcohols, including methanol, ethanol, and isopropanol, for the precipitation of organic acids and inorganic salts. Isopropanol showed the highest removal efficiency, in which 92.5% and 99.8% of organic acids and inorganic salts were precipitated, respectively. At a final step, a vacuum distillation process enabled the recovery of 76.2% of the treated 2,3-BD, with 96.1% purity, indicating that fermentatively produced 2,3-BD is effectively recovered by a simple alcohol precipitation and vacuum distillation. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Single-Molecule Imaging of Cellular Signaling

NASA Astrophysics Data System (ADS)

De Keijzer, Sandra; Snaar-Jagalska, B. Ewa; Spaink, Herman P.; Schmidt, Thomas

Single-molecule microscopy is an emerging technique to understand the function of a protein in the context of its natural environment. In our laboratory this technique has been used to study the dynamics of signal transduction in vivo. A multitude of signal transduction cascades are initiated by interactions between proteins in the plasma membrane. These cascades start by binding a ligand to its receptor, thereby activating downstream signaling pathways which finally result in complex cellular responses. To fully understand these processes it is important to study the initial steps of the signaling cascades. Standard biological assays mostly call for overexpression of the proteins and high concentrations of ligand. This sets severe limits to the interpretation of, for instance, the time-course of the observations, given the large temporal spread caused by the diffusion-limited binding processes. Methods and limitations of single-molecule microscopy for the study of cell signaling are discussed on the example of the chemotactic signaling of the slime-mold Dictyostelium discoideum. Single-molecule studies, as reviewed in this chapter, appear to be one of the essential methodologies for the full spatiotemporal clarification of cellular signaling, one of the ultimate goals in cell biology.

Software for pre-processing Illumina next-generation sequencing short read sequences

PubMed Central

2014-01-01

Background When compared to Sanger sequencing technology, next-generation sequencing (NGS) technologies are hindered by shorter sequence read length, higher base-call error rate, non-uniform coverage, and platform-specific sequencing artifacts. These characteristics lower the quality of their downstream analyses, e.g. de novo and reference-based assembly, by introducing sequencing artifacts and errors that may contribute to incorrect interpretation of data. Although many tools have been developed for quality control and pre-processing of NGS data, none of them provide flexible and comprehensive trimming options in conjunction with parallel processing to expedite pre-processing of large NGS datasets. Methods We developed ngsShoRT (next-generation sequencing Short Reads Trimmer), a flexible and comprehensive open-source software package written in Perl that provides a set of algorithms commonly used for pre-processing NGS short read sequences. We compared the features and performance of ngsShoRT with existing tools: CutAdapt, NGS QC Toolkit and Trimmomatic. We also compared the effects of using pre-processed short read sequences generated by different algorithms on de novo and reference-based assembly for three different genomes: Caenorhabditis elegans, Saccharomyces cerevisiae S288c, and Escherichia coli O157 H7. Results Several combinations of ngsShoRT algorithms were tested on publicly available Illumina GA II, HiSeq 2000, and MiSeq eukaryotic and bacteria genomic short read sequences with the focus on removing sequencing artifacts and low-quality reads and/or bases. Our results show that across three organisms and three sequencing platforms, trimming improved the mean quality scores of trimmed sequences. Using trimmed sequences for de novo and reference-based assembly improved assembly quality as well as assembler performance. In general, ngsShoRT outperformed comparable trimming tools in terms of trimming speed and improvement of de novo and reference-based assembly as measured by assembly contiguity and correctness. Conclusions Trimming of short read sequences can improve the quality of de novo and reference-based assembly and assembler performance. The parallel processing capability of ngsShoRT reduces trimming time and improves the memory efficiency when dealing with large datasets. We recommend combining sequencing artifacts removal, and quality score based read filtering and base trimming as the most consistent method for improving sequence quality and downstream assemblies. ngsShoRT source code, user guide and tutorial are available at http://research.bioinformatics.udel.edu/genomics/ngsShoRT/. ngsShoRT can be incorporated as a pre-processing step in genome and transcriptome assembly projects. PMID:24955109

Sediment Budgeting in Dam-Affected Rivers: Assessing the Influence of Damming, Tributaries, and Alluvial Valley Sediment Storage on Sediment Regimes

NASA Astrophysics Data System (ADS)

Wilcox, A. C.; Dekker, F. J.; Riebe, C. S.

2014-12-01

Although sediment supply is recognized as a fundamental driver of fluvial processes, measuring how dams affect sediment regimes and incorporating such knowledge into management strategies remains challenging. To determine the influences of damming, tributary supply, and valley morphology and sediment storage on downstream sediment supply in a dryland river, the Bill Williams River (BWR) in western Arizona, we measured basin erosion rates using cosmogenic nuclide analysis of beryllium-10 (10Be) at sites upstream and downstream of a dam along the BWR, as well as from tributaries downstream of the dam. Riverbed sediment mixing calculations were used to test if the dam, which blocks sediment supply from the upper 85% of the basin's drainage area, increases the proportion of tributary sediment to residual upstream sediment in mainstem samples downstream of the dam. Erosion rates in the BWR watershed are more than twice as large in the upper catchment (136 t km-2 yr-1) than in tributaries downstream of Alamo Dam (61 t km-2 yr-1). Tributaries downstream of the dam have little influence on mainstem sediment dynamics. The effect of the dam on reducing sediment supply is limited, however, because of the presence of large alluvial valleys along the mainstem BWR downstream of the dam that store substantial sediment and mitigate supply reductions from the upper watershed. These inferences, from our 10Be derived erosion rates and mixing calculations, are consistent with field observations of downstream changes in bed material size, which suggest that sediment-deficit conditions are restricted to a 10 km reach downstream of the dam, and limited reservoir bathymetry data. Many studies have suggested that tributary sediment inputs downstream of dams play a key role in mitigating dam-induced sediment deficits, but here we show that in a dryland river with ephemeral tributaries, sediment stored in alluvial valleys can also play a key role and in some cases trumps the role of tributaries.

Investigation of merging/reconnection heating during solenoid-free startup of plasmas in the MAST Spherical Tokamak

NASA Astrophysics Data System (ADS)

Tanabe, H.; Yamada, T.; Watanabe, T.; Gi, K.; Inomoto, M.; Imazawa, R.; Gryaznevich, M.; Scannell, R.; Conway, N. J.; Michael, C.; Crowley, B.; Fitzgerald, I.; Meakins, A.; Hawkes, N.; McClements, K. G.; Harrison, J.; O'Gorman, T.; Cheng, C. Z.; Ono, Y.; The MAST Team

2017-05-01

We present results of recent studies of merging/reconnection heating during central solenoid (CS)-free plasma startup in the Mega Amp Spherical Tokamak (MAST). During this process, ions are heated globally in the downstream region of an outflow jet, and electrons locally around the X-point produced by the magnetic field of two internal P3 coils and of two plasma rings formed around these coils, the final temperature being proportional to the reconnecting field energy. There is an effective confinement of the downstream thermal energy, due to a thick layer of reconnected flux. The characteristic structure is sustained for longer than an ion-electron energy relaxation time, and the energy exchange between ions and electrons contributes to the bulk electron heating in the downstream region. The peak electron temperature around the X-point increases with toroidal field, but the downstream electron and ion temperatures do not change.

Cellular reprogramming through mitogen-activated protein kinases.

PubMed

Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

2015-01-01

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression-including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

Enhancing productivity for cascade biotransformation of styrene to (S)-vicinal diol with biphasic system in hollow fiber membrane bioreactor.

PubMed

Gao, Pengfei; Wu, Shuke; Praveen, Prashant; Loh, Kai-Chee; Li, Zhi

2017-03-01

Biotransformation is a green and useful tool for sustainable and selective chemical synthesis. However, it often suffers from the toxicity and inhibition from organic substrates or products. Here, we established a hollow fiber membrane bioreactor (HFMB)-based aqueous/organic biphasic system, for the first time, to enhance the productivity of a cascade biotransformation with strong substrate toxicity and inhibition. The enantioselective trans-dihydroxylation of styrene to (S)-1-phenyl-1,2-ethanediol, catalyzed by Escherichia coli (SSP1) coexpressing styrene monooxygenase and an epoxide hydrolase, was performed in HFMB with organic solvent in the shell side and aqueous cell suspension in the lumen side. Various organic solvents were investigated, and n-hexadecane was found as the best for the HFMB-based biphasic system. Comparing to other reported biphasic systems assisted by HFMB, our system not only shield much of the substrate toxicity but also deflate the product recovery burden in downstream processing as the majority of styrene stayed in organic phase while the diol product mostly remained in the aqueous phase. The established HFMB-based biphasic system enhanced the production titer to 143 mM, being 16-fold higher than the aqueous system and 1.6-fold higher than the traditional dispersive partitioning biphase system. Furthermore, the combination of biphasic system with HFMB prevents the foaming and emulsification, thus reducing the burden in downstream purification. HFMB-based biphasic system could serve as a suitable platform for enhancing the productivity of single-step or cascade biotransformation with toxic substrates to produce useful and valuable chemicals.

Load-induced modulation of signal transduction networks.

PubMed

Jiang, Peng; Ventura, Alejandra C; Sontag, Eduardo D; Merajver, Sofia D; Ninfa, Alexander J; Del Vecchio, Domitilla

2011-10-11

Biological signal transduction networks are commonly viewed as circuits that pass along information--in the process amplifying signals, enhancing sensitivity, or performing other signal-processing tasks--to transcriptional and other components. Here, we report on a "reverse-causality" phenomenon, which we call load-induced modulation. Through a combination of analytical and experimental tools, we discovered that signaling was modulated, in a surprising way, by downstream targets that receive the signal and, in doing so, apply what in physics is called a load. Specifically, we found that non-intuitive changes in response dynamics occurred for a covalent modification cycle when load was present. Loading altered the response time of a system, depending on whether the activity of one of the enzymes was maximal and the other was operating at its minimal rate or whether both enzymes were operating at submaximal rates. These two conditions, which we call "limit regime" and "intermediate regime," were associated with increased or decreased response times, respectively. The bandwidth, the range of frequency in which the system can process information, decreased in the presence of load, suggesting that downstream targets participate in establishing a balance between noise-filtering capabilities and a circuit's ability to process high-frequency stimulation. Nodes in a signaling network are not independent relay devices, but rather are modulated by their downstream targets.

Rho proteins of plants--functional cycle and regulation of cytoskeletal dynamics.

PubMed

Mucha, Elena; Fricke, Inka; Schaefer, Antje; Wittinghofer, Alfred; Berken, Antje

2011-11-01

Rho-related ROP proteins are molecular switches that essentially regulate a wide variety of processes. Of central interest is their influence on the plant cytoskeleton by which they affect vital processes like cell division, growth, morphogenesis, and pathogen defense. ROPs switch between GTP- and GDP-bound conformations by strictly regulated nucleotide exchange and GTP-hydrolysis, and only the active GTP-form interacts with downstream effectors to ultimately provoke a biological response. However, the mode of action of the engaged regulators and effectors as well as their upstream and downstream interaction partners have long been largely unknown. As opposed to analogous systems in animals and fungi, plants use specific GTPase activating proteins (RopGAPs) with a unique domain composition and novel guanine nucleotide exchange factors (RopGEFs) with a probable link to cell surface receptors. Moreover, plants comprise novel effector molecules and adapters connecting ROPs to mostly unknown downstream targets on the route to the cytoskeleton. This review aims to summarize recent knowledge on the molecular mechanisms and reaction cascades involved in ROP dependent cytoskeletal rearrangements, addressing the structure and function of the unusual RopGAPs, RopGEFs and effectors, and the upstream and downstream pathways linking ROPs to cell receptor-like kinases, actin filaments, and microtubules. Copyright © 2010 Elsevier GmbH. All rights reserved.

Understanding Coupled Earth-Surface Processes through Experiments and Models (Invited)

NASA Astrophysics Data System (ADS)

Overeem, I.; Kim, W.

2013-12-01

Traditionally, both numerical models and experiments have been purposefully designed to ';isolate' singular components or certain processes of a larger mountain to deep-ocean interconnected source-to-sink (S2S) transport system. Controlling factors driven by processes outside of the domain of immediate interest were treated and simplified as input or as boundary conditions. Increasingly, earth surface processes scientists appreciate feedbacks and explore these feedbacks with more dynamically coupled approaches to their experiments and models. Here, we discuss key concepts and recent advances made in coupled modeling and experimental setups. In addition, we emphasize challenges and new frontiers to coupled experiments. Experiments have highlighted the important role of self-organization; river and delta systems do not always need to be forced by external processes to change or develop characteristic morphologies. Similarly modeling f.e. has shown that intricate networks in tidal deltas are stable because of the interplay between river avulsions and the tidal current scouring with both processes being important to develop and maintain the dentritic networks. Both models and experiment have demonstrated that seemingly stable systems can be perturbed slightly and show dramatic responses. Source-to-sink models were developed for both the Fly River System in Papua New Guinea and the Waipaoa River in New Zealand. These models pointed to the importance of upstream-downstream effects and enforced our view of the S2S system as a signal transfer and dampening conveyor belt. Coupled modeling showed that deforestation had extreme effects on sediment fluxes draining from the catchment of the Waipaoa River in New Zealand, and that this increase in sediment production rapidly shifted the locus of offshore deposition. The challenge in designing coupled models and experiments is both technological as well as intellectual. Our community advances to make numerical model coupling more straightforward through common interfaces and standardization of time-stepping, model domains and model parameters. At the same time major steps forward require an interdisciplinary approach, wherein the source to sink system contains ecological feedbacks and human actors.

Application of quality by design principles to the development and technology transfer of a major process improvement for the manufacture of a recombinant protein.

PubMed

Looby, Mairead; Ibarra, Neysi; Pierce, James J; Buckley, Kevin; O'Donovan, Eimear; Heenan, Mary; Moran, Enda; Farid, Suzanne S; Baganz, Frank

2011-01-01

This study describes the application of quality by design (QbD) principles to the development and implementation of a major manufacturing process improvement for a commercially distributed therapeutic protein produced in Chinese hamster ovary cell culture. The intent of this article is to focus on QbD concepts, and provide guidance and understanding on how the various components combine together to deliver a robust process in keeping with the principles of QbD. A fed-batch production culture and a virus inactivation step are described as representative examples of upstream and downstream unit operations that were characterized. A systematic approach incorporating QbD principles was applied to both unit operations, involving risk assessment of potential process failure points, small-scale model qualification, design and execution of experiments, definition of operating parameter ranges and process validation acceptance criteria followed by manufacturing-scale implementation and process validation. Statistical experimental designs were applied to the execution of process characterization studies evaluating the impact of operating parameters on product quality attributes and process performance parameters. Data from process characterization experiments were used to define the proven acceptable range and classification of operating parameters for each unit operation. Analysis of variance and Monte Carlo simulation methods were used to assess the appropriateness of process design spaces. Successful implementation and validation of the process in the manufacturing facility and the subsequent manufacture of hundreds of batches of this therapeutic protein verifies the approaches taken as a suitable model for the development, scale-up and operation of any biopharmaceutical manufacturing process. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

MoCha: Molecular Characterization of Unknown Pathways.

PubMed

Lobo, Daniel; Hammelman, Jennifer; Levin, Michael

2016-04-01

Automated methods for the reverse-engineering of complex regulatory networks are paving the way for the inference of mechanistic comprehensive models directly from experimental data. These novel methods can infer not only the relations and parameters of the known molecules defined in their input datasets, but also unknown components and pathways identified as necessary by the automated algorithms. Identifying the molecular nature of these unknown components is a crucial step for making testable predictions and experimentally validating the models, yet no specific and efficient tools exist to aid in this process. To this end, we present here MoCha (Molecular Characterization), a tool optimized for the search of unknown proteins and their pathways from a given set of known interacting proteins. MoCha uses the comprehensive dataset of protein-protein interactions provided by the STRING database, which currently includes more than a billion interactions from over 2,000 organisms. MoCha is highly optimized, performing typical searches within seconds. We demonstrate the use of MoCha with the characterization of unknown components from reverse-engineered models from the literature. MoCha is useful for working on network models by hand or as a downstream step of a model inference engine workflow and represents a valuable and efficient tool for the characterization of unknown pathways using known data from thousands of organisms. MoCha and its source code are freely available online under the GPLv3 license.

An additional k-means clustering step improves the biological features of WGCNA gene co-expression networks.

PubMed

Botía, Juan A; Vandrovcova, Jana; Forabosco, Paola; Guelfi, Sebastian; D'Sa, Karishma; Hardy, John; Lewis, Cathryn M; Ryten, Mina; Weale, Michael E

2017-04-12

Weighted Gene Co-expression Network Analysis (WGCNA) is a widely used R software package for the generation of gene co-expression networks (GCN). WGCNA generates both a GCN and a derived partitioning of clusters of genes (modules). We propose k-means clustering as an additional processing step to conventional WGCNA, which we have implemented in the R package km2gcn (k-means to gene co-expression network, https://github.com/juanbot/km2gcn ). We assessed our method on networks created from UKBEC data (10 different human brain tissues), on networks created from GTEx data (42 human tissues, including 13 brain tissues), and on simulated networks derived from GTEx data. We observed substantially improved module properties, including: (1) few or zero misplaced genes; (2) increased counts of replicable clusters in alternate tissues (x3.1 on average); (3) improved enrichment of Gene Ontology terms (seen in 48/52 GCNs) (4) improved cell type enrichment signals (seen in 21/23 brain GCNs); and (5) more accurate partitions in simulated data according to a range of similarity indices. The results obtained from our investigations indicate that our k-means method, applied as an adjunct to standard WGCNA, results in better network partitions. These improved partitions enable more fruitful downstream analyses, as gene modules are more biologically meaningful.

High-Spatial-Resolution OH and CH2O PLIF Visualization in a Dual-Mode Scramjet Combustor

NASA Technical Reports Server (NTRS)

Geipel, Clayton M.

2017-01-01

A high-spatial-resolution planar laser-induced fluorescence (PLIF) imaging system was constructed and used to image a cavity-stabilized, premixed ethylene-air flame. The flame was created within a continuous flow, electrically-heated supersonic combustion facility consisting of a Mach 2 nozzle, an isolator with flush-wall fuel injectors, a combustor with a cavity flameholder of height 9 mm and optical access, and an extender. Tests were conducted at total temperature 1200 K, total pressure 300 kPa, equivalence ratio near 0.4 in the combustor, and Mach number near 0.6 in the combustor. A frequency-doubled Nd:YAG laser pumped a dye laser, which produced light at 283.55 nm. The beam was shaped into a light sheet with full width half-maximum 25 microns, which illuminated a streamwise plane that bisected the cavity. An intensified camera system imaged OH in this plane with a square 6.67 mm field of view and in-plane resolution 39 microns. Images were taken between the backward-facing step and 120 mm downstream of the step. OH structures as small as 110 microns were observed. CH2O was excited using 352.48 nm light; the smallest observed CH2O structures were approximately 200 microns wide. Approximately 15,000 images per species were processed and used to compute composite images.

GapBlaster-A Graphical Gap Filler for Prokaryote Genomes.

PubMed

de Sá, Pablo H C G; Miranda, Fábio; Veras, Adonney; de Melo, Diego Magalhães; Soares, Siomar; Pinheiro, Kenny; Guimarães, Luis; Azevedo, Vasco; Silva, Artur; Ramos, Rommel T J

2016-01-01

The advent of NGS (Next Generation Sequencing) technologies has resulted in an exponential increase in the number of complete genomes available in biological databases. This advance has allowed the development of several computational tools enabling analyses of large amounts of data in each of the various steps, from processing and quality filtering to gap filling and manual curation. The tools developed for gap closure are very useful as they result in more complete genomes, which will influence downstream analyses of genomic plasticity and comparative genomics. However, the gap filling step remains a challenge for genome assembly, often requiring manual intervention. Here, we present GapBlaster, a graphical application to evaluate and close gaps. GapBlaster was developed via Java programming language. The software uses contigs obtained in the assembly of the genome to perform an alignment against a draft of the genome/scaffold, using BLAST or Mummer to close gaps. Then, all identified alignments of contigs that extend through the gaps in the draft sequence are presented to the user for further evaluation via the GapBlaster graphical interface. GapBlaster presents significant results compared to other similar software and has the advantage of offering a graphical interface for manual curation of the gaps. GapBlaster program, the user guide and the test datasets are freely available at https://sourceforge.net/projects/gapblaster2015/. It requires Sun JDK 8 and Blast or Mummer.

Decomposition behavior of hemicellulose and lignin in the step-change flow rate liquid hot water.

PubMed

Zhuang, Xinshu; Yu, Qiang; Wang, Wen; Qi, Wei; Wang, Qiong; Tan, Xuesong; Yuan, Zhenhong

2012-09-01

Hemicellulose and lignin are the main factors limiting accessibility of hydrolytic enzymes besides the crystallinity of cellulose. The decomposition behavior of hemicellulose and lignin in the step-change flow rate hot water system was investigated. Xylan removal increased from 64.53% for batch system (solid concentration 4.25% w/v, 18 min, 184°C) to 83.78% at high flow rates of 30 ml/min for 8 min, and then 10 ml/min for 10 min. Most of them (80-90%) were recovered as oligosaccharide. It was hypothesized that the flowing water could enhance the mass transfer to improve the sugars recovery. In addition, the solubilization mechanism of lignin in the liquid hot water was proposed according to the results of Fourier transform-infrared spectroscopy and scanning electron microscopy of the water-insoluble fraction and gas chromatography-mass spectrometry of the water-soluble fraction. It was proposed that lignin in the liquid hot water first migrated out of the cell wall in the form of molten bodies, and then flushed out of the reactor. A small quantity of them was further degraded into monomeric products such as vanillin, syringe aldehyde, coniferyl aldehyde, ferulic acid, and p-hydroxy-cinnamic acid. All of these observations would provide important information for the downstream processing, such as purification and concentration of sugars and the enzymatic digestion of residual solid.

Sequence-specific sepsis-related DNA capture and fluorescent labeling in monoliths prepared by single-step photopolymerization in microfluidic devices.

PubMed

Knob, Radim; Hanson, Robert L; Tateoka, Olivia B; Wood, Ryan L; Guerrero-Arguero, Israel; Robison, Richard A; Pitt, William G; Woolley, Adam T

2018-05-21

Fast determination of antibiotic resistance is crucial in selecting appropriate treatment for sepsis patients, but current methods based on culture are time consuming. We are developing a microfluidic platform with a monolithic column modified with oligonucleotides designed for sequence-specific capture of target DNA related to the Klebsiella pneumoniae carbapenemase (KPC) gene. We developed a novel single-step monolith fabrication method with an acrydite-modified capture oligonucleotide in the polymerization mixture, enabling fast monolith preparation in a microfluidic channel using UV photopolymerization. These prepared columns had a threefold higher capacity compared to monoliths prepared in a multistep process involving Schiff-base DNA attachment. Conditions for denaturing, capture and fluorescence labeling using hybridization probes were optimized with synthetic 90-mer oligonucleotides. These procedures were applied for extraction of a PCR amplicon from the KPC antibiotic resistance gene in bacterial lysate obtained from a blood sample spiked with E. coli. The results showed similar eluted peak areas for KPC amplicon extracted from either hybridization buffer or bacterial lysate. Selective extraction of the KPC DNA was verified by real time PCR on eluted fractions. These results show great promise for application in an integrated microfluidic diagnostic system that combines upstream blood sample preparation and downstream single-molecule counting detection. Copyright © 2018 Elsevier B.V. All rights reserved.

An integratable microfluidic cartridge for forensic swab samples lysis.

PubMed

Yang, Jianing; Brooks, Carla; Estes, Matthew D; Hurth, Cedric M; Zenhausern, Frederic

2014-01-01

Fully automated rapid forensic DNA analysis requires integrating several multistep processes onto a single microfluidic platform, including substrate lysis, extraction of DNA from the released lysate solution, multiplexed PCR amplification of STR loci, separation of PCR products by capillary electrophoresis, and analysis for allelic peak calling. Over the past several years, most of the rapid DNA analysis systems developed started with the reference swab sample lysate and involved an off-chip lysis of collected substrates. As a result of advancement in technology and chemistry, addition of a microfluidic module for swab sample lysis has been achieved in a few of the rapid DNA analysis systems. However, recent reports on integrated rapid DNA analysis systems with swab-in and answer-out capability lack any quantitative and qualitative characterization of the swab-in sample lysis module, which is important for downstream forensic sample processing. Maximal collection and subsequent recovery of the biological material from the crime scene is one of the first and critical steps in forensic DNA technology. Herein we present the design, fabrication and characterization of an integratable swab lysis cartridge module and the test results obtained from different types of commonly used forensic swab samples, including buccal, saliva, and blood swab samples, demonstrating the compatibility with different downstream DNA extraction chemistries. This swab lysis cartridge module is easy to operate, compatible with both forensic and microfluidic requirements, and ready to be integrated with our existing automated rapid forensic DNA analysis system. Following the characterization of the swab lysis module, an integrated run from buccal swab sample-in to the microchip CE electropherogram-out was demonstrated on the integrated prototype instrument. Therefore, in this study, we demonstrate that this swab lysis cartridge module is: (1) functionally, comparable with routine benchtop lysis, (2) compatible with various types of swab samples and chemistries, and (3) integratable to achieve a micro total analysis system (μTAS) for rapid DNA analysis. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Connecting the Dots between PubMed Abstracts

PubMed Central

Hossain, M. Shahriar; Gresock, Joseph; Edmonds, Yvette; Helm, Richard; Potts, Malcolm; Ramakrishnan, Naren

2012-01-01

Background There are now a multitude of articles published in a diversity of journals providing information about genes, proteins, pathways, and diseases. Each article investigates subsets of a biological process, but to gain insight into the functioning of a system as a whole, we must integrate information from multiple publications. Particularly, unraveling relationships between extra-cellular inputs and downstream molecular response mechanisms requires integrating conclusions from diverse publications. Methodology We present an automated approach to biological knowledge discovery from PubMed abstracts, suitable for “connecting the dots” across the literature. We describe a storytelling algorithm that, given a start and end publication, typically with little or no overlap in content, identifies a chain of intermediate publications from one to the other, such that neighboring publications have significant content similarity. The quality of discovered stories is measured using local criteria such as the size of supporting neighborhoods for each link and the strength of individual links connecting publications, as well as global metrics of dispersion. To ensure that the story stays coherent as it meanders from one publication to another, we demonstrate the design of novel coherence and overlap filters for use as post-processing steps. Conclusions We demonstrate the application of our storytelling algorithm to three case studies: i) a many-one study exploring relationships between multiple cellular inputs and a molecule responsible for cell-fate decisions, ii) a many-many study exploring the relationships between multiple cytokines and multiple downstream transcription factors, and iii) a one-to-one study to showcase the ability to recover a cancer related association, viz. the Warburg effect, from past literature. The storytelling pipeline helps narrow down a scientist's focus from several hundreds of thousands of relevant documents to only around a hundred stories. We argue that our approach can serve as a valuable discovery aid for hypothesis generation and connection exploration in large unstructured biological knowledge bases. PMID:22235301

Urban Wastewater Impacts on the Spatial Distribution of Solutes and Microbial Constituents in the Musi River, India

NASA Astrophysics Data System (ADS)

Ensink, J.; Scott, C. A.; Cairncross, S.

2006-05-01

Wastewater discharge from expanding urban centers deteriorates the quality of receiving waters, a trend that has management and investment implications for cities around the world. This paper presents the results of a 14-month water quality evaluation over a 40-km longitudinal profile downstream of the city of Hyderabad, India (population 7 million) on the Musi River, a tributary to the Krishna River. Upstream to downstream improvements in Musi water quality for microbial constituents (nematode egg, faecal coliform), dissolved oxygen, and nitrate are attributed to natural attenuation processes (dilution, die-off, sedimentation and biological processes) coupled with the effects of in-stream hydraulic infrastructure (weirs and reservoirs). Conversely, upstream to downstream increases in total dissolved solids concentrations are caused by off- stream infrastructure and agricultural water use resulting in crop evapotranspiration and increased solute concentration in the return flow of irrigation diverted upstream in the wastewater system. Future water quality management challenges resulting from rampant urban growth, particularly in developing countries, are discussed.

Influence of Wastewater Discharge on the Metabolic Potential of the Microbial Community in River Sediments.

PubMed

Li, Dong; Sharp, Jonathan O; Drewes, Jörg E

2016-01-01

To reveal the variation of microbial community functions during water filtration process in river sediments, which has been utilized widely in natural water treatment systems, this study investigates the influence of municipal wastewater discharge to streams on the phylotype and metabolic potential of the microbiome in upstream and particularly various depths of downstream river sediments. Cluster analyses based on both microbial phylogenetic and functional data collectively revealed that shallow upstream sediments grouped with those from deeper subsurface downstream regions. These sediment samples were distinct from those found in shallow downstream sediments. Functional genes associated with carbohydrate, xenobiotic, and certain amino acid metabolisms were overrepresented in upstream and deep downstream samples. In contrast, the more immediate contact with wastewater discharge in shallow downstream samples resulted in an increase in the relative abundance of genes associated with nitrogen, sulfur, purine and pyrimidine metabolisms, as well as restriction-modification systems. More diverse bacterial phyla were associated with upstream and deep downstream sediments, mainly including Actinobacteria, Planctomycetes, and Firmicutes. In contrast, in shallow downstream sediments, genera affiliated with Betaproteobacteria and Gammaproteobacteria were enriched with putative functions that included ammonia and sulfur oxidation, polyphosphate accumulation, and methylotrophic bacteria. Collectively, these results highlight the enhanced capabilities of microbial communities residing in deeper stream sediments for the transformation of water contaminants and thus provide a foundation for better design of natural water treatment systems to further improve the removal of contaminants.

Data Evaluation of Actinide Cross Sections: 238Pu, 237Pu, and 236Pu

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

Guaglioni, S.; Jurgenson, E.; Descalle, M. A.

This report documents the recent evaluation of the 236Pu, 237Pu, and 238Pu cross section sets. Nuclear data evaluation is the fundamental interface that takes measured nuclear cross section data and turns them into a continuous curve that 1) is consistent with other measurements and nuclear reaction theory/models, and 2) is required by down-stream users. All experiments that generate nuclear data need to include an evaluation step for their data to be broadly useful to the end users.

Recycling of glass: accounting of greenhouse gases and global warming contributions.

PubMed

Larsen, Anna W; Merrild, Hanna; Christensen, Thomas H

2009-11-01

Greenhouse gas (GHG) emissions related to recycling of glass waste were assessed from a waste management perspective. Focus was on the material recovery facility (MRF) where the initial sorting of glass waste takes place. The MRF delivers products like cullet and whole bottles to other industries. Two possible uses of reprocessed glass waste were considered: (i) remelting of cullet added to glass production; and (ii) re-use of whole bottles. The GHG emission accounting included indirect upstream emissions (provision of energy, fuels and auxiliaries), direct activities at the MRF and bottle-wash facility (combustion of fuels) as well as indirect downstream activities in terms of using the recovered glass waste in other industries and, thereby, avoiding emissions from conventional production. The GHG accounting was presented as aggregated global warming factors (GWFs) for the direct and indirect upstream and downstream processes, respectively. The range of GWFs was estimated to 0-70 kg CO(2)eq. tonne( -1) of glass waste for the upstream activities and the direct emissions from the waste management system. The GWF for the downstream effect showed some significant variation between the two cases. It was estimated to approximately -500 kg CO(2)-eq. tonne(- 1) of glass waste for the remelting technology and -1500 to -600 kg CO(2)-eq. tonne(-1) of glass waste for bottle re-use. Including the downstream process, large savings of GHG emissions can be attributed to the waste management system. The results showed that, in GHG emission accounting, attention should be drawn to thorough analysis of energy sources, especially electricity, and the downstream savings caused by material substitution.

Effects of soluble and particulate substrate on the carbon and energy footprint of wastewater treatment processes.

PubMed

Gori, Riccardo; Jiang, Lu-Man; Sobhani, Reza; Rosso, Diego

2011-11-15

Most wastewater treatment plants monitor routinely carbonaceous and nitrogenous load parameters in influent and effluent streams, and often in the intermediate steps. COD fractionation discriminates the selective removal of VSS components in different operations, allowing accurate quantification of the energy requirements and mass flows for secondary treatment, sludge digestion, and sedimentation. We analysed the different effects of COD fractions on carbon and energy footprint in a wastewater treatment plant with activated sludge in nutrient removal mode and anaerobic digestion of the sludge with biogas energy recovery. After presenting a simple rational procedure for COD and solids fractions quantification, we use our carbon and energy footprint models to quantify the effects of varying fractions on carbon equivalent flows, process energy demand and recovery. A full-scale real process was modelled with this procedure and the results are reported in terms of energy and carbon footprint. For a given process, the increase of the ratio sCOD/COD increases the energy demand on the aeration reactors, the associated CO(2) direct emission from respiration, and the indirect emission for power generation. Even though it appears as if enhanced primary sedimentation is a carbon and energy footprint mitigation practice, care must be used since the nutrient removal process downstream may suffer from an excessive bCOD removal and an increased mean cell retention time for nutrient removal may be required. Copyright © 2011 Elsevier Ltd. All rights reserved.

Effects of episodic sediment supply on bedload transport rate in mountain rivers. Detecting debris flow activity using continuous monitoring

NASA Astrophysics Data System (ADS)

Uchida, Taro; Sakurai, Wataru; Iuchi, Takuma; Izumiyama, Hiroaki; Borgatti, Lisa; Marcato, Gianluca; Pasuto, Alessandro

2018-04-01

Monitoring of sediment transport from hillslopes to channel networks as a consequence of floods with suspended and bedload transport, hyperconcentrated flows, debris and mud flows is essential not only for scientific issues, but also for prevention and mitigation of natural disasters, i.e. for hazard assessment, land use planning and design of torrent control interventions. In steep, potentially unstable terrains, ground-based continuous monitoring of hillslope and hydrological processes is still highly localized and expensive, especially in terms of manpower. In recent years, new seismic and acoustic methods have been developed for continuous bedload monitoring in mountain rivers. Since downstream bedload transport rate is controlled by upstream sediment supply from tributary channels and bed-external sources, continuous bedload monitoring might be an effective tool for detecting the sediments mobilized by debris flow processes in the upper catchment and thus represent an indirect method to monitor slope instability processes at the catchment scale. However, there is poor information about the effects of episodic sediment supply from upstream bed-external sources on downstream bedload transport rate at a single flood time scale. We have examined the effects of sediment supply due to upstream debris flow events on downstream bedload transport rate along the Yotagiri River, central Japan. To do this, we have conducted continuous bedload observations using a hydrophone (Japanese pipe microphone) located 6.4 km downstream the lower end of a tributary affected by debris flows. Two debris flows occurred during the two-years-long observation period. As expected, bedload transport rate for a given flow depth showed to be larger after storms triggering debris flows. That is, although the magnitude of sediment supply from debris flows is not large, their effect on bedload is propagating >6 km downstream at a single flood time scale. This indicates that continuous bedload observations could be effective for detecting sediment supply as a consequence of debris flow events.

Modeling the GLOF Hazard Process Chain at Imja Lake in the Nepal Himalaya

NASA Astrophysics Data System (ADS)

Lala, J.; McKinney, D. C.; Rounce, D.

2017-12-01

The Hindu Kush-Himalaya region contains more glacial ice than any other non-polar region on earth. Many glacial lakes in Nepal are held in place by natural moraine dams, which are inherently unstable. Avalanches or landslides entering glacial lakes can cause tsunami-like waves that can overtop the moraines and trigger glacial lake outburst floods (GLOF). Mass loss at the Imja glacier is the highest in the Mount Everest region, and contributes to the expansion of Imja Tsho, a lake with several villages downstream. A GLOF from the lake might destroy both property and human life, making an understanding of flood triggering processes beneficial for both the downstream villages and other GLOF-prone areas globally. The process chain for an avalanche-induced GLOF was modeled numerically. The volume and velocity of debris from avalanches entering various future lake extents were calculated using RAMMS. Resulting waves and downstream flooding were simulated using BASEMENT to evaluate erosion at the terminal moraine. Wave characteristics in BASEMENT were validated with empirical equations to ensure the proper transfer of momentum from the avalanche to the lake. Moraine erosion was determined for two geomorphologic scenarios: a site-specific scenario using field samples, and a worst-case scenario based on past literature. Both cases resulted in no flooding outside the river channel at downstream villages. Worst-case scenario geomorphology resulted in increased channelization of the lake outlet and some moraine erosion but no catastrophic collapse. Site-specific data yielded similar results but with even less erosion and downstream discharge. While the models confirmed that Imja Tsho is unlikely to produce a catastrophic GLOF in the near future, they also highlight the importance of continued monitoring of the lake. Furthermore, the ease and flexibility of these methods allows for their adoption by a wide range of stakeholders for modeling other high-risk lakes.

The Impact of Affect on Out-Group Judgments Depends on Dominant Information-Processing Styles: Evidence From Incidental and Integral Affect Paradigms.

PubMed

Isbell, Linda M; Lair, Elicia C; Rovenpor, Daniel R

2016-04-01

Two studies tested the affect-as-cognitive-feedback model, in which positive and negative affective states are not uniquely associated with particular processing styles, but rather serve as feedback about currently accessible processing styles. The studies extend existing work by investigating (a) both incidental and integral affect, (b) out-group judgments, and (c) downstream consequences. We manipulated processing styles and either incidental (Study 1) or integral (Study 2) affect and measured perceptions of out-group homogeneity. Positive (relative to negative) affect increased out-group homogeneity judgments when global processing was primed, but under local priming, the effect reversed (Studies 1 and 2). A similar interactive effect emerged on attributions, which had downstream consequences for behavioral intentions (Study 2). These results demonstrate that both incidental and integral affect do not directly produce specific processing styles, but rather influence thinking by providing feedback about currently accessible processing styles. © 2016 by the Society for Personality and Social Psychology, Inc.

Novel Strategies for Upstream and Downstream Processing of Tannin Acyl Hydrolase

PubMed Central

Rodríguez-Durán, Luis V.; Valdivia-Urdiales, Blanca; Contreras-Esquivel, Juan C.; Rodríguez-Herrera, Raúl; Aguilar, Cristóbal N.

2011-01-01

Tannin acyl hydrolase also referred as tannase is an enzyme with important applications in several science and technology fields. Due to its hydrolytic and synthetic properties, tannase could be used to reduce the negative effects of tannins in beverages, food, feed, and tannery effluents, for the production of gallic acid from tannin-rich materials, the elucidation of tannin structure, and the synthesis of gallic acid esters in nonaqueous media. However, industrial applications of tannase are still very limited due to its high production cost. Thus, there is a growing interest in the production, recovery, and purification of this enzyme. Recently, there have been published a number of papers on the improvement of upstream and downstream processing of the enzyme. These papers dealt with the search for new tannase producing microorganisms, the application of novel fermentation systems, optimization of culture conditions, the production of the enzyme by recombinant microorganism, and the design of efficient protocols for tannase recovery and purification. The present work reviews the state of the art of basic and biotechnological aspects of tannin acyl hydrolase, focusing on the recent advances in the upstream and downstream processing of the enzyme. PMID:21941633

An exploratory investigation of polar organic compounds in waters from a lead–zinc mine and mill complex

USGS Publications Warehouse

Rostad, Colleen E.; Schmitt, Christopher J.; Schumacher, John G.; Leiker, Thomas J.

2011-01-01

Surface water samples were collected in 2006 from a lead mine-mill complex in Missouri to investigate possible organic compounds coming from the milling process. Water samples contained relatively high concentrations of dissolved organic carbon (DOC; greater than 20 mg/l) for surface waters but were colorless, implying a lack of naturally occurring aquatic humic or fulvic acids. Samples were extracted by three different types of solid-phase extraction and analyzed by electrospray ionization/mass spectrometry. Because large amounts of xanthate complexation reagents are used in the milling process, techniques were developed to extract and analyze for sodium isopropyl xanthate and sodium ethyl xanthate. Although these xanthate reagents were not found, trace amounts of the degradates, isopropyl xanthyl thiosulfonate and isopropyl xanthyl sulfonate, were found in most locations sampled, including the tailings pond downstream. Dioctyl sulfosuccinate, a surfactant and process filtering aid, was found at concentrations estimated at 350 μg/l at one mill outlet, but not downstream. Release of these organic compounds downstream from lead-zinc mine and milling areas has not previously been reported. A majority of the DOC remains unidentified.

FAD oxidizes the ERO1-PDI electron transfer chain: The role of membrane integrity

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

Papp, Eszter; Nardai, Gabor; Mandl, Jozsef

2005-12-16

The molecular steps of the electron transfer in the endoplasmic reticulum from the secreted proteins during their oxidation are relatively unknown. We present here that flavine adenine dinucleotide (FAD) is a powerful oxidizer of the oxidoreductase system, Ero1 and PDI, besides the proteins of rat liver microsomes and HepG2 hepatoma cells. Inhibition of FAD transport hindered the action of FAD. Microsomal membrane integrity was mandatory for all FAD-related oxidation steps downstream of Ero1. The PDI inhibitor bacitracin could inhibit FAD-mediated oxidation of microsomal proteins and PDI, but did not hinder the FAD-driven oxidation of Ero1. Our data demonstrated that Ero1more » can utilize FAD as an electron acceptor and that FAD-driven protein oxidation goes through the Ero1-PDI pathway and requires the integrity of the endoplasmic reticulum membrane. Our findings prompt further studies to elucidate the membrane-dependent steps of PDI oxidation and the role of FAD in redox folding.« less

Flocculation increases the efficacy of depth filtration during the downstream processing of recombinant pharmaceutical proteins produced in tobacco.

PubMed

Buyel, Johannes F; Fischer, Rainer

2014-02-01

Flocculation is a cost-effective method that is used to improve the efficiency of clarification by causing dispersed particles to clump together, allowing their removal by sedimentation, centrifugation or filtration. The efficacy of flocculation for any given process depends on the nature and concentration of the particulates in the feed stream, the concentration, charge density and length of the flocculant polymer, the shear rate, the properties of the feed stream (e.g. pH and ionic strength) and the properties of the target products. We tested a range of flocculants and process conditions using a design of experiments approach to identify the most suitable polymers for the clarification step during the production of a HIV-neutralizing monoclonal antibody (2G12) and a fluorescent marker protein (DsRed) expressed in transgenic tobacco leaves. Among the 23 different flocculants we tested, the greatest reduction in turbidity was achieved with Polymin P, a branched, cationic polyethylenimine with a charge density of 13.0 meq/g. This flocculant reduced turbidity by more than 90% under a wide range of process conditions. We developed a model that predicted its performance under different process conditions, and this enabled us to increase the depth filter capacity three-sevenfold depending on the process scale, depth filter type and plant species. The costs of filter consumables were reduced by more than 50% compared with a process without flocculant, and there was no loss of recovery for either 2G12 or DsRed. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Genetic manipulation of STEP reverses behavioral abnormalities in a fragile X syndrome mouse model.

PubMed

Goebel-Goody, S M; Wilson-Wallis, E D; Royston, S; Tagliatela, S M; Naegele, J R; Lombroso, P J

2012-07-01

Fragile X syndrome (FXS), the most common inherited form of intellectual disability and prevailing known genetic basis of autism, is caused by an expansion in the Fmr1 gene that prevents transcription and translation of fragile X mental retardation protein (FMRP). FMRP binds to and controls translation of mRNAs downstream of metabotropic glutamate receptor (mGluR) activation. Recent work shows that FMRP interacts with the transcript encoding striatal-enriched protein tyrosine phosphatase (STEP; Ptpn5). STEP opposes synaptic strengthening and promotes synaptic weakening by dephosphorylating its substrates, including ERK1/2, p38, Fyn and Pyk2, and subunits of N-methyl-d-aspartate (NMDA) and AMPA receptors. Here, we show that basal levels of STEP are elevated and mGluR-dependent STEP synthesis is absent in Fmr1(KO) mice. We hypothesized that the weakened synaptic strength and behavioral abnormalities reported in FXS may be linked to excess levels of STEP. To test this hypothesis, we reduced or eliminated STEP genetically in Fmr1(KO) mice and assessed mice in a battery of behavioral tests. In addition to attenuating audiogenic seizures and seizure-induced c-Fos activation in the periaqueductal gray, genetically reducing STEP in Fmr1(KO) mice reversed characteristic social abnormalities, including approach, investigation and anxiety. Loss of STEP also corrected select nonsocial anxiety-related behaviors in Fmr1(KO) mice, such as light-side exploration in the light/dark box. Our findings indicate that genetically reducing STEP significantly diminishes seizures and restores select social and nonsocial anxiety-related behaviors in Fmr1(KO) mice, suggesting that strategies to inhibit STEP activity may be effective for treating patients with FXS. © 2012 The Authors. Genes, Brain and Behavior © 2012 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.

Investigating selective transport and abrasion on an alluvial fan using quantitative grain size and shape analysis

NASA Astrophysics Data System (ADS)

Litwin, K. L.; Jerolmack, D. J.

2011-12-01

Selective sorting and abrasion are the two major fluvial processes that are attributed to the downstream fining of sediments in rivers and alluvial fans. Selective transport is the process by which smaller grains are preferentially transported downstream while larger grains are deposited closer to the source. Abrasion is defined by the production of fine sediments and sand that occurs by saltation of gravel, where particle-to-particle collisions supply the energy required to break apart grains. We hypothesize that abrasion results in the gradual fining of large grains and the production of fine sands and silts, while sorting accounts for the differences in transport of these two grain-size fractions produced from abrasion, thereby creating the abrupt gravel-sand transition observed in many channel systems. In this research, we explore both selective transport and abrasion processes on the Dog Canyon alluvial fan near Alamogordo, New Mexico. We complete an extensive grain size analysis down the main channel of the fan employing an image-based technique that utilizes an autocorrelation process. We also characterize changes in grain shape using standard shape parameters, as well as Fourier analysis, which allows the study of contributions of grain roughness on a variety of length scales. Sorting appears to dominate the upper portion of the fan; the grain-size distribution narrows moving downstream until reaching a point of equal mobility, at which point sorting ceases. Abrasion exerts a subtle but persistent effect on grains during transport down the fan. Shape analysis reveals that particles become more rounded by the removal of small-scale textural features, a process that is expected to only modestly influence grain size of gravel, but should produce significant quantities of sand. This study provides a better understanding of the importance of grain abrasion and sorting on the downstream fining of channel grains in an alluvial fan, as well as an improved knowledge about the abrupt gravel-sand transition observed in a majority of alluvial fans.

An assessment of sediment-transport processes in the Lower Mekong River based on deposit grain sizes, the CM technique and flow-energy data

NASA Astrophysics Data System (ADS)

Bravard, Jean-Paul; Goichot, Marc; Tronchère, Hervé

2014-02-01

The Lower Mekong River has been an important research topic for at least 15 years, notably in the fields of hydrology, fluvial geomorphology, and the impact of dams. Recent papers refer mostly to the impact of the Lancang chain of hydroelectric dams constructed on the Chinese section of the river. Among the pending scientific questions are (1) the upstream-downstream variations in the concentration and yield of suspended sediment and (2) the relative importance of sand in the total yield. The general consensus among the scientific community is that the relative importance of sand in suspended load is not the main scientific concern despite its extensive presence in the Mekong River channel, as noted by geomorphologists, and despite its extreme importance for the stability of the delta shoreline in Viet Nam. This paper contradicts the general consensus. Its objective is to present new information on the processes of sand transport in the Lower Mekong channel. Imagery, field observations, sampling on the river banks, and grain size analysis of sand deposits have been carried out between Chiang Sean (downstream of the Chinese border) and the delta. The C-M image technique served to discriminate between the various transport processes (bedload and different types of suspension). This technique helps in understanding the changing processes responsible for downstream sediment transfer in river channels. The results of this study are the following.

Mountains, Climate Change and North American Water Security

NASA Astrophysics Data System (ADS)

Pomeroy, J. W.; Fang, X.; Whitfield, P. H.; Rasouli, K.; Harder, P.; Siemens, E.; Pradhananga, D.

2016-12-01

The juxtaposition of cold high precipitation catchments in mountains and low precipitation in downstream lowlands means that mountain water supplies support over half the world's population and sustain most irrigation agriculture. How secure is this mountain water in northern North America? Irrigation and other consumptive downstream uses have put immense pressure on water supplied from the Canadian Rockies. Excess water from these rivers also carries risk. Downstream communities are often located in the flood plains of mountain rivers, making them subject to the extreme hydrometeorology of the headwaters as was evident in the BC/Alberta/Saskatchewan floods of 2013 and droughts of 2015/2016. Climate change is disproportionately warming high mountain areas and the impacts of warming on water are magnified in high mountains because seasonal snowpacks, perennial snowfields and glaciers form important stores of water and control the timing of release of water and the seasonal and annual discharge of major mountain rivers. Changes in mountain snow and glacial regimes are rapidly occurring in Western Canada and this is already impacting downstream water security by changing flood risk, streamflow timing and volume. Hydrological process modelling is diagnosing the causes of intensification of hydrological cycling and coupled to climate models suggesting that the timing and quantity of mountain waters will shift under certain climate, glacier cover and forest cover scenarios and so impact the water security of downstream food production. So far, changes in precipitation are matched by evapotranspiration and sublimation providing some resilience to change in streamflow due to intensification of hydrological cycling. Faster glacier melt in drought periods has buffered low flows but this capacity id dwindling as glaciers ablate. The International Network for Alpine Research Catchment Hydrology (INARCH) project of GEWEX is quantifying water resiliency and risk in mountain headwaters so as to better assess the water security of downstream regions. INARCH results from Western Canada suggest current mountain river resiliency is at risk from increased climate variability as rainfall runoff replaces snowmelt and glacier melt runoff processes.

Uptake of Space Technologies - An Educational Programme

NASA Astrophysics Data System (ADS)

Bacai, Hina; Zolotikova, Svetlana; Young, Mandy; Cowsill, Rhys; Wells, Alan; Monks, Paul; Archibald, Alexandra; Smith, Teresa

2013-04-01

Earth Observation data and remote sensing technologies have been maturing into useful tools that can be utilised by local authorities and businesses to aid in activates such as monitoring climate change trends and managing agricultural land and water uses. The European Earth observation programme Copernicus, previously known as GMES (Global Monitoring for Environment and Security), provides the means to collect and process multi-source EO and environmental data that supports policy developments at the European level. At the regional and local level, the Copernicus programme has been initiated through Regional Contact Office (RCO), which provide knowledge, training, and access to expertise both locally and at a European level through the network of RCOs established across Europe in the DORIS_Net (Downstream Observatory organised by Regions active In Space - Network) project (Grant Agreement No. 262789 Coordination and support action (Coordinating) FP7 SPA.2010.1.1-07 "Fostering downstream activities and links with regions"). In the East Midlands UK RCO, educational and training workshops and modules have been organised to highlight the wider range of tools and application available to businesses and local authorities in the region. Engagement with businesses and LRA highlighted the need to have a tiered system of training to build awareness prior to investigating innovative solutions and space technology uses for societal benefits. In this paper we outline education and training programmes which have been developed at G-STEP (GMES - Science and Technology Education Partnership), University of Leicester, UK to open up the Copernicus programme through the Regional Contact Office to downstream users such as local businesses and LRAs. Innovative methods to introduce the operational uses of Space technologies in real cases through e-learning modules and web-based tools will be described and examples of good practice for educational training in these sectors will be demonstrated. The results from these workshops and awareness building campaigns will show the end-user 'pull' in the uptake of remote sensing and Earth Observation data to implement successful Local Authority action plans and projects developing innovative solutions to critical Local Authority issues.

Flood hydrology and dam-breach hydraulic analyses of four reservoirs in the Black Hills, South Dakota

USGS Publications Warehouse

Hoogestraat, Galen K.

2011-01-01

Extensive information about the construction of dams or potential downstream hazards in the event of a dam breach is not available for many small reservoirs within the Black Hills National Forest. In 2009, the U.S. Forest Service identified the need for reconnaissance-level dam-breach assessments for four of these reservoirs within the Black Hills National Forest (Iron Creek, Horsethief, Lakota, and Mitchell Lakes) with the potential to flood downstream structures. Flood hydrology and dam-breach hydraulic analyses for the four selected reservoirs were conducted by the U.S. Geological Survey in cooperation with the U.S. Forest service to estimate the areal extent of downstream inundation. Three high-flow breach scenarios were considered for cases when the dam is in place (overtopped) and when a dam break (failure) occurs: the 100-year recurrence 24-hour precipitation, 500-year recurrence peak flow, and the probable maximum precipitation. Inundation maps were developed that show the estimated extent of downstream floodwaters from simulated scenarios. Simulation results were used to determine the hazard classification of a dam break (high, significant, or low), based primarily on the potential for loss of life or property damage resulting from downstream inundation because of the flood surge.The inflow design floods resulting from the two simulated storm events (100-year 24-hour and probable maximum precipitation) were determined using the U.S. Army Corps of Engineers Hydrologic Engineering Center Hydrologic Modeling System (HEC-HMS). The inflow design flood for the 500-year recurrence peak flow was determined by using regional regression equations developed for streamflow-gaging stations with similar watershed characteristics. The step-backwater hydraulic analysis model, Hydrologic Engineering Center's River Analysis System (HEC-RAS), was used to determine water-surface profiles of in-place and dam-break scenarios for the three inflow design floods that were simulated. Inundation maps for in-place and dam-break scenarios were developed for the area downstream from the dam to the mouth of each stream.Dam-break scenarios for three of the four reservoirs assessed in this study were rated as low hazards owing to absence of permanent structures downstream from the dams. Iron Creek Lake's downstream channel to its mouth does not include any permanent structures within the inundation flood plains. For the two reservoirs with the largest watershed areas, Lakota and Mitchell Lake, the additional floodwater surge resulting from a dam break would be minor relative to the magnitude of the large flood streamflow into the reservoirs, based on the similar areal extent of inundation for the in-place and dam-break scenarios as indicated by the developed maps. A dam-break scenario at Horsethief Lake is rated as a significant hazard because of potential lives-in-jeopardy in downstream dwellings and appreciable economic loss.

Simulation of stream discharge and transport of nitrate and selected herbicides in the Mississippi River Basin

USGS Publications Warehouse

Broshears, R.E.; Clark, G.M.; Jobson, H.E.

2001-01-01

Stream discharge and the transport of nitrate, atrazine, and metolachlor in the Mississippi River Basin were simulated using the DAFLOW/BLTM hydrologic model. The simulated domain for stream discharge included river reaches downstream from the following stations in the National Stream Quality Accounting Network: Mississippi River at Clinton, IA; Missouri River at Hermann, MO: Ohio River at Grand Chain, IL: And Arkansas River at Little Rock, AR. Coefficients of hydraulic geometry were calibrated using data from water year 1996; the model was validated by favourable simulation of observed discharges in water years 1992-1994. The transport of nitrate, atrazine, and metolachlor was simulated downstream from the Mississippi River at Thebes, IL, and the Ohio River at Grand Chain. Simulated concentrations compared favourably with observed concentrations at Baton Rouge, LA. Development of this model is a preliminary step in gaining a more quantitative understanding of the sources and fate of nutrients and pesticides delivered from the Mississippi River Basin to the Gulf of Mexico.

METHOD AND APPARATUS FOR THE DETECTION OF LEAKS IN PIPE LINES

DOEpatents

Jefferson, S.; Cameron, J.F.

1961-11-28

A method is described for detecting leaks in pipe lines carrying fluid. The steps include the following: injecting a radioactive solution into a fluid flowing in the line; flushing the line clear of the radioactive solution; introducing a detector-recorder unit, comprising a radioactivity radiation detector and a recorder which records the detector signal over a time period at a substantially constant speed, into the line in association with a go-devil capable of propelling the detector-recorder unit through the line in the direction of the fluid flow at a substantia1ly constant velocity; placing a series of sources of radioactivity at predetermined distances along the downstream part of the line to make a characteristic signal on the recorder record at intervals corresponding to the location of said sources; recovering the detector-recorder unit at a downstream point along the line; transcribing the recorder record of any radioactivity detected during the travel of the detector- recorder unit in terms of distance along the line. (AEC)

Arkas: Rapid reproducible RNAseq analysis

PubMed Central

Colombo, Anthony R.; J. Triche Jr, Timothy; Ramsingh, Giridharan

2017-01-01

The recently introduced Kallisto pseudoaligner has radically simplified the quantification of transcripts in RNA-sequencing experiments.  We offer cloud-scale RNAseq pipelines Arkas-Quantification, and Arkas-Analysis available within Illumina’s BaseSpace cloud application platform which expedites Kallisto preparatory routines, reliably calculates differential expression, and performs gene-set enrichment of REACTOME pathways .  Due to inherit inefficiencies of scale, Illumina's BaseSpace computing platform offers a massively parallel distributive environment improving data management services and data importing.   Arkas-Quantification deploys Kallisto for parallel cloud computations and is conveniently integrated downstream from the BaseSpace Sequence Read Archive (SRA) import/conversion application titled SRA Import.  Arkas-Analysis annotates the Kallisto results by extracting structured information directly from source FASTA files with per-contig metadata, calculates the differential expression and gene-set enrichment analysis on both coding genes and transcripts. The Arkas cloud pipeline supports ENSEMBL transcriptomes and can be used downstream from the SRA Import facilitating raw sequencing importing, SRA FASTQ conversion, RNA quantification and analysis steps. PMID:28868134

Research on shock wave characteristics in the isolator of central strut rocket-based combined cycle engine under Ma5.5

NASA Astrophysics Data System (ADS)

Wei, Xianggeng; Xue, Rui; Qin, Fei; Hu, Chunbo; He, Guoqiang

2017-11-01

A numerical calculation of shock wave characteristics in the isolator of central strut rocket-based combined cycle (RBCC) engine fueled by kerosene was carried out in this paper. A 3D numerical model was established by the DES method. The kerosene chemical kinetic model used the 9-component and 12-step simplified mechanism model. Effects of fuel equivalence ratio, inflow total temperature and central strut rocket on-off on shock wave characteristics were studied under Ma5.5. Results demonstrated that with the increase of equivalence ratio, the leading shock wave moves toward upstream, accompanied with higher possibility of the inlet unstart. However, the leading shock wave moves toward downstream as the inflow total temperature rises. After the central strut rocket is closed, the leading shock wave moves toward downstream, which can reduce risks of the inlet unstart. State of the shear layer formed by the strut rocket jet flow and inflow can influence the shock train structure significantly.

Use of Bayesian Networks to Probabilistically Model and Improve the Likelihood of Validation of Microarray Findings by RT-PCR

PubMed Central

English, Sangeeta B.; Shih, Shou-Ching; Ramoni, Marco F.; Smith, Lois E.; Butte, Atul J.

2014-01-01

Though genome-wide technologies, such as microarrays, are widely used, data from these methods are considered noisy; there is still varied success in downstream biological validation. We report a method that increases the likelihood of successfully validating microarray findings using real time RT-PCR, including genes at low expression levels and with small differences. We use a Bayesian network to identify the most relevant sources of noise based on the successes and failures in validation for an initial set of selected genes, and then improve our subsequent selection of genes for validation based on eliminating these sources of noise. The network displays the significant sources of noise in an experiment, and scores the likelihood of validation for every gene. We show how the method can significantly increase validation success rates. In conclusion, in this study, we have successfully added a new automated step to determine the contributory sources of noise that determine successful or unsuccessful downstream biological validation. PMID:18790084

The Major Apoptotic Pathway Activated and Suppressed by Poliovirus

PubMed Central

Belov, George A.; Romanova, Lyudmila I.; Tolskaya, Elena A.; Kolesnikova, Marina S.; Lazebnik, Yuri A.; Agol, Vadim I.

2003-01-01

Cells respond to poliovirus infection by switching on the apoptotic program, implementation of which is usually suppressed by viral antiapoptotic functions. We show here that poliovirus infection of HeLa cells or derivatives of MCF-7 cells was accompanied by the efflux of cytochrome c from mitochondria. This efflux occurred during both abortive infection (e.g., interrupted by guanidine-HCl and ending with apoptosis) and productive infection (leading to cytopathic effect). The former type of infection, but not the latter, was accompanied by truncation of the proapoptotic protein Bid. The virus-triggered cytochrome c efflux was suppressed by overexpression of Bcl-2. Both abortive and productive infections also resulted in a decreased level of procaspase-9, as revealed by Western blotting. In the former case, this decrease was accompanied by the accumulation of a protein with the electrophoretic mobility of active caspase-9. In contrast, in the productively infected cells, the latter protein was absent but caspase-9-related polypeptides with altered mobility could be detected. Both caspase-9 and caspase-3 were shown to be essential for the development of such hallmarks of virus-induced apoptosis as chromatin condensation, DNA degradation, and nuclear fragmentation. These and some other results suggest the following scenario. Poliovirus infection activates the apoptotic pathway, involving mitochondrial damage, cytochrome c efflux, and consecutive activation of caspase-9 and caspase-3. The apoptotic signal appears to be amplified by a loop which includes secondary processing of Bid. The implementation of the apoptotic program in productively infected cells may be suppressed, however, by the viral antiapoptotic functions, which act at a step(s) downstream of the cytochrome c efflux. The suppression appears to be caused, at least in part, by aberrant processing and degradation of procaspase-9. PMID:12477809

A bottom up approach to implementing multi-purpose mitigation measures for reducing flood risk and improving water quality in agricultural catchments

NASA Astrophysics Data System (ADS)

Wilkinson, M. E.; Quinn, P. F.; Jonczyk, J.; Burke, S.; Nicholson, A.; Barber, N.; Owen, G.; Palmer, M.

2012-04-01

A number of studies have suggested that there is evidence that modern land-use management practices have increased surface runoff at the local scale. There is an urgent need for interventions to reduce the risk of flooding whilst also delivering multiple benefits (doing more for less). There are many settlements, which regularly suffer from flooding, which would benefit from upstream mitigation measures. Interventions at the source of runoff generation can have a positive impact on the flood hydrograph downstream. An integrated approach to managing runoff can also have multiple benefits on pollution and ecology, which could lead to beneficial impacts at the catchment scale. Belford, a small community in Northumberland, UK has suffered from an increased number of flood events over the past ten years. There is currently support within the English and Welsh Environment Agency for sustainable flood management solutions such as storage ponds, wetlands, beaver dams and willow riparian features which are being trialled at Belford. These runoff attenuation features (RAFs) also have benefits to water quality, capture sediment and create new ecological zones. Although the process by which numerous RAFs were deployed in Belford proved initially difficult to achieve within the existing regulatory framework, an efficient uptake process is now supported by local regulators including several branches of the Environment Agency. The Belford runoff management framework provides a step by step guide to implementing mitigation measures in the Belford burn catchment and could be easily applied to other catchments at a similar scale. The approach is based on implementing mitigation measures through engaging with catchment stakeholders and using solid field science and management protocols.

Renaissance of protein crystallization and precipitation in biopharmaceuticals purification.

PubMed

Dos Santos, Raquel; Carvalho, Ana Luísa; Roque, A Cecília A

The current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand. With the upstream improvements, the bottleneck shifted towards the downstream process. New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective. Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules, overcoming the current chromatographic bottlenecks. Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules. This review focuses in the role of these two methodologies in current industrial purification processes, and highlights their potential implementation in the purification pipeline of high value therapeutic molecules, overcoming chromatographic holdups. Copyright © 2016 Elsevier Inc. All rights reserved.

Ion and electron dynamics generating the Hall current in the exhaust far downstream of the reconnection x-line

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

Fujimoto, Keizo, E-mail: keizo.fujimoto@nao.ac.jp; Takamoto, Makoto

2016-01-15

We have investigated the ion and electron dynamics generating the Hall current in the reconnection exhaust far downstream of the x-line where the exhaust width is much larger than the ion gyro-radius. A large-scale particle-in-cell simulation shows that most ions are accelerated through the Speiser-type motion in the current sheet formed at the center of the exhaust. The transition layers formed at the exhaust boundary are not identified as slow mode shocks. (The layers satisfy mostly the Rankine-Hugoniot conditions for a slow mode shock, but the energy conversion hardly occurs there.) We find that the ion drift velocity is modifiedmore » around the layer due to a finite Larmor radius effect. As a result, the ions are accumulated in the downstream side of the layer, so that collimated ion jets are generated. The electrons experience two steps of acceleration in the exhaust. The first is a parallel acceleration due to the out-of-plane electric field E{sub y} which has a parallel component in most area of the exhaust. The second is a perpendicular acceleration due to E{sub y} at the center of the current sheet and the motion is converted to the parallel direction. Because of the second acceleration, the electron outflow velocity becomes almost uniform over the exhaust. The difference in the outflow profile between the ions and electrons results in the Hall current in large area of the exhaust. The present study demonstrates the importance of the kinetic treatments for collisionless magnetic reconnection even far downstream from the x-line.« less

Impacts of large dams on downstream flow conditions of rivers: Aggradation and reduction of the Medjerda channel capacity downstream of the Sidi Salem dam (Tunisia)

NASA Astrophysics Data System (ADS)

Zahar, Yadh; Ghorbel, Abdelmajid; Albergel, Jean

2008-04-01

SummarySince the opening of the Sidi Salem dam on the watercourse of the Medjerda, in 1981, an alarming narrowing of the riverbed in the lower valley has been observed. This geo-morphological change is attributed to different factors ranking from the reduction in the discharge flows, which used to clean out the riverbed to the periodic releases of turbid water undertaken to remove the silt deposition inside the reservoir, which increased the sediment deposition in the downstream channel. Other smaller hydraulic projects are also held responsible for the loss of the water velocity including a series of concrete sills meant to raise water levels, numerous cross bridges and the management of the downstream Laroussia dam regulating the discharge from the Cap Bon canal. The above anthropogenic factors, in conjunction with natural topographical conditions characterized by a generally shallow slope and a very sinuous watercourse, led to an extremely rapid aggradation of the downstream channel-bed. This paper proposes an analysis of this process and argues that the resulting reduction in channel capacity is one of the major causes of the large floods experienced in the country since 1996.

Study of Unsteady Flows with Concave Wall Effect

NASA Technical Reports Server (NTRS)

Wang, Chi R.

2003-01-01

This paper presents computational fluid dynamic studies of the inlet turbulence and wall curvature effects on the flow steadiness at near wall surface locations in boundary layer flows. The time-stepping RANS numerical solver of the NASA Glenn-HT RANS code and a one-equation turbulence model, with a uniform inlet turbulence modeling level of the order of 10 percent of molecular viscosity, were used to perform the numerical computations. The approach was first calibrated for its predictabilities of friction factor, velocity, and temperature at near surface locations within a transitional boundary layer over concave wall. The approach was then used to predict the velocity and friction factor variations in a boundary layer recovering from concave curvature. As time iteration proceeded in the computations, the computed friction factors converged to their values from existing experiments. The computed friction factors, velocity, and static temperatures at near wall surface locations oscillated periodically in terms of time iteration steps and physical locations along the span-wise direction. At the upstream stations, the relationship among the normal and tangential velocities showed vortices effects on the velocity variations. Coherent vortices effect on the velocity components broke down at downstream stations. The computations also predicted the vortices effects on the velocity variations within a boundary layer flow developed along a concave wall surface with a downstream recovery flat wall surface. It was concluded that the computational approach might have the potential to analyze the flow steadiness in a turbine blade flow.

Spatiotemporal variability of inorganic nutrients during wastewater effluent dominated streamflow conditions in Indian Creek, Johnson County, Kansas, 2012–15

USGS Publications Warehouse

Foster, Guy M.; Graham, Jennifer L.; Williams, Thomas J.; King, Lindsey R.

2016-10-31

Nutrients, particularly nitrogen and phosphorus, are a leading cause of water-quality impairment in Kansas and the Nation. Indian Creek is one of the most urban drainage basins in Johnson County, Kansas, and environmental and biological conditions are affected by contaminants from point and other urban sources. The Johnson County Douglas L. Smith Middle Basin (hereinafter Middle Basin) wastewater treatment facility (WWTF) is the largest point-source discharge on Indian Creek. A second facility, the Tomahawk Creek WWTF, discharges into Indian Creek approximately 11.6 kilometers downstream from the Middle Basin WWTF. To better characterize the spatiotemporal variability of nutrients in Indian Creek, the U.S. Geological Survey, in cooperation with the Kansas Department of Health and Environment and Johnson County Wastewater, collected high-resolution spatial and temporal (a large number of samples collected over the entire reach or at single locations over a long period of time) inorganic nutrient (nitrate plus nitrite and orthophosphorus) data using a combination of discrete samples and sensor-measured data during 2012 through 2015.Nutrient patterns observed in Indian Creek along the upstream-downstream gradient during wastewater effluent dominated streamflow conditions were largely affected by the WWTFs and by travel time of the parcels of water. Nitrate plus nitrite concentrations in the Middle Basin WWTF effluent and at downstream sites varied by as much as 6 milligrams per liter over a 24-hour period. The cyclical variability in the Middle Basin WWTF effluent generated a nitrate plus nitrite pulse that could be tracked for approximately 11.5 kilometers downstream in Indian Creek, until the effect was masked by the Tomahawk Creek WWTF effluent discharge. All longitudinal surveys showed the same general patterns along the upstream-downstream gradient, though streamflows, wastewater effluent contributions to streamflow, and nutrient concentrations spanned a wide range. Differences in orthophosphorus and nitrate plus nitrite patterns were clear along the upstream-downstream gradient in Indian Creek, and orthophosphorus concentrations were not as variable as nitrate plus nitrite concentrations. In general, nitrate plus nitrite concentrations decreased downstream from the Middle Basin WWTF to minima near the confluence with Tomahawk Creek, increased downstream from the Tomahawk Creek WWTF, and then varied little within the study reach. Orthophosphorus concentrations generally decreased downstream from the Middle Basin WWTF.Despite the marked variability in nitrate plus nitrite concentrations caused by the Middle Basin WWTF effluent discharges, decreases in nitrate plus nitrite concentrations were discernable along the study reach between the two WWTFs. Decreases in nitrate plus nitrite concentrations along study reach were less variable than the cyclical variability typically measured, reiterating the effect of the Middle Basin WWTF effluent discharges on the spatiotemporal variability of nitrate plus nitrite in Indian Creek. Although decreases and rates of change in nitrate plus nitrite concentration were similar between the upper and lower reaches of Indian Creek, relations with initial nitrate plus nitrite concentrations and seasonal patterns were different between the upper (from College to the Marty study sites) and lower reaches (from Marty to the Mission Farms study sites) and did not reflect patterns observed for the overall reach. Quantifying the decreases in nitrate plus nitrite concentration caused by dilution and other in-stream processes were beyond the scope of this study, and were limited by available data. The data that are available suggest that dilution and other in-stream processes play a role in decreasing nitrate plus nitrite concentrations downstream from the Middle Basin WWTF in Indian Creek.Analysis of the spatiotemporal variability of nutrients focused on below-normal and normal streamflow conditions, when streamflow and nutrient conditions in Indian Creek were largely controlled by WWTF effluent flows and nutrient removal processes. Spatial and temporal data indicate there are decreases in nutrient concentrations along the upstream-downstream gradient in Indian Creek, but quantifying decreases is complicated by the variability in nutrient concentrations caused by the WWTFs. During below-normal and normal streamflow conditions, Indian Creek nutrient concentrations downstream from the Middle Basin WWTF primarily reflect effluent concentrations in the hours or days before depending on relative distance downstream.

Integrated optimisation technique based on computer-aided capacity and safety evaluation for managing downstream lane-drop merging area of signalised junctions

NASA Astrophysics Data System (ADS)

Chen, CHAI; Yiik Diew, WONG

2017-02-01

This study provides an integrated strategy, encompassing microscopic simulation, safety assessment, and multi-attribute decision-making, to optimize traffic performance at downstream merging area of signalized intersections. A Fuzzy Cellular Automata (FCA) model is developed to replicate microscopic movement and merging behavior. Based on simulation experiment, the proposed FCA approach is able to provide capacity and safety evaluation of different traffic scenarios. The results are then evaluated through data envelopment analysis (DEA) and analytic hierarchy process (AHP). Optimized geometric layout and control strategies are then suggested for various traffic conditions. An optimal lane-drop distance that is dependent on traffic volume and speed limit can thus be established at the downstream merging area.

Optimized scalable network switch

DOEpatents

Blumrich, Matthias A [Ridgefield, CT; Chen, Dong [Croton On Hudson, NY; Coteus, Paul W [Yorktown Heights, NY; Gara, Alan G [Mount Kisco, NY; Giampapa, Mark E [Irvington, NY; Heidelberger, Philip [Cortlandt Manor, NY; Steinmacher-Burow, Burkhard D [Mount Kisco, NY; Takken, Todd E [Mount Kisco, NY; Vranas, Pavlos M [Bedford Hills, NY

2007-12-04

In a massively parallel computing system having a plurality of nodes configured in m multi-dimensions, each node including a computing device, a method for routing packets towards their destination nodes is provided which includes generating at least one of a 2m plurality of compact bit vectors containing information derived from downstream nodes. A multilevel arbitration process in which downstream information stored in the compact vectors, such as link status information and fullness of downstream buffers, is used to determine a preferred direction and virtual channel for packet transmission. Preferred direction ranges are encoded and virtual channels are selected by examining the plurality of compact bit vectors. This dynamic routing method eliminates the necessity of routing tables, thus enhancing scalability of the switch.

Optimized scalable network switch

DOEpatents

Blumrich, Matthias A.; Chen, Dong; Coteus, Paul W.

2010-02-23

In a massively parallel computing system having a plurality of nodes configured in m multi-dimensions, each node including a computing device, a method for routing packets towards their destination nodes is provided which includes generating at least one of a 2m plurality of compact bit vectors containing information derived from downstream nodes. A multilevel arbitration process in which downstream information stored in the compact vectors, such as link status information and fullness of downstream buffers, is used to determine a preferred direction and virtual channel for packet transmission. Preferred direction ranges are encoded and virtual channels are selected by examining the plurality of compact bit vectors. This dynamic routing method eliminates the necessity of routing tables, thus enhancing scalability of the switch.

Continuous downstream processing for high value biological products: A Review.

PubMed

Zydney, Andrew L

2016-03-01

There is growing interest in the possibility of developing truly continuous processes for the large-scale production of high value biological products. Continuous processing has the potential to provide significant reductions in cost and facility size while improving product quality and facilitating the design of flexible multi-product manufacturing facilities. This paper reviews the current state-of-the-art in separations technology suitable for continuous downstream bioprocessing, focusing on unit operations that would be most appropriate for the production of secreted proteins like monoclonal antibodies. This includes cell separation/recycle from the perfusion bioreactor, initial product recovery (capture), product purification (polishing), and formulation. Of particular importance are the available options, and alternatives, for continuous chromatographic separations. Although there are still significant challenges in developing integrated continuous bioprocesses, recent technological advances have provided process developers with a number of attractive options for development of truly continuous bioprocessing operations. © 2015 Wiley Periodicals, Inc.

Effect of wastewater treatment facility closure on endocrine disrupting chemicals in a Coastal Plain stream

USGS Publications Warehouse

Bradley, Paul M.; Journey, Celeste A.; Clark, Jimmy M.

2016-01-01

Wastewater treatment facility (WWTF) closures are rare environmental remediation events; offering unique insight into contaminant persistence, long-term wastewater impacts, and ecosystem recovery processes. The U.S. Geological Survey assessed the fate of select endocrine disrupting chemicals (EDC) in surface water and streambed sediment one year before and one year after closure of a long-term WWTF located within the Spirit Creek watershed at Fort Gordon, Georgia. Sample sites included a WWTF-effluent control located upstream from the outfall, three downstream effluent-impacted sites located between the outfall and Spirit Lake, and one downstream from the lake's outfall. Prior to closure, the 2.2-km stream segment downstream from the WWTF outfall was characterized by EDC concentrations significantly higher (α = 0.05) than at the control site; indicating substantial downstream transport and limited in-stream attenuation of EDC, including pharmaceuticals, estrogens, alkylphenol ethoxylate (APE) metabolites, and organophosphate flame retardants (OPFR). Wastewater-derived pharmaceutical, APE metabolites, and OPFR compounds were also detected in the outflow of Spirit Lake, indicating the potential for EDC transport to aquatic ecosystems downstream of Fort Gordon under effluent discharge conditions. After the WWTF closure, no significant differences in concentrations or numbers of detected EDC compounds were observed between control and downstream locations. The results indicated EDC pseudo-persistence under preclosure, continuous supply conditions, with rapid attenuation following WWTF closure. Low concentrations of EDC at the control site throughout the study and comparable concentrations in downstream locations after WWTF closure indicated additional, continuing, upstream contaminant sources within the Spirit Creek watershed. 

Research on the coordination framework for water resources utilization on the interests of mutual compensation in Lancang-Mekong River

NASA Astrophysics Data System (ADS)

Wang, Y.; Fang, D., VI; Xu, J.; Dong, Q.

2017-12-01

The Lancang-Mekong River is an important international river, cascaded hydropower stations development in which attracts the attention of downstream countries. In this paper, we proposed a coordination framework for water resources utilization on the interests of mutual compensation to relieve the conflict of upstream and downstream countries. Firstly, analyze the benefits and risks caused by the cascaded hydropower stations development and the evolution process of water resources use conflict between upstream and downstream countries. Secondly, evaluate the benefits and risks of flood control, water supply, navigation and power generation based on the energy theory of cascaded hydropower stations development in Lancang-Mekong River. Thirdly, multi-agent cooperation motivation and cooperation conditions between upstream and downstream countries in Lancang-Mekong River is given. Finally, the coordination framework for water resources utilization on the interests of mutual compensation in Lancang-Mekong River is presented. This coordination framework for water resources utilization can increase comprehensive benefits in Lancang-Mekong River.

Use of continuous monitoring to assess stream nitrate flux and transformation patterns.

PubMed

Jones, Christopher; Kim, Sea-Won; Schilling, Keith

2017-01-01

Delivery of nitrogen from farmed fields to the stream network is an ongoing water quality issue in central North America and other parts of the world. Although fertilization and other farming practices have been refined to produce environmental improvements, stemming loss of nitrogen, especially in the soluble nitrate form, is a problem that has seemingly defied solution. The Iowa Nutrient Reduction Strategy is a policy initiative designed to implement conservation and other farm management practices to produce reductions in nitrate loading. The strategy does not focus on how the streams themselves may or may not be processing nitrogen and reducing downstream loading. We used continuous high-frequency nitrate and discharge monitoring over 3 years at two sites separated by 18 km in a low-order, agricultural stream in eastern Iowa to estimate how nitrogen is processed, and whether or not these processes are reducing downstream loading. We conclude that the upstream to downstream nitrate concentration decline between the two sites was not driven by denitrification. These data also show that nitrate concentrations are closely coupled to discharge during periods of adequate moisture, but decoupling of concentration from discharge occurs during dry periods. This decoupling is a possible indicator of in-stream nitrate processing. Finally, nitrate concentrations are likely diluted by water sourced from non-row crop land covers in the lower reaches of the watershed.

Hydroxyl carboxylate based non-phosphorus corrosion inhibition process for reclaimed water pipeline and downstream recirculating cooling water system.

PubMed

Wang, Jun; Wang, Dong; Hou, Deyin

2016-01-01

A combined process was developed to inhibit the corrosion both in the pipeline of reclaimed water supplies (PRWS) and in downstream recirculating cooling water systems (RCWS) using the reclaimed water as makeup. Hydroxyl carboxylate-based corrosion inhibitors (e.g., gluconate, citrate, tartrate) and zinc sulfate heptahydrate, which provided Zn(2+) as a synergistic corrosion inhibition additive, were added prior to the PRWS when the phosphate (which could be utilized as a corrosion inhibitor) content in the reclaimed water was below 1.7 mg/L, and no additional corrosion inhibitors were required for the downstream RCWS. Satisfactory corrosion inhibition was achieved even if the RCWS was operated under the condition of high numbers of concentration cycles. The corrosion inhibition requirement was also met by the appropriate combination of PO4(3-) and Zn(2+) when the phosphate content in the reclaimed water was more than 1.7 mg/L. The process integrated not only water reclamation and reuse, and the operation of a highly concentrated RCWS, but also the comprehensive utilization of phosphate in reclaimed water and the application of non-phosphorus corrosion inhibitors. The proposed process reduced the operating cost of the PRWS and the RCWS, and lowered the environmental hazard caused by the excessive discharge of phosphate. Furthermore, larger amounts of water resources could be conserved as a result. Copyright © 2015. Published by Elsevier B.V.

Geothermal Thermoelectric Generation (G-TEG) with Integrated Temperature Driven Membrane Distillation and Novel Manganese Oxide for Lithium Extraction

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

Renew, Jay; Hansen, Tim

Southern Research Institute (Southern) teamed with partners Novus Energy Technologies (Novus), Carus Corporation (Carus), and Applied Membrane Technology, Inc. (AMT) to develop an innovative Geothermal ThermoElectric Generation (G-TEG) system specially designed to both generate electricity and extract high-value lithium (Li) from low-temperature geothermal brines. The process combined five modular technologies including – silica removal, nanofiltration (NF), membrane distillation (MD), Mn-oxide sorbent for Li recovery, and TEG. This project provides a proof of concept for each of these technologies. The first step in the process is silica precipitation through metal addition and pH adjustment to prevent downstream scaling in membrane processes.more » Next, the geothermal brine is concentrated with the first of a two stage MD system. The first stage MD system is made of a high-temperature material to withstand geothermal brine temperatures up to 150C.° The first stage MD is integrated with a G-TEG module for simultaneous energy generation. The release of energy from the MD permeate drives heat transfer across the TE module, producing electricity. The first stage MD concentrate is then treated utilizing an NF system to remove Ca 2+ and Mg 2+. The NF concentrate will be disposed in the well by reinjection. The NF permeate undergoes concentration in a second stage of MD (polymeric material) to further concentrate Li in the NF permeate and enhance the efficiency of the downstream Li recovery process utilizing a Mn-oxide sorbent. Permeate from both the stages of the MD can be beneficially utilized as the permeates will contain less contaminants than the feed water. The concentrated geothermal brines are then contacted with the Mn-oxide sorbent. After Li from the geothermal brine is adsorbed on the sorbent, HCl is then utilized to regenerate the sorbent and recover the Li. The research and development project showed that the Si removal goal (>80%) could be achieved by increasing the pH of the brine and adding Fe 3+ under several scenarios. The NF was also successful in achieving significant Ca 2+ and Mg 2+ removal (~80%) while retaining most Li in the permeate for high strength brines. MD experiments showed that geothermal brines could be significantly concentrated with little fouling due to pre-treatment.« less

Analyzing the Impacts of Dams on Riparian Ecosystems: A Review of Research Strategies and Their Relevance to the Snake River Through Hells Canyon

PubMed Central

Braatne, Jeffrey H.; Goater, Lori A.; Blair, Charles L.

2007-01-01

River damming provides a dominant human impact on river environments worldwide, and while local impacts of reservoir flooding are immediate, subsequent ecological impacts downstream can be extensive. In this article, we assess seven research strategies for analyzing the impacts of dams and river flow regulation on riparian ecosystems. These include spatial comparisons of (1) upstream versus downstream reaches, (2) progressive downstream patterns, or (3) the dammed river versus an adjacent free-flowing or differently regulated river(s). Temporal comparisons consider (4) pre- versus post-dam, or (5) sequential post-dam conditions. However, spatial comparisons are complicated by the fact that dams are not randomly located, and temporal comparisons are commonly limited by sparse historic information. As a result, comparative approaches are often correlative and vulnerable to confounding factors. To complement these analyses, (6) flow or sediment modifications can be implemented to test causal associations. Finally, (7) process-based modeling represents a predictive approach incorporating hydrogeomorphic processes and their biological consequences. In a case study of Hells Canyon, the upstream versus downstream comparison is confounded by a dramatic geomorphic transition. Comparison of the multiple reaches below the dams should be useful, and the comparison of Snake River with the adjacent free-flowing Salmon River may provide the strongest spatial comparison. A pre- versus post-dam comparison would provide the most direct study approach, but pre-dam information is limited to historic reports and archival photographs. We conclude that multiple study approaches are essential to provide confident interpretations of ecological impacts downstream from dams, and propose a comprehensive study for Hells Canyon that integrates multiple research strategies. PMID:18043964

Economic Analysis of an Integrated Annatto Seeds-Sugarcane Biorefinery Using Supercritical CO2 Extraction as a First Step

PubMed Central

Albarelli, Juliana Q.; Santos, Diego T.; Cocero, María José; Meireles, M. Angela A.

2016-01-01

Recently, supercritical fluid extraction (SFE) has been indicated to be utilized as part of a biorefinery, rather than as a stand-alone technology, since besides extracting added value compounds selectively it has been shown to have a positive effect on the downstream processing of biomass. To this extent, this work evaluates economically the encouraging experimental results regarding the use of SFE during annatto seeds valorization. Additionally, other features were discussed such as the benefits of enhancing the bioactive compounds concentration through physical processes and of integrating the proposed annatto seeds biorefinery to a hypothetical sugarcane biorefinery, which produces its essential inputs, e.g., CO2, ethanol, heat and electricity. For this, first, different configurations were modeled and simulated using the commercial simulator Aspen Plus® to determine the mass and energy balances. Next, each configuration was economically assessed using MATLAB. SFE proved to be decisive to the economic feasibility of the proposed annatto seeds-sugarcane biorefinery concept. SFE pretreatment associated with sequential fine particles separation process enabled higher bixin-rich extract production using low-pressure solvent extraction method employing ethanol, meanwhile tocotrienols-rich extract is obtained as a first product. Nevertheless, the economic evaluation showed that increasing tocotrienols-rich extract production has a more pronounced positive impact on the economic viability of the concept. PMID:28773616

Validity of the Symbol Digit Modalities Test as a cognition performance outcome measure for multiple sclerosis

PubMed Central

Benedict, Ralph HB; DeLuca, John; Phillips, Glenn; LaRocca, Nicholas; Hudson, Lynn D; Rudick, Richard

2017-01-01

Cognitive and motor performance measures are commonly employed in multiple sclerosis (MS) research, particularly when the purpose is to determine the efficacy of treatment. The increasing focus of new therapies on slowing progression or reversing neurological disability makes the utilization of sensitive, reproducible, and valid measures essential. Processing speed is a basic elemental cognitive function that likely influences downstream processes such as memory. The Multiple Sclerosis Outcome Assessments Consortium (MSOAC) includes representatives from advocacy organizations, Food and Drug Administration (FDA), European Medicines Agency (EMA), National Institute of Neurological Disorders and Stroke (NINDS), academic institutions, and industry partners along with persons living with MS. Among the MSOAC goals is acceptance and qualification by regulators of performance outcomes that are highly reliable and valid, practical, cost-effective, and meaningful to persons with MS. A critical step for these neuroperformance metrics is elucidation of clinically relevant benchmarks, well-defined degrees of disability, and gradients of change that are deemed clinically meaningful. This topical review provides an overview of research on one particular cognitive measure, the Symbol Digit Modalities Test (SDMT), recognized as being particularly sensitive to slowed processing of information that is commonly seen in MS. The research in MS clearly supports the reliability and validity of this test and recently has supported a responder definition of SDMT change approximating 4 points or 10% in magnitude. PMID:28206827

Validity of the Symbol Digit Modalities Test as a cognition performance outcome measure for multiple sclerosis.

PubMed

Benedict, Ralph Hb; DeLuca, John; Phillips, Glenn; LaRocca, Nicholas; Hudson, Lynn D; Rudick, Richard

2017-04-01

Cognitive and motor performance measures are commonly employed in multiple sclerosis (MS) research, particularly when the purpose is to determine the efficacy of treatment. The increasing focus of new therapies on slowing progression or reversing neurological disability makes the utilization of sensitive, reproducible, and valid measures essential. Processing speed is a basic elemental cognitive function that likely influences downstream processes such as memory. The Multiple Sclerosis Outcome Assessments Consortium (MSOAC) includes representatives from advocacy organizations, Food and Drug Administration (FDA), European Medicines Agency (EMA), National Institute of Neurological Disorders and Stroke (NINDS), academic institutions, and industry partners along with persons living with MS. Among the MSOAC goals is acceptance and qualification by regulators of performance outcomes that are highly reliable and valid, practical, cost-effective, and meaningful to persons with MS. A critical step for these neuroperformance metrics is elucidation of clinically relevant benchmarks, well-defined degrees of disability, and gradients of change that are deemed clinically meaningful. This topical review provides an overview of research on one particular cognitive measure, the Symbol Digit Modalities Test (SDMT), recognized as being particularly sensitive to slowed processing of information that is commonly seen in MS. The research in MS clearly supports the reliability and validity of this test and recently has supported a responder definition of SDMT change approximating 4 points or 10% in magnitude.

Modelling of sedimentation and remobilization in in-line storage sewers for stormwater treatment.

PubMed

Frehmann, T; Flores, C; Luekewille, F; Mietzel, T; Spengler, B; Geiger, W F

2005-01-01

A special arrangement of combined sewer overflow tanks is the in-line storage sewer with downstream discharge (ISS-down). This layout has the advantage that, besides the sewer system, no other structures are required for stormwater treatment. The verification of the efficiency with respect to the processes of sedimentation and remobilization of sediment within the in-line storage sewer with downstream discharge is carried out in a combination of a field and a pilot plant study. The model study was carried out using a pilot plant model scaled 1:13. The following is intended to present some results of the pilot plant study and the mathematical empirical modelling of the sedimentation and remobilization process.

Assessment of turbulent models for scramjet flowfields

NASA Technical Reports Server (NTRS)

Sindir, M. M.; Harsha, P. T.

1982-01-01

The behavior of several turbulence models applied to the prediction of scramjet combustor flows is described. These models include the basic two equation model, the multiple dissipation length scale variant of the two equation model, and the algebraic stress model (ASM). Predictions were made of planar backward facing step flows and axisymmetric sudden expansion flows using each of these approaches. The formulation of each of these models are discussed, and the application of the different approaches to supersonic flows is described. A modified version of the ASM is found to provide the best prediction of the planar backward facing step flow in the region near the recirculation zone, while the basic ASM provides the best results downstream of the recirculation. Aspects of the interaction of numerica modeling and turbulences modeling as they affect the assessment of turbulence models are discussed.

An experimental study of combustion: The turbulent structure of a reacting shear layer formed at a rearward-facing step. Ph.D. Thesis. Final Report

NASA Technical Reports Server (NTRS)

Pitz, R. W.

1981-01-01

A premixed propane-air flame is stabilized in a turbulent free shear layer formed at a rearward-facing step. The mean and rms averages of the turbulent velocity flow field were determined by LDV for both reacting and non-reacting flows. The reaching flow was visualized by high speed schlieren photography. Large scale structures dominate the reacting shear layer. The growth of the large scale structures is tied to the propagation of the flame. The linear growth rate of the reacting shear layer defined by the mean velocity profiles is unchanged by combustion but the virtual origin is shifted downstream. The reacting shear layer based on the mean velocity profiles is shifted toward the recirculation zone and the reattachments lengths are shortened by 30%.

It's DE-licious: A Recipe for Differential Expression Analyses of RNA-seq Experiments Using Quasi-Likelihood Methods in edgeR.

PubMed

Lun, Aaron T L; Chen, Yunshun; Smyth, Gordon K

2016-01-01

RNA sequencing (RNA-seq) is widely used to profile transcriptional activity in biological systems. Here we present an analysis pipeline for differential expression analysis of RNA-seq experiments using the Rsubread and edgeR software packages. The basic pipeline includes read alignment and counting, filtering and normalization, modelling of biological variability and hypothesis testing. For hypothesis testing, we describe particularly the quasi-likelihood features of edgeR. Some more advanced downstream analysis steps are also covered, including complex comparisons, gene ontology enrichment analyses and gene set testing. The code required to run each step is described, along with an outline of the underlying theory. The chapter includes a case study in which the pipeline is used to study the expression profiles of mammary gland cells in virgin, pregnant and lactating mice.

EVALUATION OF AN ELECTRONIC PAEDIATRIC INTENSIVE CARE UNIT (PICU) MEDICATION RECONCILIATION (MR) FORM.

PubMed

Murray, Diane; Sedgeworth, Cathy; Kinnear, Moira; Diack, Lesley

2016-09-01

To gather opinions from doctors and pharmacists to improve the design of the PICU MR form generated by the electronic prescribing and clinical notes system to support transfer of care from PICU to downstream wards that use paper systems. A purposive sample of 10 forms covering a comprehensive range of medication information common to PICU patients was selected from practice between March 2014 and May 2014. Pharmacists (n=7) and doctors (n=9) who received these forms on downstream wards were invited to participate in semi-structured one-to-one interviews (n=20) with the PICU pharmacist within 48 hrs of receipt to explore their views about the form. The interview schedule was informed from literature and peer review. Comments and suggestions about layout and the MR process were invited. Two pilot interviews (1 pharmacist, 1 doctor) were conducted to test a priori themes were covered and that the questioning style was open, avoided leading and the participant was given time to consider their response. Interviews were recorded using an encrypted digital recorder, transcribed and checked (10%) for accuracy and coding. Framework analysis focused on documentation and work processes. A 110 bed paediatric hospital with critical care, medical, neurology, haematology, oncology and mixed speciality surgical services. The 8-bedded PICU uses the electronic clinical information system (CIS) MetaVision® provided by iMDsoft® and includes electronic prescribing and clinical notes. Downstream wards using paper systems are provided with a new paper drug chart and printed CIS documentation on transfer. New themes extracted during analysis included misunderstanding of the purpose of the form, barriers to use and accessibility of the form. Despite positive comments about the form "…just pull the sheet and you know the medication is confirmed…" (Pharmacist) and "…I think it's a really good system. I think it's quite user friendly and it prints it out in an easy to read way…" (Dr). Issues were identified including unfamiliar documentation "on a paper kardex you would see straight away [medication was discontinued]… you don't know that you need to look somewhere else until you've missed it and it's along with some other chart…" (Pharmacist). Suggestions were made to alter the layout of the form to follow the logical order of steps in the MR process. Non-standard terminology introduced by the computer system was considered ambiguous. Barriers to using the form included misunderstandings in relation to the stage of the patient's journey - was it referring to admission to hospital, admission to PICU, during PICU admission or transfer from PICU? Educational needs were identified during the interviews. Missing forms were a problem. "…our biggest issue is that they come down without having had the discharge printed off…" (Dr). User input informed recommendations for improvements such as clarity of wording and layout of the form, ensuring the MR form is available downstream and highlighted areas for user education. Further evaluation will be undertaken following implementation of these changes. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Integration process of fermentation and liquid biphasic flotation for lipase separation from Burkholderia cepacia.

PubMed

Sankaran, Revathy; Show, Pau Loke; Lee, Sze Ying; Yap, Yee Jiun; Ling, Tau Chuan

2018-02-01

Liquid Biphasic Flotation (LBF) is an advanced recovery method that has been effectively applied for biomolecules extraction. The objective of this investigation is to incorporate the fermentation and extraction process of lipase from Burkholderia cepacia using flotation system. Initial study was conducted to compare the performance of bacteria growth and lipase production using flotation and shaker system. From the results obtained, bacteria shows quicker growth and high lipase yield via flotation system. Integration process for lipase separation was investigated and the result showed high efficiency reaching 92.29% and yield of 95.73%. Upscaling of the flotation system exhibited consistent result with the lab-scale which are 89.53% efficiency and 93.82% yield. The combination of upstream and downstream processes in a single system enables the acceleration of product formation, improves the product yield and facilitates downstream processing. This integration system demonstrated its potential for biomolecules fermentation and separation that possibly open new opportunities for industrial production. Copyright © 2017 Elsevier Ltd. All rights reserved.

BIN1/M-Amphiphysin2 induces clustering of phosphoinositides to recruit its downstream partner dynamin

NASA Astrophysics Data System (ADS)

Picas, Laura; Viaud, Julien; Schauer, Kristine; Vanni, Stefano; Hnia, Karim; Fraisier, Vincent; Roux, Aurélien; Bassereau, Patricia; Gaits-Iacovoni, Frédérique; Payrastre, Bernard; Laporte, Jocelyn; Manneville, Jean-Baptiste; Goud, Bruno

2014-12-01

Phosphoinositides play a central role in many physiological processes by assisting the recruitment of proteins to membranes through specific phosphoinositide-binding motifs. How this recruitment is coordinated in space and time is not well understood. Here we show that BIN1/M-Amphiphysin2, a protein involved in T-tubule biogenesis in muscle cells and frequently mutated in centronuclear myopathies, clusters PtdIns(4,5)P2 to recruit its downstream partner dynamin. By using several mutants associated with centronuclear myopathies, we find that the N-BAR and the SH3 domains of BIN1 control the kinetics and the accumulation of dynamin on membranes, respectively. We show that phosphoinositide clustering is a mechanism shared by other proteins that interact with PtdIns(4,5)P2, but do not contain a BAR domain. Our numerical simulations point out that clustering is a diffusion-driven process in which phosphoinositide molecules are not sequestered. We propose that this mechanism plays a key role in the recruitment of downstream phosphoinositide-binding proteins.

Inactivation of p53 in pterygium influence miR-200a expression resulting in ZEB1/ZEB2 up-regulation and EMT processing.

PubMed

Wu, Chueh-Wei; Peng, Mei-Ling; Yeh, Ken-Tu; Tsai, Yi-Yu; Chiang, Chun-Chi; Cheng, Ya-Wen

2016-05-01

Loss of p53 function has been linked to progression of pterygium. MiR-200a is known to be controlled by p53. Here, we hypothesize that expression of miR-200a and downstream ZEB1/ZEB2 genes are regulated epithelial-mesenchymal transition (EMT) involved in the pathogenesis and recurrence of pterygium. For this study, 120 primary pterygial samples were collected. Immunohistochemistry and real-time RT-PCR were performed to determine the expression of p53, p53 down-stream EMT associated protein and miR-200a. The molecular correlation of p53, miR-200a and downstream genes were confirmed using primary pterygium cells (PECs). Expression of miR-200a in pterygium tissues was significantly lower than in conjunctiva controls (p = 0.015). Up-regulated miR-200a levels were positively correlated with and p53 protein expression (p < 0.001). The miR-200a downstream ZEB1/ZEB1 protein expression were negative correlated with miR-200a expression. Cell model studies demonstrated that miR-200a controlled the EMT of PECs through up-regulated ZEB1, ZEB2 and Snail gene expression. Our study demonstrated that inactivation of p53 in pterygium may influence miR-200a, resulting in ZEB1/ZEB2 up-regulation and EMT processing of pterygium. Therefore, we suggest that expression of miR-200a play an important role in EMT processing and recurrence of pterygium. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cell disruption and lipid extraction for microalgal biorefineries: A review.

PubMed

Lee, Soo Youn; Cho, Jun Muk; Chang, Yong Keun; Oh, You-Kwan

2017-11-01

The microalgae-based biorefinement process has attracted much attention from academic and industrial researchers attracted to its biofuel, food and nutraceutical applications. In this paper, recent developments in cell-disruption and lipid-extraction methods, focusing on four biotechnologically important microalgal species (namely, Chlamydomonas, Haematococcus, Chlorella, and Nannochloropsis spp.), are reviewed. The structural diversity and rigidity of microalgal cell walls complicate the development of efficient downstream processing methods for cell-disruption and subsequent recovery of intracellular lipid and pigment components. Various mechanical, chemical and biological cell-disruption methods are discussed in detail and compared based on microalgal species and status (wet/dried), scale, energy consumption, efficiency, solvent extraction, and synergistic combinations. The challenges and prospects of the downstream processes for the future development of eco-friendly and economical microalgal biorefineries also are outlined herein. Copyright © 2017 Elsevier Ltd. All rights reserved.

Problems of drinking water treatment along Ismailia Canal Province, Egypt.

PubMed

Geriesh, Mohamed H; Balke, Klaus-Dieter; El-Rayes, Ahmed E

2008-03-01

The present drinking water purification system in Egypt uses surface water as a raw water supply without a preliminary filtration process. On the other hand, chlorine gas is added as a disinfectant agent in two steps, pre- and post-chlorination. Due to these reasons most of water treatment plants suffer low filtering effectiveness and produce the trihalomethane (THM) species as a chlorination by-product. The Ismailia Canal represents the most distal downstream of the main Nile River. Thus its water contains all the proceeded pollutants discharged into the Nile. In addition, the downstream reaches of the canal act as an agricultural drain during the closing period of the High Dam gates in January and February every year. Moreover, the wide industrial zone along the upstream course of the canal enriches the canal water with high concentrations of heavy metals. The obtained results indicate that the canal gains up to 24.06x10(6) m3 of water from the surrounding shallow aquifer during the closing period of the High Dam gates, while during the rest of the year, the canal acts as an influent stream losing about 99.6x10(6) m3 of its water budget. The reduction of total organic carbon (TOC) and suspended particulate matters (SPMs) should be one of the central goals of any treatment plan to avoid the disinfectants by-products. The combination of sedimentation basins, gravel pre-filtration and slow sand filtration, and underground passage with microbiological oxidation-reduction and adsorption criteria showed good removal of parasites and bacteria and complete elimination of TOC, SPM and heavy metals. Moreover, it reduces the use of disinfectants chemicals and lowers the treatment costs. However, this purification system under the arid climate prevailing in Egypt should be tested and modified prior to application.

Problems of drinking water treatment along Ismailia Canal Province, Egypt*

PubMed Central

Geriesh, Mohamed H.; Balke, Klaus-Dieter; El-Rayes, Ahmed E.

2008-01-01

The present drinking water purification system in Egypt uses surface water as a raw water supply without a preliminary filtration process. On the other hand, chlorine gas is added as a disinfectant agent in two steps, pre- and post-chlorination. Due to these reasons most of water treatment plants suffer low filtering effectiveness and produce the trihalomethane (THM) species as a chlorination by-product. The Ismailia Canal represents the most distal downstream of the main Nile River. Thus its water contains all the proceeded pollutants discharged into the Nile. In addition, the downstream reaches of the canal act as an agricultural drain during the closing period of the High Dam gates in January and February every year. Moreover, the wide industrial zone along the upstream course of the canal enriches the canal water with high concentrations of heavy metals. The obtained results indicate that the canal gains up to 24.06×106 m3 of water from the surrounding shallow aquifer during the closing period of the High Dam gates, while during the rest of the year, the canal acts as an influent stream losing about 99.6×106 m3 of its water budget. The reduction of total organic carbon (TOC) and suspended particulate matters (SPMs) should be one of the central goals of any treatment plan to avoid the disinfectants by-products. The combination of sedimentation basins, gravel pre-filtration and slow sand filtration, and underground passage with microbiological oxidation-reduction and adsorption criteria showed good removal of parasites and bacteria and complete elimination of TOC, SPM and heavy metals. Moreover, it reduces the use of disinfectants chemicals and lowers the treatment costs. However, this purification system under the arid climate prevailing in Egypt should be tested and modified prior to application. PMID:18357626

PIV measurements in the near wakes of hollow cylinders with holes

NASA Astrophysics Data System (ADS)

Firat, Erhan; Ozkan, Gokturk M.; Akilli, Huseyin

2017-05-01

The wake flows behind fixed, hollow, rigid circular cylinders with two rows of holes connecting the front and rear stagnation lines were investigated using particle image velocimetry (PIV) for various combinations of three hole diameters, d = 0.1 D, 0.15 D, and 0.20 D, six hole-to-hole distances, l = 2 d, 3 d, 4 d, 5 d, 6 d, and 7 d, and ten angles of incidence ( α), from 0° to 45° in steps of 5°, at a Reynolds number of Re = 6,900. Time-averaged velocity distributions, instantaneous and time-averaged vorticity patterns, time-averaged streamline topology, and hot spots of turbulent kinetic energy occurred through the interaction of shear layers from the models were presented to show how the wake flow was modified by the presence of the self-issuing jets with various momentums emanating from the downstream holes. In general, as hole diameter which is directly related to jet momentum increased, the values of time-averaged wake characteristics (length of time-averaged recirculation region, vortex formation length, length of shear layers, and gap between the shear layers) increased. Irrespective to d and l tested, the values of the vortex formation length of the models are greater than that of the cylinder without hole (reference model). That is, vortex formation process was shifted downstream by aid of jets. It was found that time-averaged wake characteristics were very sensitive to α. As α increased, the variation of these characteristics can be modeled by exponential decay functions. The effect of l on the three-dimensional vortex shedding patterns in the near wake of the models was also discussed.

Impacts of Woody Debris on Fluvial Processes and Channel Morphology in Stable and Unstable Streams

DTIC Science & Technology

1996-05-01

flotation of emergent and riparian trees (Howan, 1987), (Figure 2.1). 0 Fetherston et al. (1995) suggest that debris inputs are either "’chronic or episodic...the channel. Jams are therefore commonly located in bend apices or in unstable reaches downstream of knickpoints. Figure 4.2 demonstrates this...observation, showing debris jam locations just downstream of bend apices on a planform plot of Abiaca Creek. Jams do not, however, appear to have a regular

Method for automatically evaluating a transition from a batch manufacturing technique to a lean manufacturing technique

DOEpatents

Ivezic, Nenad; Potok, Thomas E.

2003-09-30

A method for automatically evaluating a manufacturing technique comprises the steps of: receiving from a user manufacturing process step parameters characterizing a manufacturing process; accepting from the user a selection for an analysis of a particular lean manufacturing technique; automatically compiling process step data for each process step in the manufacturing process; automatically calculating process metrics from a summation of the compiled process step data for each process step; and, presenting the automatically calculated process metrics to the user. A method for evaluating a transition from a batch manufacturing technique to a lean manufacturing technique can comprise the steps of: collecting manufacturing process step characterization parameters; selecting a lean manufacturing technique for analysis; communicating the selected lean manufacturing technique and the manufacturing process step characterization parameters to an automatic manufacturing technique evaluation engine having a mathematical model for generating manufacturing technique evaluation data; and, using the lean manufacturing technique evaluation data to determine whether to transition from an existing manufacturing technique to the selected lean manufacturing technique.

Channel morphology and patterns of bedload transport in fluvial, formerly-glaciated, forested headwater streams of the Columbia Mountains, Canada

NASA Astrophysics Data System (ADS)

Green, Kim; Brardinoni, Francesco; Alila, Younes

2013-04-01

This study examines channel-reach morphology and bedload transport dynamics in relation to landscape structure and snowmelt hydrology in Cotton and Elk Creek, two headwater streams of the southern Columbia Mountains, Canada. Data collection is based on field surveys and GIS analysis in conjunction with a nested monitoring network of water discharge and bed load transfer. The nested monitoring network is designed to examine the effects of channel bed texture, and the influence of free-formed (i.e., boulder cascades, step pools, and riffle pools) and forced-alluvial morphologies (i.e., forced step pools) on bedload entrainment and transport. The landscape is characterized by subdued glaciated topography in which sediment is primarily supplied by bank failures and fluvial transfer dominates the channelized sediment cascade. The spatial distribution of channel types is mainly controlled by glacially imposed local slope together with availability of wood and glacigenic materials. Interestingly, downstream hydraulic geometry as well as downstream patterns of the coarse channel bed fraction and stream power are all insensitive to systematic changes of local slope along the typically stepped longitudinal profiles. An indication that the study alluvial systems are adjusted to the contemporary hydrologic and sedimentary regimes, and as such through post-LGM times have been able to compensate for the glacially-imposed boundary conditions. Stepwise multiple regression indicates that annual bedload yield is chiefly controlled by the number of peak events over threshold discharge. During such high flows, repeated destabilization of channel bed armouring and re-mobilization of sediment stored behind logjams can ensure sediment supply for bedload transport across the entire snowmelt season. In particular, channel morphology affects distinctively the variability of bed load response to hydrologic forcing. The observed spatial variability in annual bedload yield appears to correlate with inter-basin differences in basic morphometric attributes, among which slope aspect plays a prominent role.

Molecular Steps in the Immune Signaling Pathway Evoked by Plant Elicitor Peptides: Ca2+-Dependent Protein Kinases, Nitric Oxide, and Reactive Oxygen Species Are Downstream from the Early Ca2+ Signal1[OPEN

PubMed Central

Ma, Yi; Zhao, Yichen; Walker, Robin K.; Berkowitz, Gerald A.

2013-01-01

Endogenous plant elicitor peptides (Peps) can act to facilitate immune signaling and pathogen defense responses. Binding of these peptides to the Arabidopsis (Arabidopsis thaliana) plasma membrane-localized Pep receptors (PEPRs) leads to cytosolic Ca2+ elevation, an early event in a signaling cascade that activates immune responses. This immune response includes the amplification of signaling evoked by direct perception of pathogen-associated molecular patterns by plant cells under assault. Work included in this report further characterizes the Pep immune response and identifies new molecular steps in the signal transduction cascade. The PEPR coreceptor BRASSINOSTEROID-INSENSITIVE1 Associated Kinase1 contributes to generation of the Pep-activated Ca2+ signal and leads to increased defense gene expression and resistance to a virulent bacterial pathogen. Ca2+-dependent protein kinases (CPKs) decode the Ca2+ signal, also facilitating defense gene expression and enhanced resistance to the pathogen. Nitric oxide and reduced nicotinamide adenine dinucleotide phosphate oxidase-dependent reactive oxygen species generation (due to the function of Respiratory Burst Oxidase Homolog proteins D and F) are also involved downstream from the Ca2+ signal in the Pep immune defense signal transduction cascade, as is the case with BRASSINOSTEROID-INSENSITIVE1 Associated Kinase1 and CPK5, CPK6, and CPK11. These steps of the pathogen defense response are required for maximal Pep immune activation that limits growth of a virulent bacterial pathogen in the plant. We find a synergism between function of the PEPR and Flagellin Sensing2 receptors in terms of both nitric oxide and reactive oxygen species generation. Presented results are also consistent with the involvement of the secondary messenger cyclic GMP and a cyclic GMP-activated Ca2+-conducting channel in the Pep immune signaling pathway. PMID:24019427

Stochastic modeling of Cryptosporidium parvum to predict transport, retention, and downstream exposure

NASA Astrophysics Data System (ADS)

Drummond, J. D.; Boano, F.; Atwill, E. R.; Li, X.; Harter, T.; Packman, A. I.

2016-12-01

Rivers are a means of rapid and long-distance transmission of pathogenic microorganisms from upstream terrestrial sources. Thus, significant fluxes of pathogen loads from agricultural lands can occur due to transport in surface waters. Pathogens enter streams and rivers in a variety of processes, notably overland flow, shallow groundwater discharge, and direct inputs from host populations such as humans and other vertebrate species. Viruses, bacteria, and parasites can enter a stream and persist in the environment for varying amounts of time. Of particular concern is the protozoal parasite, Cryptosporidium parvum, which can remain infective for weeks to months under cool and moist conditions, with the infectious state (oocysts) largely resistant to chlorination. In order to manage water-borne diseases more effectively we need to better predict how microbes behave in freshwater systems, particularly how they are transported downstream in rivers and in the process interact with the streambed and other solid surfaces. Microbes continuously immobilize and resuspend during downstream transport due to a variety of processes, such as gravitational settling, attachment to in-stream structures such as submerged macrophytes, and hyporheic exchange and filtration within underlying sediments. These various interactions result in a wide range of microbial residence times in the streambed and therefore influence the persistence of pathogenic microbes in the stream environment. We developed a stochastic mobile-immobile model to describe these microbial transport and retention processes in streams and rivers that also accounts for microbial inactivation. We used the model to assess the transport, retention, and inactivation of C. parvum within stream environments, specifically under representative flow conditions of California streams where C. parvum exposure can be at higher risk due to agricultural nonpoint sources. The results demonstrate that the combination of stream reach-scale analysis and multi-scale stochastic modeling improves assessment of C. parvum transport and retention in streams in order to predict downstream exposure to human communities, wildlife, and livestock.

Occipital Alpha Activity during Stimulus Processing Gates the Information Flow to Object-Selective Cortex

PubMed Central

Zumer, Johanna M.; Scheeringa, René; Schoffelen, Jan-Mathijs; Norris, David G.; Jensen, Ole

2014-01-01

Given the limited processing capabilities of the sensory system, it is essential that attended information is gated to downstream areas, whereas unattended information is blocked. While it has been proposed that alpha band (8–13 Hz) activity serves to route information to downstream regions by inhibiting neuronal processing in task-irrelevant regions, this hypothesis remains untested. Here we investigate how neuronal oscillations detected by electroencephalography in visual areas during working memory encoding serve to gate information reflected in the simultaneously recorded blood-oxygenation-level-dependent (BOLD) signals recorded by functional magnetic resonance imaging in downstream ventral regions. We used a paradigm in which 16 participants were presented with faces and landscapes in the right and left hemifields; one hemifield was attended and the other unattended. We observed that decreased alpha power contralateral to the attended object predicted the BOLD signal representing the attended object in ventral object-selective regions. Furthermore, increased alpha power ipsilateral to the attended object predicted a decrease in the BOLD signal representing the unattended object. We also found that the BOLD signal in the dorsal attention network inversely correlated with visual alpha power. This is the first demonstration, to our knowledge, that oscillations in the alpha band are implicated in the gating of information from the visual cortex to the ventral stream, as reflected in the representationally specific BOLD signal. This link of sensory alpha to downstream activity provides a neurophysiological substrate for the mechanism of selective attention during stimulus processing, which not only boosts the attended information but also suppresses distraction. Although previous studies have shown a relation between the BOLD signal from the dorsal attention network and the alpha band at rest, we demonstrate such a relation during a visuospatial task, indicating that the dorsal attention network exercises top-down control of visual alpha activity. PMID:25333286

Nitric oxide: a multitasked signaling gas in plants.

PubMed

Domingos, Patricia; Prado, Ana Margarida; Wong, Aloysius; Gehring, Christoph; Feijo, Jose A

2015-04-01

Nitric oxide (NO) is a gaseous reactive oxygen species (ROS) that has evolved as a signaling hormone in many physiological processes in animals. In plants it has been demonstrated to be a crucial regulator of development, acting as a signaling molecule present at each step of the plant life cycle. NO has also been implicated as a signal in biotic and abiotic responses of plants to the environment. Remarkably, despite this plethora of effects and functional relationships, the fundamental knowledge of NO production, sensing, and transduction in plants remains largely unknown or inadequately characterized. In this review we cover the current understanding of NO production, perception, and action in different physiological scenarios. We especially address the issues of enzymatic and chemical generation of NO in plants, NO sensing and downstream signaling, namely the putative cGMP and Ca(2+) pathways, ion-channel activity modulation, gene expression regulation, and the interface with other ROS, which can have a profound effect on both NO accumulation and function. We also focus on the importance of NO in cell-cell communication during developmental processes and sexual reproduction, namely in pollen tube guidance and embryo sac fertilization, pathogen defense, and responses to abiotic stress. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

Zebrafish Pronephros Development.

PubMed

Naylor, Richard W; Qubisi, Sarah S; Davidson, Alan J

The pronephros is the first kidney type to form in vertebrate embryos. The first step of pronephrogenesis in the zebrafish is the formation of the intermediate mesoderm during gastrulation, which occurs in response to secreted morphogens such as BMPs and Nodals. Patterning of the intermediate mesoderm into proximal and distal cell fates is induced by retinoic acid signaling with downstream transcription factors including wt1a, pax2a, pax8, hnf1b, sim1a, mecom, and irx3b. In the anterior intermediate mesoderm, progenitors of the glomerular blood filter migrate and fuse at the midline and recruit a blood supply. More posteriorly localized tubule progenitors undergo epithelialization and fuse with the cloaca. The Notch signaling pathway regulates the formation of multi-ciliated cells in the tubules and these cells help propel the filtrate to the cloaca. The lumenal sheer stress caused by flow down the tubule activates anterior collective migration of the proximal tubules and induces stretching and proliferation of the more distal segments. Ultimately these processes create a simple two-nephron kidney that is capable of reabsorbing and secreting solutes and expelling excess water-processes that are critical to the homeostasis of the body fluids. The zebrafish pronephric kidney provides a simple, yet powerful, model system to better understand the conserved molecular and cellular progresses that drive nephron formation, structure, and function.

Ultrasound assisted three phase partitioning of a fibrinolytic enzyme.

PubMed

Avhad, Devchand N; Niphadkar, Sonali S; Rathod, Virendra K

2014-03-01

The present investigation is aimed at ultrasound assisted three phase partitioning (UATPP) of a fibrinolytic enzyme from Bacillus sphaericus MTCC 3672. Three phase partitioning integrates the concentration and partial purification step of downstream processing of a biomolecule. Three phase system is formed with simultaneous addition of ammonium sulfate to crude broth and followed by t-butanol. UATPP of a fibrinolytic enzyme was studied by varying different process parameters such as ammonium sulfate saturation concentration, pH, broth to t-butanol ratio, temperature, ultrasound frequency, ultrasonication power, and duty cycle. The optimized parameters yielding maximum purity of 16.15-fold of fibrinolytic enzyme with 65% recovery comprised of 80% ammonium sulfate saturation, pH 9, temperature 30 °C, broth to t-butanol ratio 0.5 (v/v), at 25 kHz frequency and 150 W ultrasonication power with 40% duty cycle for 5 min irradiation time. SDS PAGE analysis of partitioned enzyme shows partial purification with a molecular weight in the range of 55-70 kDa. Enhanced mass transfer of UATPP resulted in higher fold purity of fibrinolytic enzyme with reduced time of operation from 1 h to 5 min as compared to conventional TPP. Outcome of our findings highlighted the use of UATPP as an efficient biosepartion technique. Copyright © 2013 Elsevier B.V. All rights reserved.

Fuel processors for fuel cell APU applications

NASA Astrophysics Data System (ADS)

Aicher, T.; Lenz, B.; Gschnell, F.; Groos, U.; Federici, F.; Caprile, L.; Parodi, L.

The conversion of liquid hydrocarbons to a hydrogen rich product gas is a central process step in fuel processors for auxiliary power units (APUs) for vehicles of all kinds. The selection of the reforming process depends on the fuel and the type of the fuel cell. For vehicle power trains, liquid hydrocarbons like gasoline, kerosene, and diesel are utilized and, therefore, they will also be the fuel for the respective APU systems. The fuel cells commonly envisioned for mobile APU applications are molten carbonate fuel cells (MCFC), solid oxide fuel cells (SOFC), and proton exchange membrane fuel cells (PEMFC). Since high-temperature fuel cells, e.g. MCFCs or SOFCs, can be supplied with a feed gas that contains carbon monoxide (CO) their fuel processor does not require reactors for CO reduction and removal. For PEMFCs on the other hand, CO concentrations in the feed gas must not exceed 50 ppm, better 20 ppm, which requires additional reactors downstream of the reforming reactor. This paper gives an overview of the current state of the fuel processor development for APU applications and APU system developments. Furthermore, it will present the latest developments at Fraunhofer ISE regarding fuel processors for high-temperature fuel cell APU systems on board of ships and aircrafts.

pH-induced conformational change of the rotavirus VP4 spike: implications for cell entry and antibody neutralization.

PubMed

Pesavento, Joseph B; Crawford, Sue E; Roberts, Ed; Estes, Mary K; Prasad, B V Venkataram

2005-07-01

The rotavirus spike protein, VP4, is a major determinant of infectivity and neutralization. Previously, we have shown that trypsin-enhanced infectivity of rotavirus involves a transformation of the VP4 spike from a flexible to a rigid bilobed structure. Here we show that at elevated pH the spike undergoes a drastic, irreversible conformational change and becomes stunted, with a pronounced trilobed appearance. These particles with altered spikes, at a normal pH of 7.5, despite the loss of infectivity and the ability to hemagglutinate, surprisingly exhibit sialic acid (SA)-independent cell binding in contrast to the SA-dependent cell binding exhibited by native virions. Remarkably, a neutralizing monoclonal antibody that remains bound to spikes throughout the pH changes (pH 7 to 11 and back to pH 7) completely prevents this conformational change, preserving the SA-dependent cell binding and hemagglutinating functions of the virion. A hypothesis that emerges from the present study is that high-pH treatment triggers a conformational change that mimics a post-SA-attachment step to expose an epitope recognized by a downstream receptor in the rotavirus cell entry process. This process involves sequential interactions with multiple receptors, and the mechanism by which the antibody neutralizes is by preventing this conformational change.

Results of the Alternative Water Processor Test, A Novel Technology for Exploration Wastewater Remediation

NASA Technical Reports Server (NTRS)

Vega, Leticia; Meyer, Caitlin

2016-01-01

Biologically-based water recovery systems are a regenerative, low energy alternative to physiochemical processes to reclaim water from wastewater. This paper summarizes the results of the Alternative Water Processor (AWP) test conducted over one year. The AWP recovered 90% of water from four crewmembers using (4) membrane aerated bioreactors (MABRs) to remove carbon and nitrogen from an exploration mission wastewater, including urine, hygiene, laundry and humidity condensate. Downstream, a coupled forward and reverse osmosis system removed large organics and inorganic salts from the biological system effluent. The system exceeded the overall objectives of the test by recovering 90% of the influent wastewater processed and a 29% reduction of consumables from the current state of the art water recovery system on the International Space Station (ISS). However the biological system fell short of its test goals, failing to remove 75% and 90% of the influent ammonium and organic carbon, respectively. Despite not meeting its test goals, the BWP demonstrated the feasibility of an attached-growth biological system for simultaneous nitrification and denitrification, an innovative, volume and consumable-saving design that doesn't require toxic pretreatment. This paper will explain the reasons for this and will discuss steps to optimize each subsystem to increase effluent quality from the MABRs and the FOST to advance the system.

Results of the Alternative Water Processor Test, A Novel Technology for Exploration Wastewater Remediation

NASA Technical Reports Server (NTRS)

Vega, Leticia; Meyer, Caitlin

2015-01-01

Biologically-based water recovery systems are a regenerative, low energy alternative to physiochemical processes to reclaim water from wastewater. This paper summarizes the results of the Alternative Water Processor (AWP) test conducted over one year. The AWP recovered 90% of water from four crewmembers using (4) membrane aerated bioreactors (MABRs) to remove carbon and nitrogen from an exploration mission wastewater, including urine, hygiene, laundry and humidity condensate. Downstream, a coupled forward and reverse osmosis system removed large organics and inorganic salts from the biological system effluent. The system exceeded the overall objectives of the test by recovering 90% of the influent wastewater processed and a 29% reduction of consumables from the current state of the art water recovery system on the International Space Station (ISS). However the biological system fell short of its test goals, failing to remove 75% and 90% of the influent ammonium and organic carbon, respectively. Despite not meeting its test goals, the BWP demonstrated the feasibility of an attachedgrowth biological system for simultaneous nitrification and denitrification, an innovative, volume and consumable-saving design that doesn't require toxic pretreatment. This paper will explain the reasons for this and will discuss steps to optimize each subsystem to increase effluent quality from the MABRs and the FOST to advance the system.

A Simple Field Guides to Identify Fire Effects on Soils

NASA Astrophysics Data System (ADS)

Robichaud, Peter

2016-04-01

Following wildfires post fire assessment personnel or teams assess immediate post-fire watershed conditions. These assessment teams must determine threats from flooding, soil erosion, and instability in a relatively short time period. Various tools and guides have been developed to assist in that process. A soil burn severity map is often the first step in the rapid assessment process. It enables BAER teams to prioritize field reviews and locate burned areas that may pose a risk to critical values within or downstream of the burned area. Five field parameters are easily determined in the field 1) remaining ground cover and characteristic, 2) ash color and depth, 3) soil structure, 4) fine roots, and 5) soil water repellency. All parameters are visual identified except water repellency which can be determined by the Water Drop Penetration Time (WDPT) test or Mini-Disk Infiltrometer (MDI). Often times the MDI test takes less time, is less subjective, and provides a relative infiltration rate which the WDPT test does not. The MDI test results are often put into "degree of soil water repellency" categories (strong, weak, and none). These field procedures that indicate the fire effects on the soil conditions help assessment teams consistently interpret, field validate and map soil burn severity.

Perspective: Towards environmentally acceptable criteria for downstream fish passage through mini hydro and irrigation infrastructure in the Lower Mekong River Basin

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

Baumgartner, Lee J.; Daniel Deng, Z.; Thorncraft, Garry

2014-01-01

Tropical rivers have high annual discharges optimal for hydropower and irrigation development. The Mekong River is one of the largest tropical river systems, supporting a unique mega-diverse fish community. Fish are an important commodity in the Mekong, contributing a large proportion of calcium, protein, and essential nutrients to the diet of the local people and providing a critical source of income for rural households. Many of these fish migrate not only upstream and downstream within main-channel habitats but also laterally into highly productive floodplain habitat to both feed and spawn. Most work to date has focused on providing for upstreammore » fish passage, but downstream movement is an equally important process to protect. Expansion of hydropower and irrigation weirs can disrupt downstream migrations and it is important to ensure that passage through regulators or mini hydro systems is not harmful or fatal. Many new infrastructure projects (<6 m head) are proposed for the thousands of tributary streams throughout the Lower Mekong Basin and it is important that designs incorporate the best available science to protect downstream migrants. Recent advances in technology have provided new techniques which could be applied to Mekong fish species to obtain design criteria that can facilitate safe downstream passage. Obtaining and applying this knowledge to new infrastructure projects is essential in order to produce outcomes that are more favorable to local ecosystems and fisheries.« less

Pollutant Dilution and Diffusion in Urban Street Canyon Neighboring Streets

NASA Astrophysics Data System (ADS)

Sun, Z.; Fu, Zh. M.

2011-09-01

In the present study we investigated the airflow patterns and air quality of a series of typical street canyon combinations, developed a mass balance model to determine the local pollutant dilution rate, and discuss the impact of upstream canyon on the air quality of downstream canyon. The results indicated that the geometrical size of upstream and downstream buildings have significant impacts on the ambient airflow patterns. The pollution distribution within the canyons varies with different building combinations and flow patterns. Within the upstream canyon, pollution always accumulates to the low building side for non-symmetrical canyon, and for symmetrical canyon high level of pollution occurs at the leeward side. The height of the middle and downstream buildings can evidently change the pollutant dispersion direction during the transport process. Within the polluted canyon, the pollutant dilution rate (PDR) also varies with different street canyon combinations. The highest PDR is observed when the upstream buildings are both low buildings no matter the height of downstream building. However, the two cases are likely to contribution pollution to the downstream canyon. The H-L-H combination is mostly against local pollution remove, while the L-H-L case is considered the best optimistic building combination with both the ability of diluting local pollution and not remarkably decreasing air quality of downstream canyon. The current work is expected instructive for city designers to optimize traffic patterns under typical existing geometry or in the development of urban geometry modification for air quality control.

Integrated continuous processing of proteins expressed as inclusion bodies: GCSF as a case study.

PubMed

Kateja, Nikhil; Agarwal, Harshit; Hebbi, Vishwanath; Rathore, Anurag S

2017-07-01

Affordability of biopharmaceuticals continues to be a challenge, particularly in developing economies. This has fuelled advancements in manufacturing that can offer higher productivity and better economics without sacrificing product quality in the form of an integrated continuous manufacturing platform. While platform processes for monoclonal antibodies have existed for more than a decade, development of an integrated continuous manufacturing process for bacterial proteins has received relatively scant attention. In this study, we propose an end-to-end integrated continuous downstream process (from inclusion bodies to unformulated drug substance) for a therapeutic protein expressed in Escherichia coli as inclusion body. The final process consisted of a continuous refolding in a coiled flow inverter reactor directly coupled to a three-column periodic counter-current chromatography for capture of the product followed by a three-column con-current chromatography for polishing. The continuous bioprocessing train was run uninterrupted for 26 h to demonstrate its capability and the resulting output was analyzed for the various critical quality attributes, namely product purity (>99%), high molecular weight impurities (<0.5%), host cell proteins (<100 ppm), and host cell DNA (<10 ppb). All attributes were found to be consistent over the period of operation. The developed assembly offers smaller facility footprint, higher productivity, fewer hold steps, and significantly higher equipment and resin utilization. The complexities of process integration in the context of continuous processing have been highlighted. We hope that the study presented here will promote development of highly efficient, universal, end-to-end, fully continuous platforms for manufacturing of biotherapeutics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:998-1009, 2017. © 2016 American Institute of Chemical Engineers.

Seasonally-varying mechanical impact of the Tibetan Plateau on the South Asian Monsoon

NASA Astrophysics Data System (ADS)

Bordoni, S.; Park, H.

2011-12-01

Land-sea thermal contrast and heating of the atmosphere over the Tibetan Plateau have long been considered the main driving of the large-scale South-Asian monsoon circulation. Recent works (e.g., Bordoni and Schneider 2008, Boos and Kuang 2010) have challenged this prevailing view, by suggesting that monsoons can occur even in the absence of zonal inhomogeneities and that the Tibetan Plateau might be acting more as a mechanical obstacle to the circulation than as its main heat source. Elucidating the role of land-sea contrast and of the Tibetan Plateau on the current South Asian climate is the first step to understand how this might have evolved on geological time-scales and how it might respond to changing radiative forcing and land surface conditions in future decades. In this work, we examine the mechanical impact of the Tibetan Plateau on the South Asian monsoon in a hierarchy of atmospheric general circulations models. During the pre-monsoon season and monsoon onset (April-May-June), when westerlies over the southern Tibetan Plateau are still strong, the Tibetan Plateau triggers early monsoon rainfall downstream. The downstream moist convection is accompanied by strong monsoonal low-level winds and subsidence upstream of the Tibetan Plateau. In experiments where the Tibetan Plateau is removed, monsoon onset occurs about one month later, but the circulation becomes progressively stronger and reaches comparable strength during the mature phase. During the mature and decaying phase of the monsoon (July-August-September), when westerlies over the southern Tibetan Plateau almost disappear, the strength of the monsoon circulation is largely unaffected by the presence of the Plateau. A dry dynamical core with east-west oriented narrow mountains in the subtropics consistently simulates downstream convergence with background zonal westerlies over the mountain range. In a moist atmosphere, the mechanically-driven downstream convergence is expected to be associated with significant moisture convergence. We argue that the mechanically-driven downstream convergence in the presence of the Tibetan Plateau is responsible for the zonally asymmetric monsoon onset, particularly over the Bay of Bengal and South China.

Confusion—specimen mix-up in dermatopathology and measures to prevent and detect it

PubMed Central

Weyers, Wolfgang

2014-01-01

Maintaining patient identity throughout the biopsy pathway is critical for the practice of dermatology and dermatopathology. From the biopsy procedure to the acquisition of the pathology report, a specimen may pass through the hands of more than twenty individuals in several workplaces. The risk of a mix-up is considerable and may account for more serious mistakes than diagnostic errors. To prevent specimen mix-up, work processes should be standardized and automated wherever possible, e.g., by strict order in the operating room and in the laboratory and by adoption of a bar code system to identify specimens and corresponding request forms. Mutual control of clinicians, technicians, histopathologists, and secretaries, both simultaneously and downstream, is essential to detect errors. The most vulnerable steps of the biopsy pathway, namely, labeling of specimens and request forms and accessioning of biopsy specimens in the laboratory, should be carried out by two persons simultaneously. In preceding work steps, clues must be provided that allow a mix-up to be detected later on, such as information about clinical diagnosis, biopsy technique, and biopsy site by the clinician, and a sketch of the specimen by the technician grossing it. Awareness of the danger of specimen mix-up is essential for preventing and detecting it. The awareness can be heightened by documentation of any error in the biopsy pathway. In case of suspicion, a mix-up of specimens from different patients can be confirmed by DNA analysis. PMID:24520511

Confusion-specimen mix-up in dermatopathology and measures to prevent and detect it.

PubMed

Weyers, Wolfgang

2014-01-01

Maintaining patient identity throughout the biopsy pathway is critical for the practice of dermatology and dermatopathology. From the biopsy procedure to the acquisition of the pathology report, a specimen may pass through the hands of more than twenty individuals in several workplaces. The risk of a mix-up is considerable and may account for more serious mistakes than diagnostic errors. To prevent specimen mix-up, work processes should be standardized and automated wherever possible, e.g., by strict order in the operating room and in the laboratory and by adoption of a bar code system to identify specimens and corresponding request forms. Mutual control of clinicians, technicians, histopathologists, and secretaries, both simultaneously and downstream, is essential to detect errors. The most vulnerable steps of the biopsy pathway, namely, labeling of specimens and request forms and accessioning of biopsy specimens in the laboratory, should be carried out by two persons simultaneously. In preceding work steps, clues must be provided that allow a mix-up to be detected later on, such as information about clinical diagnosis, biopsy technique, and biopsy site by the clinician, and a sketch of the specimen by the technician grossing it. Awareness of the danger of specimen mix-up is essential for preventing and detecting it. The awareness can be heightened by documentation of any error in the biopsy pathway. In case of suspicion, a mix-up of specimens from different patients can be confirmed by DNA analysis.

Particle Image Velocimetry studies of bicuspid aortic valve hemodynamics

NASA Astrophysics Data System (ADS)

Saikrishnan, Neelakantan; Yap, Choon-Hwai; Yoganathan, Ajit P.

2010-11-01

Bicuspid aortic valves (BAVs) are a congenital anomaly of the aortic valve with two fused leaflets, affecting about 1-2% of the population. BAV patients have much higher incidence of valve calcification & aortic dilatation, which may be related to altered mechanical forces from BAV hemodynamics. This study aims to characterize BAV hemodynamics using Particle Image Velocimetry(PIV). BAV models are constructed from normal explanted porcine aortic valves by suturing two leaflets together. The valves are mounted in an acrylic chamber with two sinuses & tested in a pulsatile flow loop at physiological conditions. 2D PIV is performed to obtain flow fields in three planes downstream of the valve. The stenosed BAV causes an eccentric jet, resulting in a very strong vortex in the normal sinus. The bicuspid sinus vortex appears much weaker, but more unstable. Unsteady oscillatory shear stresses are also observed, which have been associated with adverse biological response; characterization of the hemodynamics of BAVs will provide the first step to understanding these processes better. Results from multiple BAV models of varying levels of stenosis will be presented & higher stenosis corresponded to stronger jets & increased aortic wall shear stresses.

Non-Transition-Metal Catalytic System for N 2 Reduction to NH 3: A Density Functional Theory Study of Al-Doped Graphene

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

Tian, Yong-Hui; Hu, Shuangli; Sheng, Xiaolan

The prevalent catalysts for natural and artificial N 2 fixation are known to hinge upon transition-metal (TM) elements. In this paper, we demonstrate by density functional theory that Al-doped graphene is a potential non-TM catalyst to convert N 2 to NH 3 in the presence of relatively mild proton/electron sources. In the integrated structure of the catalyst, the Al atom serves as a binding site and catalytic center while the graphene framework serves as an electron buffer during the successive proton/electron additions to N 2 and its various downstream N xH y intermediates. The initial hydrogenation of N 2 canmore » readily take place via an internal H-transfer process with the assistance of a Li + ion as an additive. Finally, in view of the recurrence of H transfer in the first step of N 2 reduction observed in biological nitrogenases and other synthetic catalysts, this finding highlights the significance of heteroatom-assisted H transfer in the design of synthetic catalysts for N 2 fixation.« less

A novel intermembrane space–targeting signal docks cysteines onto Mia40 during mitochondrial oxidative folding

PubMed Central

Sideris, Dionisia P.; Petrakis, Nikos; Katrakili, Nitsa; Mikropoulou, Despina; Gallo, Angelo; Ciofi-Baffoni, Simone; Banci, Lucia; Bertini, Ivano

2009-01-01

Mia40 imports Cys-containing proteins into the mitochondrial intermembrane space (IMS) by ensuring their Cys-dependent oxidative folding. In this study, we show that the specific Cys of the substrate involved in docking with Mia40 is substrate dependent, the process being guided by an IMS-targeting signal (ITS) present in Mia40 substrates. The ITS is a 9-aa internal peptide that (a) is upstream or downstream of the docking Cys, (b) is sufficient for crossing the outer membrane and for targeting nonmitochondrial proteins, (c) forms an amphipathic helix with crucial hydrophobic residues on the side of the docking Cys and dispensable charged residues on the other side, and (d) fits complementary to the substrate cleft of Mia40 via hydrophobic interactions of micromolar affinity. We rationalize the dual function of Mia40 as a receptor and an oxidase in a two step–specific mechanism: an ITS-guided sliding step orients the substrate noncovalently, followed by docking of the substrate Cys now juxtaposed to pair with the Mia40 active Cys. PMID:20026652

Right-hemispheric dominance for visual remapping in humans.

PubMed

Pisella, L; Alahyane, N; Blangero, A; Thery, F; Blanc, S; Pelisson, D

2011-02-27

We review evidence showing a right-hemispheric dominance for visuo-spatial processing and representation in humans. Accordingly, visual disorganization symptoms (intuitively related to remapping impairments) are observed in both neglect and constructional apraxia. More specifically, we review findings from the intervening saccade paradigm in humans--and present additional original data--which suggest a specific role of the asymmetrical network at the temporo-parietal junction (TPJ) in the right hemisphere in visual remapping: following damage to the right dorsal posterior parietal cortex (PPC) as well as part of the corpus callosum connecting the PPC to the frontal lobes, patient OK in a double-step saccadic task exhibited an impairment when the second saccade had to be directed rightward. This singular and lateralized deficit cannot result solely from the patient's cortical lesion and, therefore, we propose that it is due to his callosal lesion that may specifically interrupt the interhemispheric transfer of information necessary to execute accurate rightward saccades towards a remapped target location. This suggests a specialized right-hemispheric network for visuo-spatial remapping that subsequently transfers target location information to downstream planning regions, which are symmetrically organized.

Atropos: specific, sensitive, and speedy trimming of sequencing reads.

PubMed

Didion, John P; Martin, Marcel; Collins, Francis S

2017-01-01

A key step in the transformation of raw sequencing reads into biological insights is the trimming of adapter sequences and low-quality bases. Read trimming has been shown to increase the quality and reliability while decreasing the computational requirements of downstream analyses. Many read trimming software tools are available; however, no tool simultaneously provides the accuracy, computational efficiency, and feature set required to handle the types and volumes of data generated in modern sequencing-based experiments. Here we introduce Atropos and show that it trims reads with high sensitivity and specificity while maintaining leading-edge speed. Compared to other state-of-the-art read trimming tools, Atropos achieves significant increases in trimming accuracy while remaining competitive in execution times. Furthermore, Atropos maintains high accuracy even when trimming data with elevated rates of sequencing errors. The accuracy, high performance, and broad feature set offered by Atropos makes it an appropriate choice for the pre-processing of Illumina, ABI SOLiD, and other current-generation short-read sequencing datasets. Atropos is open source and free software written in Python (3.3+) and available at https://github.com/jdidion/atropos.

A cascading activity-based probe sequentially targets E1–E2–E3 ubiquitin enzymes

PubMed Central

Mulder, Monique P.C.; Witting, Katharina; Berlin, Ilana; Pruneda, Jonathan N.; Wu, Kuen-Phon; Chang, Jer-Gung; Merkx, Remco; Bialas, Johanna; Groettrup, Marcus; Vertegaal, Alfred C.O.; Schulman, Brenda A.; Komander, David; Neefjes, Jacques; Oualid, Farid El; Ovaa, Huib

2016-01-01

Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers, orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a staggering breadth of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Akin to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe ‘hops’ and ‘traps’ catalytically active ubiquitin-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activities in living cells, presents novel and versatile tools to interrogate the Ub/Ubl cascades. PMID:27182664

Ordered Conformational Changes in Damaged DNA Induced by Nucleotide Excision Repair Factors*

PubMed Central

Tapias, Angels; Auriol, Jerome; Forget, Diane; Enzlin, Jacqueline H.; Schärer, Orlando D; Coin, Frederic; Coulombe, Benoit; Egly, Jean-Marc

2015-01-01

In response to genotoxic attacks, cells activate sophisticated DNA repair pathways such as nucleotide excision repair (NER), which consists of damage removal via dual incision and DNA resynthesis. Using permanganate footprinting as well as highly purified factors, we show that NER is a dynamic process that takes place in a number of successive steps during which the DNA is remodeled around the lesion in response to the various NER factors. XPC/HR23B first recognizes the damaged structure and initiates the opening of the helix from position −3 to +6. TFIIH is then recruited and, in the presence of ATP, extends the opening from position −6 to +6; it also displaces XPC downstream from the lesion, thereby providing the topological structure for recruiting XPA and RPA, which will enlarge the opening. Once targeted by XPG, the damaged DNA is further melted from position −19 to +8. XPG and XPF/ERCC1 endo-nucleases then cut the damaged DNA at the limit of the opened structure that was previously “labeled” by the positioning of XPC/HR23B and TFIIH. PMID:14981083

Reinventing R&D in an open innovation ecosystem.

PubMed

Traitler, Helmut; Watzke, Heribert J; Saguy, I Sam

2011-03-01

Today, the idea that random collisions and interactions offer solutions and business opportunities is no longer acceptable. Instead, partnerships and alignments, both downstream and upstream, are paramount for cross-fertilization and synergy. To survive, and thrive, in today's world of global innovation, alliances based on compatible differences must be sought. Innovation Partnerships and the Sharing-is-Winning model represent a paradigm shift toward accelerating co-development of sustainable innovation, with alignment of the entire value chain with consumer-centric innovations being one of its main pillars. It includes 3 levels of typical joint development: universities, research institutes, and centers; start-ups and individual inventors; a select number of key strategic suppliers. Reinventing R&D in an open innovation ecosystem and increasing success rates in an increasingly competitive marketplace require implementing significant steps--both perceived and tangible. Specific recommendations are provided for 10 major identified topics: leadership, strategy, the consumer, the value chain, internal experts and championship, metrics, IP, culture, academia, and passion. The Sharing-is-Winning model extends the scope of open innovation to sustainable and enhanced processes of co-innovation.

Mass and momentum turbulent transport experiments with confined swirling coaxial jets

NASA Technical Reports Server (NTRS)

Roback, R.; Johnson, B. V.

1983-01-01

Swirling coaxial jets mixing downstream, discharging into an expanded duct was conducted to obtain data for the evaluation and improvement of turbulent transport models currently used in a variety of computational procedures throughout the combustion community. A combination of laser velocimeter (LV) and laser induced fluorescence (LIF) techniques was employed to obtain mean and fluctuating velocity and concentration distributions which were used to derive mass and momentum turbulent transport parameters currently incorporated into various combustor flow models. Flow visualization techniques were also employed to determine qualitatively the time dependent characteristics of the flow and the scale of turbulence. The results of these measurements indicated that the largest momentum turbulent transport was in the r-z plane. Peak momentum turbulent transport rates were approximately the same as those for the nonswirling flow condition. The mass turbulent transport process for swirling flow was complicated. Mixing occurred in several steps of axial and radial mass transport and was coupled with a large radial mean convective flux. Mixing for swirling flow was completed in one-third the length required for nonswirling flow.

Construction of large signaling pathways using an adaptive perturbation approach with phosphoproteomic data.

PubMed

Melas, Ioannis N; Mitsos, Alexander; Messinis, Dimitris E; Weiss, Thomas S; Rodriguez, Julio-Saez; Alexopoulos, Leonidas G

2012-04-01

Construction of large and cell-specific signaling pathways is essential to understand information processing under normal and pathological conditions. On this front, gene-based approaches offer the advantage of large pathway exploration whereas phosphoproteomic approaches offer a more reliable view of pathway activities but are applicable to small pathway sizes. In this paper, we demonstrate an experimentally adaptive approach to construct large signaling pathways from phosphoproteomic data within a 3-day time frame. Our approach--taking advantage of the fast turnaround time of the xMAP technology--is carried out in four steps: (i) screen optimal pathway inducers, (ii) select the responsive ones, (iii) combine them in a combinatorial fashion to construct a phosphoproteomic dataset, and (iv) optimize a reduced generic pathway via an Integer Linear Programming formulation. As a case study, we uncover novel players and their corresponding pathways in primary human hepatocytes by interrogating the signal transduction downstream of 81 receptors of interest and constructing a detailed model for the responsive part of the network comprising 177 species (of which 14 are measured) and 365 interactions.

Atropos: specific, sensitive, and speedy trimming of sequencing reads

PubMed Central

Collins, Francis S.

2017-01-01

A key step in the transformation of raw sequencing reads into biological insights is the trimming of adapter sequences and low-quality bases. Read trimming has been shown to increase the quality and reliability while decreasing the computational requirements of downstream analyses. Many read trimming software tools are available; however, no tool simultaneously provides the accuracy, computational efficiency, and feature set required to handle the types and volumes of data generated in modern sequencing-based experiments. Here we introduce Atropos and show that it trims reads with high sensitivity and specificity while maintaining leading-edge speed. Compared to other state-of-the-art read trimming tools, Atropos achieves significant increases in trimming accuracy while remaining competitive in execution times. Furthermore, Atropos maintains high accuracy even when trimming data with elevated rates of sequencing errors. The accuracy, high performance, and broad feature set offered by Atropos makes it an appropriate choice for the pre-processing of Illumina, ABI SOLiD, and other current-generation short-read sequencing datasets. Atropos is open source and free software written in Python (3.3+) and available at https://github.com/jdidion/atropos. PMID:28875074

Intermolecular Interactions and the Viscosity of Highly Concentrated Monoclonal Antibody Solutions.

PubMed

Binabaji, Elaheh; Ma, Junfen; Zydney, Andrew L

2015-09-01

The large increase in viscosity of highly concentrated monoclonal antibody solutions can be challenging for downstream processing, drug formulation, and delivery steps. The objective of this work was to examine the viscosity of highly concentrated solutions of a high purity IgG1 monoclonal antibody over a wide range of protein concentrations, solution pH, ionic strength, and in the presence / absence of different excipients. Experiments were performed with an IgG1 monoclonal antibody provided by Amgen. The steady-state viscosity was evaluated using a Rheometrics strain-controlled rotational rheometer with a concentric cylinder geometry. The viscosity data were well-described by the Mooney equation. The data were analyzed in terms of the antibody virial coefficients obtained from osmotic pressure data evaluated under the same conditions. The viscosity coefficient in the absence of excipients was well correlated with the third osmotic virial coefficient, which has a negative value (corresponding to short range attractive interactions) at the pH and ionic strength examined in this work. These results provide important insights into the effects of intermolecular protein-protein interactions on the behavior of highly concentrated antibody solutions.

Acid-induced aggregation propensity of nivolumab is dependent on the Fc.

PubMed

Liu, Boning; Guo, Huaizu; Xu, Jin; Qin, Ting; Xu, Lu; Zhang, Junjie; Guo, Qingcheng; Zhang, Dapeng; Qian, Weizhu; Li, Bohua; Dai, Jianxin; Hou, Sheng; Guo, Yajun; Wang, Hao

2016-01-01

Nivolumab, an anti-programmed death (PD)1 IgG4 antibody, has shown notable success as a cancer treatment. Here, we report that nivolumab was susceptible to aggregation during manufacturing, particularly in routine purification steps. Our experimental results showed that exposure to low pH caused aggregation of nivolumab, and the Fc was primarily responsible for an acid-induced unfolding phenomenon. To compare the intrinsic propensity of acid-induced aggregation for other IgGs subclasses, tocilizumab (IgG1), panitumumab (IgG2) and atezolizumab (aglyco-IgG1) were also investigated. The accurate pH threshold of acid-induced aggregation for individual IgG Fc subclasses was identified and ranked as: IgG1 < aglyco-IgG1 < IgG2 < IgG4. This result was cross-validated by thermostability and conformation analysis. We also assessed the effect of several protein stabilizers on nivolumab, and found mannitol ameliorated the acid-induced aggregation of the molecule. Our results provide valuable insight into downstream manufacturing process development, especially for immune checkpoint modulating molecules with a human IgG4 backbone.

Intraflagellar transport protein 122 antagonizes Sonic Hedgehog signaling and controls ciliary localization of pathway components.

PubMed

Qin, Jian; Lin, Yulian; Norman, Ryan X; Ko, Hyuk W; Eggenschwiler, Jonathan T

2011-01-25

Primary cilia are required for proper Sonic Hedgehog (Shh) signaling in mammals. However, their role in the signal transduction process remains unclear. We have identified sister of open brain (sopb), a null allele of mouse Intraflagellar transport protein 122 (Ift122). IFT122 negatively regulates the Shh pathway in the cilium at a step downstream of the Shh ligand and the transmembrane protein Smoothened, but upstream of the Gli2 transcription factor. Ift122(sopb) mutants generate primary cilia, but they show features of defective retrograde intraflagellar transport. IFT122 controls the ciliary localization of Shh pathway regulators in different ways. Disruption of IFT122 leads to accumulation of Gli2 and Gli3 at cilia tips while blocking the ciliary localization of the antagonist TULP3. Suppressor of Fused and Smoothened localize to the cilium through an IFT122-independent mechanism. We propose that the balance between positive and negative regulators of the Shh pathway at the cilium tip controls the output of the pathway and that Shh signaling regulates this balance through intraflagellar transport.

Differential Gene Expression (DEX) and Alternative Splicing Events (ASE) for Temporal Dynamic Processes Using HMMs and Hierarchical Bayesian Modeling Approaches.

PubMed

Oh, Sunghee; Song, Seongho

2017-01-01

In gene expression profile, data analysis pipeline is categorized into four levels, major downstream tasks, i.e., (1) identification of differential expression; (2) clustering co-expression patterns; (3) classification of subtypes of samples; and (4) detection of genetic regulatory networks, are performed posterior to preprocessing procedure such as normalization techniques. To be more specific, temporal dynamic gene expression data has its inherent feature, namely, two neighboring time points (previous and current state) are highly correlated with each other, compared to static expression data which samples are assumed as independent individuals. In this chapter, we demonstrate how HMMs and hierarchical Bayesian modeling methods capture the horizontal time dependency structures in time series expression profiles by focusing on the identification of differential expression. In addition, those differential expression genes and transcript variant isoforms over time detected in core prerequisite steps can be generally further applied in detection of genetic regulatory networks to comprehensively uncover dynamic repertoires in the aspects of system biology as the coupled framework.

Dynamic behaviors of cavitation bubble for the steady cavitating flow

NASA Astrophysics Data System (ADS)

Cai, Jun; Huai, Xiulan; Li, Xunfeng

2009-12-01

In this paper, by introducing the flow velocity item into the classical Rayleigh-Plesset dynamic equation, a new equation, which does not involve the time term and can describe the motion of cavitation bubble in the steady cavitating flow, has been obtained. By solving the new motion equation using Runge-Kutta fourth order method with adaptive step size control, the dynamic behaviors of cavitation bubble driven by the varying pressure field downstream of a venturi cavitation reactor are numerically simulated. The effects of liquid temperature (corresponding to the saturated vapor pressure of liquid), cavitation number and inlet pressure of venturi on radial motion of bubble and pressure pulse due to the radial motion are analyzed and discussed in detail. Some dynamic behaviors of bubble different from those in previous papers are displayed. In addition, the internal relationship between bubble dynamics and process intensification is also discussed. The simulation results reported in this work reveal the variation laws of cavitation intensity with the flow conditions of liquid, and will lay a foundation for the practical application of hydrodynamic cavitation technology.

Right-hemispheric dominance for visual remapping in humans

PubMed Central

Pisella, L.; Alahyane, N.; Blangero, A.; Thery, F.; Blanc, S.; Pelisson, D.

2011-01-01

We review evidence showing a right-hemispheric dominance for visuo-spatial processing and representation in humans. Accordingly, visual disorganization symptoms (intuitively related to remapping impairments) are observed in both neglect and constructional apraxia. More specifically, we review findings from the intervening saccade paradigm in humans—and present additional original data—which suggest a specific role of the asymmetrical network at the temporo-parietal junction (TPJ) in the right hemisphere in visual remapping: following damage to the right dorsal posterior parietal cortex (PPC) as well as part of the corpus callosum connecting the PPC to the frontal lobes, patient OK in a double-step saccadic task exhibited an impairment when the second saccade had to be directed rightward. This singular and lateralized deficit cannot result solely from the patient's cortical lesion and, therefore, we propose that it is due to his callosal lesion that may specifically interrupt the interhemispheric transfer of information necessary to execute accurate rightward saccades towards a remapped target location. This suggests a specialized right-hemispheric network for visuo-spatial remapping that subsequently transfers target location information to downstream planning regions, which are symmetrically organized. PMID:21242144

Non-Transition-Metal Catalytic System for N 2 Reduction to NH 3: A Density Functional Theory Study of Al-Doped Graphene

DOE PAGES

Tian, Yong-Hui; Hu, Shuangli; Sheng, Xiaolan; ...

2018-01-16

The prevalent catalysts for natural and artificial N 2 fixation are known to hinge upon transition-metal (TM) elements. In this paper, we demonstrate by density functional theory that Al-doped graphene is a potential non-TM catalyst to convert N 2 to NH 3 in the presence of relatively mild proton/electron sources. In the integrated structure of the catalyst, the Al atom serves as a binding site and catalytic center while the graphene framework serves as an electron buffer during the successive proton/electron additions to N 2 and its various downstream N xH y intermediates. The initial hydrogenation of N 2 canmore » readily take place via an internal H-transfer process with the assistance of a Li + ion as an additive. Finally, in view of the recurrence of H transfer in the first step of N 2 reduction observed in biological nitrogenases and other synthetic catalysts, this finding highlights the significance of heteroatom-assisted H transfer in the design of synthetic catalysts for N 2 fixation.« less

RNA Framework: an all-in-one toolkit for the analysis of RNA structures and post-transcriptional modifications.

PubMed

Incarnato, Danny; Morandi, Edoardo; Simon, Lisa Marie; Oliviero, Salvatore

2018-06-09

RNA is emerging as a key regulator of a plethora of biological processes. While its study has remained elusive for decades, the recent advent of high-throughput sequencing technologies provided the unique opportunity to develop novel techniques for the study of RNA structure and post-transcriptional modifications. Nonetheless, most of the required downstream bioinformatics analyses steps are not easily reproducible, thus making the application of these techniques a prerogative of few laboratories. Here we introduce RNA Framework, an all-in-one toolkit for the analysis of most NGS-based RNA structure probing and post-transcriptional modification mapping experiments. To prove the extreme versatility of RNA Framework, we applied it to both an in-house generated DMS-MaPseq dataset, and to a series of literature available experiments. Notably, when starting from publicly available datasets, our software easily allows replicating authors' findings. Collectively, RNA Framework provides the most complete and versatile toolkit to date for a rapid and streamlined analysis of the RNA epistructurome. RNA Framework is available for download at: http://www.rnaframework.com.

Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signaling.

PubMed

Kleino, Anni; Ramia, Nancy F; Bozkurt, Gunes; Shen, Yanfang; Nailwal, Himani; Huang, Jing; Napetschnig, Johanna; Gangloff, Monique; Chan, Francis Ka-Ming; Wu, Hao; Li, Jixi; Silverman, Neal

2017-10-17

In the Drosophila immune response, bacterial derived diaminopimelic acid-type peptidoglycan binds the receptors PGRP-LC and PGRP-LE, which through interaction with the adaptor protein Imd leads to activation of the NF-κB homolog Relish and robust antimicrobial peptide gene expression. PGRP-LC, PGRP-LE, and Imd each contain a motif with some resemblance to the RIP Homotypic Interaction Motif (RHIM), a domain found in mammalian RIPK proteins forming functional amyloids during necroptosis. Here we found that despite sequence divergence, these Drosophila cryptic RHIMs formed amyloid fibrils in vitro and in cells. Amyloid formation was required for signaling downstream of Imd, and in contrast to the mammalian RHIMs, was not associated with cell death. Furthermore, amyloid formation constituted a regulatable step and could be inhibited by Pirk, an endogenous feedback regulator of this pathway. Thus, diverse sequence motifs are capable of forming amyloidal signaling platforms, and the formation of these platforms may present a regulatory point in multiple biological processes. Copyright © 2017 Elsevier Inc. All rights reserved.

Wave-Particle Interactions and Particle Acceleration in Turbulent Plasmas: Hybrid Simulations

NASA Astrophysics Data System (ADS)

Kucharek, Harald; Pogorelov, Nikolai; Mueller, Hans; Gamayunov, Konstantin; Farrugia, Charles

2015-04-01

Wave-particle interactions and acceleration processes are present in all key regions inside and outside of the heliosphere. Spacecraft observations measure ion distributions and accelerated ion populations, which are the result of one or several processes. For instance STEREO measures energetic particles associated with interplanetary discontinuities and in the solar wind. Voyager and IBEX provide unique data of energetic particles from the termination shock and the inner and outer heliopause. The range of plasma conditions covered by observations is enormous. However, the physical processes causing particle acceleration and wave-particle interaction and determining the particle distributions are still unknown. Currently two mechanisms, the so-called pumping mechanism (Fisk and Gloeckler, 2010) and merging/contracting island (Fermo, Drake & Swisdak, 2010) are discussed as promising models. In order to determine these individual processes, numerical models or theoretical considerations are needed. Hybrid simulations, which include all kinetic processes self-consistently on the ion level, are a very proven, powerful tool to investigate wave-particle interaction, turbulence, and phase-space evolution of pickup and solar wind ions. In the framework of this study we performed 3D multi-species hybrid simulations for an ion/ion beam instability to study the temporal evolution of ion distributions, their stability, and the influence of self-generated waves. We investigated the energization of ions downstream of interplanetary discontinuities and shocks and downstream of the termination shock, the turbulence, and growth rate of instabilities and compared the results with theoretical predictions. The simulations show that ions can be accelerated downstream of collisionless shocks by trapping of charged particles in coherent wave fronts.

The neural basis of parallel saccade programming: an fMRI study.

PubMed

Hu, Yanbo; Walker, Robin

2011-11-01

The neural basis of parallel saccade programming was examined in an event-related fMRI study using a variation of the double-step saccade paradigm. Two double-step conditions were used: one enabled the second saccade to be partially programmed in parallel with the first saccade while in a second condition both saccades had to be prepared serially. The intersaccadic interval, observed in the parallel programming (PP) condition, was significantly reduced compared with latency in the serial programming (SP) condition and also to the latency of single saccades in control conditions. The fMRI analysis revealed greater activity (BOLD response) in the frontal and parietal eye fields for the PP condition compared with the SP double-step condition and when compared with the single-saccade control conditions. By contrast, activity in the supplementary eye fields was greater for the double-step condition than the single-step condition but did not distinguish between the PP and SP requirements. The role of the frontal eye fields in PP may be related to the advanced temporal preparation and increased salience of the second saccade goal that may mediate activity in other downstream structures, such as the superior colliculus. The parietal lobes may be involved in the preparation for spatial remapping, which is required in double-step conditions. The supplementary eye fields appear to have a more general role in planning saccade sequences that may be related to error monitoring and the control over the execution of the correct sequence of responses.

Fate of antimony and arsenic in contaminated waters at the abandoned Su Suergiu mine (Sardinia, Italy)

USGS Publications Warehouse

Cidu, Rosa; Dore, Elisabetta; Biddau, Riccardo; Nordstrom, D. Kirk

2018-01-01

We investigated the fate of Sb and As downstream of the abandoned Su Suergiu mine (Sardinia, Italy) and surrounding areas. The mined area is a priority in the Sardinian remediation plan for contaminated sites due to the high concentrations of Sb and As in the mining-related wastes, which may impact the Flumendosa River that supplies water for agriculture and domestic uses. Hydrogeochemical surveys conducted from 2005 to 2015 produced time-series data and downstream profiles of water chemistry at 46 sites. Water was sampled at: springs and streams unaffected by mining; adits and streams in the mine area; drainage from the slag heaps; stream water downstream of the slag drainages; and the Flumendosa River downstream from the confluence of the contaminated waters. At specific sites, water sampling was repeated under different flow conditions, resulting in a total of 99 samples. The water samples were neutral to slightly alkaline. Elevated Sb (up to 30 mg L−1) and As (up to 16 mg L−1) concentrations were observed in water flowing from the slag materials from where the Sb ore was processed. These slag materials were the main Sb and As source at Su Suergiu. A strong base, Na-carbonate, from the foundry wastes, had a major influence on mobilizing Sb and As. Downstream contamination can be explained by considering that: (1) the predominant aqueous species, Sb(OH)6 − and HAsO4 −2, are not favored in sorption processes at the observed pH conditions; (2) precipitation of Sb- and As-bearing solid phases was not observed, which is consistent with modeling results indicating undersaturation; and (3) the main decrease in dissolved Sb and As concentrations was by dilution. Dissolved As concentrations in the Flumendosa River did not generally exceed the EU limit of 10 µg L−1, whereas dissolved Sb in the river downstream of the contamination source always exceeded the EU limit of 5 µg L−1. Recent actions aimed at retaining runoff from the slag heaps are apparently not sufficiently mitigating contamination in the Flumendosa River.

Assessing the cumulative impacts of geographically isolated wetlands on watershed hydrology using the SWAT model coupled with improved wetland modules.

PubMed

Lee, S; Yeo, I-Y; Lang, M W; Sadeghi, A M; McCarty, G W; Moglen, G E; Evenson, G R

2018-06-07

Despite recognizing the importance of wetlands in the Coastal Plain of the Chesapeake Bay Watershed (CBW) in terms of ecosystem services, our understanding of wetland functions has mostly been limited to individual wetlands and overall catchment-scale wetland functions have rarely been investigated. This study is aimed at assessing the cumulative impacts of wetlands on watershed hydrology for an agricultural watershed within the Coastal Plain of the CBW using the Soil and Water Assessment Tool (SWAT). We employed two improved wetland modules for enhanced representation of physical processes and spatial distribution of riparian wetlands (RWs) and geographically isolated wetlands (GIWs). This study focused on GIWs as their hydrological impacts on watershed hydrology are poorly understood and GIWs are poorly protected. Multiple wetland scenarios were prepared by removing all or portions of the baseline GIW condition indicated by the U.S. Fish and Wildlife Service National Wetlands Inventory geospatial dataset. We further compared the impacts of GIWs and RWs on downstream flow (i.e., streamflow at the watershed outlet). Our simulation results showed that GIWs strongly influenced downstream flow by altering water transport mechanisms in upstream areas. Loss of all GIWs reduced both water routed to GIWs and water infiltrated into the soil through the bottom of GIWs, leading to an increase in surface runoff of 9% and a decrease in groundwater flow of 7% in upstream areas. These changes resulted in increased variability of downstream flow in response to extreme flow conditions. GIW loss also induced an increase in month to month variability of downstream flow and a decrease in the baseflow contribution to streamflow. Loss of all GIWs was shown to cause a greater fluctuation of downstream flow than loss of all RWs for this study site, due to a greater total water storage capacity of GIWs. Our findings indicate that GIWs play a significant role in controlling hydrological processes in upstream areas and downstream flow and, therefore, protecting GIWs is important for enhanced hydrological resilience to extreme flow conditions in this region. Copyright © 2018 Elsevier Ltd. All rights reserved.

A transport model for the deterministic stresses associated with turbomachinery blade row interactions

NASA Astrophysics Data System (ADS)

van de Wall, Allan George

The unsteady process resulting from the interaction of upstream vortical structures with a downstream blade row in turbomachines can have a significant impact on the machine efficiency. A transport model assuming incompressible flow and using linear theory was developed to take this process into account in the computation of time-average multistage turbomachinery flows. The upstream vortical structures are transported by the mean flow of the downstream blade row, redistributing the time-average unsteady kinetic energy (Uke ) associated with the incoming disturbance. The model was applied to compressor and turbine geometry. For compressors, the Uke associated with upstream 2-D wakes and 3-D tip clearance flows is reduced as a result of the interaction with a downstream blade row. This reduction results from inviscid effects as well as viscous effects and reduces the loss associated with the upstream disturbance. Any disturbance passing through a compressor blade row results in a smaller loss than if the disturbance was mixed-out prior to entering the blade row. For turbines, the Uke associated with upstream 2-D wakes and 3-D tip clearance flows are significantly amplified by inviscid effects as a result of the interaction with a downstream turbine blade row. Viscous effects act to reduce the amplification of the Uke by inviscid effects but results in a substantial loss. Any disturbance passing through a turbine blade row results in a larger loss than if the disturbance was mixedout prior to entering the blade row.

The integrated role of ACh, ERK and mTOR in the mechanisms of hippocampal inhibitory avoidance memory.

PubMed

Giovannini, Maria Grazia; Lana, Daniele; Pepeu, Giancarlo

2015-03-01

The purpose of this review is to summarize the present knowledge on the interplay among the cholinergic system, Extracellular signal-Regulated Kinase (ERK) and Mammalian Target of Rapamycin (mTOR) pathways in the development of short and long term memories during the acquisition and recall of the step-down inhibitory avoidance in the hippocampus. The step-down inhibitory avoidance is a form of associative learning that is acquired in a relatively simple one-trial test through several sensorial inputs. Inhibitory avoidance depends on the integrated activity of hippocampal CA1 and other brain areas. Recall can be performed at different times after acquisition, thus allowing for the study of both short and long term memory. Among the many neurotransmitter systems involved, the cholinergic neurons that originate in the basal forebrain and project to the hippocampus are of crucial importance in inhibitory avoidance processes. Acetylcholine released from cholinergic fibers during acquisition and/or recall of behavioural tasks activates muscarinic and nicotinic acetylcholine receptors and brings about a long-lasting potentiation of the postsynaptic membrane followed by downstream activation of intracellular pathway (ERK, among others) that create conditions favourable for neuronal plasticity. ERK appears to be salient not only in long term memory, but also in the molecular mechanisms underlying short term memory formation in the hippocampus. Since ERK can function as a biochemical coincidence detector in response to extracellular signals in neurons, the activation of ERK-dependent downstream effectors is determined, in part, by the duration of ERK phosphorylation itself. Long term memories require protein synthesis, that in the synapto-dendritic compartment represents a direct mechanism that can produce rapid changes in protein content in response to synaptic activity. mTOR in the brain regulates protein translation in response to neuronal activity, thereby modulating synaptic plasticity and long term memory formation. Some studies demonstrate a complex interplay among the cholinergic system, ERK and mTOR. It has been shown that co-activation of muscarinic acetylcholine receptors and β-adrenergic receptors facilitates the conversion of short term to long term synaptic plasticity through an ERK- and mTOR-dependent mechanism which requires translation initiation. It seems therefore that the complex interplay among the cholinergic system, ERK and mTOR is crucial in the development of new inhibitory avoidance memories in the hippocampus. Copyright © 2015 Elsevier Inc. All rights reserved.

Spatial and temporal variability in nutrient concentrations in surface waters of the Chattahoochee River basin near Atlanta, Georgia, USA

USGS Publications Warehouse

Peters, N.E.; Buell, G.R.; Frick, E.A.

1997-01-01

Nutrient concentrations from the early 1970s through 1995 were evaluated at several sites along the Chattahoochee River and its tributaries near Atlanta, to determine general patterns and processes controlling nutrient concentrations in the river. A spatial analysis was conducted on data collected in 1994 and 1995 from an intensive nutrient study of the Chattahoochee River and its tributaries by the Georgia Department of Natural Resources, Environmental Protection Division. The 1994-1995 data show step increases in ammonium (NH4-N), nitrite plus nitrate (NO2 + NO3-N), and total-phosphorus (Tot-P) concentrations in the river. The step increases occur downstream of two wastewater treatment facilities (WWTFs) and Peachtree Creek, a small tributary inflow with degraded water quality draining a predominantly urban and industrial area. Median NO2 + NO3-N and Tot-P concentrations in the mainstem increase downstream of these inputs from 0.5 to 1 mg 1-1 and from 0.04 to 0.13 mg 1-1, respectively. NH4-N concentrations were typically low with 95% of the 2575 observations less than 0.2 mg 1-1 throughout the river system, except some high values (>1 mg 1-1) in some tributaries, particularly near the central part of Atlanta. High NH4-N concentrations are attributed to sewage discharge as they also are associated with high biological oxygen demand and faecal coliform bacteria concentrations. Nutrient concentrations vary temporally. An assessment of four sites, two mainstem and two tributaries, from 1970 to 1995 indicates a progressive increase and variability in NO2 + NO3-N concentrations during the period. The progressive increase in NO2 + NO3-N concentrations and their variability is similar to that reported for surface waters throughout the world and for which increased fertilizer usage has been attributed. Tot-P concentrations increase at mainstem sites through the middle to late 1980s and decrease markedly thereafter, due to improvements to WWTFs and a 1990 phosphate detergent ban. NH4-N concentrations, although less pronounced than Tot-P, display a similar decrease from the late 1980s to 1995 at the four sites. Tot-P concentration variability has increased at the tributary sites since 1993, although recent concentrations, on average, are the lowest since 1970 at each of the four sites.

In silico Analysis of 3′-End-Processing Signals in Aspergillus oryzae Using Expressed Sequence Tags and Genomic Sequencing Data

PubMed Central

Tanaka, Mizuki; Sakai, Yoshifumi; Yamada, Osamu; Shintani, Takahiro; Gomi, Katsuya

2011-01-01

To investigate 3′-end-processing signals in Aspergillus oryzae, we created a nucleotide sequence data set of the 3′-untranslated region (3′ UTR) plus 100 nucleotides (nt) sequence downstream of the poly(A) site using A. oryzae expressed sequence tags and genomic sequencing data. This data set comprised 1065 sequences derived from 1042 unique genes. The average 3′ UTR length in A. oryzae was 241 nt, which is greater than that in yeast but similar to that in plants. The 3′ UTR and 100 nt sequence downstream of the poly(A) site is notably U-rich, while the region located 15–30 nt upstream of the poly(A) site is markedly A-rich. The most frequently found hexanucleotide in this A-rich region is AAUGAA, although this sequence accounts for only 6% of all transcripts. These data suggested that A. oryzae has no highly conserved sequence element equivalent to AAUAAA, a mammalian polyadenylation signal. We identified that putative 3′-end-processing signals in A. oryzae, while less well conserved than those in mammals, comprised four sequence elements: the furthest upstream U-rich element, A-rich sequence, cleavage site, and downstream U-rich element flanking the cleavage site. Although these putative 3′-end-processing signals are similar to those in yeast and plants, some notable differences exist between them. PMID:21586533

Implication of two in-stream processes in the fate of nutrients discharged by sewage system into a temporary river.

PubMed

David, Arthur; Perrin, Jean-Louis; Rosain, David; Rodier, Claire; Picot, Bernadette; Tournoud, Marie-George

2011-10-01

The aim of this study was to better understand the fate of nutrients discharged by sewage treatment plants into an intermittent Mediterranean river, during a low-flow period. Many pollutants stored in the riverbed during the low-flow period can be transferred to the downstream environments during flood events. The study focused on two processes that affect the fate and the transport of nutrients, a physical process (retention in the riverbed sediments) and a biological process (denitrification). A spatial campaign was carried out during a low-flow period to characterize the nutrient contents of both water and sediments in the Vène River. The results showed high nutrient concentrations in the water column downstream of the treated wastewater disposal (up to 13,315 μg N/L for ammonium and 2,901 μg P/L for total phosphorus). Nutrient concentrations decreased rapidly downstream of the disposal whereas nutrient contents in the sediments increased (up to 1,898 and 784 μg/g for total phosphorus and Kjeldahl nitrogen, respectively). According to an in situ experiment using sediment boxes placed in the riverbed for 85 days, we estimated that the proportion of nutrients trapped in the sediments represents 25% (respectively 10%) of phosphorus (respectively nitrogen) loads lost from the water column. In parallel, laboratory tests indicated that denitrification occurred in the Vène River, and we estimated that denitrification likely coupled to nitrification processes during the 85 days of the experiment was significantly involved in the removal of nitrogen loads (up to 38%) from the water column and was greater than accumulation processes.

Enhancing the aggressive intensity of hydrodynamic cavitation through a Venturi tube by increasing the pressure in the region where the bubbles collapse

NASA Astrophysics Data System (ADS)

Soyama, H.; Hoshino, J.

2016-04-01

In this paper, we used a Venturi tube for generating hydrodynamic cavitation, and in order to obtain the optimum conditions for this to be used in chemical processes, the relationship between the aggressive intensity of the cavitation and the downstream pressure where the cavitation bubbles collapse was investigated. The acoustic power and the luminescence induced by the bubbles collapsing were investigated under various cavitating conditions, and the relationships between these and the cavitation number, which depends on the upstream pressure, the downstream pressure at the throat of the tube and the vapor pressure of the test water, was found. It was shown that the optimum downstream pressure, i.e., the pressure in the region where the bubbles collapse, increased the aggressive intensity by a factor of about 100 compared to atmospheric pressure without the need to increase the input power. Although the optimum downstream pressure varied with the upstream pressure, the cavitation number giving the optimum conditions was constant for all upstream pressures.

Endogenous peptide profile for elucidating biosynthetic processing of the ghrelin precursor.

PubMed

Tsuchiya, Takashi; Iwakura, Hiroshi; Minamino, Naoto; Kangawa, Kenji; Sasaki, Kazuki

2017-09-02

Ghrelin is an orexigenic peptide primarily produced by gastric endocrine cells. The biosynthetic cleavage site of ghrelin has been well documented, but how its downstream region undergoes proteolytic processing remains poorly explored. Here, we provide the first snapshot of endogenous peptides from the ghrelin precursor by profiling the secretopeptidome of cultured mouse ghrelin-producing cells during exocytosis. Mapping of MS/MS sequenced peptides to the precursor highlighted three atypical monobasic processing sites, including the established C-terminus of ghrelin and the N-terminal cleavage site for obestatin, a putative 23-amino-acid C-terminally amidated peptide. However, we found that mouse obestatin does not occur in the form originally reported, but that a different amidation site is used to generate a shorter peptide. These data can be extended to study and characterize the precursor-derived peptides located downstream of ghrelin in different biological contexts. Copyright © 2017 Elsevier Inc. All rights reserved.

Creative PDB`s (parts databases)

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

Cote, T.J.

1998-12-31

PDB component property entries and creative picklists can make the schematic entry process and downstream tools such as BOM generation more useful. This presentation will show how creative PDB`s can enhance the design process. Examples of PDB entries developed at Los Alamos National Laboratory will be discussed.

Investigation of Particle Sampling Bias in the Shear Flow Field Downstream of a Backward Facing Step

NASA Technical Reports Server (NTRS)

Meyers, James F.; Kjelgaard, Scott O.; Hepner, Timothy E.

1990-01-01

The flow field about a backward facing step was investigated to determine the characteristics of particle sampling bias in the various flow phenomena. The investigation used the calculation of the velocity:data rate correlation coefficient as a measure of statistical dependence and thus the degree of velocity bias. While the investigation found negligible dependence within the free stream region, increased dependence was found within the boundary and shear layers. Full classic correction techniques over-compensated the data since the dependence was weak, even in the boundary layer and shear regions. The paper emphasizes the necessity to determine the degree of particle sampling bias for each measurement ensemble and not use generalized assumptions to correct the data. Further, it recommends the calculation of the velocity:data rate correlation coefficient become a standard statistical calculation in the analysis of all laser velocimeter data.

Analysis of two-equation turbulence models for recirculating flows

NASA Technical Reports Server (NTRS)

Thangam, S.

1991-01-01

The two-equation kappa-epsilon model is used to analyze turbulent separated flow past a backward-facing step. It is shown that if the model constraints are modified to be consistent with the accepted energy decay rate for isotropic turbulence, the dominant features of the flow field, namely the size of the separation bubble and the streamwise component of the mean velocity, can be accurately predicted. In addition, except in the vicinity of the step, very good predictions for the turbulent shear stress, the wall pressure, and the wall shear stress are obtained. The model is also shown to provide good predictions for the turbulence intensity in the region downstream of the reattachment point. Estimated long time growth rates for the turbulent kinetic energy and dissipation rate of homogeneous shear flow are utilized to develop an optimal set of constants for the two equation kappa-epsilon model. The physical implications of the model performance are also discussed.

Velocity-Field Measurements of an Axisymmetric Separated Flow Subjected to Amplitude-Modulated Excitation

NASA Technical Reports Server (NTRS)

Trosin, Barry James

2007-01-01

Active flow control was applied at the point of separation of an axisymmetric, backward-facing-step flow. The control was implemented by employing a Helmholtz resonator that was externally driven by an amplitude-modulated, acoustic disturbance from a speaker located upstream of the wind tunnel. The velocity field of the separating/reattaching flow region downstream of the step was characterized using hotwire velocity measurements with and without flow control. Conventional statistics of the data reveal that the separating/reattaching flow is affected by the imposed forcing. Triple decomposition along with conditional averaging was used to distinguish periodic disturbances from random turbulence in the fluctuating velocity component. A significant outcome of the present study is that it demonstrates that amplitude-modulated forcing of the separated flow alters the flow in the same manner as the more conventional method of periodic excitation.

Sources, spatial variation, and speciation of heavy metals in sediments of the Tamagawa River in Central Japan.

PubMed

Shikazono, N; Tatewaki, K; Mohiuddin, K M; Nakano, T; Zakir, H M

2012-01-01

Sediments of the Tamagawa River in central Japan were studied to explain the spatial variation, to identify the sources of heavy metals, and to evaluate the anthropogenic influence on these pollutants in the river. Sediment samples were collected from 20 sites along the river (five upstream, four midstream, and 11 downstream). Heavy metal concentrations, viz. chromium, nickel, copper, zinc, lead, cadmium, and molybdenum, in the samples were measured using inductively coupled plasma-mass spectroscopy. The chemical speciations of heavy metals in the sediments were identified by the widely used five-step Hall method. Lead isotopes were analyzed to identify what portion is contributed by anthropogenic sources. The total heavy metal concentrations were compared with global averages for continental crust (shale) and average values for Japanese river sediments. The mean heavy metal concentrations were higher in downstream sediments than in upstream and midstream samples, and the concentrations in the silt samples were higher than those in the sand samples. Speciation results demonstrate that, for chromium and nickel, the residual fractions were dominant. These findings imply that the influence of anthropogenic chromium and nickel contamination is negligible, while copper, zinc, and lead were mostly extracted in the non-residual fraction (metals in adsorbed/exchangeable/carbonate forms or bound to amorphous Fe oxyhydroxides, crystalline Fe oxides, or organic matter), indicating that these elements have high chemical mobility. The proportion of lead (Pb) isotopes in the downstream silt samples indicates that Pb accumulation is primarily derived from anthropogenic sources.

Design methodology for integrated downstream separation systems in an ethanol biorefinery

NASA Astrophysics Data System (ADS)

Mohammadzadeh Rohani, Navid

Energy security and environmental concerns have been the main drivers for a historic shift to biofuel production in transportation fuel industry. Biofuels should not only offer environmental advantages over the petroleum fuels they replace but also should be economically sustainable and viable. The so-called second generation biofuels such as ethanol which is the most produced biofuel are mostly derived from lignocellulosic biomasses. These biofuels are more difficult to produce than the first generation ones mainly due to recalcitrance of the feedstocks in extracting their sugar contents. Costly pre-treatment and fractionation stages are required to break down lignocellulosic feedstocks into their constituent elements. On the other hand the mixture produced in fermentation step in a biorefinery contains very low amount of product which makes the subsequent separation step more difficult and more energy consuming. In an ethanol biorefinery, the dilute fermentation broth requires huge operating cost in downstream separation for recovery of the product in a conventional distillation technique. Moreover, the non-ideal nature of ethanol-water mixture which forms an iseotrope at almost 95 wt%, hinders the attainment of the fuel grade ethanol (99.5 wt%). Therefore, an additional dehydration stage is necessary to purify the ethanol from its azeotropic composition to fuel-grade purity. In order to overcome the constraint pertaining to vapor-liquid equilibrium of ethanol-water separation, several techniques have been investigated and proposed in the industry. These techniques such as membrane-based technologies, extraction and etc. have not only sought to produce a pure fuel-grade ethanol but have also aimed at decreasing the energy consumption of this energy-intensive separation. Decreasing the energy consumption of an ethanol biorefinery is of paramount importance in improving its overall economics and in facilitating the way to displacing petroleum transportation fuel and obtaining energy security. On the other hand, Process Integration (PI) as defined by Natural Resource Canada as the combination of activities which aim at improving process systems, their unit operations and their interactions in order to maximize the efficiency of using water, energy and raw materials can also help biorefineries lower their energy consumptions and improve their economics. Energy integration techniques such as pinch analysis adopted by different industries over the years have ensured using heat sources within a plant to supply the demand internally and decrease the external utility consumption. Therefore, adopting energy integration can be one of the ways biorefinery technology owners can consider in their process development as well as their business model in order to improve their overall economics. The objective of this thesis is to propose a methodology for designing integrated downstream separation in a biorefinery. This methodology is tested in an ethanol biorefinery case study. Several alternative separation techniques are evaluated in their energy consumption and economics in three different scenarios; stand-alone without energy integration, stand-alone with internal energy integration and integrated-with Kraft. The energy consumptions and capital costs of separation techniques are assessed in each scenario and the cost and benefit of integration are determined and finally the best alternative is found through techno-economic metrics. Another advantage of this methodology is the use of a graphical tool which provides insights on decreasing energy consumption by modifying the process condition. The pivot point of this work is the use of a novel energy integration method called Bridge analysis. This systematic method which originally is intended for retrofit situation is used here for integration with Kraft process. Integration potentials are identified through this method and savings are presented for each design. In stand-alone with internal integration scenario, the conventional pinch method is used for energy analysis. The results reveal the importance of energy integration in reducing energy consumption. They also show that in an ethanol biorefinery, by adopting energy integration in the conventional distillation separation, we can achieve greater energy saving compared to other alternative techniques. This in turn suggests that new alternative technologies which imply big risks for the company might not be an option for reducing the energy consumption as long as an internal and external integration is incorporated in the business model of an ethanol biorefinery. It is also noteworthy that the methodology developed in this work can be extended as a future work to include a whole biorefinery system. (Abstract shortened by UMI.).

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

Tan, Eric C. D.; Talmadge, Michael; Dutta, Abhijit

This report was developed as part of the U.S. Department of Energy’s Bioenergy Technologies Office’s (BETO’s) efforts to enable the development of technologies for the production of infrastructure-compatible, cost-competitive liquid hydrocarbon fuels from lignocellulosic biomass feedstocks. The research funded by BETO is designed to advance the state of technology of biomass feedstock supply and logistics, conversion, and overall system sustainability. It is expected that these research improvements will be made within the 2022 timeframe. As part of their involvement in this research and development effort, the National Renewable Energy Laboratory and the Pacific Northwest National Laboratory investigate the economics ofmore » conversion pathways through the development of conceptual biorefinery process models and techno-economic analysis models. This report describes in detail one potential conversion process for the production of high-octane gasoline blendstock via indirect liquefaction of biomass. The processing steps of this pathway include the conversion of biomass to synthesis gas or syngas via indirect gasification, gas cleanup, catalytic conversion of syngas to methanol intermediate, methanol dehydration to dimethyl ether (DME), and catalytic conversion of DME to high-octane, gasoline-range hydrocarbon blendstock product. The conversion process configuration leverages technologies previously advanced by research funded by BETO and demonstrated in 2012 with the production of mixed alcohols from biomass. Biomass-derived syngas cleanup via reforming of tars and other hydrocarbons is one of the key technology advancements realized as part of this prior research and 2012 demonstrations. The process described in this report evaluates a new technology area for the downstream utilization of clean biomass-derived syngas for the production of high-octane hydrocarbon products through methanol and DME intermediates. In this process, methanol undergoes dehydration to DME, which is subsequently converted via homologation reactions to high-octane, gasoline-range hydrocarbon products.« less

Temperature, Genes, and Sex: a Comparative View of Sex Determination in Trachemys scripta and Mus musculus

PubMed Central

Yao, Humphrey H-C; Capel, Blanche

2014-01-01

Sex determination, the step at which differentiation of males and females is initiated in the embryo, is of central importance to the propagation of species. There is a remarkable diversity of mechanisms by which sex determination is accomplished. In general these mechanisms fall into two categories: Genetic Sex Determination (GSD), which depends on genetic differences between the sexes, and Environmental Sex Determination (ESD), which depends on extrinsic cues. In this review we will consider these two means of determining sex with particular emphasis on two species: a species that depends on GSD, Mus musculus, and a species that depends on ESD, Trachemys scripta. Because the structural organization of the adult testis and ovary is very similar across vertebrates, most biologists had expected that the pathways downstream of the sex-determining switch would be conserved. However, emerging data indicate that not only are the initial sex determining mechanisms different, but the downstream pathways and morphogenetic events leading to the development of a testis or ovary also are different. PMID:16046442

Simulation of stream discharge and transport of nitrate and selected herbicides in the Mississippi River Basin

NASA Astrophysics Data System (ADS)

Broshears, Robert E.; Clark, Gregory M.; Jobson, Harvey E.

2001-05-01

Stream discharge and the transport of nitrate, atrazine, and metolachlor in the Mississippi River Basin were simulated using the DAFLOW/BLTM hydrologic model. The simulated domain for stream discharge included river reaches downstream from the following stations in the National Stream Quality Accounting Network: Mississippi River at Clinton, IA; Missouri River at Hermann, MO; Ohio River at Grand Chain, IL; and Arkansas River at Little Rock, AR. Coefficients of hydraulic geometry were calibrated using data from water year 1996; the model was validated by favourable simulation of observed discharges in water years 1992-1994. The transport of nitrate, atrazine, and metolachlor was simulated downstream from the Mississippi River at Thebes, IL, and the Ohio River at Grand Chain. Simulated concentrations compared favourably with observed concentrations at Baton Rouge, LA. Development of this model is a preliminary step in gaining a more quantitative understanding of the sources and fate of nutrients and pesticides delivered from the Mississippi River Basin to the Gulf of Mexico. Published in 2001 by John Wiley & Sons, Ltd.

Multi-site modulation of flux during monolignol formation in loblolly pine (Pinus taeda)

NASA Technical Reports Server (NTRS)

Anterola, A. M.; van Rensburg, H.; van Heerden, P. S.; Davin, L. B.; Lewis, N. G.

1999-01-01

Loblolly pine (Pinus taeda L.) cell suspension cultures secrete monolignols when placed in 8% sucrose/20 mM KI solution, and these were used to identify phenylpropanoid pathway flux-modulating steps. When cells were provided with increasing amounts of either phenylalanine (Phe) or cinnamic acid, cellular concentrations of immediate downstream products (cinnamic and p-coumaric acids, respectively) increased, whereas caffeic and ferulic acid pool sizes were essentially unaffected. Increasing Phe concentrations resulted in increased amounts of p-coumaryl alcohol relative to coniferyl alcohol. However, exogenously supplied cinnamic, p-coumaric, caffeic, and ferulic acids resulted only in increases in their intercellular concentrations, but not that of downstream cinnamyl aldehydes and monolignols. Supplying p-coumaryl and coniferyl aldehydes up to 40, 000-320,000-fold above the detection limits resulted in rapid, quantitative conversion into the monolignols. Only at nonphysiological concentrations was transient accumulation of intracellular aldehydes observed. These results indicate that cinnamic and p-coumaric acid hydroxylations assume important regulatory positions in phenylpropanoid metabolism, whereas cinnamyl aldehyde reduction does not serve as a control point. Copyright 1999 Academic Press.

High resolution morphobathymetric analysis and short-term evolution of the upper part of the Capbreton submarine canyon (south-east Bay of Biscay - French Atlantic coast)

NASA Astrophysics Data System (ADS)

Gillet, Hervé; Mazières, Alaïs; Mulder, Thierry; Cremer, Michel

2013-04-01

The Capbreton Canyon stands out by its deep incision through continental shelf and slope and its present turbidite activity. The head of the canyon is anthropically disconnected from the Adour River since 1310 AD, but is located close enough to the coast to allow a direct supply by longshore drift. Sedimentary processes in upper part of the Capbreton Canyon are poorly documented. Several evidences, including sandy slide scars in the head, suggest that this area plays a major role in triggering downstream gravity currents). However, no modern sedimentary activity in the upper canyon had so far been evidenced. Our study is based on the analysis and comparison of several sets of multibeam bathymetric data acquired in 1998, 2010 and 2012 (up to 1.5 m resolution). The morphobathymetric analysis brought the following key observations: - The upper part of the canyon is characterised by a meandering talweg underlined by two kinds of terraces: (1) small elongated terraces standing only 10 to 15 m above the talweg axis and (2) large terraces standing 45 to 100 m above the talweg axis. - The regular 1° longitudinal slope of the talweg is interrupted by several 10 m high knickpoints. - The floor of the talweg shows some rough areas scattered with transversal bedforms similar to the sediment waves described in the Monterey Canyon upper part (Smith et al, 2005). The morphological evolutions in the upper part of the canyon over the last 14 years especially affect the floor of the talweg: - Between 1998 and 2010, we observe a downstream succession of accretion areas (up to 11m thick) and erosion areas (reaching -25 m). The largest and highest terraces remain stable over this period, whereas the smallest and lowest elongated terraces show active sedimentation (+5 to +8 m). - Difference between 2010 and 2012 DEMs reveals three localized erosion spots corresponding to 200 m backward stepping of the knickpoints. Such observation confirms the active headward erosion in this part of the canyon. - Conversely, the flanks of this part of the canyon do not show significant evolution. We did not observe any large lateral slide such as the canyon flank collapse recently recognised in the upper part of the Monterey or Cap Lopez canyons. (1)Since the lateral sediment supply in the canyon seems to be limited (no significant evolution of the canyon wall), we consider that most of the sediments deposited in this area is supplied from the canyon head. (2)We propose that the lowest elongated terraces are the remnant of sandy slides confined in the upper talweg and later overdeepened by the regressive erosion. This process contrasts with the downstream part of the canyon, where the terraces are constructed by the spilling of turbidity current. (3)These results are consistent with the process evidenced in the head of the canyon and support the assumption that the turbidite processes in modern canyons are related to sandy mass sliding from the head of the canyon.

Wildfire effects on source-water quality--Lessons from Fourmile Canyon fire, Colorado, and implications for drinking-water treatment

USGS Publications Warehouse

Writer, Jeffrey H.; Murphy, Sheila F.

2012-01-01

Forested watersheds provide high-quality source water for many communities in the western United States. These watersheds are vulnerable to wildfires, and wildfire size, fire severity, and length of fire season have increased since the middle 1980s (Westerling and others, 2006). Burned watersheds are prone to increased flooding and erosion, which can impair water-supply reservoirs, water quality, and drinking-water treatment processes. Limited information exists on the degree, timing, and duration of the effects of wildfire on water quality, making it difficult for drinking-water providers to evaluate the risk and develop management options. In order to evaluate the effects of wildfire on water quality and downstream ecosystems in the Colorado Front Range, the U.S. Geological Survey initiated a study after the 2010 Fourmile Canyon fire near Boulder, Colorado. Hydrologists frequently sampled Fourmile Creek at monitoring sites upstream and downstream of the burned area to study water-quality changes during hydrologic conditions such as base flow, spring snowmelt, and summer thunderstorms. This fact sheet summarizes principal findings from the first year of research. Stream discharge and nitrate concentrations increased downstream of the burned area during snowmelt runoff, but increases were probably within the treatment capacity of most drinking-water plants, and limited changes were observed in downstream ecosystems. During and after high-intensity thunderstorms, however, turbidity, dissolved organic carbon, nitrate, and some metals increased by 1 to 4 orders of magnitude within and downstream of the burned area. Increases of such magnitude can pose problems for water-supply reservoirs, drinking-water treatment plants, and downstream aquatic ecosystems.

Rhizosecretion improves the production of Cyanovirin-N in Nicotiana tabacum through simplified downstream processing.

PubMed

Madeira, Luisa M; Szeto, Tim H; Ma, Julian K-C; Drake, Pascal M W

2016-07-01

Rhizosecretion has many advantages for the production of recombinant pharmaceuticals, notably facile downstream processing from hydroponic medium. The aim of this study was to increase yields of the HIV microbicide candidate, Cyanovirin-N (CV-N), obtained using this production platform and to develop a simplified methodology for its downstream processing from hydroponic medium. Placing hydroponic cultures on an orbital shaker more than doubled the concentration of CV-N in the hydroponic medium compared to plants which remained stationary, reaching a maximum of approximately 20μg/ml in one week, which is more than 3 times higher than previously reported yields. The protein composition of the hydroponic medium, the rhizosecretome, was characterised in plants cultured with or without the plant growth regulator alpha-napthaleneacetic acid by LC-ESI-MS/MS, and CV-N was the most abundant protein. The issue of large volumes in the rhizosecretion system was addressed by using ion exchange chromatography to concentrate CV-N and partially remove impurities. The semi-purified CV-N was demonstrated to bind to HIV gp120 in an ELISA and to neutralise HIVBa-L with an IC50 of 6nM in a cell-based assay. Rhizosecretion is therefore a practicable and inexpensive method for the production of functional CV-N. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Static Longitudinal Stability of a Rocket Vehicle Having a Rear-Facing Step Ahead of the Stabilizing Fins

NASA Technical Reports Server (NTRS)

Keynton, Robert J.

1961-01-01

Tests were conducted at Mach numbers of 3.96 and 4.65 in the Langley Unitary Plan wind tunnel to determine the static longitudinal stability characteristics of a fin-stabilized rocket-vehicle configuration which had a rearward facing step located upstream of the fins. Two fin sizes and planforms, a delta and a clipped delta, were tested. The angle of attack was varied from 6 deg to -6 deg and the Reynolds number based on model 6 length was about 10 x 10. The configuration with the larger fins (clipped delta) had a center of pressure slightly rearward of and an initial normal-force-curve slope slightly higher than that of the configuration with the smaller fins (delta) as would be expected. Calculations of the stability parameters gave a slightly lower initial slope of the normal-force curve than measured data, probably because of boundary-layer separation ahead of the step. The calculated center of pressure agreed well with the measured data. Measured and calculated increments in the initial slope of the normal-force curve and in the center of pressure, due to changing fins, were in excellent agreement indicating that separated flow downstream of the step did not influence flow over the fins. This result was consistent with data from schlieren photographs.

Structure-guided Mutational Analysis of the Nucleotidyltransferase Domain of Escherichia coli DNA Ligase (LigA).

PubMed

Wang, Li Kai; Zhu, Hui; Shuman, Stewart

2009-03-27

NAD(+)-dependent DNA ligases (LigA) are ubiquitous in bacteria, where they are essential for growth and present attractive targets for antimicrobial drug discovery. LigA has a distinctive modular structure in which a nucleotidyltransferase catalytic domain is flanked by an upstream NMN-binding module and by downstream OB-fold, zinc finger, helix-hairpin-helix, and BRCT domains. Here we conducted a structure-function analysis of the nucleotidyltransferase domain of Escherichia coli LigA, guided by the crystal structure of the LigA-DNA-adenylate intermediate. We tested the effects of 29 alanine and conservative mutations at 15 amino acids on ligase activity in vitro and in vivo. We thereby identified essential functional groups that coordinate the reactive phosphates (Arg(136)), contact the AMP adenine (Lys(290)), engage the phosphodiester backbone flanking the nick (Arg(218), Arg(308), Arg(97) plus Arg(101)), or stabilize the active domain fold (Arg(171)). Finer analysis of the mutational effects revealed step-specific functions for Arg(136), which is essential for the reaction of LigA with NAD(+) to form the covalent ligase-AMP intermediate (step 1) and for the transfer of AMP to the nick 5'-PO(4) to form the DNA-adenylate intermediate (step 2) but is dispensable for phosphodiester formation at a preadenylylated nick (step 3).

Impact of pretreatment and downstream processing technologies on economics and energy in cellulosic ethanol production.

PubMed

Kumar, Deepak; Murthy, Ganti S

2011-09-05

While advantages of biofuel have been widely reported, studies also highlight the challenges in large scale production of biofuel. Cost of ethanol and process energy use in cellulosic ethanol plants are dependent on technologies used for conversion of feedstock. Process modeling can aid in identifying techno-economic bottlenecks in a production process. A comprehensive techno-economic analysis was performed for conversion of cellulosic feedstock to ethanol using some of the common pretreatment technologies: dilute acid, dilute alkali, hot water and steam explosion. Detailed process models incorporating feedstock handling, pretreatment, simultaneous saccharification and co-fermentation, ethanol recovery and downstream processing were developed using SuperPro Designer. Tall Fescue (Festuca arundinacea Schreb) was used as a model feedstock. Projected ethanol yields were 252.62, 255.80, 255.27 and 230.23 L/dry metric ton biomass for conversion process using dilute acid, dilute alkali, hot water and steam explosion pretreatment technologies respectively. Price of feedstock and cellulose enzymes were assumed as $50/metric ton and 0.517/kg broth (10% protein in broth, 600 FPU/g protein) respectively. Capital cost of ethanol plants processing 250,000 metric tons of feedstock/year was $1.92, $1.73, $1.72 and $1.70/L ethanol for process using dilute acid, dilute alkali, hot water and steam explosion pretreatment respectively. Ethanol production cost of $0.83, $0.88, $0.81 and $0.85/L ethanol was estimated for production process using dilute acid, dilute alkali, hot water and steam explosion pretreatment respectively. Water use in the production process using dilute acid, dilute alkali, hot water and steam explosion pretreatment was estimated 5.96, 6.07, 5.84 and 4.36 kg/L ethanol respectively. Ethanol price and energy use were highly dependent on process conditions used in the ethanol production plant. Potential for significant ethanol cost reductions exist in increasing pentose fermentation efficiency and reducing biomass and enzyme costs. The results demonstrated the importance of addressing the tradeoffs in capital costs, pretreatment and downstream processing technologies.

Impact of pretreatment and downstream processing technologies on economics and energy in cellulosic ethanol production

PubMed Central

2011-01-01

Background While advantages of biofuel have been widely reported, studies also highlight the challenges in large scale production of biofuel. Cost of ethanol and process energy use in cellulosic ethanol plants are dependent on technologies used for conversion of feedstock. Process modeling can aid in identifying techno-economic bottlenecks in a production process. A comprehensive techno-economic analysis was performed for conversion of cellulosic feedstock to ethanol using some of the common pretreatment technologies: dilute acid, dilute alkali, hot water and steam explosion. Detailed process models incorporating feedstock handling, pretreatment, simultaneous saccharification and co-fermentation, ethanol recovery and downstream processing were developed using SuperPro Designer. Tall Fescue (Festuca arundinacea Schreb) was used as a model feedstock. Results Projected ethanol yields were 252.62, 255.80, 255.27 and 230.23 L/dry metric ton biomass for conversion process using dilute acid, dilute alkali, hot water and steam explosion pretreatment technologies respectively. Price of feedstock and cellulose enzymes were assumed as $50/metric ton and 0.517/kg broth (10% protein in broth, 600 FPU/g protein) respectively. Capital cost of ethanol plants processing 250,000 metric tons of feedstock/year was $1.92, $1.73, $1.72 and $1.70/L ethanol for process using dilute acid, dilute alkali, hot water and steam explosion pretreatment respectively. Ethanol production cost of $0.83, $0.88, $0.81 and $0.85/L ethanol was estimated for production process using dilute acid, dilute alkali, hot water and steam explosion pretreatment respectively. Water use in the production process using dilute acid, dilute alkali, hot water and steam explosion pretreatment was estimated 5.96, 6.07, 5.84 and 4.36 kg/L ethanol respectively. Conclusions Ethanol price and energy use were highly dependent on process conditions used in the ethanol production plant. Potential for significant ethanol cost reductions exist in increasing pentose fermentation efficiency and reducing biomass and enzyme costs. The results demonstrated the importance of addressing the tradeoffs in capital costs, pretreatment and downstream processing technologies. PMID:21892958